Biol 251 exam 3
same. In E. coli, the binding of the 50S ribosomal subunit to produce the intact ribosome forms three functionally important ribosomal sites:
A (aminoacyl) site P (peptidyl) site E (exit) site
The primary stain, which fluoresces green, can penetrate
intact cytoplasmic membranes, staining both live and dead cells.
The three main types of RNA directly involved in protein synthesis are
messenger RNA (mRNA), ribosomal RNA (rRNA), and transfer RNA (tRNA).
Some of the practical applications of the destructive effects of heat on microbes are
sterilization by steam, pasteurization, and incineration of inoculating loops.
In some cases, if the nucleotide in the third position is changed, the same amino acid is
still incorporated.
Types of culture media
1. Defined or synthetic medium 2. Complex media 3. Supportive media 4.Enriched media 5. Selective media 6. Differential media
Base analogs
"Structurally similar to normal nitrogenous bases" Exhibit base pairing properties different from the bases they replace Cause a stable mutation 5-bromouracil (5BU) is a nucleoside analog to thymine
These degerming methods remove most
(but not all) microbes from the skin's surface.
nosocomial
(hospital-acquired) infection
Temperature requirements: low temp
-Affect membranes fluidity -Slow down chemical reactions and diffusion -Proteins become too rigid
A protein sequence consists of
20 commonly occurring amino acids.
Since the first case of infection with human immunodeficiency virus (HIV) was reported in 1981, nearly
40 million people have died from HIV infection,2 the virus that causes acquired immune deficiency syndrome (AIDS).
Today, women still remain underrepresented in many fields of science and medicine. While more than half of the undergraduate degrees in science are awarded to women, only
46% of doctoral degrees in science are awarded to women.
Spontaneous Mutations
May result from errors in DNA replication Transition mutations - Purine to purine or pyrimidine to pyrimidine (common) Transversion mutations - Purine to pyrimidine or pyrimidine to purine (rare)
tube D are indifferent to the presence of oxygen.
The aerotolerant anaerobes do not use oxygen because they usually have a fermentative metabolism, but they are not harmed by the presence of oxygen as obligate anaerobes are.
Radiation
Nonionizing Ultraviolet light Induces the formation of covalent bond between adjacent TpT Alter the structure of DNA DNA polymerase replicate the dimer incorrectly (frameshift or point mutation) Dimers can inhibit progress polymerases and arrest replication
Point Mutations
Nonsense mutations - cause the early termination of translation Result in a shortened polypeptide Phenotype may be more or less severely affected E.g. UGG tryptophan codon to UGA stop codon
The formation of a biofilm essentially involves the
attachment of planktonic cells to a substrate, where they become sessile (attached to a surface).
bactericides kill
bacteria
Because ribosomes read the mRNA in triplet codons, frameshift mutations can
change every amino acid after the point of the mutation. The new reading frame may also include a stop codon before the end of the coding sequence. Consequently, proteins made from genes containing frameshift mutations are nearly always nonfunctional.
The P (peptidyl) site binds
charged tRNAs carrying amino acids that have formed peptide bonds with the growing polypeptide chain but have not yet dissociated from their corresponding tRNA.
The discovery of the enzyme telomerase clarified our understanding of how
chromosome ends are maintained.
In a closed environment, the culture density is also a measure of the
number of cells in the population.
Obligate anaerobes usually lack all
three enzymes.
A prime example of an all-purpose medium is
tryptic soy broth (TSB).
adenine and thymine form
two hydrogen bonds between them,
Mutations are higher in viruses!
~1 in 10,000 nucleotides Low fidelity DNA and or RNA polymerases Lack proof-reading capability Mutations accumulate Reassortment is also common Exchange nucleic acid segments with a different strains of the same virus Human rate ~1 in billion nucleotides Influenza reassortment is the picture
The elongation in transcription phase begins when the
σ subunit dissociates from the polymerase, allowing the core enzyme to synthesize RNA complementary to the DNA template in a 5' to 3' direction at a rate of approximately 40 nucleotides per second.
Five types of histones
H1, H2A, H2B, H3, and H4
Research indicates that the mutation has been present since before
HIV appeared and may have been selected for in European populations as a result of exposure to the plague or smallpox.
This exciting finding has led to new avenues in
HIV research, including looking for drugs to block CCR5 binding to HIV in individuals who lack the mutation.
Effects of Mutations
Impact is readily noticed only if it produces a change in phenotype Wild type - prevalent form of a gene and its associated phenotype Mutant type - altered form of a gene and its associated phenotype Forward mutation Backward mutation
RNA polymerases catalyzes RNA synthesis
In E. coli RNA polymerase comprises six subunit Holoenzyme - ααββ'ω and σ Core enzyme - ααββ'ω σ factor enables RNA polymerase to bind to a promoter
Avery, Macleod, McCarty experiment
Inactivated various components of heat-killed S cells (sugars, proteins, RNA and DNA) They used enzymes that specifically degraded proteins, RNA, and DNA and mixed with R strain to transform the R strain
Formation of the hairpin structures halts the forward moment of the RNA pol Interaction between U-A bonds RNA-DNA are weakest of all base pairs RNA, DNA, and RNA polymerase dissociate, and transcription stops
Intrinsic Termination Base pair strength of interaction G:C strongest A:T moderate A:U weakest
RNAs play important role in protein synthesis mRNA
Jacob and Monod suggested the existence of intermediate molecule during the protein synthesis - mRNA mRNA carries the message from the DNA Contains complementary bases to the genes on DNA mRNA is relatively unstable and short-lived in the cell
Okazaki fragments are named after the
Japanese research team and married couple Reiji and Tsuneko Okazaki, who first discovered them in 1966.
listeriosis, an infection caused by
L. monocytogenes. Listeriosis is a serious infection with a 20% mortality rate and is a particular risk to a women's fetus.
Hans and Zacharias Janssen
Late 15th or early 16th century Invented the telescope, the simple microscope, and the compound microscope during the late 1500s or early 1600s Secretive about their work and never published
Mutations
Latin mutare - to change Stable, heritable alterations in DNA sequence Repair mechanisms correct for any errors Some remains uncorrected Provides opportunity for the evolutionary changes
Mendel proposed two general rules of inheritance
Law of Dominance and Law of Segregation
Two well known experiments that demonstrate the importance of mutation and natural selection in the evolution of bacteria
Lederberg Experiment - 1952 Lenski Experiment - 1988 to present
Natural Selection - Lederberg Experiment
Lederberg investigated whether mutations occurred before or after natural selection (in absence of selection force) Bacteria were grown on agar plates without an antibiotic Colonies were transferred via replica plating to a medium with an antibiotic = natural selection
An example of a selective medium is
MacConkey agar. It contains bile salts and crystal violet, which interfere with the growth of many gram-positive bacteria and favor the growth of gram-negative bacteria, particularly the Enterobacteriaceae. These species are commonly named enterics, reside in the intestine, and are adapted to the presence of bile salts.
Microorganisms require a balanced mixture of nutrients
Macroelements and Micronutrients
Life in extreme environments raises fascinating questions about the adaptation of macromolecules and metabolic processes. Very low temperatures affect cells in many ways such as
Membranes lose their fluidity and are damaged by ice crystal formation. Chemical reactions and diffusion slow considerably. Proteins become too rigid to catalyze reactions and may undergo denaturation.
Streak Plate Technique
Microbial mixture is transferred onto the edge of an agar plate with an inoculating loop and then streaked out over the surface in one of several patterns
Coulter Counter
Microbial suspension is forced through a small hole or orifice Every time a microbial cell passes through the orifice, electrical resistance increases (or the conductivity drops) and the cell is counted
Mismatch Repair
Mismatch repair system help to remove mismatches It make use of differences in methylation state of old and new DNA strand DNA is hemimethylated (parental strand is methylated; new strand is not)
Point Mutations
Missense mutations - Changes a codon for one amino acid into a codon for another. E.g. GAGà glutamic acid; changed to GUG à valine Effect: range from complete loss of activity to no change at all
Spread Plate Method
Mixture of cells is spread out on an agar surface at a relatively low density, every cell grows into a separate colony
Replica plate technique developed by Joshua Lederberg (1925-2008)
Molecular biologist Celebrity of bacterial genetics! Novel contribution: Transduction, Conjugation Replica plating
insertion
Mutations also result from the addition of one or more bases,
Most pathogens grow rapidly, like E. coli, but there are exceptions. For example,
Mycobacterium tuberculosis, the causative agent of tuberculosis, has a generation time of between 15 and 20 hours. On the other hand, M. leprae, which causes Hansen's disease (leprosy), grows much more slowly, with a doubling time of 14 days.
binary fission first step
Next, the replication of DNA starts at a location on the circular chromosome called the origin of replication, where the chromosome is attached to the inner cell membrane.
In our example, we used one cell as the initial number of cells. For any number of starting cells, the formula is adapted as follows:
Nn=N02^n Nn is the number of cells at any generation n, N0 is the initial number of cells, and n is the number of generations.
Point Mutations
Occurs at only one nucleotide in the gene Silent mutations - Nucleotide in codon changes, no change in amino acid encoded by that codon E.g. CGU changed to CGC both codes for arginine No change in protein or phenotype
Termination of transcription
Occurs at the end of the gene (terminator sequence) Core RNA polymerase dissociates from the template DNA Newly synthesized RNA is released DNA duplex is restored Can happen by either of the two mechanisms, depending on the gene being transcribed - 1.Intrinsic Termination or 2.rho-dependent termination
Biochemical mutations
Often inactivate a biosynthetic pathway Mutant fail to make an essential macromolecule such as an amino acid or nucleotide Wild-type: Prototroph Mutant: Auxotroph Conditional phenotype; unable to grow on medium lacking that molecule, but grows when it is provided
These steps produce small DNA sequence fragments known as
Okazaki fragments, each separated by RNA primer.
Foundation of genetics - Mendel's monohybrid cross
Only one of the parental traits was expressed in the F1 generation At the F2 stage, both the traits were expressed in the proportion 3:1 Similar observations were made for other pairs of traits (Seed shape, seed color, pod shape, pod color, flower position, stem length)
Antiparallel nature of DNA helix creates another problem
Only one strand is synthesized continuously (leading strand) Other strand is synthesized discontinuously (lagging strand) Lagging strand synthesis requires priming action for each fragment referred to as Okazaki fragments
Microbes uses different transport mechanisms
Passive Diffusion and Facilitated Diffusion
Elucidation of DNA structure James Watson and Francis Crick (team Cambridge)
Performed no experiments! Used data from Chargaff and Franklin's study. Build model
Known volumes are vacuum-filtered aseptically through a membrane with a pore size small enough to trap microorganisms. The membrane is transferred to a
Petri plate containing an appropriate growth medium. Colonies are counted after incubation. Calculation of the cell density is made by dividing the cell count by the volume of filtered liquid.
The calibrated slide is called a
Petroff-Hausser chamber and is similar to a hemocytometer used to count red blood cells.
Termination of translation
Protein synthesis stops when the ribosome reaches one of three nonsense codons—UAA, UAG, and UGA These codons don't code for amino acids, any of the three 'release factors' enter the A site instead of tRNA Small and large ribosomal subunits dissociate from the mRNA
Petroff-Hausser chamber can be used for direct counting of microbes
Provide an estimate of the total number of cells The number of microorganisms in a sample can be calculated by considering the chamber's volume and any sample dilution
Microbe usually grow as mixed populations in natural habitats
Pure culture techniques allow separation of microbes from mixed populations
Comparison of DNA and RNA polymerase: RNA polymerase
RNA polymerase Synthesizes the RNA Functions during the gene expression process Adds ribose nucleotides A, U, G and C Produce single-stranded RNA molecule Can added nucleotide directly on the template DNA Speed - ~40 nucleotides per second Copies small parts on chromosome (genes) Can unwind the DNA on its own Relatively higher error rate
Eukaryotic transcription occurs in nucleus
RNA polymerase II that transcribes mRNA genes requires extra transcription factors to recognize its promoter Eukaryotic promoters more complex - the TATA box (30 bases upstream), and the GC and CAAT boxes located between 50 to 100 bases Additional general transcription factors, promoter specific factors, and promoter elements may be needed for transcription
Eukaryotes use three different polymerases,
RNA polymerases I, II, and III, all structurally distinct from the bacterial RNA polymerase. Each transcribes a different subset of genes.
The primer is five to 10 nucleotides long and complementary to the parental or template DNA. It is synthesized by
RNA primase, which is an RNA polymerase. Unlike DNA polymerases, RNA polymerases do not need a free 3'-OH group to synthesize an RNA molecule.
The process of removing intron-encoded RNA sequences and reconnecting those encoded by exons is called____ and is facilitated by the action of a
RNA splicing spliceosome containing small nuclear ribonucleo proteins (snRNPs).
Although polyribosomes also form in eukaryotes, they cannot do so until
RNA synthesis is complete and the RNA molecule has been modified and transported out of the nucleus.
Cells access the information stored in DNA by creating
RNA to direct the synthesis of proteins through the process of translation.
During the process of transcription, the information encoded within the DNA sequence of one or more genes is transcribed into a strand of RNA, also called an
RNA transcript
Mutation: Physical agents
Radiation
Proofreading
Removal of a mismatched base immediately after it has been added Polymerization: 5' à 3' Proofreading: 3' à 5' DNA polymerase uses exonuclease activity for proofreading
binary fission step two
Replication continues in opposite directions along the chromosome until the terminus is reached
B form of DNA is most common form
Right-handed helix Diameter = 20 °A One helix turn = 3.4 nm No of bases per turn = ~10 Base pair rotation = 36° Distance between bp = 0.34 nm
Other parameters affecting microbial growth
Salt requirements Barophiles Photoautotrophs
Microbes and viruses in genetic research involved
Search of the genetic material which was narrowed down to chromosome
Point Mutations
Silent mutations Missense mutations nonsense mutations
examples of ROS
Singlet oxygen (O2•), superoxide (O2−),(O2−), peroxides (H2O2), hydroxyl radical (OH•), and hypochlorite ion (OCl−), the active ingredient of household bleach,
Complex media
Some ingredients of unknown chemical composition. E.g. Nutrient broth, tryptic soy broth, and MacConkey agar
Enriched media
Special nutrients added media. E.g. blood agar
acidophiles. exmples
Species of the archaean genus Ferroplasma live in acid mine drainage at pH values of 0-2.9. Lactobacillus bacteria, which are an important part of the normal microbiota of the vagina, can tolerate acidic environments at pH values 3.5-6.8 and also contribute to the acidity of the vagina (pH of 4, except at the onset of menstruation) through their metabolic production of lactic acid. The vagina's acidity plays an important role in inhibiting other microbes that are less tolerant of acidity.
Mutations Occur in one of two ways:
Spontaneous mutations - arise occasionally in all cells and occur in the absence of any added agent Induced mutations - exposure to a mutagen (physical or chemical agent)
Radiation
Strong ionizing radiation (X-rays and gamma rays) - single- and double-stranded breaks in DNA Ionizing radiations can also modify bases (E.g. deamination of cytosine to uracil Uses - kill microbes to sterilize medical devices and foods
Supportive media
Support the growth of many microorganisms. E.g. Tryptic broth
RNA Transcription
Synthesis of RNA under the direction of DNA - RNA transcript RNA polymerase catalyzes transcription process Bacteria use the same RNA polymerase to transcribe all their genes (mRNA, rRNA, & tRNA) Eukaryotes have multiple RNA polymerase! RNA polymerase I - rRNA RNA polymerase II - mRNA RNA polymerase III - tRNA
The particular bacteriophage they(Hershey and Chase) were studying was the
T2 bacteriophage, which infects E. coli cells. As we now know today, T2 attaches to the surface of the bacterial cell and then it injects its nucleic acids inside the cell. The phage DNA makes multiple copies of itself using the host machinery, and eventually the host cell bursts, releasing a large number of bacteriophages.
In most cases, promoters are located just upstream of the genes they regulate. Although promoter sequences vary among bacterial genomes, a few elements are conserved. At the -10 and -35 positions within the DNA prior to the initiation site (designated +1), there are two promoter consensus sequences, or regions that are similar across all promoters and across various bacterial species. The -10 consensus sequence, called the
TATA box, is TATAAT. The -35 sequence is recognized and bound by σ.
Eukaryotic Pre-RNA is modified in various ways
The RNA pol II produce primary transcript - pre-mRNA A special 7-methylguanosine nucleotide, called the 5' cap, is added to the 5' end of the growing transcript A string of approximately 200 adenine nucleotides to the 3' end, called the poly-A tail Non-coding intron are removed, and exons are spliced together - RNA splicing (occurs in spliceosome)
Can there be life without oxygen?
The answer is that molecular oxygen (O2) is not always needed. The earliest signs of life are dated to a period when conditions on earth were highly reducing and free oxygen gas was essentially nonexistent.
binary fission step three
The center of the enlarged cell constricts until two daughter cells are formed, each offspring receiving a complete copy of the parental genome and a division of the cytoplasm (cytokinesis). This process of cytokinesis and cell division is directed by a protein called FtsZ.
binary fission step six
The divisome activates to produce a peptidoglycan cell wall and build a septum that divides the two daughter cells. The daughter cells are separated by the division septum, where all of the cells' outer layers (the cell wall and outer membranes, if present) must be remodeled to complete division. For example, we know that specific enzymes break bonds between the monomers in peptidoglycans and allow addition of new subunits along the division septum.
Most prokaryotes reproduce by binary fission
The entire process is very complex Studied in very few representatives Still poorly understood 1.Chromosome replication and partitioning 2.Cytokinesis
Two factors heavily influence the level of cleanliness required for a particular fomite and, hence, the protocol chosen to achieve this level.
The first factor is the application for which the item will be used. For example, invasive applications that require insertion into the human body require a much higher level of cleanliness than applications that do not. The second factor is the level of resistance to antimicrobial treatment by potential pathogens. For example, foods preserved by canning often become contaminated with the bacterium Clostridium botulinum, which produces the neurotoxin that causes botulism.
In eukaryotes, initiation complex formation is similar, with the following differences:
The initiator tRNA is a different specialized tRNA carrying methionine, called Met-tRNAi Instead of binding to the mRNA at the Shine-Dalgarno sequence, the eukaryotic initiation complex recognizes the 5' cap of the eukaryotic mRNA, then tracks along the mRNA in the 5' to 3' direction until the AUG start codon is recognized. At this point, the 60S subunit binds to the complex of Met-tRNAi, mRNA, and the 40S subunit.
Lederberg Experiment
The majority of the colonies did not grow on the medium with penicillin Colonies X & Y are derived from cells that contained one or more mutations that conferred resistance to penicillin Lederberg also washed the original plate with penicillin to make sure that the colonies were resistant before the transfer Investigate mutation, change in phenotype in a long-term evolution experiment Starting in 1988, E. coli strains were grown in minimal medium containing 139 μM glucose and 1,700 μM citrate (required for iron acquisition and not a carbon source) Every day 1 ml of the culture was transferred to 99 ml of fresh medium (1/100)
Macromolecules in thermophiles and hyperthermophiles show some notable structural differences from what is observed in the mesophiles such as
The ratio of saturated to polyunsaturated lipids increases to limit the fluidity of the cell membranes. Their DNA sequences show a higher proportion of guanine-cytosine nitrogenous bases, which are held together by three hydrogen bonds in contrast to adenine and thymine, which are connected in the double helix by two hydrogen bonds. Additional secondary ionic and covalent bonds, as well as the replacement of key amino acids to stabilize folding, contribute to the resistance of proteins to denaturation.
Natural Selection
There is variation in traits Some beetles are green and some are brown There is differential reproduction Greens tend to get eaten by birds and survive to reproduce less often than brown beetles do There is heredity Surviving brown beetles have brown baby beetles End result More advantageous trait becomes more common in the population. If continues, all individuals in the population will be brown
Examples of thermophiles include
Thermus aquaticus and Geobacillus spp.
Intrinsic Termination
These termination sequences have two characteristic features: 1.A series of U residues in the transcribed RNA and, 2.Preceding a self-complementary region Intrinsic terminator of tryptophan operon in the picture
rho-dependent termination
These terminator lacks a poly-U region Requires a special protein, the rho factor (ρ) - rho-dependent termination rho factor has RNA:DNA helicase activity It causes the polymerase to dissociate from the mRNA
In 1944, Oswald Avery, Colin MacLeod, and Maclyn McCarty were interested in exploring Griffith's transforming principle further.
They isolated the S strain from infected dead mice, heat-killed it, and inactivated various components of the S extract, conducting a systematic elimination study (Figure 10.8). They used enzymes that specifically degraded proteins, RNA, and DNA and mixed the S extract with each of these individual enzymes. Then, they tested each extract/enzyme combination's resulting ability to transform the R strain, as observed by the diffuse growth of the S strain in culture media and confirmed visually by growth on plates. They found that when DNA was degraded, the resulting mixture was no longer able to transform the R strain bacteria, whereas no other enzymatic treatment was able to prevent transformation. This led them to conclude that DNA was the transforming principle. Despite their results, many scientists did not accept their conclusion, instead believing that there were protein contaminants within their extracts.
Mismatch Repair in E. coli
Three proteins MutS, MutL, and MutH 1.MutS detects mismatch 2.MutH cut the nonmethylated strand 3.An exonuclease removes a portion of the strand (including the incorrect nucleotide) 4.DNA pol fills the gap 5.Ligase seals the gap
Repair of Thymine Dimers
Thymine dimers are common (many organisms cannot avoid ultraviolet light) Nucleotide excision repair remove the pyrimidine dimer and replace it with the correct nucleotides A repair enzyme called UvrABC endonuclease removes damaged bases Gap is filled by DNA polymerase I, and ligase joins the fragments Photoreactivation - Enzyme photolyase detect thymine dimers In presence of visible light it breaks thymine dimer and restore A:T base pairing Photoreactivation appears to be present in all organisms
Some have suggested that women do not belong in the laboratory, including Nobel Prize winner
Tim Hunt, whose 2015 public comments suggesting that women are too emotional for science were met with widespread condemnation.
Initiation of transcription
Transcription begins at a promoter RNA polymerase holoenzyme binds to the promoter Next, it unwinds a short region of DNA within the promoter (-10 site, 'TATAAT') RNA polymerase then start adding ribonucleotides and forming a phosphodiester bond between them (~ 10 nucleotides) Sigma factor is released, and elongation complex is formed Sigma factor is required only for initiation!
Asexual reproduction in prokaryotes
Transfer a nearly identical copy of their genetic material to their offspring Horizontal gene transfer (HGT) is the key to the genetic diversity in prokaryotes Occur by the three primary mechanisms 1.Transformation 2.Transduction 3.Conjugation
Structure of RNA
Typically single stranded Forms complex secondary structures due to internal base pairing Presence of ribose sugar Thymine is replaced by Uracil Three types = mRNA, tRNA and rRNA Differs in function
Pyrrolysine uses a different stop codon,
UAG. The incorporation of pyrrolysine requires the pylS gene and a unique transfer RNA (tRNA) with a CUA anticodon.
In the case of selenocysteine, the codon used is
UGA (normally a stop codon).
A wide variety of bacteria may cause
UTIs, which typically occur when bacteria from the lower gastrointestinal tract are introduced to the urinary tract.
Elucidation of DNA structure Morris Wilkins and Rosalind Franklin were apart of the Team King's College
Used x-ray crystallography To obtain DNA structure
Alkaliphile examples
Vibrio cholerae, the pathogenic agent of cholera, grows best at the slightly basic pH of 8.0; it can survive pH values of 11.0 but is inactivated by the acid of the stomach. When it comes to survival at high pH, the bright pink archaean Natronobacterium, found in the soda lakes of the African Rift Valley, may hold the record at a pH of 10.5
As the number of cells increases through the log phase, several factors contribute to a slowing of the growth rate including
Waste products accumulate and nutrients are gradually used up. In addition, gradual depletion of oxygen begins to limit aerobic cell growth. This combination of unfavorable conditions slows and finally stalls population growth.
In a series of experiments, Griffith analyzed the effects of live R, live S, and heat-killed S strains of S. pneumoniae on live mice.
When mice were injected with the live S strain, the mice died. When he injected the mice with the live R strain or the heat-killed S strain, the mice survived. But when he injected the mice with a mixture of live R strain and heat-killed S strain, the mice died. Upon isolating the live bacteria from the dead mouse, he only recovered the S strain of bacteria. When he then injected this isolated S strain into fresh mice, the mice died. Griffith concluded that something had passed from the heat-killed S strain into the live R strain and "transformed" it into the pathogenic S strain; he called this the "transforming principle." These experiments are now famously known as Griffith's transformation experiments.
Hershey-Chase Experiment
Worked on E. coli and T2 phage
Griffith's Transformation experiment
Worked on Streptococcus pneumoniae Conclusion - Transforming principle from dead S cells was transformed to R cells making them turn S. Nature of transforming principle was unknown
One gene - one protein hypothesis George Wells Beadle and Edward Lawrie Tatum
Worked on Neurospora crassa, a mold Used X-rays to induce changes to a sequence of nucleic acids Mated mutant mold and grew them on Complete medium (supplemented with vitamins and amino acids) Minimal medium (lacking supplements) Looked for mutants that grew on a complete medium but not on the minimal medium Conclusion- mutants lack genes to encoded biosynthetic pathways
Biofilms are not restricted to solid surface substrates, however. Almost any surface in a liquid environment containing some minimal nutrients will eventually develop
a biofilm.
In another set of experiments, Hämmerling used two species of Acetabularia that have different cap morphologies, A. crenulata and A. mediterranea (Figure 10.4). He cut the caps from both types of cells and then grafted the stalk from an A. crenulata onto an A. mediterranea foot, and vice versa. Over time, he observed that the grafted cell with the A. crenulata foot and A. mediterranea stalk developed
a cap with the A. crenulata morphology. Conversely, the grafted cell with the A. mediterranea foot and A. crenulata stalk developed a cap with the A. mediterranea morphology. He microscopically confirmed the presence of nuclei in the feet of these cells and attributed the development of these cap morphologies to the nucleus of each grafted cell. Thus, he showed experimentally that the nucleus was the location of genetic material that dictated a cell's properties.
When the cell population reaches a critical threshold (a quorum), these autoinducers initiate
a cascade of reactions that activate genes associated with cellular functions that are beneficial only when the population reaches a critical density. For example, in some pathogens, synthesis of virulence factors only begins when enough cells are present to overwhelm the immune defenses of the host.
The secondary stain, which fluoresces red, can stain
a cell only if the cytoplasmic membrane is considerably damaged. Thus, live cells fluoresce green because they only absorb the green stain, whereas dead cells appear red because the red stain displaces the green stain on their nucleic acids
another type of point mutation, called a nonsense mutation, converts
a codon encoding an amino acid (a sense codon) into a stop codon (a nonsense codon).
Aseptic technique involves
a combination of protocols that collectively maintain sterility, or asepsis, thus preventing contamination of the patient with microbes and infectious agents.
The decrease in light passing through the sample and reaching the detector is associated with
a decrease in percent transmission and increase in absorbance measured by the spectrophotometer.
A missense mutation results in
a different amino acid being incorporated into the resulting polypeptide. The effect of a missense mutation depends on how chemically different the new amino acid is from the wild-type amino acid. The location of the changed amino acid within the protein also is important. For example, if the changed amino acid is part of the enzyme's active site, then the effect of the missense mutation may be significant.
In recent years, scientific interest has been piqued by the discovery of
a few individuals from northern Europe who are resistant to HIV infection.
Prokaryotes and eukaryotes perform fundamentally the same process of transcription, with
a few significant differences
The building blocks of nucleic acids are nucleotides. Nucleotides that compose DNA are called deoxyribonucleotides. The three components of a deoxyribonucleotide are
a five-carbon sugar called deoxyribose, a phosphate group, and a nitrogenous base, a nitrogen-containing ring structure that is responsible for complementary base pairing between nucleic acid strands. The carbon atoms of the five-carbon deoxyribose are numbered 1ʹ, 2ʹ, 3ʹ, 4ʹ, and 5ʹ (1ʹ is read as "one prime"). A nucleoside comprises the five-carbon sugar and nitrogenous base.
There are only a small number of laboratories in the United States and around the world appropriately equipped to work with these agents. In addition to BSL-3 precautions, laboratory workers in BSL-4 facilities must also change their clothing on entering the laboratory, shower on exiting, and decontaminate all material on exiting. While working in the laboratory, they must either wear
a full-body protective suit with a designated air supply or conduct all work within a biological safety cabinet with a high-efficiency particulate air (HEPA)-filtered air supply and a doubly HEPA-filtered exhaust.
BSL-3 laboratories require
a hands-free sink, an eyewash station near the exit, and two sets of self-closing and locking doors at the entrance. These laboratories are equipped with directional airflow, meaning that clean air is pulled through the laboratory from clean areas to potentially contaminated areas. This air cannot be recirculated, so a constant supply of clean air is required.
To eliminate the risk for C. botulinum contamination, commercial food-canning protocols are designed with
a large margin of error. They assume an impossibly large population of endospores (1012 per can) and aim to reduce this population to 1 endospore per can to ensure the safety of canned foods.
During replica plating,
a population of bacterial cells is mutagenized and then plated as individual cells on a complex nutritionally complete plate and allowed to grow into colonies.
The number of serial dilutions is chosen according to
a preliminary estimate of the culture density.
Archaea also have
a single RNA polymerase, it is more similar to RNA Pol II of eukaryotes
Survival at the Low pH of the Stomach Peptic ulcers (or stomach ulcers) are painful sores on the stomach lining. Until the 1980s, they were believed to be caused by spicy foods, stress, or a combination of both. Patients were typically advised to eat bland foods, take anti-acid medications, and avoid stress. These remedies were not particularly effective, and the condition often recurred. This all changed dramatically when the real cause of most peptic ulcers was discovered to be
a slim, corkscrew-shaped bacterium, Helicobacter pylori. This organism was identified and isolated by Barry Marshall and Robin Warren, whose discovery earned them the Nobel Prize in Medicine in 2005.
A few cells, the so-called persisters, are characterized by
a slow metabolic rate.
During the stationary phase, cells switch to
a survival mode of metabolism.
DNA replication models
a)semiconserative model b)conservative model c)dispersive model
Ribosomes are composed of rRNA and protein. As its name suggests, rRNA is a major constituent of ribosomes, composing up to
about 60% of the ribosome by mass and providing the location where the mRNA binds.
Because the rate of killing remains constant even when the population size varies, the percentage killed is more useful information than the
absolute number of microbes killed.
Microorganisms that grow optimally at pH less than 5.55 are called
acidophiles. For example, the sulfur-oxidizing Sulfolobus spp. isolated from sulfur mud fields and hot springs in Yellowstone National Park are extreme acidophiles. These archaea survive at pH values of 2.5-3.5. Acidophilic microorganisms display a number of adaptations to survive in strong acidic environments. For example, proteins show increased negative surface charge that stabilizes them at low pH. Pumps actively eject H+ ions out of the cells. The changes in the composition of membrane phospholipids probably reflect the need to maintain membrane fluidity at low pH.
In the logarithmic (log) growth phase, sometimes called exponential growth phase, the cells are
actively dividing by binary fission and their number increases exponentially.
A fixed volume of the original culture, 1.0 mL, is
added to and thoroughly mixed with the first dilution tube solution, which contains 9.0 mL of sterile broth. This step represents a dilution factor of 10, or 1:10, compared with the original culture. From this first dilution, the same volume, 1.0 mL, is withdrawn and mixed with a fresh tube of 9.0 mL of dilution solution. The dilution factor is now 1:100 compared with the original culture. This process continues until a series of dilutions is produced that will bracket the desired cell concentration for accurate counting.
In E. coli, RNA polymerase comprises six polypeptide subunits, five of which compose the polymerase core enzyme responsible for
adding RNA nucleotides to a growing strand.
Spontaneous Mutations Replication errors can also result in
addition and deletion of nucleotides Short stretch of the same nucleotide
The resulting single-stranded RNA molecule, composed of ribonucleotides containing the bases
adenine (A), cytosine (C), guanine (G), and uracil (U), acts as a mobile molecular copy of the original DNA sequence.
In the last two decades of the 19th century, German biochemist Albrecht Kossel (1853-1927) isolated and characterized the five different nucleotide bases composing nucleic acid. These are
adenine, guanine, cytosine, thymine (in DNA), and uracil (in RNA). Kossell received the Nobel Prize in Physiology or Medicine in 1910 for his work on nucleic acids and for his considerable work on proteins, including the discovery of histidine.
point mutation
affects a single base and most commonly occurs when one base is substituted or replaced by another.
The bacterium Pseudomonas aeruginosa often colonizes biofilms in the
airways of patients with cystic fibrosis, causing chronic and sometimes fatal infections of the lungs.
Because Listeria bacteria can easily be confused with diphtheroids, another common group of gram-positive rods, it is important to
alert the laboratory when listeriosis is suspected.
At the other end of the spectrum are
alkaliphiles: microorganisms that grow best at pH between 8.0 and 10.5.
Recall that acidity is a function of the concentration of hydrogen ions [H+] and is measured as pH. Environments with pH values below 7.0 are considered acidic, whereas those with pH values above 7.0 are considered basic. Extreme pH affects the structure of
all macromolecules.
As the numbers of bacteria in a suspension increase, the turbidity
also increases and causes less light to reach the detector.
A change in the DNA sequence is conferred to mRNA through transcription, and may lead to an altered amino acid sequence in a protein on translation. Because proteins carry out the vast majority of cellular functions, a change in
amino acid sequence in a protein may lead to an altered phenotype for the cell and organism.
The tRNA molecule interacts with three factors:
aminoacyl tRNA synthetases, ribosomes, and mRNA.
An amino acid is added to the end of a tRNA molecule through the process of tRNA "charging," during which each tRNA molecule is linked to its correct or cognate amino acid by a group of enzymes called
aminoacyl tRNA synthetases.
The preferred drugs for the treatment of listeriosis are
ampicillin or penicillin G with an aminoglycoside antibiotic.
Free-floating microbial cells that live in
an aquatic environment are called planktonic cells.
In some cyanobacteria, many nucleoids may accumulate in
an enlarged round cell or along a filament, leading to the generation of many new cells at once.
The serial dilution of a culture is
an important first step before proceeding to either the pour plate or spread plate method.
The number of bacteria in a clinical sample serves as
an indication of the extent of an infection.
When used to treat an infection, -static treatments are typically sufficient in
an otherwise healthy individual, preventing the pathogen from multiplying, thus allowing the individual's immune system to clear the infection.
Many obligate anaerobes are found in the environment where
anaerobic conditions exist, such as in deep sediments of soil, still waters, and at the bottom of the deep ocean where there is no photosynthetic life. Anaerobic conditions also exist naturally in the intestinal tract of animals. Obligate anaerobes, mainly Bacteroidetes, represent a large fraction of the microbes in the human gut. Transient anaerobic conditions exist when tissues are not supplied with blood circulation; they die and become an ideal breeding ground for obligate anaerobes.
The study of obligate anaerobes requires special equipment. Obligate anaerobic bacteria must be grown under conditions devoid of oxygen. The most common approach is culture in an
anaerobic jar (Figure 9.21). Anaerobic jars include chemical packs that remove oxygen and release carbon dioxide (CO2).
Cells in the deep layers of a biofilm are metabolically inactive and may be less susceptible to the action of
antibiotics that disrupt metabolic activities.
Unlike disinfectants, antiseptics are
antimicrobial chemicals safe for use on living skin or tissues.
Watson and Crick proposed that DNA is made up of two strands that are twisted around each other to form a right-handed helix. The two DNA strands are
antiparallel, such that the 3ʹ end of one strand faces the 5ʹ end of the other. The 3ʹ end of each strand has a free hydroxyl group, while the 5ʹ end of each strand has a free phosphate group. The sugar and phosphate of the polymerized nucleotides form the backbone of the structure, whereas the nitrogenous bases are stacked inside. These nitrogenous bases on the interior of the molecule interact with each other, base pairing.
Transcription of a particular gene always proceeds from one of the two DNA strands that acts as a template, the so-called
antisense strand.
The process of applying an antiseptic is called
antisepsis.
Genes must be expressed at the
appropriate time and place Gene structure of eukaryotes is more complex
Analysis of the diffraction patterns of DNA has determined that there are
approximately 10 bases per turn in DNA. The asymmetrical spacing of the sugar-phosphate backbones generates major grooves (where the backbone is far apart) and minor grooves (where the backbone is close together) (Figure 10.16). These grooves are locations where proteins can bind to DNA. The binding of these proteins can alter the structure of DNA, regulate replication, or regulate transcription of DNA into RNA.
unusual amino acids such as selenocysteine and pyrrolysine have been observed in
archaea and bacteria.
Examples of obligate aerobes
are Mycobacterium tuberculosis, the causative agent of tuberculosis and Micrococcus luteus, a gram-positive bacterium that colonizes the skin. Neisseria meningitidis, the causative agent of severe bacterial meningitis, and N. gonorrhoeae, the causative agent of sexually transmitted gonorrhea, are also obligate aerobes.
BSL-1 agents
are those that generally do not cause infection in healthy human adults. These include noninfectious bacteria, such as nonpathogenic strains of Escherichia coli and Bacillus subtilis, and viruses known to infect animals other than humans, such as baculoviruses (insect viruses). Because working with BSL-1 agents poses very little risk, few precautions are necessary. Laboratory workers use standard aseptic technique and may work with these agents at an open laboratory bench or table, wearing personal protective equipment (PPE) such as a laboratory coat, goggles, and gloves, as needed. Other than a sink for handwashing and doors to separate the laboratory from the rest of the building, no additional modifications are needed.
Counts of live cells are needed when
assessing the extent of an infection, the effectiveness of antimicrobial compounds and medication, or contamination of food and water.
Psychrophiles are microorganisms that can grow
at 0 °C and below, have an optimum growth temperature close to15 °C, and usually do not survive at temperatures above 20 °C. They are found in permanently cold environments such as the deep waters of the oceans. Because they are active at low temperature, psychrophiles and psychrotrophs are important decomposers in cold climates.
The other strand, complementary to the 5' to 3' parental DNA, grows away from the replication fork, so the polymerase must move back toward the replication fork to begin adding bases to a new primer, again in the direction
away from the replication fork. It does so until it bumps into the previously synthesized strand and then it moves back again
Although mostly studied in bacterial populations, quorum sensing takes place between
bacteria and eukaryotes and between eukaryotic cells such as the fungus Candida albicans, a common member of the human microbiota that can cause infections in immunocompromised individuals.
The Ames test, developed by Bruce Ames (1928-) in the 1970s, is a method that uses
bacteria for rapid, inexpensive screening of the carcinogenic potential of new chemical compounds.
the general structures and functions of the protein synthesis machinery are comparable from
bacteria to human cells.
causative agents examples
bacteria, cysts
Estimating the number of bacterial cells in a sample, known as a
bacterial count, is a common task performed by microbiologists.
Interestingly, archaea contain a single RNA polymerase that is more closely related to eukaryotic RNA polymerase II than to its
bacterial counterpart.
urinary tract infection (UTI)
bacterial infection was responsible for her sudden-onset high fever, abdominal pain, and bloody urine.
In a spectrophotometer, a light beam is transmitted through a
bacterial suspension, the light passing through the suspension is measured by a detector, and the amount of light passing through the sample and reaching the detector is converted to either percent transmission or a logarithmic value called absorbance (optical density).
Ionizing radiation can also modify
bases; for example, the deamination of cytosine to uracil, analogous to the action of nitrous acid.
In addition to the characteristics of a good disinfectant, antiseptics must also
be selectively effective against microorganisms and able to penetrate tissue deeply without causing tissue damage.
Semicritical items do not typically need to
be sterilized but do require a high level of disinfection.
Because of its involvement in initiation, fMet is inserted at the
beginning (N terminus) of every polypeptide chain synthesized by E. coli. In E. coli mRNA, a leader sequence upstream of the first AUG codon, called the Shine-Dalgarno sequence (also known as the ribosomal binding site AGGAGG), interacts through complementary base pairing with the rRNA molecules that compose the ribosome.
Rarely, a missense mutation may be
beneficial.
Quorum sensing—which can occur
between cells of different species within a biofilm—enables microorganisms to detect their cell density through the release and binding of small, diffusible molecules called autoinducers
The most common mechanism of cell replication in bacteria is a process called
binary fission
The small subunit is responsible for
binding the mRNA template, whereas the large subunit binds tRNAs (discussed in the next subsection).
In nature, microorganisms grow mainly in
biofilms, complex and dynamic ecosystems that form on a variety of environmental surfaces, from industrial conduits and water treatment pipelines to rocks in river beds.
The human body harbors many types of
biofilms, some beneficial and some harmful. For example, the layers of normal microbiota lining the intestinal and respiratory mucosa play a role in warding off infections by pathogens.
In 1865, Mendel presented the results of his experiments with nearly 30,000 pea plants to the local natural history society. He demonstrated that traits are transmitted faithfully from parents to offspring independently of other traits. In 1866, he published his work, "Experiments in Plant Hybridization,"1 in the Proceedings of the Natural History Society of Brünn. Mendel's work went virtually unnoticed by the scientific community, which believed, incorrectly, in the theory of
blending of traits in continuous variation.
certain types of infectious agents (like C. jejuni, Salmonella, and E. histolytica) are associated with the production of
bloody stools.
Shellfish beds must be regularly monitored for the presence of the toxins, and harvests are often shut down when it is present, incurring economic costs to the fishery. Cyanobacteria, which can form
blooms in marine and freshwater ecosystems, produce toxins called microcystins, which can cause allergic reactions and liver damage when ingested in drinking water or during swimming.
HIV targets helper T cells that play a key role in bridging the innate and adaptive immune response, infecting and killing cells normally involved in the
body's response to infection.
Recently, indirect ways of measuring live cells have been developed that are____. These methods measure cell activity by____.
both fast and easy to implement. following the production of metabolic products or disappearance of reactants.
The reason why transcription and translation can occur simultaneously is because
both of these processes occur in the same 5' to 3' direction, they both occur in the cytoplasm of the cell, and because the RNA transcript is not processed once it is transcribed. This allows a prokaryotic cell to respond to an environmental signal requiring new proteins very quickly. In contrast, in eukaryotic cells, simultaneous transcription and translation is not possible.
Even organisms that do not use aerobic respiration need some way to
break down some of the ROS that may form from atmospheric oxygen.
A sample of culture rapidly mixed in a drop of 3% hydrogen peroxide will release
bubbles if the culture is catalase positive.
Other species may form a long narrow extension at one pole in a process called
budding. The tip of the extension swells and forms a smaller cell, the bud that eventually detaches from the parent cell. Budding is most common in yeast (picture b), but it is also observed in prosthecate bacteria and some cyanobacteria.
Proteins within a cell have many functions, including
building cellular structures and serving as enzyme catalysts for cellular chemical reactions that give cells their specific characteristics.
Metal cans of food contaminated with C. botulinum will
bulge due to the microbe's production of gases; contaminated jars of food typically bulge at the metal lid.
DNA strands can be separated in the laboratory
by heating or treating it with certain chemicals. DNA denaturation
Nucleotides preceding the initiation site are designated "upstream," whereas nucleotides following the initiation site are called
called "downstream" nucleotides.
Once single-stranded DNA is accessible at the origin of replication, DNA replication
can begin.
maximum growth pH, minimum growth pH, and optimum growth pH
can cover a wide range, which is important for the preservation of food and to microorganisms' survival in the stomach. For example, the optimum growth pH of Salmonella spp. is 7.0-7.5, but the minimum growth pH is closer to 4.2.
One common approach to grow capnophiles is to use a
candle jar. A candle jar consists of a jar with a tight-fitting lid that can accommodate the cultures and a candle. After the cultures are added to the jar, the candle is lit and the lid closed. As the candle burns, it consumes most of the oxygen present and releases CO2.
Bacteria that grow best in a higher concentration of CO2 and a lower concentration of oxygen than present in the atmosphere are called
capnophiles
Exposure to a mutagen can increase the rate of mutation more than 1000-fold. Mutagens are often also
carcinogens, agents that cause cancer.
nearly all carcinogens are mutagenic, not all mutagens are necessarily
carcinogens.
The base sequence of deoxyribonucleic acid (DNA) is responsible for
carrying and retaining the hereditary information in a cell.
Aerotolerant anaerobes do have SOD but no
catalase.
Samples from the salad showed the presence of gram-positive cocci bacteria in clusters. The colonies were positive for
catalase. The bacteria grew on mannitol salt agar fermenting mannitol, as shown by the change to yellow of the medium. The pH indicator in mannitol salt agar is phenol red, which turns to yellow when the medium is acidified by the products of fermentation.
Telomerase contains a
catalytic part and a built-in RNA template. It attaches to the end of the chromosome, and complementary bases to the RNA template are added on the 3' end of the DNA strand. Once the 3' end of the lagging strand template is sufficiently elongated, DNA polymerase can add the nucleotides complementary to the ends of the chromosomes. In this way, the ends of the chromosomes are replicated.
Measuring turbidity is a fast method to estimate
cell density as long as there are enough cells in a sample to produce turbidity.
Binary fission is the most common pattern of
cell division in prokaryotes, but it is not the only one. Other mechanisms usually involve asymmetrical division (as in budding) or production of spores in aerial filaments.
Before dividing, the cell grows and increases its number of
cellular components.
Sometimes the effects of missense mutations may be only apparent under
certain environmental conditions; such missense mutations are called conditional mutations.
The area of the squares and the height at which the coverslip is positioned are specified for the
chamber. The concentration must be corrected for dilution if the sample was diluted before enumeration.
Surfaces in hospital rooms are commonly sanitized using a
chemical disinfectant to prevent disease transmission between patients.
When the complete chemical composition of a medium is known, it is called a
chemically defined medium. For example, in EZ medium, all individual chemical components are identified and the exact amounts of each is known.
Mendel carried out his experiments long before chromosomes were visualized under a microscope. However, with the improvement of microscopic techniques during the late 1800s, cell biologists could stain and visualize subcellular structures with dyes and observe their actions during meiosis. They were able to observe
chromosomes replicating, condensing from an amorphous nuclear mass into distinct X-shaped bodies and migrating to separate cellular poles. The speculation that chromosomes might be the key to understanding heredity led several scientists to examine Mendel's publications and re-evaluate his model in terms of the behavior of chromosomes during mitosis and meiosis.
Following replication, the resulting complete circular genomes of prokaryotes are concatenated, meaning that the
circular DNA chromosomes are interlocked and must be separated from each other. This is accomplished through the activity of bacterial topoisomerase IV, which introduces double-stranded breaks into DNA molecules, allowing them to separate from each other; the enzyme then reseals the circular chromosomes.
Chemical disinfectants, such as chlorine bleach or products containing chlorine, are used to
clean nonliving surfaces such as laboratory benches, clinical surfaces, and bathroom sinks.
Conditions that limit contact between the agent and the targeted cells cells—for example, the presence of bodily fluids, tissue, organic debris (e.g., mud or feces), or biofilms on surfaces—increase the
cleaning time or intensity of the microbial control protocol required to reach the desired level of cleanliness. All these factors must be considered when choosing the appropriate protocol to control microbial growth in a given situation.
Various other methods are used in
clinical and nonclinical settings to reduce the microbial load on items.
The growth pattern taken place in the picture takes place in a
closed environment; nutrients are not added and waste and dead cells are not removed.
Detailed observations of biofilms under confocal laser and scanning electron microscopes reveal
clusters of microorganisms embedded in a matrix interspersed with open water channels.
Each amino acid is defined within the mRNA by a triplet of nucleotides called a
codon
Basic proteins helps
coiling
The proton motive force responsible for production of ATP in cellular respiration depends on the concentration gradient of H+ across the plasma membrane. If H+ ions are neutralized by hydroxide ions, the concentration gradient
collapses and impairs energy production. But the component most sensitive to pH in the cell is its workhorse, the protein. Moderate changes in pH modify the ionization of amino-acid functional groups and disrupt hydrogen bonding, which, in turn, promotes changes in the folding of the molecule, promoting denaturation and destroying activity.
The results of a viable plate count are usually expressed as
colony-forming units per milliliter (CFU/mL) rather than cells per milliliter because more than one cell may have landed on the same spot to give rise to a single colony. Furthermore, samples of bacteria that grow in clusters or chains are difficult to disperse and a single colony may represent several cells.
Antifreeze proteins and solutes that decrease the freezing temperature of the cytoplasm are
common. The lipids in the membranes tend to be unsaturated to increase fluidity. Growth rates are much slower than those encountered at moderate temperatures.
The RNA-specific pyrimidine uracil forms a
complementary base pair with adenine and is used instead of the thymine used in DNA.
In DNA, adenine (A) and thymine (T) are
complementary base pairs, and cytosine (C) and guanine (G) are also complementary base pairs, explaining Chargaff's rules
Beadle and Tatum irradiated the mold with X-rays to induce changes to a sequence of nucleic acids, called mutations. They mated the irradiated mold spores and attempted to grow them on both a
complete medium and a minimal medium.
A ribosome is a
complex macromolecule composed of catalytic rRNAs (called ribozymes) and structural rRNAs, as well as many distinct polypeptides.
Ribosomal movements, or steps, are induced by
conformational changes that advance the ribosome by three bases in the 3' direction.
LT and ST cause the excretion of chloride ions from intestinal cells to the intestinal lumen, causing a
consequent loss of water from intestinal cells, resulting in diarrhea. CF encodes a bacterial protein that aids in allowing the bacterium to adhere to the lining of the small intestine.
There were two competing models also suggested:
conservative and dispersive,
It is possible to predict the number of cells in a population when they divide by binary fission at a
constant rate. As an example, consider what happens if a single cell divides every 30 minutes for 24 hours.
The culture density in a stationary culture is_____.
constant.
Although RNA does not serve as the hereditary information in most cells, RNA does hold this function for many viruses that do not
contain DNA. Thus, RNA clearly does have the additional capacity to serve as genetic information.
In complex media, which
contain extracts and digests of yeasts, meat, or plants, the precise chemical composition of the medium is not known. Amounts of individual components are undetermined and variable. Nutrient broth, tryptic soy broth, and brain heart infusion, are all examples of complex media.
Because C. botulinum and its endospores are commonly found in soil, they may easily
contaminate crops during harvesting, and these endospores can later germinate within the anaerobic environment once foods are canned.
Each BSL requires a different level of biocontainment to prevent
contamination and spread of infectious agents to laboratory personnel and, ultimately, the community. For example, the lowest BSL, BSL-1, requires the fewest precautions because it applies to situations with the lowest risk for microbial infection.
For many clinical purposes, aseptic technique is necessary to prevent
contamination of sterile surfaces.
The scientific community believed, incorrectly, that the process of inheritance involved a blending of parental traits that produced an intermediate physical appearance in offspring; this hypothetical process appeared to be correct because of what we know now as
continuous variation, which results from the action of many genes to determine a particular characteristic, like human height.
To prevent the spread of human disease, it is necessary to
control the growth and abundance of microbes in or on various items frequently used by humans.
DNA is replicated when a cell makes a duplicate copy of its DNA, then the cell divides, resulting in the
correct distribution of one DNA copy to each resulting cell.
This interaction anchors the 30S ribosomal subunit at the
correct location on the mRNA template. At this point, the 50S ribosomal subunit then binds to the initiation complex, forming an intact ribosome.
Cells in several small squares must be
counted and the average taken to obtain a reliable measurement.
The direct methods involve
counting the cells in a liquid culture or colonies on a plate. It is a direct way of estimating how many organisms are present in a sample.
A sample of the culture suspension is added to the chamber under a
coverslip that is placed at a specific height from the surface of the grid.
Scientists in the laboratories of Thomas Steitz (1940-) and Peter Moore (1939-) at Yale University were able to
crystallize the ribosome structure from Haloarcula marismortui, a halophilic archaeon isolated from the Dead Sea. Because of the importance of this work, Steitz shared the 2009 Nobel Prize in Chemistry with other scientists who made significant contributions to the understanding of ribosome structure.
The study of microorganisms is greatly facilitated if we are able to
culture them, that is, to keep reproducing populations alive under laboratory conditions.
It is possible to correlate turbidity readings to the actual number of cells by performing a viable plate count of samples taken from
cultures having a range of absorbance values.
In E. coli, the DNA is scanned by an enzyme complex. If a distortion in the double helix is found that was introduced by the pyrimidine dimer, the enzyme complex
cuts the sugar-phosphate backbone several bases upstream and downstream of the dimer, and the segment of DNA between these two cuts is then enzymatically removed. DNA pol I replaces the missing nucleotides with the correct ones and DNA ligase seals the gap in the sugar-phosphate backbone.
This modification further protects the pre-mRNA from degradation and signals to cellular factors that the transcript needs to be exported to the
cytoplasm.
Transfer RNAs (tRNAs) are structural RNA molecules and, depending on the species, many different types of tRNAs exist in the
cytoplasm.
viability stains (or live stains) bind to nucleic acids, but the primary and secondary stains differ in their ability to cross the
cytoplasmic membrane.
pathogens that cross the placenta can be very
dangerous for the fetus.
. Combustion products like polycyclic aromatic hydrocarbons are particularly
dangerous intercalating agents that can lead to mutation-caused cancers. The intercalating agents ethidium bromide and acridine orange are commonly used in the laboratory to stain DNA for visualization and are potential mutagens.
As a culture medium accumulates toxic waste and nutrients are exhausted, cells die in greater and greater numbers. Soon, the number of dying cells exceeds the number of dividing cells, leading to an exponential decrease in the number of cells. This is the aptly named
death phase, sometimes called the decline phase. Many cells lyse and release nutrients into the medium, allowing surviving cells to maintain viability and form endospores.
The diabetic foot will have to be surgically
debrided (debridement refers to the removal of dead and infected tissue) and a sample sent for microbiological lab analysis. as long as the bone has not yet been infected.
This redundancy in the genetic code is called
degeneracy
The act of handwashing is an example of
degerming, in which microbial numbers are significantly reduced by gently scrubbing living tissue, most commonly skin, with a mild chemical (e.g., soap) to avoid the transmission of pathogenic microbes.
Wiping the skin with an alcohol swab at an injection site is another example of
degerming.
During transcription, a ribonucleotide complementary to the DNA template strand is added to the growing RNA strand and a covalent phosphodiester bond is formed by
dehydration synthesis between the new nucleotide and the last one added.
Because of the additional hydrogen bonding between the C = G base pair, DNA with a high GC content is more difficult to
denature than DNA with a lower GC content.
At the opposite end of the temperature spectrum, heat
denatures proteins and nucleic acids. Increased fluidity impairs metabolic processes in membranes.
During the polymerization process,
deoxynucleotide triphosphates (dNTP) are used.
Photoautotrophs
depend on sufficient light to grow and multiply (cyanobacteria or green sulfur bacteria)
Often used for water and food samples, the MPN method evaluates
detectable growth by observing changes in turbidity or color due to metabolic activity.
Microbes and their viruses played a pivotal role in
determining DNA as genetic material
Aerobic respiration constantly generates reactive oxygen species (ROS), byproducts that must be
detoxified.
Prolonged use of antibiotics for other infections increases the probability of a patient
developing a secondary C. difficile infection. Antibiotic treatment disrupts the balance of microorganisms in the intestine and allows the colonization of the gut by C. difficile, causing a significant inflammation of the colon.
When exposed to a particular microbial control protocol, a fixed percentage of the microbes within the population will
die
Other chemical mutagens can modify normal DNA bases, resulting in
different base-pairing rules. For example, nitrous acid deaminates cytosine, converting it to uracil.
The composition of each component varies across taxa; for instance, ribosomes may consist of
different numbers of ribosomal RNAs (rRNAs) and polypeptides depending on the organism.
Nonionizing radiation, like ultraviolet light, is not energetic enough to initiate these types of chemical changes. However, nonionizing radiation can induce
dimer formation between two adjacent pyrimidine bases, commonly two thymines, within a nucleotide strand.
Two major approaches are used to measure cell number, including
direct and indirect methods
The simplest way to count bacteria is called the
direct microscopic cell count, which involves transferring a known volume of a culture to a calibrated slide and counting the cells under a light microscope.
For example, higher temperatures and higher concentrations of
disinfectants kill microbes more quickly and effectively.
The E (exit) site releases
dissociated tRNAs so that they can be recharged with free amino acids.
Differential media
distinguish among different groups of microbes. E.g. MacConkey agar
The differential media make it easy to
distinguish colonies of different bacteria by a change in the color of the colonies or the color of the medium. Color changes are the result of end products created by interaction of bacterial enzymes with differential substrates in the medium or, in the case of hemolytic reactions, the lysis of red blood cells in the medium.
Thermophiles are widely
distributed in hot springs, geothermal soils, and manmade environments such as garden compost piles where the microbes break down kitchen scraps and vegetal material.
Samples with too few colonies (<30)
do not give statistically reliable numbers, and overcrowded plates (>300 colonies) make it difficult to accurately count individual colonies. Also, counts in this range minimize occurrences of more than one bacterial cell forming a single colony. Thus, the calculated CFU is closer to the true number of live bacteria in the population.
On the downside, the counting chamber
does not work well with dilute cultures because there may not be enough cells to count.
The deoxyribonucleotide is named according to the nitrogenous bases. ). The nitrogenous bases adenine (A) and guanine (G) are the purines; they have a
double-ring structure with a six-carbon ring fused to a five-carbon ring.
The ssDNA strands can also be put back together as
double-stranded DNA (dsDNA), through reannealing or renaturing by cooling or removing the chemical denaturants, allowing these hydrogen bonds to reform.
Rotaviruses, which cause severe gastroenteritis in children and other immunocompromised individuals, are examples of
double-stranded RNA viruses.
In prokaryotes (Bacteria and Archaea), the generation time is also called the
doubling time and is defined as the time it takes for the population to double through one round of binary fission. Bacterial doubling times vary enormously. Whereas Escherichia coli can double in as little as 20 minutes under optimal growth conditions in the laboratory, bacteria of the same species may need several days to double in especially harsh environments.
The small and large ribosomal subunits dissociate from the mRNA and from
each other; they are recruited almost immediately into another translation initiation complex.
The plates are incubated until colonies appear. Two to three plates are usually prepared from each dilution and the numbers of colonies counted on
each plate are averaged. In all cases, thorough mixing of samples with the dilution medium (to ensure the cell distribution in the tube is random) is paramount to obtaining reliable results.
With some knowledge of the arginine biosynthesis pathway, they identified three classes of arginine mutants by supplementing the minimal medium with intermediates (citrulline or ornithine) in the pathway. The three mutants differed in their abilities to grow in each of the media, which led the group of scientists to propose, in 1945, that
each type of mutant had a defect in a different gene in the arginine biosynthesis pathway. This led to the so-called one gene-one enzyme hypothesis, which suggested that each gene encodes one enzyme.
The advantages of the chamber are that the direct method is
easy to use, relatively fast, and inexpensive.
Because C. botulinum can produce endospores that can survive harsh conditions, extreme temperatures and pressures must be used to
eliminate the endospores.
For example, low- and medium-acid foods are heated to 121 °C for a minimum of 2.52 minutes, which is the time it would take to reduce a population of 1012 endospores per can down to 1 endospore at this temperature. Even so, commercial sterilization does not
eliminate the presence of all microbes; rather, it targets those pathogens that cause spoilage and foodborne diseases, while allowing many nonpathogenic organisms to survive.
An anaerobic chamber is an
enclosed box from which all oxygen is removed. Gloves sealed to openings in the box allow handling of the cultures without exposing the culture to air
Transcribed RNA sequences corresponding to introns do not
encode regions of the functional polypeptide and are removed from the pre-mRNA during processing.
Several of the steps during elongation, including binding of a charged aminoacyl tRNA to the A site and translocation, require
energy derived from GTP hydrolysis, which is catalyzed by specific elongation factors.
The addition of these nucleotides require
energy. This energy is present in the bonds of three phosphate groups attached to each nucleotide (a triphosphate nucleotide), similar to how energy is stored in the phosphate bonds of adenosine triphosphate (ATP)
One type of media is
enriched media, contains growth factors, vitamins, and other essential nutrients to promote the growth of fastidious organisms, organisms that cannot make certain nutrients and require them to be added to the medium.
traveler's diarrhea caused by
enterotoxigenic E. coli (ETEC), the causative agent of most traveler's diarrhea.
bacterial infection examples
enterotoxigenic E. coli, Vibrio cholerae, Campylobacter jejuni, Salmonella
Observations using confocal microscopy have shown that
environmental conditions influence the overall structure of biofilms.
The rRNA ensures the proper alignment of the mRNA, tRNA, and the ribosomes; the rRNA of the ribosome also has an
enzymatic activity (peptidyl transferase) and catalyzes the formation of the peptide bonds between two aligned amino acids during protein synthesis.
The nicks that remain between the newly synthesized DNA (that replaced the RNA primer) and the previously synthesized DNA are sealed by the
enzyme DNA ligase that catalyzes the formation of covalent phosphodiester linkage between the 3'-OH end of one DNA fragment and the 5' phosphate end of the other fragment, stabilizing the sugar-phosphate backbone of the DNA molecule.
Besides direct methods of counting cells, other methods, based on an indirect detection of cell density, are commonly used to
estimate and compare cell densities in a culture.
Once the calibration curve has been produced, it can be used to
estimate cell counts for all samples obtained or cultured under similar conditions and with densities within the range of values used to construct the curve.
Quality control of drinking water, food, medication, and even cosmetics relies on
estimates of bacterial counts to detect contamination and prevent the spread of disease.
The leading strand is continuously synthesized by the
eukaryotic polymerase enzyme pol δ, while the lagging strand is synthesized by pol ε. A sliding clamp protein holds the DNA polymerase in place so that it does not fall off the DNA. The enzyme ribonuclease H (RNase H), instead of a DNA polymerase as in bacteria, removes the RNA primer, which is then replaced with DNA nucleotides. The gaps that remain are sealed by DNA ligase.
Eukaryotic genes that encode polypeptides are composed of coding sequences called
exons (ex-on signifies that they are expressed) and intervening sequences called introns (int-ron denotes their intervening role).
As synthesis proceeds, the RNA primers are replaced by DNA. The primers are removed by the
exonuclease activity of DNA polymerase I, and the gaps are filled in.
Thomas Morgan provided
experimental evidence to support the chromosomal theory of inheritance
During the log phase, the relationship between time and number of cells is not linear but
exponential; however, the growth curve is often plotted on a semilogarithmic graph, which gives the appearance of a linear relationship.
In certain pathogenic bacteria, the stationary phase is also associated with the
expression of virulence factors, products that contribute to a microbe's ability to survive, reproduce, and cause disease in a host organism. For example, quorum sensing in Staphylococcus aureus initiates the production of enzymes that can break down human tissue and cellular debris, clearing the way for bacteria to spread to new tissue where nutrients are more plentiful.
Even though RNA is single stranded, most types of RNA molecules show
extensive intramolecular base pairing between complementary sequences within the RNA strand, creating a predictable three-dimensional structure essential for their function
The extracellular matrix consists of
extracellular polymeric substances (EPS) secreted by the organisms in the biofilm.
Finally, biofilms provide an ideal environment for the exchange of
extrachromosomal DNA, which often includes genes that confer antibiotic resistance.
The hydrothermal vents at the bottom of the ocean are a prime example of
extreme environments, with temperatures reaching an estimated 340 °C. Noteworthy examples of such microbes are Pyrobolus and Pyrodictium, archaea that grow at 105 °C and survive autoclaving. Figure 9.29 shows the typical skewed curves of temperature-dependent growth for the categories of microorganisms we have discussed.
The organisms retrieved from arctic lakes such as Lake Whillans are considered extreme
extreme psychrophiles (cold loving).
Lipids are hydrolyzed by an
extremely basic pH.
Staphylococci and Enterobacteriaceae are examples of
facultative anaerobes.
Tube C shows heavy growth at the top of the tube and growth throughout the tube, a typical result with
facultative anaerobes. Facultative anaerobes are organisms that thrive in the presence of oxygen but also grow in its absence by relying on fermentation or anaerobic respiration, if there is a suitable electron acceptor other than oxygen and the organism is able to perform anaerobic respiration.
Historically, women have been underrepresented in the sciences and in medicine, and often their pioneering contributions have gone relatively unnoticed. For example, although Rosalind Franklin performed the X-ray diffraction studies demonstrating the double helical structure of DNA, it is Watson and Crick who became
famous for this discovery, building on her data. There still remains great controversy over whether their acquisition of her data was appropriate and whether personality conflicts and gender bias contributed to the delayed recognition of her significant contributions.
Ideally, disinfectants should be
fast acting, stable, easy to prepare, inexpensive, and easy to use.
Selective media
favor the growth of a particular microorganism. Bile salts or dyes like crystal violet favor the growth of gram-negative bacteria. E.g. MacConkey agar
The presence of coliforms in water is considered a sign of contamination by
fecal matter.
Linear sequence of nucleotides with
fixed start and end points
Inanimate items, such as doorknobs, toys, or towels, which may harbor microbes and aid in disease transmission, are called
fomites
Artificial fertilizers have become an important tool in
food production around the world. They are responsible for many of the gains of the so-called green revolution of the 20th century, which has allowed the planet to feed many of its more than 7 billion people.
Because Listeria can infect animals, it often contaminates
food such as meat, fish, or dairy products. Contamination of commercial foods can often be traced to persistent biofilms that form on manufacturing equipment that is not sufficiently cleaned.
The initiation of protein synthesis begins with the
formation of an initiation complex.
By the early 1950s, considerable evidence had accumulated indicating that DNA was the genetic material of cells, and now the race was on to discover its three-dimensional structure. Around this time, Austrian biochemist Erwin Chargaff5 (1905-2002) examined the content of DNA in different species and discovered that adenine, thymine, guanine, and cytosine were not
found in equal quantities, and that it varied from species to species, but not between individuals of the same species. He found that the amount of adenine was very close to equaling the amount of thymine, and the amount of cytosine was very close to equaling the amount of guanine, or A = T and G = C. These relationships are also known as Chargaff's rules.
The new cells often split from the parent filament and float away in a process called
fragmentation(picture a)
Using these values, a calibration curve is generated by plotting turbidity as a
function of cell density.
fungicides kill
fungi.
While Miescher was isolating and discovering DNA in the 1860s, Austrian monk and botanist Johann Gregor Mendel (1822-1884) was experimenting with
garden peas, demonstrating and documenting basic patterns of inheritance, now known as Mendel's laws.
Intron-encoded RNA sequences are removed from the pre-mRNA while it is still in the nucleus. Although they are not translated, introns appear to have various functions, including_______. On completion of these modifications, the mature transcript, the mRNA that encodes a
gene regulation and mRNA transport. polypeptide, is transported out of the nucleus, destined for the cytoplasm for translation.
The number of available media to grow bacteria is considerable. Some media are considered
general all-purpose media and support growth of a large variety of organisms.
The genetic information stored in DNA is divided into units called
genes
Transduction in prokaryotes
genes are transferred between cells in a virus It can be generalized transduction or specialized transduction based on genes being transferred
The relationship between an mRNA codon and its corresponding amino acid is called the
genetic code.
Genes are basic unit of
genetic information
Alfred Hershey and Martha Chase performed their own experiments in 1952 and were able to provide confirmatory evidence that DNA, not protein, was the
genetic material
Barbara McClintock demonstrated
genetic recombination event occurring during meiosis
For any given bacterial species, the generation time under specific growth conditions (nutrients, temperature, pH, and so forth) is
genetically determined, and this generation time is called the intrinsic growth rate.
In humans, telomerase is typically active in
germ cells and adult stem cells; it is not active in adult somatic cells and may be associated with the aging of these cells.
Any mutations in the tRNA or rRNA can result in
global problems for the cell because both are necessary for proper protein synthesis.
The amino acid bound to the P-site tRNA is also linked to the
growing polypeptide chain.
Infections of the body do not always follow the
growth curve, but correlations can exist depending upon the site and type of infection. When the number of live cells is plotted against time, distinct phases can be observed in the curve
Microorganisms grown in closed culture (also known as a batch culture), in which no nutrients are added and most waste is not removed, follow a reproducible growth pattern referred to as the
growth curve. An example of a batch culture in nature is a pond in which a small number of cells grow in a closed environment.
bacteriostatic treatments inhibit
growth of bacteria,
Most algal blooms are not directly harmful to humans or wildlife; however, they can cause
harm indirectly.
Extreme alkaliphiles
have adapted to their harsh environment through evolutionary modification of lipid and protein structure and compensatory mechanisms to maintain the proton motive force in an alkaline environment. For example, the alkaliphile Bacillus firmus derives the energy for transport reactions and motility from a Na+ ion gradient rather than a proton motive force. Many enzymes from alkaliphiles have a higher isoelectric point, due to an increase in the number of basic amino acids, than homologous enzymes from neutrophiles.
However, other biofilms in the body can have a detrimental effect on
health. For example, the plaque that forms on teeth is a biofilm that can contribute to dental and periodontal disease.
One food sterilization protocol, commercial sterilization, uses
heat at a temperature low enough to preserve food quality but high enough to destroy common pathogens responsible for food poisoning, such as C. botulinum.
A mutation is a
heritable change in the DNA sequence of an organism. The resulting organism, called a mutant, may have a recognizable change in phenotype compared to the wild type, which is the phenotype most commonly observed in nature.
Yogurt, pickles, sauerkraut, and lime-seasoned dishes all owe their tangy taste to a
high acid content
Barophiles
high atmospheric pressure (bacteria that live at the bottom of the ocean)
The organisms that were able to detoxify reactive oxygen species harnessed the
high electronegativity of oxygen to produce free energy for their metabolism and thrived in the new environment.
The hydrogen bonds holding together strands of DNA break up at
high pH
Organisms called psychrotrophs, also known as psychrotolerant, prefer cooler environments, from a
high temperature of 25 °C to refrigeration temperature about 4 °C. They are found in many natural environments in temperate climates. They are also responsible for the spoilage of refrigerated food.
Pathogens embedded within biofilms exhibit a
higher resistance to antibiotics than their free-floating counterparts. Several hypotheses have been proposed to explain why.
Microbes isolated from the vents achieve optimal growth at temperatures
higher than 100 °C.
If wearing a suit, the air pressure within the suit must be
higher than that outside the suit, so that if a leak in the suit occurs, laboratory air that may be contaminated cannot be drawn into the suit
The process of DNA replication is
highly accurate, but mistakes can occur spontaneously or be induced by mutagens. Uncorrected mistakes can lead to serious consequences for the phenotype.
Culturing many microorganisms is challenging because of
highly specific nutritional and environmental requirements and the diversity of these requirements among different species.
Chromosomal DNA is typically wrapped around
histones (in eukaryotes and archaea) or histone-like proteins (in bacteria), and is supercoiled, or extensively wrapped and twisted on itself. This packaging makes the information in the DNA molecule inaccessible.
In prokaryotes, reproduction is always asexual, although extensive genetic recombination in the form of
horizontal gene transfer takes place.
In Figure 9.32, the differential fermentation of lactose can be observed on MacConkey agar. The lactose fermenters produce acid, which turns the medium and the colonies of strong fermenters
hot pink. The medium is supplemented with the pH indicator neutral red, which turns to hot pink at low pH. Selective and differential media can be combined and play an important role in the identification of bacteria by biochemical methods.
The base pairs are stabilized by
hydrogen bonds
In the laboratory, exposing the two DNA strands of the double helix to high temperatures or to certain chemicals can break the
hydrogen bonds between complementary bases, thus separating the strands into two separate single strands of DNA (single-stranded DNA [ssDNA]). This process is called DNA denaturation
Examples of antiseptics include
hydrogen peroxide and isopropyl alcohol.
Proteins in psychrophiles are, in general, rich in
hydrophobic residues, display an increase in flexibility, and have a lower number of secondary stabilizing bonds when compared with homologous proteins from mesophiles.
Higher up on the extreme temperature scale we find the
hyperthermophiles, which are characterized by growth ranges from 80 °C to a maximum of 110 °C, with some extreme examples that survive temperatures above 121 °C, the average temperature of an autoclave.
Nitrous acid also deaminates adenine to
hypoxanthine, which base pairs with cytosine instead of thymine, resulting in the conversion of a TA base pair to a CG base pair.
The optimum oxygen concentration, as the name implies, is the
ideal concentration of oxygen for a particular microorganism.
Although the terms for these methods are often used interchangeably, there are
important distinctions
The number of CFU per mL is equal to 50 × 10 × 10,000 = 5,000,000. The number of bacteria in the culture is estimated as 5 million cells/mL. The colony count obtained from the 1:1000 dilution was 389, well below the expected 500 for a 10-fold difference in dilutions. This highlights the issue of
inaccuracy when colony counts are greater than 300 and more than one bacterial cell grows into a single colony.
The A (aminoacyl) site binds
incoming charged aminoacyl tRNAs.
The enzymes involved in this mechanism recognize the
incorrectly added nucleotide, excise it, and replace it with the correct base. One example is the methyl-directed mismatch repair in E. coli. The DNA is hemimethylated. This means that the parental strand is methylated while the newly synthesized daughter strand is not. It takes several minutes before the new strand is methylated. Proteins MutS, MutL, and MutH bind to the hemimethylated site where the incorrect nucleotide is found. MutH cuts the nonmethylated strand (the new strand).
Phenotypic changes may also contribute to the
increased resistance exhibited by bacterial cells in biofilms. For example, the increased production of efflux pumps, membrane-embedded proteins that actively extrude antibiotics out of bacterial cells, have been shown to be an important mechanism of antibiotic resistance among biofilm-associated bacteria.
Measuring dry weight of a culture sample is another
indirect method of evaluating culture density without directly measuring cell counts.
It is possible to estimate the concentration of cells in the original sample by counting
individual cells in a number of squares and determining the volume of the sample observed.
The error rate of DNA polymerase is one incorrect base per billion base pairs replicated. Exposure to mutagens can cause
induced mutations, which are various types of chemical agents or radiation
Transcription proceeds in three steps
initiation elongation termination
At the origin of replication, a prereplication complex composed of several proteins, including helicase, forms and recruits other enzymes involved in the
initiation of replication, including topoisomerase to relax supercoiling, single-stranded binding protein, RNA primase, and DNA polymerase.
Ribosomes dissociate into large and small subunits when they are not synthesizing proteins and reassociate during the
initiation of translation
The nucleotide pair in the DNA double helix that corresponds to the site from which the first 5' RNA nucleotide is transcribed is the
initiation site.
For the method illustrated in the picture, a series of three dilutions of the water sample is tested by
inoculating five lactose broth tubes with 10 mL of sample, five lactose broth tubes with 1 mL of sample, and five lactose broth tubes with 0.1 mL of sample. The lactose broth tubes contain a pH indicator that changes color from red to yellow when the lactose is fermented. After inoculation and incubation, the tubes are examined for an indication of coliform growth by a color change in media from red to yellow. The first set of tubes (10-mL sample) showed growth in all the tubes; the second set of tubes (1 mL) showed growth in two tubes out of five; in the third set of tubes, no growth is observed in any of the tubes (0.1-mL dilution). The numbers 5, 2, and 0 are compared with Figure B1 in Appendix B, which has been constructed using a probability model of the sampling procedure. From our reading of the table, we conclude that 49 is the most probable number of bacteria per 100 mL of pond water.no lo
The beginning of the growth curve represents a small number of cells, referred to as an
inoculum, that are added to a fresh culture medium, a nutritional broth that supports growth.
frameshift mutations, caused by
insertions or deletions of a number of nucleotides that are not a multiple of three are extremely problematic because a shift in the reading frame results
Critical items must be sterile because they will be used
inside the body, often penetrating sterile tissues or the bloodstream; examples of critical items include surgical instruments, catheters, and intravenous fluids.
no signs of bacterial infection, which further suggests that this was a case of
intoxication. Since intoxication is due to secreted toxins, bacteria are not usually detected in blood or stool samples.
Subsequent work by Beadle, Tatum, and colleagues showed that they could
isolate different classes of mutants that required a particular supplement, like the amino acid arginine
Modern understanding of DNA has evolved from the discovery of nucleic acid to the development of the double-helix model. In the 1860s, Friedrich Miescher (1844-1895), a physician by profession, was the first person to
isolate phosphorus-rich chemicals from leukocytes (white blood cells) from the pus on used bandages from a local surgical clinic. He named these chemicals (which would eventually be known as RNA and DNA) "nuclein" because they were isolated from the nuclei of the cells.
Spontaneous Mutations: Replication errors occur when bases in template strand takes a rare tautomeric form
keto (common) or enol (rare) "Tautomers are isomers of a compound which differ only in the position of the protons and electrons" Bases such as guanine and thymine can occur in two tautomeric forms
Ionizing radiation exposure is used to
kill microbes to sterilize medical devices and foods, because of its dramatic nonspecific effect in damaging DNA, proteins, and other cellular components
As we have seen, methods to estimate viable cell numbers can be
labor intensive and take time because cells must be grown.
Sterilization protocols are generally reserved for
laboratory, medical, manufacturing, and food industry settings, where it may be imperative for certain items to be completely free of potentially infectious agents.
Examples of aerotolerant anaerobes include
lactobacilli and streptococci, both found in the oral microbiota. Campylobacter jejuni, which causes gastrointestinal infections, is an example of a microaerophile and is grown under low-oxygen conditions.
The initial phase of the growth curve is called the
lag phase, during which cells are gearing up for the next phase of growth.
The number of cells does not change during the
lag phase; however, cells grow larger and are metabolically active, synthesizing proteins needed to grow within the medium.
The strand with the Okazaki fragments is known as the
lagging strand, and its synthesis is said to be discontinuous.
The extracellular matrix represents a
large fraction of the biofilm, accounting for 50%-90% of the total dry mass.
Microbial mats that float on water, for example, are biofilms that contain
large populations of photosynthetic microorganisms. Biofilms found in the human mouth may contain hundreds of bacterial species.
The most important difference between prokaryotes and eukaryotes is the
latter's membrane-bound nucleus, which influences the ease of use of RNA molecules for protein synthesis.
During replication, one strand, which is complementary to the 3' to 5' parental DNA strand, is synthesized continuously toward the replication fork because polymerase can add nucleotides in this direction. This continuously synthesized strand is known as the
leading strand
Although -static treatments do not actually kill infectious agents, they are often
less toxic to humans and other animals, and may also better preserve the integrity of the item treated. Such treatments are typically sufficient to keep the microbial population of an item in check.
For researchers or laboratory personnel working with pathogens, the risks associated with specific pathogens determine the
levels of cleanliness and control required.
Subsequent knowledge about the processes of transcription and translation led scientists to revise this to the "one gene-one polypeptide" hypothesis. Although there are some genes that do not encode polypeptides (but rather encode for transfer RNAs [tRNAs] or ribosomal RNAs [rRNAs], which we will discuss later), the one gene-one enzyme hypothesis is true in many cases, especially in microbes. Beadle and Tatum's discovery of the
link between genes and corresponding characteristics earned them the 1958 Nobel Prize in Physiology and Medicine and has since become the basis for modern molecular genetics.
The foremost approach is to measure the turbidity (cloudiness) of a sample of bacteria in a
liquid suspension.
in many situations, it is important to know the number of
live, or viable, cells.
In many cases, though, it is advantageous to maintain cells in the
logarithmic phase of growth.
rRNA and tRNA are stable types of RNA. In prokaryotes and eukaryotes, tRNA and rRNA are encoded in the DNA, then copied into
long RNA molecules that are cut to release smaller fragments containing the individual mature RNA species.
The soil bacteria Actinomyces grow in long filaments divided by septa, similar to the mycelia seen in fungi, resulting in
long cells with multiple nucleoids.
The telomeres protect coding sequences from being
lost as cells continue to divide.
Ribosomes are the site of translation
mRNA has Shine-Dalgarno sequence that bind to 16S rRNA in the small subunit of ribosomes
Translation requires the input of an
mRNA template, ribosomes, tRNAs, and various enzymatic factors.
Multiple ribosomes can translate the same mRNA molecule
mRNA with multiple associated ribosomes- polyribosome (polysome) Polysomes are found in bacteria, archaea, and eukaryotes
Genes can code for
mRNA, rRNA or tRNA
The essential steps of replication in eukaryotes are the same as in prokaryotes. Before replication can start, the DNA has to be
made available as a template.
One example is in industries that harvest microbial products. A chemostat is used to
maintain a continuous culture in which nutrients are supplied at a steady rate. A controlled amount of air is mixed in for aerobic processes. Bacterial suspension is removed at the same rate as nutrients flow in to maintain an optimal growth environment.
Other protocols are followed to
maintain the sterile field while the medical procedure is being performed.
Each mRNA molecule is simultaneously translated by
many ribosomes, all synthesizing protein in the same direction: reading the mRNA from 5' to 3' and synthesizing the polypeptide from the N terminus to the C terminus.
the highest pH is the
maximum growth pH
The highest temperature at which growth can occur is its
maximum growth temperature. The following ranges of permissive growth temperatures are approximate only and can vary according to other environmental factors.
The highest tolerated concentration of oxygen is the
maximum permissive oxygen concentration.
Most bacteria are neutrophiles,
meaning they grow optimally at a pH within one or two pH units of the neutral pH of 7 (see Figure 9.26). Most familiar bacteria, like Escherichia coli, staphylococci, and Salmonella spp. are neutrophiles and do not fare well in the acidic pH of the stomach. However, there are pathogenic strains of E. coli, S. typhi, and other species of intestinal pathogens that are much more resistant to stomach acid. In comparison, fungi thrive at slightly acidic pH values of 5.0-6.0.
Biofilms can also form on
medical devices used in or on the body, causing infections in patients with in-dwelling catheters, artificial joints, or contact lenses.
This can be accomplished using a modification of the plate count technique called the
membrane filtration technique
Within a biofilm, different species of microorganisms establish
metabolic collaborations in which the waste product of one organism becomes the nutrient for another. For example, aerobic microorganisms consume oxygen, creating anaerobic regions that promote the growth of anaerobes. This occurs in many polymicrobial infections that involve both aerobic and anaerobic pathogens.
The synthesis of proteins consumes more of a cell's energy than any other_____. In turn, proteins account for more mass than any other macromolecule of living organisms. They perform virtually every function of a cell, serving as both functional (e.g., enzymes) and structural elements. The process of translation, or
metabolic process. protein synthesis, the second part of gene expression, involves the decoding by a ribosome of an mRNA message into a polypeptide product.
Tube E on the right shows a "Goldilocks" culture. The oxygen level has to be just right for growth, not too much and not too little. These
microaerophiles are bacteria that require a minimum level of oxygen for growth, about 1%-10%, well below the 21% found in the atmosphere.
The process of disinfection inactivates most
microbes on the surface of a fomite by using antimicrobial chemicals or heat.
Failure to practice aseptic technique during many types of clinical procedures may introduce
microbes to the patient's body and put the patient at risk for sepsis, a systemic inflammatory response to an infection that results in high fever, increased heart and respiratory rates, shock, and, possibly, death.
The degree of microbial control can be evaluated using a
microbial death curve to describe the progress and effectiveness of a particular protocol.
Two separate lines of research, begun in the mid to late 1800s, ultimately led to the discovery and characterization of DNA and the foundations of genetics, the science of heredity. These lines of research began to converge in the 1920s, and research using
microbial systems ultimately resulted in significant contributions to elucidating the molecular basis of genetics.
Through the early 20th century, DNA was not yet recognized as the genetic material responsible for heredity, the passage of traits from one generation to the next. In fact, much of the research was dismissed until the
mid-20th century.
Cells have developed several repair mechanisms to
minimize the number of mutations that persist.
The organism will not grow outside the range of oxygen levels found between the
minimum and maximum permissive oxygen concentrations.
The lowest pH value that an organism can tolerate is called the
minimum growth pH
The lowest temperature at which the organism can survive and replicate is its
minimum growth temperature
The lowest concentration of oxygen that allows growth is called the
minimum permissive oxygen concentration.
Spontaneous Mutations Can occur due to the
mobile genetic elements such as insertion sequences and transposons "DNA that can move and integrate into different sites in the chromosomes" Usually inactivate genes
Organisms categorized as mesophiles ("middle loving") are adapted to
moderate temperatures, with optimal growth temperatures ranging from room temperature (about 20 °C) to about 45 °C. As would be expected from the core temperature of the human body, 37 °C (98.6 °F), normal human microbiota and pathogens (e.g., E. coli, Salmonella spp., and Lactobacillus spp.) are mesophiles.
Chemicals that are more mutagenic will bring about
more mutants with restored histidine synthesis in the Ames test.
The optimum growth pH is the
most favorable pH for the growth of an organism.
For these specimens, microbiologists routinely use the
most probable number (MPN) method, a statistical procedure for estimating of the number of viable microorganisms in a sample.
Structurally speaking, ribonucleic acid (RNA), is quite similar to DNA. However, whereas DNA molecules are typically long and double stranded, RNA molecules are
much shorter and are typically single stranded. RNA molecules perform a variety of roles in the cell but are mainly involved in the process of protein synthesis (translation) and its regulation.
Eukaryotic genomes are much more complex and larger than prokaryotic genomes and are typically composed of
multiple linear chromosomes
The human genome, for example, has 3 billion base pairs per haploid set of chromosomes, and 6 billion base pairs are inserted during replication. There are
multiple origins of replication on each eukaryotic chromosome; the human genome has 30,000 to 50,000 origins of replication.
Recurring cyanobacterial algal blooms in Lake Erie have forced
municipalities to issue drinking water bans for days at a time because of unacceptable toxin levels. This is just a small sampling of the negative consequences of algal blooms, red tides, and dead zones.
induced Mutations Caused by
mutagens "Any agent that directly damages DNA, alters its chemistry, or interferes with its functioning" Chemical Mutagen - base analogs, DNA-modifying agents, and intercalating agent Physical agents - radiation
The Ames test measures the
mutation rate associated with exposure to the compound, which, if elevated, may indicate that exposure to this compound is associated with greater cancer risk.
Exposure to either ionizing or nonionizing radiation can each induce
mutations in DNA, although by different mechanisms.
Consumption of contaminated shellfish can cause severe
neurological and gastrointestinal symptoms in humans.
The ability of H. pylori to survive the low pH of the stomach would seem to suggest that it is an extreme acidophile. As it turns out, this is not the case. In fact, H. pylori is a
neutrophile. So, how does it survive in the stomach? Remarkably, H. pylori creates a microenvironment in which the pH is nearly neutral. It achieves this by producing large amounts of the enzyme urease, which breaks down urea to form NH4+ and CO2. The ammonium ion raises the pH of the immediate environment.
The bacterial cell cycle involves the formation of
new cells through the replication of DNA and partitioning of cellular components into two daughter cells.
Fertilizers that are applied at inappropriate times or in too-large quantities allow
nitrogen and phosphorus compounds to escape use by crop plants and enter drainage systems.
Artificial fertilizers provide
nitrogen and phosphorus, key limiting nutrients, to crop plants, removing the normal barriers that would otherwise limit the rate of growth. Thus, fertilized crops grow much faster, and farms that use fertilizer produce higher crop yields.
Items that may contact but not penetrate intact skin are
noncritical items; examples are bed linens, furniture, crutches, stethoscopes, and blood pressure cuffs. These articles need to be clean but not highly disinfected.
Salt requirements
nonhalophiles, halotolerant, moderate halophiles, and extreme halophiles
In 1928, British bacteriologist Frederick Griffith he reported the first demonstration of bacterial transformation, a process in which external DNA is taken up by a cell, thereby changing its characteristics.3 He was working with two strains of Streptococcus pneumoniae, a bacterium that causes pneumonia: a rough (R) strain and a smooth (S) strain. The R strain is
nonpathogenic and lacks a capsule on its outer surface; as a result, colonies from the R strain appear rough when grown on plates. The S strain is pathogenic and has a capsule outside its cell wall, allowing it to escape phagocytosis by the host immune system. The capsules cause colonies from the S strain to appear smooth when grown on plates.
The termination of translation occurs when a
nonsense codon (UAA, UAG, or UGA) is encountered for which there is no complementary tRNA.
Chemicals called nucleoside analogs are structurally similar to
normal nucleotide bases and can be incorporated into DNA during replication
Collinearity of the genetic code perpetuate mutation to protein
normal(wild type) altered (mutant type)
Using a counting chamber does not necessarily yield an accurate count of the number of live cells because it is
not always possible to distinguish between live cells, dead cells, and debris of the same size under the microscope. newly developed fluorescence staining techniques make it possible to distinguish viable and dead bacteria.
Because some microbes remain, the disinfected item is
not considered sterile.
Sterilization protocols require procedures that are
not practical, or necessary, in many settings.
MacConkey agar and sorbitol-MacConkey agar plates and xylose-lysine-deoxycholate (XLD) plates were inoculated with stool samples and did
not reveal any unusually colored colonies, and no black colonies or white colonies were observed on XLD. All lactose fermenters on MacConkey agar also ferment sorbitol. These results ruled out common agents of food-borne illnesses: E. coli, Salmonella spp., and Shigella spp.
By experimenting with true-breeding pea plants, Mendel avoided the appearance of unexpected traits in offspring that might occur if he used plants that were
not true-breeding. Mendel performed hybridizations, which involve mating two true-breeding individuals (P generation) that have different traits, and examined the characteristics of their offspring (first filial generation, F1) as well as the offspring of self-fertilization of the F1 generation (second filial generation, F2)
the genetic role of a fourth class of molecules:
nucleic acids. Like other macromolecules, nucleic acids are composed of monomers, called nucleotides, which are polymerized to form large strands. Each nucleic acid strand contains certain nucleotides that appear in a certain order within the strand, called its base sequence.
In eukaryotes, synthesis, cutting, and assembly of rRNA into ribosomes takes place in the
nucleolus region of the nucleus, but these activities occur in the cytoplasm of prokaryotes. Neither of these types of RNA carries instructions to direct the synthesis of a polypeptide, but they play other important roles in protein synthesis.
DNA can also be enzymatically degraded and used as a source of
nucleosides and nucleotides for the cell. Unlike other macromolecules, DNA does not serve a structural role in cells.
The resulting strand of nucleic acid has a free phosphate group at the 5ʹ carbon end and a free hydroxyl group at the 3ʹ carbon end. The two unused phosphate groups from the nucleotide triphosphate are released as pyrophosphate during phosphodiester bond formation. Pyrophosphate is subsequently hydrolyzed, releasing the energy used to drive
nucleotide polymerization.
Translation of the mRNA template converts
nucleotide-based genetic information into the "language" of amino acids to create a protein product.
Bacteria use the same RNA polymerase to transcribe all of their genes. Like DNA polymerase, RNA polymerase adds
nucleotides one by one to the 3'-OH group of the growing nucleotide chain.
DNA pol III can add
nucleotides only in the 5' to 3' direction It requires a free 3'-OH group to which it can add nucleotides
In an early set of experiments, Hämmerling removed either the cap or the foot of the cells and observed whether new caps or feet were regenerated (Figure 10.3). He found that when the foot of these cells was removed, new feet did not grow; however, when caps were removed from the cells, new caps were regenerated. This suggested that the hereditary information was located in the
nucleus-containing foot of each cell.
In a clever set of experiments in the 1930s and 1940s, German scientist Joachim Hämmerling (1901-1980), using the single-celled alga Acetabularia as a microbial model, established that the genetic information in a eukaryotic cell is housed within the
nucleus.
The number of cells increases exponentially and can be expressed as 2n, where n is the
number of generations.
The structure of biofilms may also change with other environmental conditions such as
nutrient availability.
Inappropriate use of fertilizers in residential settings can also contribute to
nutrient loads, which find their way to lakes and coastal marine ecosystems. As water warms and nutrients are plentiful, microscopic algae bloom, often changing the color of the water because of the high cell density.
Channels in the EPS allow movement of
nutrients, waste, and gases throughout the biofilm. This keeps the cells hydrated, preventing desiccation. EPS also shelters organisms in the biofilm from predation by other microbes or cells (e.g., protozoans, white blood cells in the human body).
In tube A, all the growth is seen at the top of the tube. The bacteria are
obligate (strict) aerobes that cannot grow without an abundant supply of oxygen.
Tube B looks like the opposite of tube A. Bacteria grow at the bottom of tube B. Those are
obligate anaerobes, which are killed by oxygen.
The goal of the serial dilution process is to
obtain plates with CFUs in the range of 30-300, and the process usually involves several dilutions in multiples of 10 to simplify calculation.
DNA replication has been well studied in bacteria primarily because
of the small size of the genome and the mutants that are available. E. coli has 4.6 million base pairs (Mbp) in a single circular chromosome and all of it is replicated in approximately 42 minutes, starting from a single origin of replication and proceeding around the circle bidirectionally (i.e., in both directions). This means that approximately 1000 nucleotides are added per second. The process is quite rapid and occurs with few errors.
These base analogs induce mutations because they
often have different base-pairing rules than the bases they replace.
The pyrimidines, cytosine (C) and thymine (T), are smaller nitrogenous bases that have
only a six-carbon ring structure.
Microbes can be roughly classified according to the range of temperature at which they can grow. The growth rates are the highest at the
optimum growth temperature for the organism.
viricides kill
or inactivate viruses,
As the algal population expands and then dies, it provides a large increase in
organic matter to the bacteria that live in deep water. With this large supply of nutrients, the population of nonphotosynthetic microorganisms explodes, consuming available oxygen and creating "dead zones" where animal life has virtually disappeared.
Photoreactivation is particularly important for
organisms chronically exposed to ultraviolet radiation, like plants, photosynthetic bacteria, algae, and corals, to prevent the accumulation of mutations caused by thymine dimer formation.
Fortunately, despite significant differences in size, structure, reproduction strategies, and other biological characteristics, there is biochemical unity among all
organisms; they have in common the same underlying molecules responsible for heredity and the use of genetic material to give cells their varying characteristics.
E. coli has a single origin of replication (as do most prokaryotes), called
oriC, on its one chromosome.
The initiation of replication occurs at specific nucleotide sequence called the
origin of replication, where various proteins bind to begin the replication process.
Offspring appear to be a "blend" of their parents' traits when we look at characteristics that exhibit continuous variation. The blending theory of inheritance asserted that the
original parental traits were lost or absorbed by the blending in the offspring, but we now know that this is not the case.
The sliding clamp is a ring-shaped protein that binds to the DNA and holds the polymerase in place. Beyond its role in initiation, topoisomerase also prevents the
overwinding of the DNA double helix ahead of the replication fork as the DNA is opening up; it does so by causing temporary nicks in the DNA helix and then resealing it.
Bacterial density increases in the area where
oxygen concentration is best suited for the growth of that particular organism.
Only after cyanobacteria started releasing oxygen as a byproduct of photosynthesis and the capacity of iron in the oceans for taking up oxygen was exhausted did
oxygen levels increase in the atmosphere. This event, often referred to as the Great Oxygenation Event or the Oxygen Revolution, caused a massive extinction.
pH requirements
pH can be acidic, neutral, and alkaline Extreme pH affects the structure of all macromolecules Proteins are affected the most
ETEC produces several plasmid-encoded virulence factors that make it
pathogenic compared with typical E. coli. These include the secreted toxins heat-labile enterotoxin (LT) and heat-stabile enterotoxin (ST), as well as colonization factor (CF).
As growth slows, so too does the synthesis of
peptidoglycans, proteins, and nucleic-acids; thus, stationary cultures are less susceptible to antibiotics that disrupt these processes.
The formation of each peptide bond is catalyzed by
peptidyl transferase, an RNA-based ribozyme that is integrated into the 50S ribosomal subunit.
The enzymes play an important role by limiting the damage caused by
peroxidation of membrane lipids.
Brief history of discovery and characterization of DNA: Richard Altmann (1852-1900) - Isolated
phosphorus-rich chemicals from leukocytes, named it "nuclein"
If DNA serves as the complete library of cellular information, mRNA serves as a
photocopy of specific information needed at a particular point in time that serves as the instructions to make a protein.
The direct repair (also called light repair) of thymine dimers occurs through the process of
photoreactivation in the presence of visible light.
Sterilization can be accomplished through either
physical means, such as exposure to high heat, pressure, or filtration through an appropriate filter, or by chemical means.
Despite the discovery of DNA in the late 1800s, scientists did not make the association with heredity for many more decades. To make this connection, scientists, including a number of microbiologists, performed many experiments on
plants, animals, and bacteria.
A very dilute sample—drinking water, for example—may not contain enough organisms to use either of the
plate count methods described. In such cases, the original sample must be concentrated rather than diluted before plating.
The Ames test uses as the test organism a strain of Salmonella typhimurium that is a histidine auxotroph, unable to synthesize its own histidine because of a mutation in an essential gene required for its synthesis. After exposure to a potential mutagen, these bacteria are
plated onto a medium lacking histidine, and the number of mutants regaining the ability to synthesize histidine is recorded and compared with the number of such mutants that arise in the absence of the potential mutagen
There are several types of mutations that are classified according to how the DNA molecule is altered.
point mutation insertion deletion
Once elongation is complete, another processing enzyme then adds a string of approximately 200 adenine nucleotides to the 3' end, called the
poly-A tail
Phosphodiester bonds leads to formation of
polynucleotide
The complete structure containing an mRNA with multiple associated ribosomes is called a
polyribosome (or polysome).
EPS is a hydrated gel composed primarily of
polysaccharides and containing other macromolecules such as proteins, nucleic acids, and lipids. It plays a key role in maintaining the integrity and function of the biofilm.
Depletion of oxygen in the water is not the only damaging consequence of some algal blooms. The algae that produce red tides in the Gulf of Mexico, Karenia brevis, secrete
potent toxins that can kill fish and other organisms and also accumulate in shellfish.
From each tube, a sample is plated on solid medium using either the
pour plate method (Figure 9.12) or the spread plate method
The enrichment cultures foster the
preferential growth of a desired microorganism that represents a fraction of the organisms present in an inoculum. For example, if we want to isolate bacteria that break down crude oil, hydrocarbonoclastic bacteria, sequential subculturing in a medium that supplies carbon only in the form of crude oil will enrich the cultures with oil-eating bacteria.
Cells in the log phase show constant growth rate and uniform metabolic activity. For this reason, cells in the log phase are
preferentially used for industrial applications and research work. The log phase is also the stage where bacteria are the most susceptible to the action of disinfectants and common antibiotics that affect protein, DNA, and cell-wall synthesis.
Photoreactivation appears to be
present in all organisms, with the exception of placental mammals, including humans.
In bacteria, DNA polymerase III binds to the 3'-OH group of the nicked strand and begins to unidirectionally replicate the DNA using the un-nicked strand as a template, displacing the nicked strand as it does so. Completion of DNA replication at the site of the original nick results in full displacement of the nicked strand, which may then recircularize into a single-stranded DNA molecule. RNA primase then synthesizes a
primer to initiate DNA replication at the single-stranded origin (sso) site of the single-stranded DNA (ssDNA) molecule, resulting in a double-stranded DNA (dsDNA) molecule identical to the other circular DNA molecule.
Because this sequence allows the start of DNA synthesis, it is appropriately called the
primer.
In addition to the mRNA template, many molecules and macromolecules contribute to the
process of translation
Translation is similar in
prokaryotes and eukaryotes.
Following initiation of replication, in a process similar to that found in
prokaryotes, elongation is facilitated by eukaryotic DNA polymerases.
The resolution of concatemers is an issue unique to
prokaryotic DNA replication because of their circular chromosomes. Because both bacterial DNA gyrase and topoisomerase IV are distinct from their eukaryotic counterparts, these enzymes serve as targets for a class of antimicrobial drugs called quinolones.
Most of the mistakes introduced during DNA replication are
promptly corrected by most DNA polymerases through a function called proofreading.
Microbiologists have also played a crucial part in our understanding of genetics. Experimental organisms such as Mendel's garden peas, Morgan's fruit flies, and McClintock's corn had already been used successfully to pave the way for an understanding of genetics. However, microbes and viruses were (and still are) excellent model systems for the study of genetics because, unlike peas, fruit flies, and corn, they are
propagated more easily in the laboratory, growing to high population densities in a small amount of space and in a short time. In addition, because of their structural simplicity, microbes and viruses are more readily manipulated genetically.
It is essential that all of the intron-encoded RNA sequences are completely and precisely removed from a pre-mRNA before protein synthesis so that the exon-encoded RNA sequences are
properly joined together to code for a functional polypeptide. If the process errs by even a single nucleotide, the sequences of the rejoined exons would be shifted, and the resulting polypeptide would be nonfunctional.
The primary transcript (also called pre-mRNA) is first coated with RNA-stabilizing proteins to
protect it from degradation while it is processed and exported out of the nucleus.
Hershey and Chase were studying a bacteriophage, a virus that infects bacteria. Viruses typically have a simple structure: a
protein coat, called the capsid, and a nucleic acid core that contains the genetic material, either DNA or RNA
DNA denaturation is analogous to
protein denaturation,
Point mutations may have a wide range of effects on
protein function
The genetic code is nearly universal. With a few exceptions, virtually all species use the same genetic code for
protein synthesis, which is powerful evidence that all extant life on earth shares a common origin.
DNA replication uses a large number of
proteins and enzymes
Adenosine triphosphate (ATP) formation, biosynthesis of
proteins and nucleic acids, and consumption of oxygen can all be monitored to estimate the number of cells.
Many missense mutations result in
proteins that are still functional, at least to some degree.
Eukaryotic DNA is highly supercoiled and packaged, which is facilitated by many
proteins, including histones
In Microbial Metabolism, we discussed the microbial catabolism of three classes of macromolecules:
proteins, lipids and carbohydrates.
Sterile fields are created according to
protocols requiring the use of sterilized materials, such as packaging and drapings, and strict procedures for washing and application of sterilants.
Base pairing takes place between a
purine and pyrimidine.
James Watson (1928-), an American scientist, and Francis Crick (1916-2004), a British scientist, were working together in the 1950s to discover DNA's structure. They used Chargaff's rules and Franklin and Wilkins' X-ray diffraction images of DNA fibers to piece together the
purine-pyrimidine pairing of the double helical DNA molecule
To construct the sugar-phosphate backbone, the two terminal phosphates are released from the dNTP as a
pyrophosphate.
The mechanism by which cells in a biofilm coordinate their activities in response to environmental stimuli is called
quorum sensing.
RNAs play important role in protein synthesis rRNA
rRNA are stable types of RNA rRNA copied from DNA as a long RNA molecules that are cut to release smaller fragments rRNA is a major constituent of ribosomes (60% by mass) rRNA ensures the proper alignment of the mRNA, tRNA, and the ribosomes rRNA also has an enzymatic activity, catalyzes the formation of the peptide bonds between two amino acid
Regardless of the environment where they occur, biofilms are not
random collections of microorganisms; rather, they are highly structured communities that provide a selective advantage to their constituent microorganisms.
Filamentous biofilms called streamers form in
rapidly flowing water, such as freshwater streams, eddies, and specially designed laboratory flow cells that replicate growth conditions in fast-moving fluids.
The EPS may also slow the diffusion of antibiotics and antiseptics, preventing them from
reaching cells in the deeper layers of the biofilm.
Most organisms could not survive the powerful oxidative properties of
reactive oxygen species (ROS), highly unstable ions and molecules derived from partial reduction of oxygen that can damage virtually any macromolecule or structure with which they come in contact.
DNA renaturation
reannealing of DNA strands by complementary base pairing
Because only 0.1 mL of suspension was pipetted on the plate, the multiplier required to
reconstitute the original concentration is 10 × 10,000.
Cells from the corresponding colony on the nutritionally complete plate can be used to
recover the mutant for further study.
Thioglycolate has strong
reducing properties and autoclaving flushes out most of the oxygen. The tubes are inoculated with the bacterial cultures to be tested and incubated at an appropriate temperature. Over time, oxygen slowly diffuses throughout the thioglycolate tube culture from the top.
Because the production of thymine dimers is common (many organisms cannot avoid ultraviolet light), mechanisms have evolved to
repair these lesions.
DNA pol I and DNA pol II are primarily required for
repair.
One common technique used to identify bacterial mutants is called
replica plating. This technique is used to detect nutritional mutants, called auxotrophs, which have a mutation in a gene encoding an enzyme in the biosynthesis pathway of a specific nutrient, such as an amino acid. As a result, whereas wild-type cells retain the ability to grow normally on a medium lacking the specific nutrient, auxotrophs are unable to grow on such a medium.
The elucidation of the structure of the double helix by James Watson and Francis Crick in 1953 provided a hint as to how DNA is copied during the process of
replication
Two replication forks are formed at the origin of replication, allowing for bidirectional replication and formation of a structure that looks like a bubble when viewed with a transmission electron microscope; as a result, this structure is called a
replication bubble
Recall that AT sequences have fewer hydrogen bonds and, hence, have weaker interactions than guanine-cytosine (GC) sequences. These enzymes require ATP hydrolysis. As the DNA opens up, Y-shaped structures called
replication forks are formed.
Macroelements
required in relatively large amounts. C, O, H, N, S, and P. components of carbohydrates, lipids, proteins, and nucleic acids.
One critical difference in activity between DNA polymerase and RNA polymerase is the
requirement for a 3'-OH onto which to add nucleotides: DNA polymerase requires such a 3'-OH group, thus necessitating a primer, whereas RNA polymerase does not.
DNA pol III is able to add nucleotides only in the 5' to 3' direction (a new DNA strand can be only extended in this direction). This is because DNA polymerase
requires a free 3'-OH group to which it can add nucleotides by forming a covalent phosphodiester bond between the 3'-OH end and the 5' phosphate of the next nucleotide. This also means that it cannot add nucleotides if a free 3'-OH group is not available, which is the case for a single strand of DNA. The problem is solved with the help of an RNA sequence that provides the free 3'-OH end.
the benefits of crop fertilizer—the main cause of such blooms—are difficult to dispute. There is no easy solution to this dilemma, as a ban on fertilizers is not politically or economically feasible. In lieu of this, we must advocate for
responsible use and regulation in agricultural and residential contexts, as well as the restoration of wetlands, which can absorb excess fertilizers before they reach lakes and oceans.
RNA is typically single stranded and is made of ribonucleotides that are linked by phosphodiester bonds. A ribonucleotide in the RNA chain contains
ribose (the pentose sugar), one of the four nitrogenous bases (A, U, G, and C), and a phosphate group.
Whereas many bacterial plasmids replicate by a process similar to that used to copy the bacterial chromosome, other plasmids, several bacteriophages, and some viruses of eukaryotes use
rolling circle replication
viral infection examples
rotavirus or norovirus
A typical application of MPN method is the estimation of the number of coliforms in a
sample of pond water. Coliforms are gram-negative rod bacteria that ferment lactose.
All the tests point to Staphylococcus aureus as the organism that
secreted the toxin.
Under the right environmental conditions, this type of mutation may give the organism that harbors it a
selective advantage.
In 1856, Mendel began his decade-long research into inheritance patterns. He used the diploid garden pea, Pisum sativum, as his primary model system because it naturally
self-fertilizes and is highly inbred, producing "true-breeding" pea plant lines—plants that always produce offspring that look like the parent.
Matthew Meselson (1930-) and Franklin Stahl (1929-) experiment grew E. coli for several generations in a medium containing a "heavy" isotope of nitrogen (15N) that was incorporated into nitrogenous bases and, eventually, into the DNA. This labeled the parental DNA. The E. coli culture was then shifted into a medium containing 14N and allowed to grow for one generation. The cells were harvested and the DNA was isolated. The DNA was separated by ultracentrifugation, during which the DNA formed bands according to its density. DNA grown in 15N would be expected to form a band at a higher density position than that grown in 14N. Meselson and Stahl noted that after one generation of growth in 14N, the single band observed was intermediate in position in between DNA of cells grown exclusively in 15N or 14N. This suggested either a
semiconservative or dispersive mode of replication. Some cells were allowed to grow for one more generation in 14N and spun again. The DNA harvested from cells grown for two generations in 14N formed two bands: one DNA band was at the intermediate position between 15N and 14N, and the other corresponded to the band of 14N DNA. These results could only be explained if DNA replicates in a semiconservative manner. Therefore, the other two models were ruled out. As a result of this experiment, we now know that during DNA replication, each of the two strands that make up the double helix serves as a template from which new strands are copied. The new strand will be complementary to the parental or "old" strand. The resulting DNA molecules have the same sequence and are divided equally into the two daughter cells.
Gastrointestinal endoscopes and various types of equipment for respiratory therapies are examples of
semicritical items; they may contact mucous membranes or nonintact skin but do not penetrate tissues.
The RNA product is complementary to the template strand of DNA and is almost identical to the nontemplate DNA strand, or the
sense strand. The only difference is that in RNA, all of the T nucleotides are replaced with U nucleotides; during RNA synthesis, U is incorporated when there is an A in the complementary antisense strand.
The laboratory itself must be located either in a
separate building or in an isolated portion of a building and have its own air supply and exhaust system, as well as its own decontamination system.
DNA denaturation
separation of two DNA strand upon breaking the hydrogen bond
The ability to artificially manipulate DNA in this way is the basis for
several important techniques in biotechnology.
Protein-encoding primary transcripts, the RNA molecules directly synthesized by RNA polymerase, must undergo
several processing steps to protect these RNA molecules from degradation during the time they are transferred from the nucleus to the cytoplasm and translated into a protein. For example, eukaryotic mRNAs may last for several hours, whereas the typical prokaryotic mRNA lasts no more than 5 seconds.
The toxin secreted by S. aureus is known to cause
severe gastroenteritis. The organism was probably introduced into the salad during preparation by the food handler and multiplied while the salad was kept in the warm ambient temperature during the speeches.
The sixth subunit is known as
sigma (σ). The σ factor enables RNA polymerase to bind to a specific promoter, thus allowing for the transcription of various genes. There are various σ factors that allow for transcription of various genes.
However, careless use and overuse of artificial fertilizers have been demonstrated to have
significant negative impacts on aquatic ecosystems, both freshwater and marine.
This change would have no effect on the protein's structure, and is thus called a
silent mutation
Various organizations around the world, including the World Health Organization (WHO) and the European Union (EU), use a
similar classification scheme.
Another microbial model, the red bread mold Neurospora crassa, was used by George Beadle and Edward Tatum to demonstrate the relationship between genes and the proteins they encode. Beadle had worked with fruit flies in Morgan's laboratory but found them too complex to perform certain types of experiments. N. crassa, on the other hand, is a
simpler organism and has the ability to grow on a minimal medium because it contains enzymatic pathways that allow it to use the medium to produce its own vitamins and amino acids.
Most bacteria have a
single circular chromosome; however, some exceptions exist. For example, Borrelia burgdorferi, the causative agent of Lyme disease, has a linear chromosome.
E. coli has a
single origin of replication (as do most prokaryotes) Contains approximately 245 base pairs and is rich in adenine- thymine (AT)
Strong ionizing radiation like X-rays and gamma rays can cause
single- and double-stranded breaks in the DNA backbone through the formation of hydroxyl radicals on radiation exposure
Mature tRNAs take on a three-dimensional structure when complementary bases exposed in the
single-stranded RNA molecule hydrogen bond with each other
Rhinoviruses, which cause the common cold; influenza viruses; and the Ebola virus are
single-stranded RNA viruses.
The DNA near each replication fork is coated with
single-stranded binding proteins to prevent the single-stranded DNA from rewinding into a double helix.
Some of the proteins that bind to the origin of replication are important in making
single-stranded regions of DNA accessible for replication.
Staphylococci are found on the
skin and upper respiratory tract.
Chemical mutagens known as intercalating agents work differently. These molecules_____. As a result, during DNA replication, DNA polymerase may either
slide between the stacked nitrogenous bases of the DNA double helix, distorting the molecule and creating atypical spacing between nucleotide base pairs. skip replicating several nucleotides (creating a deletion) or insert extra nucleotides (creating an insertion). Either outcome may lead to a frameshift mutation
There is no cure for HIV infection, but many drugs have been developed to
slow or block the progression of the virus.
In E. coli, this complex involves the
small 30S ribosome, the mRNA template, three initiation factors that help the ribosome assemble correctly, guanosine triphosphate (GTP) that acts as an energy source, and a special initiator tRNA carrying N-formyl-methionine (fMet-tRNAfMet)
Pregnant women are thus advised to avoid consumption of
soft cheeses, refrigerated cold cuts, smoked seafood, and unpasteurized dairy products.
L. monocytogenes is a gram-positive short rod found in
soil, water, and food. It is classified as a psychrophile and is halotolerant. Its ability to multiply at refrigeration temperatures (4-10 °C) and its tolerance for high concentrations of salt (up to 10% sodium chloride [NaCl]) make it a frequent source of food poisoning.
This metabolic capability of H. pylori is the basis of an accurate, noninvasive test for infection. The patient is given a
solution of urea containing radioactively labeled carbon atoms. If H. pylori is present in the stomach, it will rapidly break down the urea, producing radioactive CO2 that can be detected in the patient's breath. Because peptic ulcers may lead to gastric cancer, patients who are determined to have H. pylori infections are treated with antibiotics.
Generation time for bacteria is different because
some bacteria may reproduce rapidly or remain dormant for thousands of years.
"sterilization" is somewhat of a
somewhat of a misnomer in this context, and commercial sterilization may be more accurately described as "quasi-sterilization."
Serving as adaptors, each tRNA type binds to a
specific codon on the mRNA template and adds the corresponding amino acid to the polypeptide chain. Therefore, tRNAs are the molecules that actually "translate" the language of RNA into the language of proteins.
The laboratory instrument used to measure turbidity is called a
spectrophotometer
Mistakes in the process of DNA replication can cause
spontaneous mutations to occur.
In bacteria capable of producing endospores, many cells undergo
sporulation during the stationary phase.
The central area of the counting chamber is etched into
squares of various sizes.
The DNA template includes repeated nucleotide sequences that act as termination signals, causing RNA polymerase to
stall and release from the DNA template, freeing the RNA transcript.
The degree of drying must be_____. This method is especially useful for____.
standardized to account for residual water content. filamentous microorganisms, which are difficult to enumerate by direct or viable plate count.
Another codon, AUG, also has a special function. In addition to specifying the amino acid methionine, it also typically serves as the
start codon to initiate translation.
Secondary metabolites, including antibiotics, are synthesized in the
stationary phase.
The total number of live cells reaches a plateau referred to as the
stationary phase. In this phase, the number of new cells created by cell division is now equivalent to the number of cells dying; thus, the total population of living cells is relatively stagnant.
Chemicals that can be used to achieve sterilization are called
sterilants. Sterilants effectively kill all microbes and viruses, and, with appropriate exposure time, can also kill endospores.
Medical procedures that carry risk of contamination must be performed in a
sterile field, a designated area that is kept free of all vegetative microbes, endospores, and viruses.
Cells from these colonies are removed from this master plate, often using
sterile velvet. This velvet, containing cells, is then pressed in the same orientation onto plates of various media.
Typical disinfection does not lead to
sterilization because endospores tend to survive even when all vegetative cells have been killed.
The most extreme protocols for microbial control aim to achieve
sterilization: the complete removal or killing of all vegetative cells, endospores, and viruses from the targeted item or environment.
Because eukaryotic chromosomes are linear, one might expect that their replication would be more
straightforward. As in prokaryotes, the eukaryotic DNA polymerase can add nucleotides only in the 5' to 3' direction. In the leading strand, synthesis continues until it reaches either the end of the chromosome or another replication fork progressing in the opposite direction. On the lagging strand, DNA is synthesized in short stretches, each of which is initiated by a separate primer. When the replication fork reaches the end of the linear chromosome, there is no place to make a primer for the DNA fragment to be copied at the end of the chromosome. These ends thus remain unpaired and, over time, they may get progressively shorter as cells continue to divide.
An exonuclease removes a portion of the
strand (including the incorrect nucleotide). The gap formed is then filled in by DNA pol III and ligase.
By the beginning of the 20th century, a great deal of work had already been done on characterizing DNA and establishing the foundations of genetics, including attributing heredity to chromosomes found within the nucleus. Despite all of this research, it was not until well into the 20th century that these lines of research converged and scientists began to consider that DNA could be the genetic material that offspring inherited from their parents. DNA, containing only four different nucleotides, was thought to be
structurally too simple to encode such complex genetic information. Instead, protein was thought to have the complexity required to serve as cellular genetic information because it is composed of 20 different amino acids that could be combined in a huge variety of combinations. Microbiologists played a pivotal role in the research that determined that DNA is the molecule responsible for heredity.
In addition to preventing degradation, factors involved in
subsequent protein synthesis recognize the cap, which helps initiate translation by ribosomes.
Acetabularia spp. are unusually large algal cells that grow asymmetrically, forming a "foot" containing the nucleus, which is used for
substrate attachment; a stalk; and an umbrella-like cap—structures that can all be easily seen with the naked eye.
Resistance to common antibiotics is still rare in Listeria and antibiotic treatment is usually
successful.
As the adaptor molecules of translation, it is surprising that tRNAs can fit so much specificity into
such a small package.
Many ecosystems are still free of molecular oxygen. Some are found in extreme locations_______; others are part of our everyday landscape, such as
such as deep in the ocean or in earth's crust marshes, bogs, and sewers. Within the bodies of humans and other animals, regions with little or no oxygen provide an anaerobic environment for microorganisms.
Other organisms may not require
such extreme measures and can be controlled by a procedure such as washing clothes in a laundry machine.
Individual nucleoside triphosphates combine with each other by covalent bonds known as 5ʹ-3ʹ phosphodiester bonds, or linkages whereby the phosphate group attached to the 5ʹ carbon of the sugar of one nucleotide bonds to the hydroxyl group of the 3ʹ carbon of the sugar of the next nucleotide. Phosphodiester bonding between nucleotides forms the
sugar-phosphate backbone, the alternating sugar-phosphate structure composing the framework of a nucleic acid strand
The subtle structural difference between the sugars gives DNA added stability, making DNA more
suitable for storage of genetic information, whereas the relative instability of RNA makes it more suitable for its more short-term functions.
Hershey and Chase labeled the protein coat in one batch of phage using radioactive sulfur, 35S, because
sulfur is found in the amino acids methionine and cysteine but not in nucleic acids. They labeled the DNA in another batch using radioactive phosphorus, 32P, because phosphorus is found in DNA and RNA but not typically in protein.
Each batch of phage was allowed to infect the cells separately. After infection, Hershey and Chase put each phage bacterial suspension in a blender, which detached the phage coats from the host cell, and spun down the resulting suspension in a centrifuge. The heavier bacterial cells settled down and formed a pellet, whereas the lighter phage particles stayed in the supernatant. In the tube with the protein labeled, the radioactivity remained only in the
supernatant. In the tube with the DNA labeled, the radioactivity was detected only in the bacterial cells. Hershey and Chase concluded that it was the phage DNA that was injected into the cell that carried the information to produce more phage particles, thus proving that DNA, not proteins, was the source of the genetic material. As a result of their work, the scientific community more broadly accepted DNA as the molecule responsible for heredity.
Three main enzymes break down those toxic byproducts:
superoxide dismutase, peroxidase, and catalase. Each one catalyzes a different reaction.
Media that inhibit the growth of unwanted microorganisms and support the growth of the organism of interest by
supplying nutrients and reducing competition are called selective media.
Under appropriate conditions, mesophiles and even thermophiles can
survive freezing. Liquid cultures of bacteria are mixed with sterile glycerol solutions and frozen to −80 °C for long-term storage as stocks. Cultures can withstand freeze drying (lyophilization) and then be stored as powders in sealed ampules to be reconstituted with broth when needed.
Amino acids are attached to tRNA in a separate process
tRNA charging - a process of adding amino acid to a tRNA molecule In bacteria, there can be 60 and 90 types of tRNAs tRNAs are charged by the enzyme - aminoacyl tRNA Synthetases (at least 20 aminoacyl-tRNA synthetases)
Eukaryotic microbes including fungi and protozoans also produce
telomerase to maintain chromosomal integrity. For her discovery of telomerase and its action, Elizabeth Blackburn (1948-) received the Nobel Prize for Medicine or Physiology in 2009.
The ends of the linear chromosomes are known as
telomeres and consist of noncoding repetitive sequences.
Now that the primer provides the free 3'-OH group, DNA polymerase III can now extend this RNA primer, adding DNA nucleotides one by one that are complementary to the
template strand
DNA pol III adds deoxyribonucleotides each complementary to a nucleotide on the
template strand, one by one to the 3'-OH group of the growing DNA chain.
Whereas 61 of the 64 possible triplets code for amino acids, three of the 64 codons do not code for an amino acid; they______. These are called
terminate protein synthesis, releasing the polypeptide from the translation machinery. stop codons or nonsense codons.
Once the complete chromosome has been replicated,
termination of DNA replication must occur.
Once a gene is transcribed, the bacterial polymerase must dissociate from the DNA template and liberate the newly made RNA. This is referred to as
termination of transcription.
Although much is known about initiation of replication, less is known about the
termination process.
At least one plate should also be nutritionally complete to ensure
that cells are being properly transferred between the plates. The other plates lack specific nutrients, allowing the researcher to discover various auxotrophic mutants unable to produce specific nutrients.
The advantage of alternative splicing is
that different types of mRNA transcripts can be generated, all derived from the same DNA sequence. In recent years, it has been shown that some archaea also have the ability to splice their pre-mRNA.
UGA can encode for selenocysteine using a stem-loop structure (known as the selenocysteine insertion sequence, or SECIS element), which is found at
the 3' untranslated region of the mRNA.
Despite compelling correlations between the behavior of chromosomes during meiosis and Mendel's observations, the Chromosomal Theory of Inheritance was proposed long before there was any direct evidence that traits were carried on chromosomes. Thomas Hunt Morgan (1866-1945) and his colleagues spent several years carrying out crosses with the fruit fly, Drosophila melanogaster. They performed meticulous microscopic observations of fly chromosomes and correlated these observations with resulting fly characteristics. Their work provided the first experimental evidence to support
the Chromosomal Theory of Inheritance in the early 1900s. In 1915, Morgan and his "Fly Room" colleagues published The Mechanism of Mendelian Heredity, which identified chromosomes as the cellular structures responsible for heredity. For his many significant contributions to genetics, Morgan received the Nobel Prize in Physiology or Medicine in 1933.
On aligning with the A site, these nonsense codons are recognized by release factors in prokaryotes and eukaryotes that result in
the P-site amino acid detaching from its tRNA, releasing the newly made polypeptide.
According to the CDC, the BSL is determined by
the agent's infectivity, ease of transmission, and potential disease severity, as well as the type of work being done with the agent.
Peptide bonds form between
the amino group of the amino acid attached to the A-site tRNA and the carboxyl group of the amino acid attached to the P-site tRNA.
The first stage involves
the attachment of planktonic cells to a surface coated with a conditioning film of organic material. At this point, attachment to the substrate is reversible, but as cells express new phenotypes that facilitate the formation of EPS, they transition from a planktonic to a sessile lifestyle. The biofilm develops characteristic structures, including an extensive matrix and water channels. Appendages such as fimbriae, pili, and flagella interact with the EPS, and microscopy and genetic analysis suggest that such structures are required for the establishment of a mature biofilm. In the last stage of the biofilm life cycle, cells on the periphery of the biofilm revert to a planktonic lifestyle, sloughing off the mature biofilm to colonize new sites. This stage is referred to as dispersal.
In all cases, the first step in quorum sensing consists of
the binding of the autoinducer to its specific receptor only when a threshold concentration of signaling molecules is reached. Once binding to the receptor takes place, a cascade of signaling events leads to changes in gene expression. The result is the activation of biological responses linked to quorum sensing, notably an increase in the production of signaling molecules themselves, hence the term autoinducer.
In the words of French scientist Jacques Monod, "What is true for E. coli is also true for the elephant," meaning that
the biochemistry of life has been maintained throughout evolution and is shared in all forms of life, from simple unicellular organisms to large, complex organisms. This biochemical continuity makes microbes excellent models to use for genetic studies.
Theodor Boveri and Walter Suttion prodosed
the chromosomal theory of inheritance
The term sanitization refers to
the cleansing of fomites to remove enough microbes to achieve levels deemed safe for public health.
Uracil then pairs with adenine in a subsequent round of replication, resulting in
the conversion of a GC base pair to an AT base pair.
Transfer RNA is the third main type of RNA and one of the smallest, usually only 70-90 nucleotides long. It carries the correct amino acid to the site of protein synthesis in the ribosome. It is the base pairing between the tRNA and mRNA that allows for
the correct amino acid to be inserted in the polypeptide chain being synthesized. Any mutations in the tRNA or rRNA can result in global problems for the cell because both are necessary for proper protein synthesis.
In proofreading, the DNA polymerase reads the newly added base, ensuring that it is complementary to
the corresponding base in the template strand before adding the next one. If an incorrect base has been added, the enzyme makes a cut to release the wrong nucleotide and a new base is added.
The amount of time it takes for a specific protocol to produce a one order-of-magnitude decrease in the number of organisms, or the death of 90% of the population, is called the
the decimal reduction time (DRT) or D-value.
An enzyme called photolyase recognizes
the distortion in the DNA helix caused by the thymine dimer and binds to the dimer. Then, in the presence of visible light, the photolyase enzyme changes conformation and breaks apart the thymine dimer, allowing the thymines to again correctly base pair with the adenines on the complementary strand.
Listeria infection is relatively common among pregnant women because
the elevated levels of progesterone downregulate the immune system, making them more vulnerable to infection. The pathogen can cross the placenta and infect the fetus, often resulting in miscarriage, stillbirth, or fatal neonatal infection.
In both bacteria and archaea, before transcriptional termination occurs, each protein-encoding transcript is already being used to begin synthesis of numerous copies of
the encoded polypeptide(s) because the processes of transcription and translation can occur concurrently, forming polyribosomes
The circular nature of plasmids and the circularization of some viral genomes on infection make this possible. Rolling circle replication begins with
the enzymatic nicking of one strand of the double-stranded circular molecule at the double-stranded origin (dso) site.
Albrecht Kossel - Isolated and characterized
the five different nucleotide bases
For example, commercial dishwashers used in
the food service industry typically use very hot water and air for washing and drying; the high temperatures kill most microbes, sanitizing the dishes.
When the bond between the phosphates is broken and diphosphate is released, the energy released allows for
the formation of a covalent phosphodiester bond by dehydration synthesis between the incoming nucleotide and the free 3'-OH group on the growing DNA strand.
Environmental signals, probably related to low nutrient availability, lead to
the formation of aerial filaments. Within these aerial filaments, elongated cells divide simultaneously. The new cells, which contain a single nucleoid, develop into spores that give rise to new colonies.
Another curious example of cell division in prokaryotes, reminiscent of live birth in animals, is exhibited by
the giant bacterium Epulopiscium. Several daughter cells grow fully in the parent cell, which eventually disintegrates, releasing the new cells to the environment.
Another type of obligate anaerobe encountered in the human body is
the gram-positive, rod-shaped Clostridium spp. Their ability to form endospores allows them to survive in the presence of oxygen. One of the major causes of health-acquired infections is C. difficile, known as C. diff.
fungistatic treatments inhibit
the growth of fungi.
Enterobacteriaceae are found primarily in
the gut and upper respiratory tract but can sometimes spread to the urinary tract, where they are capable of causing infections. It is not unusual to see mixed bacterial infections in which the facultative anaerobes use up the oxygen, creating an environment for the obligate anaerobes to flourish.
Specialized media are used in
the identification of bacteria and are supplemented with dyes, pH indicators, or antibiotics.
pregnancy enhances the risk for infections and makes her more vulnerable to those infections all because
the immune system is downregulated during pregnancy.
Deletions and insertions also cause various effects. Because codons are triplets of nucleotides, insertions or deletions in groups of three nucleotides may lead to
the insertion or deletion of one or more amino acids and may not cause significant effects on the resulting protein's functionality.
If any cells were damaged or shocked during the transfer to the new medium, repair takes place during
the lag phase.
the indirect methods depend on
the measurement of cell presence or activity without actually counting individual cells.
B form of DNA is
the most common form
BSL-4 agents are
the most dangerous and often fatal. These microbes are typically exotic, are easily transmitted by inhalation, and cause infections for which there are no treatments or vaccinations. Examples include Ebola virus and Marburg virus, both of which cause hemorrhagic fevers, and smallpox virus.
The dilution factor is used to calculate
the number of cells in the original cell culture. In our example, an average of 50 colonies was counted on the plates obtained from the 1:10,000 dilution.
The culture density is defined as
the number of cells per unit volume.
Initiation of DNA replication Starts at specific site called
the origin of replication (OriC)
The type of protocol required to achieve the desired level of cleanliness depends on
the particular item to be cleaned. For example, those used clinically are categorized as critical, semicritical, and noncritical.
BSL-3 agents have
the potential to cause lethal infections by inhalation. These may be either indigenous or "exotic," meaning that they are derived from a foreign location, and include pathogens such as Mycobacterium tuberculosis, Bacillus anthracis, West Nile virus, and human immunodeficiency virus (HIV). Because of the serious nature of the infections caused by BSL-3 agents, laboratories working with them require restricted access. Laboratory workers are under medical surveillance, possibly receiving vaccinations for the microbes with which they work. In addition to the standard PPE already mentioned, laboratory personnel in BSL-3 laboratories must also wear a respirator and work with microbes and infectious agents in a biological safety cabinet at all times.
There are two common approaches to inoculating plates for viable counts:
the pour plate and the spread plate methods. Although the final inoculation procedure differs between these two methods, they both start with a serial dilution of the culture.
The first type of processing begins while
the primary transcript is still being synthesized; a special 7-methylguanosine nucleotide, called the 5' cap, is added to the 5' end of the growing transcript.
In nucleotide excision repair (also called dark repair), enzymes remove
the pyrimidine dimer and replace it with the correct nucleotides
Death curves are often plotted as semilog plots just like microbial growth curves because
the reduction in microorganisms is typically logarithmic
deletion
the removal of one or more bases
Some errors introduced during replication are corrected shortly after
the replication machinery has moved. This mechanism is called mismatch repair.
The properties of the EPS vary according to
the resident organisms and environmental conditions.
In prokaryotes and eukaryotes, the basics of elongation of translation are
the same
As a consequence of the degeneracy of the genetic code, a point mutation will commonly result in
the same amino acid being incorporated into the resulting polypeptide despite the sequence change.
The leading strand can be extended from one primer alone, whereas the lagging strand needs a new primer for each of the short Okazaki fragments. The overall direction of the lagging strand will be 3' to 5', and that of the leading strand 5' to 3'. A protein called
the sliding clamp holds the DNA polymerase in place as it continues to add nucleotides.
The duration of the lag phase is determined by many factors, including
the species and genetic make-up of the cells, the composition of the medium, and the size of the original inoculum.
The initiator tRNA interacts with
the start codon AUG of the mRNA and carries a formylated methionine (fMet).
Because many chemicals are not directly mutagenic but are metabolized to mutagenic forms by liver enzymes, rat liver extract is commonly included at
the start of this experiment to mimic liver metabolism. After the Ames test is conducted, compounds identified as mutagenic are further tested for their potential carcinogenic properties by using other models, including animal models like mice and rats.
Other scientists were also actively exploring this field during the mid-20th century. In 1952, American scientist Linus Pauling (1901-1994) was the world's leading structural chemist and odds-on favorite to solve the structure of DNA. Pauling had earlier discovered
the structure of protein α helices, using X-ray diffraction, and, based upon X-ray diffraction images of DNA made in his laboratory, he proposed a triple-stranded model of DNA.6 At the same time, British researchers Rosalind Franklin (1920-1958) and her graduate student R.G. Gosling were also using X-ray diffraction to understand the structure of DNA (Figure 10.14). It was Franklin's scientific expertise that resulted in the production of more well-defined X-ray diffraction images of DNA that would clearly show the overall double-helix structure of DNA.
Effectiveness also depends on
the susceptibility of the agent to that disinfecting agent or protocol. The concentration of disinfecting agent or intensity of exposure is also important. Conditions that limit contact between the agent and the targeted cells
Nonsense mutations result in
the synthesis of proteins that are shorter than the wild type and typically not functional.
During thymine dimer formation,
the two adjacent thymines become covalently linked and, if left unrepaired, both DNA replication and transcription are stalled at this point. DNA polymerase may proceed and replicate the dimer incorrectly, potentially leading to frameshift or point mutations.
The culture's carrying capacity, or maximum culture density, depends on
the types of microorganisms in the culture and the specific conditions of the culture; however, carrying capacity is constant for a given organism grown under the same conditions.
Factors that determine whether a particular treatment is -cidal or -static include
the types of microorganisms targeted, the concentration of the chemical used, and the nature of the treatment applied.
The reading frame
the way nucleotides in mRNA are grouped into codons, for translation is set by the AUG start codon near the 5' end of the mRNA. Each set of three nucleotides following this start codon is a codon in the mRNA message.
The reduced toxicity of some of these -static chemicals also allows
them to be impregnated safely into plastics to prevent the growth of microbes on these surfaces. Such plastics are used in products such as toys for children and cutting boards for food preparation.
Organisms that grow at optimum temperatures of 50 °C to a maximum of 80 °C are called
thermophiles ("heat loving"). They do not multiply at room temperature.
Persister cells are medically important because
they are associated with certain chronic infections, such as tuberculosis, that do not respond to antibiotic treatment.
Eukaryotic mRNAs are also usually monocistronic, meaning that
they each encode only a single polypeptide, whereas prokaryotic mRNAs of bacteria and archaea are commonly polycistronic, meaning that they encode multiple polypeptides.
A test-tube culture starts with autoclaved
thioglycolate medium containing a low percentage of agar to allow motile bacteria to move throughout the medium.
We can easily observe different requirements for molecular oxygen by growing bacteria in
thioglycolate tube cultures.
Agents classified as BSL-2 include
those that pose moderate risk to laboratory workers and the community, and are typically "indigenous," meaning that they are commonly found in that geographical area. These include bacteria such as Staphylococcus aureus and Salmonella spp., and viruses like hepatitis, mumps, and measles viruses. BSL-2 laboratories require additional precautions beyond those of BSL-1, including restricted access; required PPE, including a face shield in some circumstances; and the use of biological safety cabinets for procedures that may disperse agents through the air (called "aerosolization"). BSL-2 laboratories are equipped with self-closing doors, an eyewash station, and an autoclave, which is a specialized device for sterilizing materials with pressurized steam before use or disposal. BSL-1 laboratories may also have an autoclave.
whereas cytosine and guanine form
three hydrogen bonds between them.
It is not clear why people of northern European descent, specifically, carry this mutation, but its prevalence seems
to be highest in northern Europe and steadily decreases in populations as one moves south.
The mRNA then interacts with ribosomes and other cellular machinery
to direct the synthesis of the protein it encodes during the process of translation
The number of microorganisms in dilute samples is usually
too low to be detected by the plate count methods described thus far.
Enzymes 'topoisomerases' change the shape and supercoiling of the chromosome. For bacteria it is
topoisomerase II (DNA gyrase) Enzyme 'helicase' then separates the DNA strands (require ATP) As the DNA opens, Y-shaped replication forks are formed The single stranded DNA near each replication fork is coated with single-stranded binding (SSB) proteins
However, enzymes called topoisomerases change the shape and supercoiling of the chromosome. For bacterial DNA replication to begin, the supercoiled chromosome is relaxed by
topoisomerase II, also called DNA gyrase. An enzyme called helicase then separates the DNA strands by breaking the hydrogen bonds between the nitrogenous base pairs.
Direct counts provide an estimate of the
total number of cells in a sample.
Transcription in prokaryotes and in eukaryotes requires the DNA double helix to partially unwind in the region of RNA synthesis. The unwound region is called a
transcription bubble
The initiation of transcription begins at a promoter, a DNA sequence onto which the
transcription machinery binds and initiates transcription.
British bacteriologist Frederick Griffith (1879-1941) was perhaps the first person to show that hereditary information could be
transferred from one cell to another "horizontally" (between members of the same generation), rather than "vertically" (from parent to offspring).
Elongation proceeds with single-codon movements of the ribosome each called a
translocation event.
With the genes bound in a nucleus, the eukaryotic cell must
transport protein-encoding RNA molecules to the cytoplasm to be translated.
The prefix indicates the type of microbe or infectious agent killed by the
treatment method
The signaling molecules in quorum sensing belong to
two major classes. Gram-negative bacteria communicate mainly using N-acylated homoserine lactones, whereas gram-positive bacteria mostly use small peptides
In eukaryotic organisms, the generation time is the time between the same points of the life cycle in
two successive generations. For example, the typical generation time for the human population is 25 years.
Separating the strands of the double helix would provide
two templates for the synthesis of new complementary strands, but exactly how new DNA molecules were constructed was still unclear.
In academia, the number of women at each level of career advancement continues to decrease, with women holding less than one-third of the positions of Ph.D.-level scientists in tenure-track positions, and less than one-quarter of the full professorships at 4-year colleges and universities.11 Even in the health professions, like nearly all other fields, women are often
underrepresented in many medical careers and earn significantly less than their male counterparts, as shown in a 2013 study published by the Journal of the American Medical Association.
mRNA is relatively
unstable and short-lived in the cell, especially in prokaryotic cells, ensuring that proteins are only made when needed.
As elongation proceeds, the DNA is continuously
unwound ahead of the core enzyme and rewound behind it
Reaction 3, shown occurring in Figure 9.23, is the basis of a
useful and rapid test to distinguish streptococci, which are aerotolerant and do not possess catalase, from staphylococci, which are facultative anaerobes.
To copy their nucleic acids, plasmids and viruses frequently use
variations on the pattern of DNA replication described for prokaryote genomes.
Introns can be spliced out differently, resulting in
various exons being included or excluded from the final mRNA product. This process is known as alternative splicing.
Why do such disparities continue to exist and how do we break these cycles? The situation is complex and likely results from the combination of
various factors, including how society conditions the behaviors of girls from a young age and supports their interests, both professionally and personally.
DNA stores the information needed to build and control the cell. The transmission of this information from mother to daughter cells is called
vertical gene transfer and it occurs through the process of DNA replication.
Some cells are described as
viable but nonculturable and will not form colonies on solid media. For all these reasons, the viable plate count is considered a low estimate of the actual number of live cells. These limitations do not detract from the usefulness of the method, which provides estimates of live bacterial numbers.
The viable plate count, or simply plate count, is a count of
viable or live cells. It is based on the principle that viable cells replicate and give rise to visible colonies when incubated under suitable conditions for the specimen.
An example of a natural disinfectant is
vinegar; its acidity kills most microbes.
Because double-stranded RNA is uncommon in eukaryotic cells, its presence serves as an indicator of
viral infection.
Although RNA is typically single stranded within cells, there is significant diversity in
viruses.
Ask most people "What are the major requirements for life?" and the answers are likely to include
water and oxygen. Few would argue about the need for water
The cell suspension used for
weighing must be concentrated by filtration or centrifugation, washed, and then dried before the measurements are taken.
Matthew Meselson (1930-) and Franklin Stahl (1929-) devised an experiment in 1958 to test_____.
which of these models correctly represents DNA replication.
They looked for mutants that grew on a complete medium, supplemented with vitamins and amino acids, but did not grow on the minimal medium lacking these supplements. Such molds theoretically contained mutations in the genes that encoded biosynthetic pathways. Upon finding such mutants, they systematically tested each to determine
which vitamin or amino acid it was unable to produce (Figure 10.5) and published this work in 1941.
Spontaneous Mutations
wild type mutant
Typically, whereas the first two positions in a codon are important for determining which amino acid will be incorporated into a growing polypeptide, the third position, called the
wobble position, is less critical.
Biofilms can also form in
wounds, sometimes causing serious infections that can spread.
Foundation of genetics- Johann Gregor Mendel (1856)
•Experimented on Pisum sativum (garden pea) •Performed hybridizations to understand the pattern of inheritance of character •Set up a basic framework of rules governing inheritance
Destruction/inhibition of microbial growth depends on various factors
•Population size •Population composition •Concentration an antimicrobial agent •Duration of the exposure •Temperature •Local environments
Steps of Replication of a DNA
DNA replication will begin at a specific area of the molecule called the origin of replication. the origin of replication denotes the area of active replication called the replication fork. In order to understand how complex eukaryotic organisms replicate DNA, scientists first studied replication in prokaryotic models, like E. coli. A number of enzymes are needed for replication to proceed once the replication fork is established. Helicase separates the strands of the double helix and single stranded binding proteins stabilize the newly single stranded regions. DNA gyrase is used to make sure the double stranded areas outside of the replication fork do not supercoil. Once the replication fork is stable, DNA polymerase catalyze the addition of new nucleotides to the growing daughter strand. Other proteins, such as beta clamps and the clamp loader, help hold the DNA polymerase in place in the DNA. Short sequences of RNA, called primers, have to be paired to the template strands by the enzyme primase becuse DNA polymerase cannot begin to add nucleotides without a primer. Replication of both strands occurs at the same time, one using continuous synthesis and the other, discontinuous. Continuous synthesis occurs on the 3'-5' oriented parentaal strand, referred to as the leading strand. New nucleotides are added to the 3' end moving continuously towards the expanding replication fork. Discontinuous synthesis occurs on the parent strand that is oriented 5'-3', called the lagging strand, and is completed in segments called Okazaki fragments. Replication on this strand uses primase to add primers ahead of the 5' end of the lagging strand. DNA polymerasse 3 then adds short sequences of nucleotides, the Okazaki fragments, to the primer filling the gap. As the helix is opened further, this process repeats until the entire strand is replicated. DNA polymerase 1 replaces the RNA primers with dna nucleotides and DNA ligase is used to ensure bonding between the fragments and the replaced nucleotides. Once both the leading and llagging strands have completed their replication, two identical copies of the DNA molecule result the process of DNA replication allows actively dividing bacterial cells to make sure all daughter cells have the same genetic instructions as the parent cell allowing them to function in the same manner thus bacterial populations can grow increasing the number of individuals in a colony
In one model, semiconservative replication, the two strands of the double helix separate during
DNA replication, and each strand serves as a template from which the new complementary strand is copied; after replication, each double-stranded DNA includes one parental or "old" strand and one "new" strand.
It is now known that DNA pol III is the enzyme required for
DNA synthesis;
By the time Hershey and Chase published their experiment in the early 1950s, microbiologists and other scientists had been researching heredity for over 80 years. Building on one another's research during that time culminated in the general agreement that
DNA was the genetic material responsible for heredity (Figure 10.10). This knowledge set the stage for the age of molecular biology to come and the significant advancements in biotechnology and systems biology that we are experiencing today.
Translation
Decoding mRNA and covalently linking amino acids together to form a polypeptide Translation requires - mRNA template, ribosomes, tRNAs, and various enzymatic factors ~900 residues per minute (~100 for eukaryotes) Amino terminal to carboxy terminal
Passive Diffusion
Dependent on the size of the concentration gradient. Small molecules such as H2O, O2, and CO2
Facilitated Diffusion
Depends on carrier proteins No input of energy is required - passive transport
Microbial cells are very complex
Different cell parts participate in different functions Require raw materials or nutrients and a source of energy
Replica Plating
Distinguishes between mutants and the wild-type strain based on their ability to grow in the absence of a particular biosynthetic end product
Intercalating agents
Distort DNA to induce single nucleotide pair insertions and deletions Planer structures, intercalate between the stacked bases of the helix Acridines (proflavin and acridine orange)
There is one notable exception to this assembly line of tRNAs:
During initiation complex formation, bacterial fMet−tRNAfMet or eukaryotic Met-tRNAi enters the P site directly without first entering the A site, providing a free A site ready to accept the tRNA corresponding to the first codon after the AUG.
As the ribosome steps across the mRNA, the former P-site tRNA enters the
E site, detaches from the amino acid, and is expelled.
Bacterial doubling times vary enormously.
E. coli (under optimal growth conditions in the laboratory) 20 min E. coli (natural environments) Few days Mycobacterium tuberculosis 15 and 20 hours M. leprae 14 days
DNA replication is very well studied in bacteria
E. coli has a single circular chromosome Contains 4.6 million base pairs Replicated in approximately 42 minutes
Advantages of the Petroff-Hausser chamber
Easy to use, relatively fast, and inexpensive, Size and morphology of microorganisms
Elongation
Elongation begins only when σ subunit dissociates from the core polymerase Core RNA polymerase alone can perform transcription elongation It unwind DNA downstream and rewind DNA upstream Add ribonucleotide in a 5' to 3' direction (~40 per second) Elongation continues until core enzyme reaches to terminator
Microbial mRNA is polycistronic
Eukaryotic mRNA is monocistronic
Ribosomes are the site of translation
Eukaryotic organelles contains 70S ribosomes (mitochondria and Chloroplast)
Law of Segregation
Factors do not show any blending (F1) and that both the characters are recovered as such in the F2 generation
Transformation in prokaryotes
First to demonstrated in Streptococcus pneumoniae "naked DNA is taken up from the environment" Recipient cells needs to be in 'competent' state Important mechanism for the acquisition of virulence factors and antibiotic resistance
Several factors contribute to the effectiveness of adisinfecting agent or microbial control protocol.
First, as demonstrated in Figure 13.5, the length of time of exposure is important. Longer exposure times kill more microbes. Because microbial death of a population exposed to a specific protocol is logarithmic, it takes longer to kill a high-population load than a low-population load exposed to the same protocol. A shorter treatment time (measured in multiples of the D-value) is needed when starting with a smaller number of organisms.
The so-called thermoenzymes purified from thermophiles have important practical applications.
For example, amplification of nucleic acids in the polymerase chain reaction (PCR) depends on the thermal stability of Taq polymerase, an enzyme isolated from T. aquaticus. Degradation enzymes from thermophiles are added as ingredients in hot-water detergents, increasing their effectiveness.
binary fission step four
FtsZ assembles into a Z ring on the cytoplasmic membrane
The Z ring is anchored by
FtsZ-binding proteins and defines the division plane between the two daughter cells.
Generation time
Generation time (doubling time): time required for population to double. Bacteria reproduce rapidly or remain dormant for thousands of years Bacterial doubling times vary enormously.
protozoan infection examples
Giardia lamblia, Cryptosporidium parvum, or Entamoeba histolytica
Microbes and viruses in genetic research involved what experiments
Griffith's Transformation experiment Hershey-Chase Experiment
Comparison of DNA and RNA polymerase: DNA Polymerase
DNA Polymerase Synthesizes the DNA Functions during the replication Adds deoxyribose nucleotides A, T, G and C Produce double-stranded DNA molecule Requires RNA primer to begin synthesis Speed - 1000 nucleotides per second Generally copies entire chromosome Requires helicase to unwind the DNA Very low error rate (Can proofread efficiently)
The Centers for Disease Control and Prevention (CDC) and the National Institutes of Health (NIH) have established four classification levels, called
"biological safety levels" (BSLs).
The streamers are anchored to the substrate by a
"head" and the "tail" floats downstream in the current. In still or slow-moving water, biofilms mainly assume a mushroom-like shape.
Richard Altmann (1852-1900) subsequently termed his teacher's nucline as
"nucleic acid" 20 years later when he discovered the acidic nature of nuclein
Richard Altmann (1852-1900) Friedrich Miescher's student subsequently termed "nuclein"
"nucleic acid" 20 years later when he discovered the acidic nature of nuclein.
The mRNA carries the message from the DNA, which controls all of the cellular activities in a cell. If a cell requires a certain protein to be synthesized, the gene for this product is
"turned on" and the mRNA is synthesized through the process of transcription
Reactions of type seen in Reaction 1 are catalyzed by peroxidases. In these reactions, an electron donor (reduced compound; e.g., reduced nicotinamide adenine dinucleotide [NADH]) oxidizes
(1) X−(2H+)+H2O2→oxidized-X+2H2O hydrogen peroxide, or other peroxides, to water.
Reaction 2 is mediated by the enzyme superoxide dismutase (SOD) and breaks down the powerful superoxide anions generated by aerobic metabolism:
(2) 2O2−+2H+→H2O2+O2
The enzyme catalase converts hydrogen peroxide to water and oxygen as shown in Reaction 3.
(3) 2H2O2→2H2O+O2
Micronutrients
(MN, Zn, Co, Mb, Ni, Cu ) - required in small amounts. Normally a part of enzymes and cofactor.
Law of Dominance
(i) Characters are controlled by discrete units called factors (ii) Factors occur in pairs (iii) In a dissimilar pair of factors one member of the pair dominates (dominant) the other (recessive)
Bacteria can cause gastroenteritis
(inflammation of the stomach and intestinal tract) either by colonizing and replicating in the host, which is considered an infection, or by secreting toxins, which is considered intoxication. Signs and symptoms of infections are typically delayed, whereas intoxication manifests within hours, as happened after the picnic.
In 1961, French scientists François Jacob and Jacques Monod hypothesized the existence of an intermediary between
DNA and its protein products, which they called messenger RNA.16 Evidence supporting their hypothesis was gathered soon afterwards showing that information from DNA is transmitted to the ribosome for protein synthesis using mRNA.
The bacterial cell cycle involves the formation of new cells through the replication of
DNA and partitioning of cellular components into two daughter cells.
In 1962, James Watson, Francis Crick, and Maurice Wilkins were awarded the Nobel Prize in Physiology and Medicine. Unfortunately, by then Franklin had died, and Nobel prizes at the time were not awarded posthumously. Work continued, however, on learning about the structure of DNA. In 1973, Alexander Rich (1924-2015) and colleagues were able to analyze
DNA crystals to confirm and further elucidate DNA structure.
In April 1953, Watson and Crick published their model of the
DNA double helix in Nature.7 The same issue additionally included papers by Wilkins and colleagues,8 as well as by Franklin and Gosling,9 each describing different aspects of the molecular structure of DNA.
Various types of chemical mutagens interact directly with
DNA either by acting as nucleoside analogs or by modifying nucleotide bases.
Conjugation in prokaryotes
DNA is directly transferred from one prokaryote to another by means of a conjugation pilus E. coli has F plasmid that contains genes for conjugation F plasmid occasionally integrates into host chromosome - high frequency recombination (hfr) strain
In bacteria, three main types of DNA polymerases are known:
DNA pol I, DNA pol II, and DNA pol III.
Termination of DNA replication
DNA pol III continues DNA polymerization until it reaches to the terminator site Because of the circular nature of the chromosome the two DNA are interlocked (concatenated) topoisomerase IV Introduces double-stranded breaks into DNA molecules, allowing them to separate from each other
One of the key players is the enzyme
DNA polymerase, also known as DNA pol.
Elongation of DNA replication
DNA polymerases (DNA pol) are DNA polymerizing enzymes Bacteria - DNA pol I, DNA pol II, and DNA pol III (most important) It adds deoxyribonucleotides each complementary to a nucleotide on the template strand, one by one to the 3'-OH group of the growing DNA chain
Temperature requirements: high temp
-Increases membrane fluidity impairs metabolic processes in membranes -Denatures proteins -Denatures nucleic acids
Mutations can alter the phenotype of an organism
-Morphological mutations Streptococcus pneumoniae that forms smooth and rough colonies is an example of microbial morphological mutant -Lethal mutations Result in the death or reduced longevity of an organism carrying the mutation Mutations occurs in genes that are essential for growth or development -Conditional mutations- A mutant allele causes a mutant phenotype in only a certain environment - restrictive condition It causes a wild-type phenotype in some different environment - permissive condition
RNAs play important role in protein synthesis tRNA
-One of the smallest RNAs (70-90 nucleotides) -Carries the correct amino acid to the site of protein synthesis -It can base pair with the mRNA within ribosome and guide incorporation of correct amino acid
DNA replication is extraordinary and complex process
-Process of copying the genetic information -Existing strand of DNA is used as a template for the synthesis of a new, identical strand -Error-free copying of DNA ensures inheritance of characters generations after generations -Not always error free! Rate of replication: Bacteria - 750 to 1,000 bp per sec Eukaryotes - 50 to 100 bp per sec
The Membrane Filtration Procedure
-Useful for aquatic samples with low bacterial load -Fixed volume of sample is filtered through a filter of specific pore size ( 0.2 micron) -A colony count gives the number of microorganisms
Physical and chemical methods of microbial control that kill the targeted microorganism are identified by the suffix
-cide (or -cidal).
Microbial growth can be controlled various agents
-physical agents -chemical agents -mechanical removal methods
Other methods do not kill organisms but, instead, stop their growth, making their population static; such methods are identified by the suffix
-stat (or -static).
Amazingly, the E. coli translation apparatus takes only
0.05 seconds to add each amino acid, meaning that a 200 amino-acid protein can be translated in just 10 seconds.
Plating methods are used to determine the number of viable microbes
1)Pour plate method 2) Spread Plate method Serial dilution is needed for accurate CFU measurements, both methods
diabetic foot
1. tingling and numbness, 2. scratch on foot does not seem to be healing and is becoming increasingly ugly 3. purplish discoloration spreading on the skin and oozing open sore, or ulcer
Elongation during translation Elongation proceeds with single-codon movements of the ribosome each called a translocation event Elongation occurs in three phases:
1.Aminoacyl-tRNA binding, 2.the transpeptidation reaction, and 3.translocation
Functions of the DNA
1.DNA is a genetic material of almost all known life forms (Some viruses are exception and contains RNA as genetic information) 2.It is a molecule of heredity (vertically transferred from parents to offspring) 3.The long-term storage of information 4.Provides instructions to construct other components of the cell, such as proteins and RNA molecules (a blueprint) 5.DNA plays a role in the evolution of a species (it has ability to undergo mutation)
Translation/ Protein synthesis Like transcription, translation is also divided into three stages:
1.Initiation, 2.Elongation, and 3.Termination
RNA primase synthesizes
10 nucleotide long RNA primer DNA pol III then extends the free 3'-OH group of the primer and starts adding DNA nucleotides
The rate of replication is approximately
100 nucleotides per second—10 times slower than prokaryotic replication.
In humans, a six base-pair sequence, TTAGGG, is repeated
100 to 1000 times to form the telomere.
During elongation in DNA replication, the addition of nucleotides occurs at its maximal rate of about
1000 nucleotides per second. DNA polymerase III can only extend in the 5' to 3' direction, which poses a problem at the replication fork. The DNA double helix is antiparallel; that is, one strand is oriented in the 5' to 3' direction and the other is oriented in the 3' to 5' direction
Although individuals around the world may be infected, the highest prevalence among people
15-49 years old is in sub-Saharan Africa, where nearly one person in 20 is infected, accounting for greater than 70% of the infections worldwide. Unfortunately, this is also a part of the world where prevention strategies and drugs to treat the infection are the most lacking.
A sample from the amniotic fluid cultured for the presence of Listeria gave negative results. Because the absence of organisms does not rule out the possibility of infection, a molecular test based on the nucleic acid amplification of the
16S ribosomal RNA of Listeria was performed to confirm that no bacteria crossed the placenta.
This mutation may protect individuals from plague (caused by the bacterium Yersinia pestis) and smallpox (caused by the variola virus) because this receptor may also be involved in these diseases. The age of this mutation is a matter of debate, but estimates suggest it appeared between
1875 years to 225 years ago, and may have been spread from Northern Europe through Viking invasions.
Eukaryote ribosomes have a small 40S subunit (which contains the____) and a large 60S subunit (which contains the
18S rRNA subunit 5S, 5.8S and 28S rRNA subunits), for a total of 80S.
He was not recognized for his extraordinary scientific contributions during his lifetime. In fact, it was not until
1900 that his work was rediscovered, reproduced, and revitalized by scientists on the brink of discovering the chromosomal basis of heredity.
Barbara McClintock did pioneering work in maize (corn) genetics from the 1930s through 1950s, discovering transposons (jumping genes), but she was not recognized until much later, receiving a Nobel Prize in Physiology or Medicine in
1983
At least one type of aminoacyl tRNA synthetase exists for each of the
20 amino acids. During this process, the amino acid is first activated by the addition of adenosine monophosphate (AMP) and then transferred to the tRNA, making it a charged tRNA, and AMP is released.
Although rRNA had long been thought to serve primarily a structural role, its catalytic role within the ribosome was proven in
2000
The origin of replication is approximately
245 base pairs long and is rich in adenine-thymine (AT) sequences.
Microbiologists typically count plates with
30-300 colonies.
In E. coli, the small subunit is described as
30S (which contains the 16S rRNA subunit), and the large subunit is 50S (which contains the 5S and 23S rRNA subunits), for a total of 70S (Svedberg units are not additive).
When the exploration of Lake Whillans started in Antarctica, researchers did not expect to find much life. Constant subzero temperatures and lack of obvious sources of nutrients did not seem to be conditions that would support a thriving ecosystem. To their surprise, the samples retrieved from the lake showed abundant microbial life. In a different but equally harsh setting, bacteria grow at the bottom of the ocean in sea vents where temperatures can reach
340 °C (700 °F).
In 1998, American geneticist Stephen J. O'Brien at the National Institutes of Health (NIH) and colleagues published the results of their genetic analysis of more than
4,000 individuals. These indicated that many individuals of Eurasian descent (up to 14% in some ethnic groups) have a deletion mutation, called CCR5-delta 32, in the gene encoding CCR5. CCR5 is a coreceptor found on the surface of T cells that is necessary for many strains of the virus to enter the host cell. The mutation leads to the production of a receptor to which HIV cannot effectively bind and thus blocks viral entry. People homozygous for this mutation have greatly reduced susceptibility to HIV infection, and those who are heterozygous have some protection from infection as well.
Mature rRNAs make up approximately
50% of each ribosome.
Bacterial species typically have between
60 and 90 types.
The three-nucleotide code means that there is a total of how many combinations
64 possible combinations (43, with four different nucleotides possible at each of the three different positions within the codon). This number is greater than the number of amino acids and a given amino acid is encoded by more than one codon
Prokaryotes have how many ribosomes?
70S ribosomes, whereas eukaryotes have 80S ribosomes in the cytoplasm and rough endoplasmic reticulum, and 70S ribosomes in mitochondria and chloroplasts.
Analysis of the chicken salad revealed an abnormal number of gram-positive cocci arranged in clusters
A culture of the gram-positive cocci releases bubbles when mixed with hydrogen peroxide. The culture turned mannitol salt agar yellow after a 24-hour incubation.
Invention of Microscope
Antonie van Leeuwenhoek Father of Microbiology 1674 Credited first microscopes powerful enough to view microbes Animalcule simple microscope
Frameshift mutations
Arise due insertion or deletion of one or two base pairs Cause reading frame shift Very deleterious; resulting in the synthesis of nonfunctional proteins Mutant phenotypes
Genetic Diversity in Prokaryotes
Asexual reproduction Conjugation Transduction Transformation
Elucidation of DNA structure Erwin Chargaff and Linus Pauling
Attempted to Build DNA model. FAILED
Another technique uses an electronic cell counting device (Coulter counter) to detect and count the changes in electrical resistance in a saline solution. by using
A glass tube with a small opening is immersed in an electrolyte solution. A first electrode is suspended in the glass tube. A second electrode is located outside of the tube. As cells are drawn through the small aperture in the glass tube, they briefly change the resistance measured between the two electrodes and the change is recorded by an electronic sensor; each resistance change represents a cell. The method is rapid and accurate within a range of concentrations; however, if the culture is too concentrated, more than one cell may pass through the aperture at any given time and skew the results. This method also does not differentiate between live and dead cells.
Spectrophotometers can be used to measure the bacterial cell mass by
A light beam is transmitted through a bacterial suspension Light passing through the suspension is measured by a detector Light hitting the detector is inversely proportional to the turbidity of culture
Flow of genetic information
A relationship among DNA, RNA, and protein Pathway of information is conserved in all forms of life - Central Dogma DNA to RNA RNA to protein
During each translocation event, the charged tRNAs enter at the
A site, then shift to the P site, and then finally to the E site for removal.
In the late 1920s, Barbara McClintock (1902-1992) developed chromosomal staining techniques to visualize and differentiate between the different chromosomes of maize (corn). In the 1940s and 1950s, she identified a breakage event on chromosome 9, which she named the dissociation locus (Ds). Ds could change position within the chromosome. She also identified an activator locus (Ac). Ds chromosome breakage could be activated by an
Ac element (transposase enzyme). At first, McClintock's finding of these jumping genes, which we now call transposons, was not accepted by the scientific community. It wasn't until the 1960s and later that transposons were discovered in bacteriophages, bacteria, and Drosophila. Today, we know that transposons are mobile segments of DNA that can move within the genome of an organism. They can regulate gene expression, protein expression, and virulence (ability to cause disease).
Fragmentation is commonly observed in the
Actinomycetes, a group of gram-positive, anaerobic bacteria commonly found in soil.
Microbes also use transport that requires energy
Active transport - transport against a concentration gradient. Input of metabolic energy. ATP-binding cassette transporters (ABC transporters) are important examples of active transport systems
binary fission step five
Additional proteins required for cell division are added to the Z ring to form a structure called the divisome
Streak Plate Technique steps
After the first sector is streaked, the inoculating loop is sterilized and an inoculum for the second sector is obtained from the first sector. A similar process is followed for streaking the third sector, except that the inoculum is from the second sector. Thus this is essentially a dilution process. Eventually, very few cells will be on the loop, and single cells will drop from it as it is rubbed along the agar surface. These develop into separate colonies. In both spread-plate and streak plate techniques, successful isolation depends on spatial separation of single cells.
Defined or synthetic medium
All chemical components are known. E.g. BG-11
Perhaps girls should be supported more from a young age in the areas of science and math. Science, technology, engineering, and mathematics (STEM) programs sponsored by the
American Association of University Women (AAUW)14 and National Aeronautics and Space Administration (NASA)15 are excellent examples of programs that offer such support.
Mutations are common in microbes
Bacteria & Archaea divide by = binary fission Division proceeded by chromosome replication from single origin E. coli cells can divide every 20 min DNA molecules are identical except for mutations Mutation rate - ~1 mutation/chromosome/generation With short generation time = lots of mutations ~ 107-108 mutations/12 hours
Initiation of translation
Begins with the formation of an initiation complex "the small 30S ribosome, the mRNA template, three initiation factors, and initiator tRNA" IF-1, IF-2, and IF-3 stand for initiation factors 1, 2, and 3 Initiator tRNA is N-formylmethionyl tRNA: fMet (fMet is inserted at the beginning of every polypeptide chain) ENERGY is obtained from GTP hydrolysis
Robert Hooke
Best known for identification of cellular structure of plant Applied word "cell" to describe the basic unit of life.
Other clostridia responsible for serious infections include
C. tetani, the agent of tetanus, and C. perfringens, which causes gas gangrene. In both cases, the infection starts in necrotic tissue (dead tissue that is not supplied with oxygen by blood circulation). This is the reason that deep puncture wounds are associated with tetanus. When tissue death is accompanied by lack of circulation, gangrene is always a danger.
This shape positions the amino-acid binding site, called the
CCA amino acid binding end, which is a cytosine-cytosine-adenine sequence at the 3' end of the tRNA, and the anticodon at the other end. The anticodon is a three-nucleotide sequence that bonds with an mRNA codon through complementary base pairing.
Although DNA testing to determine which individuals carry the
CCR5-delta 32 mutation is possible, there are documented cases of individuals homozygous for the mutation contracting HIV. For this reason, DNA testing for the mutation is not widely recommended by public health officials so as not to encourage risky behavior in those who carry the mutation. Nevertheless, inhibiting the binding of HIV to CCR5 continues to be a valid strategy for the development of drug therapies for those infected with HIV.
Spread Plate method
CFU=(No of Colonies)/(Dilution ∗volume plated) If 1.0 ml of a 1 X 10-6 dilution yielded 150 colonies, the original sample contained around 1.5 X108
Disadvantages of the Petroff-Hausser chamber
Can't differentiate live and dead cells Does not work well with dilute culture
Genetic code
Cells produce proteins by reading / decoding genetic code in the process of translation Relationship between mRNA codon and its corresponding amino acid - genetic code mRNA nucleotide sequence is translated into the amino acid sequence of proteins It defines the amino acid to be added during the protein synthesis
DNA-modifying agents
Change base structure, alter its base pairing characteristics Methyl-nitrosoguanidine- adds methyl groups to guanine, causing it to mispair with thymine
Natural Selection Charles Darwin
Charles Darwin Spent five weeks on Galapagos islands On the origin of species (1859) Origin of species is natural not divine
In 1902, Theodor Boveri (1862-1915) observed that in sea urchins, nuclear components (chromosomes) determined proper embryonic development. That same year, Walter Sutton (1877-1916) observed the separation of chromosomes into daughter cells during meiosis. Together, these observations led to the development of the
Chromosomal Theory of Inheritance, which identified chromosomes as the genetic material responsible for Mendelian inheritance.
Organization of DNA differ in three domains: Archaea and most bacteria
Closed circle Circles are further supercoiled Archaea also have histone proteins
In diabetic foot the concern here is that gas gangrene may have taken hold in the dead tissue. The most likely agent of gas gangrene is
Clostridium perfringens, an endospore-forming, gram-positive bacterium. It is an obligate anaerobe that grows in tissue devoid of oxygen. Since dead tissue is no longer supplied with oxygen by the circulatory system, the dead tissue provides pockets of ideal environment for the growth of C. perfringens.
Important properties of genetic code
Code is a triplet The code is degenerate (first two positions in a codon are important, the third position, called the wobble position, is less critical) The code is non-overlapping The code is comma less The code is unambiguous The code is universal The code is co-linear
Culture media are required to grow & maintain microbes
Culture medium is a solid (agar) or liquid (broth) preparation. It can be semi-solid as well! Must contain all the nutrients the microorganism requires for growths Knowledge of a microorganism's normal habitat is essential Biggest challenging task for microbiologists of all times!
