Exam 2 Chapter 8 Microbiology

DNA-mediated transformation (DNA transfer)

"naked" DNA is taken up by a cell. Naked AND transferred. About 20 genes and its sensitive to DNase Addition. (Its a mechanism of DNA transfer)

Plasmid transfer involves a series of steps.

(1.) making contact (2) initiating transfer (3) transferring DNA (4) transfer complete

Mismatch repair

(a mechanism for repair of errors in nucleotide incorporation) fixes errors missed by the proofreading of DNA polymerase. A specific protein binds to the site of the mismatched nucleobase, directing an enzyme to cut the sugar-phosphate backbone of the strand. Another enzyme then degrades a short region of that DNA strand, thereby eliminating the mis incorporated nucleotide. If the enzyme cuts the template strand and not the new one, then the disincorporated nucleotide would remain. Soon after a DNA strand is synthesized, an enzyme adds methyl groups to certain nucleases. This takes time, however, so the new strand is still unmethylated immediately after it is synthesized. Therefor, the template strand is methylated, whereas the new strand is not, allowing the repair enzyme to distinguish between the two. After the nucleotides are removed from the new strand, the combined actions of DNA polymerase and DNA ligase then fill in that section and seal the gap.

intercalating agents

increase the frequency of frameshift mutations. They do this because they are flat molecules that can intercalate between adjacent base pairs in a strand of DNA. This pushes the nucleotides apart, producing enough space between bases that errors are made during replication.

What is a strain's characteristics?

they are designated by three-letter abbreviations, with the first letter capitalized. For example, a strain that cannot make tryptophan is designated Trp-. For simplicity, only required growth facts are indicated. Only if a cell is resistant to an antibiotic is it indicated; streptomycin resistance is indicated as Str^R.

an organism with a spontaneous mutation to antimicrobial resistance

though rare, will become dominay in an environment where the medication is present. This happens because the antimicrobial kills the sensitive cells, allowing the resistance cells to row without competition.

Specialized transduction

transfers only a few specific genes.

What two kinds of radiation are used as mutagens?

ultraviolet (UV) light and X-rays

Point mutation

when only one base pair is changed.

Base substitution

the most common type of mutations, occurs during DNA synthesis when an incorrect nucleotide is incorporated.

F plasmid

the most thoroughly studied example is the F plasmid of E. coli. (F stands for fertility). Although this plasmid does not encode any notable characteristics except those required for transfer, other conjugative plasmids encode resistance to certain antibiotics, which explains how such resistance can easily spread among a population of cells.

recombinants

the resulting cells of the horizontal gene transfer experiment. Recombinants have properties of each of the original strains.

wild type

the typical phenotype of strains isolated from nature. Geneticists working with mutants compare them to wild type. A wild-type E. coli strain is a prototroph.

Proofreading by DNA Polymerase

(a mechanism for repair of errors in nucleotide incorporation)DNA polymerases are complex enzymes that not only synthesize DNA, but also verify the accuracy of their actions- a characteristic called proofreading. The enzymes can back up and remove a nucleotide not correctly hydrogen boned to the opposing nuclease in the template strand. The DNA polymerase then incorporates the correct nucleotide. Although the proofreading function of DNA polymerases is very efficient, it is not flawless.

Phenotype

A change in genotype often alters the organism's observable characteristics (phenotype). The phenotype involves more than just the genetic make up of an organismal it can involve more than just the genetic makeup of an organism; it can also be influenced by environmental conditions.

chemicals that modify nucleobases

A number of different chemicals modify the nucleobases in DNA, changing their base-pairing properties. This increases the change that the incorrect nucleotide will be incorporated during DNA replication.

Transduction (DNA transfer)

A virus injects bacterial DNA into a cell. DNA enclosed in a bacteriophage coat. Small fraction of the chromosome. It is NOT sensitive to DNase Addition. (Its a mechanism of DNA transfer)

(2) initiating transfer

After contact, the F pills retracts, pulling the two cells together. Meanwhile, a plasmid-encoded enzyme cuts one strand of the plasmid at a specific nucleotide sequence, the origin of transfer

Repair of Thymine Dimers

Bacteria have several mechanisms to combat the DNA-damaging effects of UV light, a component of sunlight. Mechanisms includes photoreactivation and excision repair.

Base substitutions, aerobic or anaerobic?

Base substitutions are more common in aerobic than anaerobic environments. This is because reactive oxygen species (ROS) such as superoxide and hydrogen peroxide are produced from O2. These chemicals can oxidize the nuclease guanine, and DNA polymerase often mis-pairs oxidized guanine and adenine rather than cytosine.

Mutation

Changes the existing nucleotide sequence of a cell's DNA, which is then passed on to the progeny (daughter cells) through vertical gene transfer. The modified organism and daughter cells are referred to as mutants.

Conjugation (DNA transfer)

DNA is transferred during cell-to-cell contact required. It is not sensitive to DNase addition.

The Process of Transformation

Double-stranded DNA molecules bind to specific receptors on the surface of competent cells.

Who studied transposition?

Dr. Barbara McClintock. She observed color variation in corn kernels as a result of transposons moving into and out of genes that control pigment synthesis.

F+ and F-

E.coli cells that harbor the F plasmid are F+ and those who do NOT harbor the F plasmid are F-.

What is an example of an intercalating agents?

Ethidium bromide, a chemical commonly used to stain DA in the laboratory, is an intercalating agent. The manufacturer now warns users that the chemical should be handled with great care because it likely is a carcinogen (cancer-causing). Another intercalating agent is chloroquine, which has been used for many years to treat malaria.

F' Donors

Hfr strains can revert to F+ because the process of F plasmid integration is reversible. In some instances, however, an error occurs during excision, and a piece of the bacterial chromosome is removed along with the F plasmid DNA. This action brings a chromosomal fragment into the F plasmid, producing a plasmid called F' ( F prime). Like the F plasmid, F'is a replicon that is rapidly and efficiently transferred to F-cells.

Repair of Damaged DNA

If damage is not repaired, it can quickly lead to cell death and cancer. Mutations in either gene result in a high probability (80%) of breast cancer. Mutations are rare because alterations in DNA are repaired before they can be passed on to progeny. Cells cannot repair all types of mutations, such as insertional inactivation cause by transposition.

Photoreactivation

In this mechanism, an enzyme uses the energy of visible light to break the covalent bonds of the thymine dimer, restoring the dan to its original state. Because light is require for this mechanism is is called photo reactivation, or light repair. The enzyme used is found ONLY in prokaryotes.

thymine dimers

Irradiation of cells with ultraviolet light causes covalent bonds to form between adjacent thymine molecules on a DNA strand, producing thymine dimers. The dimer cannot fit properly into the double helix. distorting the DNA molecule. Replication and transcription stall at the distortion and the cells will die if the damage is not repaired.

Indirect Selection

Is used to isolate an auxotroph from a prototrophic parent strain. This process is more difficult than direct selection because no medium will allow the growth of the mutant and not the parent. For example, Trp- mutants can grow only on a complex medium such as nutrient agar because this supplies the tryptophan they require, but Trp+ parent cells also grow on this same medium. To overcome this problem, a technique called replica plating is used, sometimes preceded by penicillin enrichment of mutants.

Who devised replica plating?

Joshua and Esther Lederberg in the early 1950s

Chemicals that are not carcinogenic but are converted to carcinogens

Many chemicals are not carcinogenic themselves but are converted to carcinogens by enzymatic reactions in animals. Bacterial cells may not produce these enzymes, so an extract of ground-up rat liver-which has the enzymes to carry out these conversions- is also added to the suspected mutagen. Additional testing must be done on any mutagenic chemical to determine if it is carcinogenic. Although data are not available on the percentage of mutagens that are carcinogens, it is clear that the Ames test is useful as a rapid screening test. So far, only compounds that increase the reversion rate have been shown to be carcinogenic.

Repair of Modified Nucleobases in DNA

Modified nucleases such as oxidized guanine can result in base substitutions if they are not repaired before the DNA is replaced. An important mechanism for repairing this defect uses a glycosylase, an enzyme that removes the oxidized nuclease from the sugar-phosphate backbone. Another enzyme then recognizes that a nucleobase is missing and cuts the DNA at this site. DNA polymerase degrades a short section of this strand to remove the damage. This same enzyme synthesizes another strand with the proper nucleotides, and DNA ligase seals the gap in the single-stranded DNA.

Direct Selection

Mutants that can grow under conditions in which the parent cells cannot are usually easy to isolate by direct selection. In this method, cells are inoculated onto an agar medium that supports the growth of the mutant, but not the parent. For example, antibiotic-resistant mutants can be easily selected directly by inoculating cells onto a medium contained the antibiotic. Only the resistant cells will form colonies.

What two mechanisms does genetic change in bacteria occur?

Mutation and horizontal gene transfer

Example of a phenotype

Serratia marcescens are red when incubated at 22 degrees Celsius but white when incubated at 37 degrees Celsius. The phenotype, but not the genotype, has changed. However, if the genes responsible for pigment production are removed, the organism's phenotype as well as the genotype changes.

Excision Repair

Some bacteria have an enzyme that recognizes the major distortions in DNA that result from thymine dimer formation. In this process, excision repair, or dark repair, the enzyme makes single-stranded cuts that flank both sides of the damaged region to remove the strand. The actions of DNA polymerase and DNA ligase then fill in and seal the gap left by the removal of the segment.

What is a frequent cause of skin and wound infections and it's treatment?

Staphylococcus aureus, the Gram positive coccus commonly called Staph. The usual treatment for these infections have been penicillin-like antibiotics (methicillin). Today, this treatment is likely to fail.

(1) making contact

The F pills of the donor cell binds to a specific receptor on the cell wall of the recipient

natural selection

The process if which In the ever-changing conditions that characterize more environments, all organism need to adapt in order to survive and multiply. If they fail, competing organisms more "fit" to thrive in the new setting will soon predominate.

mobile gene pool

The remaining genes, which vary considerably among different strains makes up the mobile gene pool or mobilome.

Transposition

Transposons can be introduced intentionally into a cell in order to generate mutations. The transposon, which cannot replicate on its own because it lacks an origin of replication, inserts into the cell's genome. This generally inactivates the gene into which it inserts.

Ultraviolet lights

UV mutagenizes cells indirectly. Its major mutagenic action results from the cell's attempting to repair the damage by SOS repair.

X-rays

X-rays cause single- and double-strand breaks in DNA, and alterations to the nucleases. Double-strand breaks often results in deletions that are lethal to the cell.

Replica Plating

a clever method for indirect selection of auxotrophic mutant. After this technique is done, the plates are exact replicas, so colonies on the master plate that cannot grow on glucose-salts can be identified; these are auxotrophs. The particular growth factor required can then be determined by adding nutrients individually to a glucose-salts medium and determining which one promotes growth.

alkylating agents

a group of chemicals that add alkyl groups (short chains of baron atoms) onto nucleases. An example is nitroso-guanidine, which adds a methyl group to guanine, causing it to base-pair with thymine.

SOS Repair

a last-ditch repair mechanism that copes with extensively damaged DNA. The enzymes that carry out this repair are induced when DNA is so heavily damaged by UV light that photo reactivation and excision repair may not be able to correct all of the damage. DNA and RNA polymerases then stall at sites of unprepared damage, so the cells cannot replicate or transcribe their DNA. Without SOS repair, the cells would die. Damaged DNA activates expression of the several dozen genes that encode the SOS system. One component of this system is a DNA polymerase that synthesizes DNA even in extensively damaged regions. Unlike the standard DNA polymerases, however the SOS DNA polymerase has no proofreading ability. Errors are made as a result, a process called SOS mutagenesis.

Prototroph

a mutant that does not require growth factors. pro to means "earliest form of"

auxotroph

a mutant that requires a growth factor. auxo means increase and troph means nourishment

What happens if the intercalating agents inserted into the template strand?

a pair pair will be added as the new strand is synthesized. If it intercalates into the strand being synthesized, a base pair will be deleted. As in spontaneous frameshift mutants, adding or subtracting a nucleotide in DNA often results in a stop codon being generated prematurely in the mRNA transcript, giving rise to shortened protein.

reversion

the change that mutations are passed on to a cell's progeny. On rare occasions, however, a mutation will then change back to its original, non--mutant state. Reversion, like the original mutation, occurs spontaneously at low frequencies.

(3) transferring DNA

a single strand of the F plasmid enters the F- cell, passing through the F pills. Once inside the recipient cell, that strand serves as a template for synthesis of th complementary strand, generating an F plasmid. Likewise, the strand that remains in the donor serves as a template for DNA synthesis, regenerating the F plasmid. The transfer only takes a few minutes.

What is the results of mis-pairing of nucleobases?

a slight distortion in the DNA helix, which can be recognized by enzymes within the cell that then repair the mistake. By quickly repairing the error before the DNA is replicated, the cell prevents the mutation.

competent

a specific physiological state that allows the cell to take up DNA. Most competent bacteria take up DNA regardless of its source. Some species accept DNA only from closely related bacteria, recognizing it by characteristic nucleotide sequences located throughout the genome.

Carcinogens

a substantial proportion of cancers appear to be caused by chemicals that are carcinogens (meaning "cancer generating") , and mot carcinogens are mutagens.

frameshift mutation

adding or subtracting one or two nucleotide pairs causes a frameshift mutation. This changes the reading frame of the corresponding mRNA molecule so that an entirely different set of codons is translated. Frequently, one of the resulting downstream codons will be a stop codon. A frameshift mutation likely results in a shortened non-functional protein- a knockout mutation. (nonsense mutation)

mutagen

an agent that induces the change. Geneticist often use mutagens to increase the mutation rate in bacteria, making mutants easier to find.

plasmids

are common in the microbial world. most members of the Bacteria and Archaea and in some Eucarya. Most plasmids are double-stranded DNA molecules.

Transduction

bacterial viruses can transfer bacterial genes from a donor to a recipient by transduction.

bacteriophages/ phage process

bacterial viruses, or simply phages, consists of genetic material, either RNA or DNA (never both), surrounded by a protein coat. A phage infects a bacterium by attaching to the cell and then injecting its nucleic acid. Enzymes encoded by the phage genome then cut the bacterial DNA into small pieces. Next, the cell's enzymes replicate the phage nucleic acid and synthesize proteins that make up phage coat, and the various components assemble to produce complete phages particles. These are then released, usually as a result of host cell lysis. The phage particles then attach to other bacterial cells and begin new cycles of infection.

Why is penicillin enrichment helpful?

because even when mutagenic agents are used, the frequency of mutations in a particular gene is low, sometimes less than one in 100 million cells. By increasing the proportion of mutant cells, it is easier to isolate them.

(4) transfer complete

both the donor and recipient cells are now F+ so they can act as donors of the F plasmid

who devised the Ames test?

by Bruce Ames and his colleagues in the 1900s

homologous recombination

can happen only if the donor DNA is similar in nucleotide sequence to a region in the recipient cell's genome. In homologous recombination, the donor DNA becomes positioned next to the complementary region of the recipient cell's DNA> The donor DNA then replaces a homologous segment of recipient DNA, and the DNA it replaced is degraded. The molecular mechanisms involved in homologous recombination are still not understood, and different mechanisms likely operate in different situations.

Chemical mutagens

cause base substitutions and cause gram shift mutations.

mutation rate

defined as the probability that a mutation will occur in a given gene per cell division. The mutation rate of different genes usually varies between 10^-4 and 10^-12 per cell division. The chance that a gene will undergo a mutation when a cell relocates its DNA prior to cell division is between one in 10,000 and one in a trillion. Calculated by taking the sum of the exponents.

Conjugative plasmids

direct their own transfer from donor to recipient cells.

what are the two types of transduction?

generalized and specialized transduction.

Induced mutations

genetic changes that occur due to an influence outside of a cell, such as exposure to a chemical or radiation.

Spontaneous mutations

genetic changes that result from normal cell processes. They occur randomly, and genes mutate spontaneously at infrequent but characteristic rates.

silent mutation

has a codon that still specifies the wild type amino acid The term" silent mutation" indicates a mutation that does not alter the function of the protein. Any base substitution that does not affect the phenotype would be a silent mutation.

HA-MRSA

healthcare-associated methicillin-resistant S. aureus

Hfr Cells

high frequency of recombination

the study of heredity:

how genes function, change, and are transferred to other organisms.

the Ames Test

illustrates the concept of such tests. It relies on the fact that mutagens increase the frequency of spontaneous reversions. It uses direct selection to determine the effect of a test chemical on the reversion rate of a histidine-requiring auxotroph of Salmonella. If the chemical is mutagenic, the reversion rate will increase relative to that observed when no chemical is added (the control). The test also gives some idea about how hazardous the chemical may be by the number of revertants that arise.

Competence

in order for transformation to occur, the recipient cell must be competent. It also requires a high concentration of bacteria, a role of quorum sensing. Presumably, This ensures that DNA in the medium will contact the competent bacteria. Even under optimal conditions, however, only a fraction of the population ever becomes competent. This means that seemingly identical cells in a population exposed to the same environment can differ in their physiological properties.

core genome

the conserved genes of the species.

DNA-mediated transformation

involved the uptake of "naked DNA by recipient cells. Naked DNA is simply free in the cell's surroundings it is not contained within a cell or a virus. The fact that the DNA is naked can be demonstrated by adding DNase (an enzyme that degrades DNA) to the medium. This prevents transformation, indicating that the process requires naked DNA. Naked DNA originates from cells that have either burst or secreted some DNA. When cells burst, the long chromosomes that were tightly jammed into the cells break up into hundreds of pieces. Some bacterial species secrete small piece of DNA, presumably as a means of promoting transformation.

Conjugation

is a complex process that requires contact between donor and recipient cells. Gram-postive and Gram-negative bacteria can both transfer DNA this way. We will consider Conjugation only in the more intensely studied Gram-negative bacteria.

a single mutation

is a rare even, so two mutations are even more unlikely. Physicians take advantage of this to prevent pathogens from developing resistance to certain antimicrobial medications. The change of a single cell becoming resistant spontaneously to both medications if the product of the mutations rates of the two genes.

Chromosomal DNA transfer

is less common than plasmid transfer and invokes Hfr cells. (1) making contact- The F pills contacts the recipient F-cell. (2) transferring DNA- A single strand of the donor chromosome begins to be transferred, starting at the origin of transfer. Gene A, closest to the origin, is transferred first. DNA synthesis creates complementary stands in both cells. (3.) transfer ends- The donor and recipient cells separate, interrupting DNA transfer. (4) integration of transferred DNA- The donor DNA is integrated into the recipient cell's chromosome by homologous recombination. Unincorporated DNA is degraded. The recipient cell is still F-.

Penicillin Enrichment

is sometimes used before replica plating to increase the proportion of auxotrophs in a broth culture. Penicillin enrichment relies on the fact that penicillin kills only growing cells. The mutagenized cells are incubated in glucose-salts broth containing penicillin. Prototrophs can multiply in this medium so most are killed, whereas the non-multiplying auxotrophs survive. The enzyme penicillinase is then added to destroy the penicillin, and the cells plated on nutrient agar to create the master plate used in replica plating.

Horizontal Gene Transfer

is the acquisition of genes from another organism. Like mutations, the changes are then passed on to the progeny.

Transposons

jumping genes, they are segments of DNA that can move from one location to another in a cell's genome, a process called transposition. The gene that a transposon jumps is insertionally inactivated by the event, meaning that the insertion destroys the function of the gene. Most transposons contain transcriptional terminators, so the expression of downstream genes in the same operon will stop as well.

What did McClintocks experimental system consist of?

kernels in ears of corn. She noticed that the various kernel colors were not inherited in a predictable manner. The colors seemed to come and go. Based on extensive data, McClinotck concluded that segments of DNA, now called transposons("jumping genes"), were moving into and out of genes involved with kernel color. This destroyed the function of the genes, thereby changing kernel color.

Barbard McCIintock

made very important discoveries in genetics. Her tools consisted of a clear mind and a curiosity that could make sense of confusing observations. S

replicon

meaning it has an origin of replication. Following gene transfer, recipient cells must replicate the DNA to pass it on to daughter cells. This can happen only if the DNA is a replicon Plasmids and chromosomes are replicons, but fragments of chromosomal DNA are not.

haploid

means bacteria only contain a single set of genes. In bacterial cells, such a change can have a significant impact because bacteria are haploid. No "backup copy" of a gene exists in a haploid organism.

Nonsense mutation

occurs when the altered codon is a stop codon, resulting in a shorter and often non functional protein. Any mutation that totally inactivates the gene is termed a null or know out mutation.

What are the two general means that bacteria have on which they routinely adjust to new circumstances?

regulating gene expression and genetic change.

Generalized transduction

results from a rare error that sometimes occurs during the construction of phage particles. A fragment of bacterial DNA-produced when the phage- encoded enzyme cuts the bacterial genome- mistakenly enters the protein coat. the product is called a transducing particle: it carries no phage DNA and therefore is not a phage. Like phage particles, a transducing particle will attach to another bacterial cell and inject the DNA it contains. The transducing particle, however, injects bacterial DNA. The bacterial DNA may then integrate into the host chromosome by homologous recombination.

missense mutation

results when the altered codon specifies a different amino acid. The effect of this depends on the position and the nature of the change. In many cases, cells with a missense mutation grow slowly because the encoded protein functions only partially. such a mutation is termed leaky.

genotype

sequence of nucleotides in DNA. A change in an organism's DNA alters the genotype.

what three possible mutation outcomes comes from base substitution?

silent, missense, or nonsense.

What is the results of deletion or addition of nucleotides during DNA replication?

spontaneous mutations. How serious the effect of this change is depends on its location in the encoded protein.

base analogs

structurally resemble nucleases but have different hydrogen-bonding properties. The analogs can be mistakenly used in place of the nucleases when nucleotides are made, and DNA polymerase then incorporates these into DNA. When a DNA strand has a base analog, the wrong nucleotide can be incorporated when the complementary strand is synthesized.

F pilus

the F plasmid encodes several proteins required for conjugation, including the F pills, also referred to as the sex pills.