Week 2

DNA polymerase used in PCR reaction is heat resistant. A.True B.False

A.

Oxygen serves as the terminal electron acceptor in A.aerobic respiration B.anaerobic respiration C.Fermentation D. reduction

A.

Plasmid are not required for host growth and/or reproduction A. T B. F

A.

Restriction endonucleases are usually produced by bacteria. A.True B.False

A.

Transcription is synthesis of _____, while translation is ______ synthesis. A.RNA, protein B.DNA, RNA C.Protein, DNA D.Protein, RNA

A.

Which microbial metabolism would be effective in taking up carbon dioxide pollution contributing to global climate change? A.photosynthesis B.Cellular respiration C.fermentation D. Nitrification

A.

Genetic Engineering and Biotechnology: •Genetic engineering: using in vitro techniques to alter genetic material in the laboratory •Basic techniques include: •Cloning vectors •Gel electrophoresis •Restriction enzymes •Molecular cloning •Cloning selection -Nucleic acid hybridization

KNOW

Bacteria may transfer antibiotic resistant genes via all of following methods except A.Conjugation B.Transformation C.Transduction D.Transposon E.Mitosis Binary fission

E.

Bacterial motility is provided by A.Capsule B.Cell wall C.Fimbriae D.Pilus E. Flagella

E.

For bacteria, cellular respiration and photosynthesis occur in A.Cell wall B.Mitochondria C.Chloroplast D.Capsule E.Cell membrane F. Ribosome

E.

Anaerobic metabolism/Fermentation: Pathways involved= G Final Electron Acceptor= O Molecules Products= A, C, E, LA Primary Pathway Found in= F, At, S anaerobes

Glycolysis Organic ATP CO2 ethanol Lactic Acid facultative aerotolerant strict

Proton Motive Force: ? outside cell ? inside cell ? pumped in and out

H+ OH- H+

semiconservative replication: synthesis of L strand proceeds continuously synthesis of L strand is discontinuous --reinitiated periodically, generating a series of F that are later joined

leading lagging fragments

Operon: •Operon: cluster of genes arranged in a L fashion whose expression is under control of a single operator --Polycistronic: multiple genes are under control of one promoter --RNA polymerase binds to promoter --Operator is located Ds of the promoter --RP binds to operator ----Increase transcription - A ----Decrease transcription - R Lac Operon: 1. Operon Off: No L -a R protein attaches to the operator of the operon. 2. Operon On: Lactose Present -lactose attaching to the repressor, which is rendered inactive and falls away. The RNA polymerase transcribes the structural genes, and the ?RNA is translated into enzymes that can act on the lactose substrate.

linear downstream regulatory proteins activator repressor lactose repressor mRNA

Translation: • Protein synthesis ( ?RNA --->protein) •??s Ribosome in bacteria • mRNA is translated in codons (? nucleotides for ? amino acid) • tRNA contain specific AA and Ac • Translation of mRNA begins at the start codon: ??? • Translation ends at a STOP codon: U??, U??, U??

mRNA 70 3 1 amino acids anticodons AUG UAA UAG UGA

Transcription breakdown: 1. Initiation -RNA polymerase binds to the promoter and M a short stretch of DNA. 2. Elongation -Sigma factor dissociates from RNA polymerase, leaving the core enzyme to complete transcription. RNA is synthesized in the ?′ to ?′ direction as the enzyme adds nucleotides to the 3′ end of the growing chain. 3. Termination -When RNA polymerase encounters a terminator, it falls off the template and releases the newly synthesized RNA.

melts 5 3

•Selective media --Inhibit the growth of unwanted O --T-M agar for isolation of Neisseria gonorrhoeae --MC agar for isolation of Gram-negative bacteria Differential media --Contains substance that bacteria change in recognizable way --Blood agar ----Certain bacteria produce H to break down red blood cells ----α-, β-, γ- hemolysis --MacConkey agar and Mannitol Salt agar ----Contains P indicator to identify bacteria that produce acid

organisms Thayer-Martin MacConkey hemolysin pH

Bacterial Growth in Lab: -Bacterial Growth is not in size, Bacteria Growth is in the number -Prokaryotic cells divide by BF ---One cell divides into two ---During log phase, cell growth is exponential ---Generation time: time it takes for population to D Growth can be calculated Nt = N0 x 2n (Nt ) number of cells in population (N0 ) original number of cells in the population (n) number of divisions Example: N0 = 10 cells in original population 4 hours assuming 20 minute generation time n = 4x60/20=12 Nt= 10 x 212 = 10 x 4,096= 40,960

binary fission double

Nutritional Factors on Growth: •Required elements 1. Major elements --C, O, H, N, S, Ph, Po, M, C and I 2. Trace elements --C, Z, C, Md and M ----Required in minute amounts Growth factors -Some bacteria cannot synthesize some cell constituents ---These must be added to G environment -Organisms can display wide variety of factor requirements ---Some need very few while others require many ----These termed F

carbon oxygen hydrogen nitrogen sulfur phosphorus potassium magnesium calcium iron cobalt zinc copper molybdenum manganese growth fastidious

Diauxic growth: Dozens of catabolic operons affected by C repression --Enzymes for degrading L, M, and other common carbon sources F genes are also controlled by catabolite repression --No need to swim in search of nutrients

catabolite lactose maltose flagellar

Microbial Size: •Size range for prokaryotes: ?? µm to >??? µm in diameter --Cocci- ?? µm to ? µm --Bacilli- ?? µm to ? µm in diameter and ??? µm to ?? µm in length. ----Short bacilli (coccobacilli) -?? µm to ? µm ----Long bacilli-?? µm to ?? µm --Examples of very large prokaryotes ----Epulopiscium fishelsoni ----Thiomargarita namibiensis --Size range for eukaryotic cells: ?? µm to >??? µm in diameter

0.2 >700 0.5 3 0.2 2 0.05 20 0.2 2 0.5 50 10 >200

Bacterial chromosome replication ? origin of replication goes Bd creates ? advancing forks where DNA synthesis can occur replication forks meet at a T site DNA replication is Sc, because two molecule created contains one of the original strands paired with a newly synthesized strand

1 bidirectional two terminating semiconservative

DNA sequencing: 4 flourescently labeled D (dd) nucleotides help sequence DNA replication of each new strand is terminated at the point of addition of a dd nucleotide

4 dideoxy

Biofilm formation are always detrimental for human A.True B.False

B.

Both transcription and translation occur in nucleus of bacteria: A.True B.False

B.

Isolating a pure microbial culture from a mixed culture can be best accomplished by the A.broth culture method B.streak plate method C.membrane filtration method D. MPN method

B.

The purpose of fermentation is to A.Synthesis glucose B.Regenerate NAD+ C.Generate proto motive force D.Generate alcohol for human to use

B.

The repressor can not bind to the operator of lac operon unless inducer (lactose) binds to it A.True B.False

B.

Chemoheterotrophs utilize _______ as energy source and carbon source A.CO2 B.Light C.Organic Compounds D.Inorganic compounds

C

A researcher is maintaining a liquid culture of bacteria at exponential growth. The pump providing nutrients to the culture breaks down for two days, allowing culture to run out of nutrients. It is very likely that upon return to the lab the researcher will find the culture in which of the following phases of growth? A.Lag B.Log C.Stationary D.continuous

C.

CHROMagar contains several dyes and is used to diagnose Urinary Tract Infections. The patient's sample is inoculated and based on the color of the colonies you can identify the pathogen. CHROMagar is best described as: A.Enriched B.Selective C.Differential D.Complex E.selective and differential

C.

Mannitol salt agar (MSA) only allows the growth of halophiles. Among the halophiles, mannitol fermenters release acid that turns the pH indicator yellow; mannitol nonfermenters leave the medium red. Onto MSA you inoculate a halophilic mannitol fermenter, a nonhalophilic mannitol fermenter, and a halophilic mannitol nonfermenter. Thus, the medium is acting as a __________ medium. A.differential B.Selective C.selective and differential D.neither selective nor differential

C.

The reason DNA sample migrate to positive cathode during electrophoresis is because A.DNA are positively charged B.DNA contain nitrogen bases C.DNA contain negatively charged phosphate groups D.DNA contains deoxyribose

C.

Bacillus anthracis causes the deadly disease anthrax. Organisms of the genus Bacillus may form endospores. This bacterium would be suitable for biological warfare because endospores are A.centrally located within the bacterial cells B.multilayered structures. C.difficult to stain. D.resistant to high temperatures, UV light and desiccation.

D.

When the target DNA is added in F plasmid, E. coli may take up the modified F plasmid via______________. A.transformation B.conjugation C.Specialized transduction D.Both A and B

D.

Mediums: 1. Blood agar -complex used routinely in clinical labs. D because colonies of hemolytic organisms are surrounded by a zone of red blood cell clearing. not selective 2. Chocolate agar -used to culture F bacteria, particularly those found in clinical specimens. not selective or differential 3. glucose-salts - chemically defined medium, used in lab experiments to study N requirements of bacteria. not selective or differential 4. MacConkey agar: -isolate gram ? rods that typically reside in the intestines -S (bile salts and dyes inhibit gram positive organisms and gram negative cocci) -D (pH indicator turns pink-red when the sugar (lactose) is fermented) 5. Nutrient Agar - routine lab work -supports growth of Nf bacteria -not selective or differential 6. Thayer-Martin -isolate Neisseria species (fastidious) -S (contains antibiotics that inhibit most organisms except Neisseria -not differential

Differential fastidious nutritional negative selective differential nonfastidious Neisseria Selective

Components of transcription: (-) strand -DNA strand that serves as the template for R synthesis -resulting RNA molecule is complementary to this strand (+) strand -DNA strand complementary to (-) strand -nucleotides sequence of RNA molecules is the same as this strand, except it has U rather than T Promoter -nucleotide sequence to which RP bind to initiate transcription RNA polymerase -enzyme that synthesizes RNA using S-S DNA as a template Sigma Factor -component of RNA polymerase that recognize P regions Terminator -nucleotide sequence at which RNA synthesis S -RNA polymerase falls off the DNA template and releases the newly synthesized RNA

RNA uracil thymine RNA polymerase single stranded promoter stops

mRNA= -sequence of AA in protein -carries DNA master code to R tRNA= -S a given amino acid -carries amino acids to R during translation rRNA= -several large structural rRNA molecules -forms the major part of a R and participates in protein synthesis Primer -an RNA that can begin DNA R -primes DNA *Only ?RNA is translated*

amino acids ribosomes specifying ribosome ribosome replication mRNA

Mutation: Change in the genetic material. It may be neutral, beneficial, or harmful. --Mutant that is growth factor required -> A --P does not require growth factors Spontaneous mutations: Occur in the absence of a M Base substitution (point mutation) --Missense mutation: Change in ?base; Result in a different amino acid --Nonsense mutation: Results in a SC Frameshift mutation: I or D of one or more nucleotide pairs

auxotroph prototroph mutagen one stop codon insertion deletion

Laboratory Cultivation: Physical State (medium's normal consistency)= 1. liquid 2. semisolid 3. solid (can be converted to liquid) 4. solid (cannot be liquified) Chemical Composition (type of chemicals medium contains) 1. Synthetic (C defined) 2. Nonsynthetic (complex; NC defined) Functional type (purpose of medium): 1. general P 2. E 3. S 4. D 5. A growth 6. S transport 7. A 8. E

chemically not chemically purpose enriched selective differential anaerobic specimen assay enumeration

Continous Culture: -Continuous exponential growth can be sustained by use of C ---Continually drips fresh nutrients in ---Releases same amount of waste product

chemostat

•Cells in laboratory grown in C or B system --No new input of nutrient and no release of waste •Population of cells increase in predictable fashion The Growth Curve 1. L stage 2. E or L stage ---Period of exponential growth ---Produce primary metabolites ---Cells enter late log phase synthesize secondary metabolites ----Used to enhance survival, such as A 3. S stage 4. D stage

closed batch Lag Exponential log antibiotics stationary death

Restriction and Modification Enzymes: •Restriction enzymes: recognize specific DNA sequences and C DNA at those sites --Widespread among ?; Rare in ? --Protect prokaryotes from hostile foreign D (e.g., viral genomes) --Essential for in vitro DNA manipulation -Restriction enzymes recognize IR sequences (palindromes) --Three classes of restriction enzymes ----Type II cleave DNA within their R sequence (palindromic) and are most useful for specific DNA manipulation --Typically ?-? base pairs long; EcoRI recognizes a ?-base-pair sequence --S ends or B ends •Restriction enzymes protect cell from invasion from foreign DNA --D foreign DNA --Must protect their own DNA from inadvertent destruction -Modification enzymes: protect cell's DNA for R enzymes --Chemically modify nucleotides in restriction recognition sequence --Modification generally consists of M of DNA

cut prokaryotes eukaryotes DNA inverted repeat recognition 4-8 6 sticky blunt destroy restriction methylation

Mutant Selection: Positive (D) selection detects mutant cells because they grow or appear different. --A test measures effect of chemical on reversion rate of H-requiring Salmonella auxotroph (identification of mutagens) ----If mutagenic, reversion rate ? relative to control ----Rat liver extract added since non-carcinogenic chemicals often converted to carcinogens by animal enzymes Negative (Id) selection detects mutant cells because they do not G.

direct Ames histidine increases indirect grow

Microarray: -GE Analysis Fluorescent in situ hybridization (FISH): -use D/D and H/test DNA Genetic engineering of plants (GMO): -use large P (??) ) of this bacterium can be used as a cloning vector for foreign genes that code for H or D resistance.

gene expression dye denature hybridize plasmid (Ti) herbicide disease

Microbial Genetics: Complete set of genetic information referred to as G --Functional unit of genome is the G ----Gene is the smallest unit of heredity (genotype) --Study of transfer of genes is G --Study of sequence of DNA is G Flow of genetic information: •Vertical gene transfer: R (parents to offspring) •Inside a cell: transcription translation DNA---------------->RNA------------->Protein •Horizontal gene transfer: transfer of genes between cells of the same generation. ---ie. transformation, conjugation, transduction

genome gene genetics genomics reproduction

Respiration: Pathways involved= G, KC, ET Final Electron Acceptor= various Io ions (NO2-, SO4-2, CO2-3) Products= highly varied but can include C, A, OA (H2S, CH4, N2) Primary Pathway Found in= anaerobes, some F and A

glycolysis Krebs Cycle electron transport inorganic CO2 ATP organic acids facultatives aerotolerants

Aerobic respiration: Pathways involved= G, KC, ET Final Electron Acceptor= O Products= A, C, H Primary Pathway Found in= A; facultative A

glycolysis Krebs cycle electron transport O2 ATP CO2 H2O aerobes anaerobes

Myobacteria: •glide over M surface, form a swarm of cells --Allows for the release of enzymes which degrade O material --In the absence of water cells form F bodies

moist organic fruiting

Nucleic Acid Hybridization: •Nucleic acid hybridization: base pairing of single strands of DNA or RNA from two different sources to give a hybrid double helix --Segment of single-stranded DNA that is used in hybridization and has a predetermined identity is called a NAP •Southern blot: a hybridization procedure where ? is in the gel and probe is RNA or DNA •Northern blot: ? is in the gel

nucleic acid probe DNA RNA

Ribonucleic Acid (RNA): -Polymer of N -Contains the bases adenine, guanine, cytosine and uracil -Sugar is R -Most RNA molecules are ? stranded Transcription: •DNA is transcribed to make R • RNA polymerase and sigma factors • Transcription begins when RNA polymerase binds to the P sequence • Transcription proceeds in the ?' -> ?' direction • Transcription stops when it reaches the T sequence

nucleotides ribose single RNA promoter 5 3 terminator

Direct microscopic count: used to determine total ? of cells; counts include living and dead cells Plate Count: time-consuming but technically simple method that does not require sophisticated equipment. Generally used only if the sample has at least ?? cells/ml Turbidity Measurement: very rapid method; used routinely. A one-term correlation with PC is required in order to use turbidity for determining cell number Most Probable number (MPN): statistical E of likely number; it is not a precise measurement. Can be used to estimate numbers of bacteria in relatively dilute solutions.

number 10^2 plate counts estimation

Biofilm formation begins when P bacteria attach to surfaces, other bacteria attach and grow on initial layer --Contains open channels for movement of nutrients and waste --Cells within biofilms can cause D ----Architecture resists immune response and antimicrobials ----Treatment becomes difficult --Br is beneficial use of biofilm •Interactions of mixed microbial communities --Prokaryotes live in M communities ----Many interactions are cooperative -------Waste of one organism nutrient for another ----Some cells compete for nutrient -------Synthesize toxic substance to inhibit growth of competitors

planktonic disease bioremediation mixed

transposon: •Segments of DNA that can move from one region of DNA to another •Contain I sequences for cutting and resealing DNA (transposase) •Complex transposons carry other genes *Gene transfer between microbes lead to spread antibiotic R*

region insertion resistance

Prok vs Euk Transcription and translation: 1. (Prok) mRNA not P (Euk) a cap is added to ?' end of mRNA and poly A tail is added to the ?' end 2. (prok) mRNA ? contain introns (euk) mRNA contains introns which are removed by S 3. (prok) translation of mRNA begins as it is being T (euk) mRNA trancript is transported out of the nucleus so that it can be translated in the C 4. (prok) mRNA is often Pc- translation begins at first AUG that follows R-binding site (euk) mRNA is Mc-translation begins at first AUG

processed 5 3 does not spicing transcribed cytoplasm polycistronic ribosome monocistronic

Endospores: •Resting structure formed by some bacteria for survival during adverse environmental conditions (R, desiccation, H, C). i.e. Bacillus spp, Clostridium spp. •Features of spores, including size, shape, and position in the V cell are useful in identifying some species of bacteria. What makes an endospore so resistant? -C (complexed with dipicolinic acid) -A-soluble, D-binding proteins -D core -S coat -D repair enzymes G= endospore to vegetative state S= vegetative state to endospore

radiation heat chemicals vegetative calcium acid DNA dehydrated spore DNA germination sporulation

Streak-plate method: -Object is to ? number of cells being spread ---S surface dilution ---Each successive spread decreases number of cells per S

reduce solid streak

Obtaining Pure Culture: •Pure culture defined as population of cells derived from S cell •Cells grown in pure culture to study the activities of specific S •Pure culture obtained using special techniques --Aseptic technique minimizes potential contamination •Cells grown on culture M --Can be broth (liquid) or solid form --Inoculation: purposefully adding Mo into a lab nutrient medium

single species media microorganisms

Gel Electrophoresis: separates DNA molecules based on S --Gels are usually made of A, a polysaccharide --Nucleic acids migrate through gel toward the positive electrode due to their negatively charged P groups --Gels can be stained with ethidium bromide or fluorescent dyeand DNA can be visualized under ?? light

size agarose phosphate UV

Shape and Arrangement of Bacteria: 1. cocci (s., coccus) - S •diplococci (s., diplococcus) - pairs; •streptococci - C •staphylococci - G-like clusters; •tetrads - 4 cocci in a S •sarcinae - cubic configuration of ? cocci 2. bacilli (s., bacillus) -R -coccobacilli - very ? rods; -vibrios -C shaped; -P arrangement -Streptobacilli 3. spirilla (s., spirillum) - R Helices 4. spirochetes - F helices with A filaments 5. mycelium - network of long, M filaments 6. pleomorphic - organisms that are variable in shape

spheres chains grape square 8 rods short comma palisades rigid flexible axial multinucleated

Sporulation: 1. Vegetative cell 2. Chromosome is duplicated and separated. 3. Cell is septated into a S and F. 4. Sporangium E forespore for further development. 5. Sporangium begins to actively synthesize S layers around forespore. 6. C and ? coat layers are deposited. 7. Spore completes maturation; sporangium starts to disintegrate. 8. The free spore is released when the dead sporangium falls away. 9. During germination, the spore S and releases a vegetative cell.

sporangium forespore engulfs spore cortex outer swells

1. Photoautotroph: Energy source= S Example= P organisms, such as A, P, Cb 2. Chemoautotroph: Energy source= Simple Io chemical Example= Only certain bacteria and archaea, such as M and deep- sea V bacteria 3. Chemoheterotroph: energy source= M conversion of the other organisms Example= P, F, many B, A A. Saprobe Energy source= metabolize the organic matter from D organisms Example= F, B, some protozoa (Dc) B. Symbiotic microbes Energy source= Obtains organic matter from L organisms Example= P, commensals, M microbes 4. Photoheterotroph: Energy source= S or O matter Example= P and G photosynthetic bacteria

sunlight photosynthetic algae plants cyanobacteria inorganic methanogens vent metabolic protozoa fungi bacteria animals dead fungi bacteria decomposers living parasites mutualistic sunlight organic purple green

The Polymerase Chain Reaction: •The polymerase chain reaction (PCR) is basically DNA replication in a TT --Also called DNA A --Denaturation: heat, typically ???C --Priming: primers base pairing with specific DNA sequence at ???C-??C --Extension: DNA polymerase (such as Taq polymerase) synthesize DNA at ???C Applications of PCR --A small amounts of DNA --Identifying a specific B --Pg studies --Looking for a specific G --Surveying different groups of environmental organisms

test tube amplification 940 500 650 720 amplifying bacteria phylogenetic gene

Horizontal Genetic Transfer and Recombination: Genetic Recombination: Exchange of genes between ? DNA molecules to form new combinations of genes on a chromosome Transformation: genes transferred into bacteria as 'N' DNA in solution •G experiment demonstrate genetic transformation •Using various components of the Killed S. pneumoniae proved DNA was the carrier of genetic information. Conjugation: genes transferred through FP from donor bacteria to recipient bacteria Transduction: genes transferred into bacteria through Bp.

two naked Griffith f pili bacteriophages

Enzymes involved in DNA Replication and Their Functions: Helicase- Uz the DNA helix Primase- synthesizing an RP DNA Polymerase II- adding B to the new DNA chain, proofreading the chain for mistakes DNA Polymerase I- removing RNA primers, replacing gaps between O fragments with correct nucleotides, repairing mismatched bases Ligase- final B of nicks in DNA during synthesis and repair Gyrase- Sc

unzipping RNA primer bases RNA primers okazaki binding supercoiling

Molecular Cloning: •Isolation and incorporation of a piece of DNA into a V so it can be replicated and manipulated •Three main steps of gene cloning: 1. I and F of source DNA --Source DNA can be genomic DNA, RNA, or PCR-amplified fragments ----Genomic DNA must first be restriction En digested ----Gel electrophoresis may be used to isolate the DNA 2.Insertion of DNA fragment into cloning vector --Most vectors are derived from P or V --DNA is generally inserted in vitro --DNA L: enzyme that joins two DNA molecules ----Works with sticky or blunt ends 3.Introduction of cloned DNA into host organism --T is often used to get recombinant DNA into host --Some cells will contain desired cloned gene, while other cells will have other cloned genes ----Gene L: mixture of cells containing a variety of genes --S cloning: gene libraries made by cloning random genome fragments

vector isolation fragmentation endonuclease plasmids viruses ligase transformation library shotgun