Chapter 8

Microbial Genetics

genetics

the study of genes, how they carry information, how information is expressed, and how genes are replicated

genome

all the genetic information in a cell

chromosomes

structures containing DNA that physically carry hereditary info → contain genes

genes

segments of DNA that encode functional products, usually proteins

genetic code

set of rules that determines how a nucleotide sequence is converted to an amino acid sequence of a protein

central dogma

• developed by Francis Crick

• path: DNA → RNA → protein

• gene in DNA is copied to make mRNA, which directs the synthesis of a protein

• when the molecule encoded by the gene is produced → the gene has been expressed

genotype

• the genetic makeup of an organism

• represents the potential properties that DNA can give the organism IF it were to be expressed

phenotype

• expression of the genes

• collection of proteins that have been produced and their function

alteration of bacterial genes and/or gene expression may

• cause disease

• prevent disease treatment

• be manipulated for human benefit

circular chromosome

• found in bacteria

• made of DNA and associated proteins

• chromosome is looped and folded → attached at several points at PM → supercoiled

• consists of protein-encoding genes and noncoding regions

short tandem repeats (STRs)

repeating sequences (2-5 base pairs/repeat) of noncoding DNA

flow of info: replication

genetic info can be transferred vertically to the next generation of cells

flow of info: recombination

genetic info can be transferred horizontally between cells of the same generation

flow of info: expression

genetic info is used within a cell to produce the proteins needed for the cell to function

semi-conservative replication

• each original strand serves as a template for the production of a new strand

• newly replicated DNA contains one original strand (conserved) and one new strand

DNA

• double helix

• backbone: deoxyribose and phosphate

• strands are complementary

• nucleotides held together by hydrogen bonds

  • A - T

  • G - C

• strands are antiparallel-parallel

• order of bases forms the genetic instructions of the organism

• has a 3’ end (with OH- attached to 3’ carbon)

• has a 5’ end (with PO₄- attached to 5’ carbon)

topoisomerase and gyrase

relax the DNA strands

helicase

separates the DNA strands to form a replication fork

DNA polymerase

• adds nucleotides to the growing DNA strand

• only adds 5’ complimentary attached to the 3’ on the original strand

• builds only in the 5’ to 3’ direction so it must start at 3’

• initiated by an RNA primer

• nucleotides that are used come from the cytoplasm

• leading strand synthesized continuously

• lagging strand synthesized discontinuously

• DNA polymerase removes primers and joins Okazaki fragments together

making a strand complementary to the leading strand of DNA

• leading strand is the one with the 3’ furthest from the replication fork

• DNA polymerase starts at that 3’

• adds a complimentary 5’ nucleotide sequence to that 3’

• keeps adding nucleotides in the 5’ to 3’ orientation towards the replication fork

• synthesis is continuous

making a strand complementary to the lagging strand of DNA

• lagging strand is the one with the 5’ furthest from the replication fork

• but DNA polymerase cannot start at that 5’ and move towards the replication fork → they want to move towards the fork, but need to start at 3’ on the original only

• RNA primase puts down a 3’ OH- in sections

• DNA poly starts at the 3’ primer furthest from the fork and puts down a 5’ to start building in 5’ to 3’

• reaches the end → DNA poly moves backwards (towards the replication fork) to the next 3’ primer

• repeats until entire strand is synthesized

• DNA poly and DNA ligase joins Okazaki fragments of new strand together

DNA ligase

makes covalent bonds to join DNA strands, Okazaki, and segments in excision repair

endonucleases

cut DNA backbone internally within a strand of DNA → facilitate repair and insertions

exonucleases

cut DNA from an exposed end of DNA → facilitate repair

photolyase

uses visible light energy to separate UV-induced pyrimidine dimers

primase

RNA polymerase that makes RNA primers from a DNA template

RNA polymerase

copies RNA from a DNA template

summary of DNA replication

1. enzymes unwind the parental double helix

   1. weak hydrogen bonds break between the nucleotides on opposite strands

2. proteins stabilize the unwound parental DNA

3. leading strand is synthesized continuously from the primer by DNA polymerase

4. lagging strand is synthesized discontinuously using short RNA primers that are extended by DNA polymerase

5. hydrogen bonds form between complementary nucleotides

6. DNA polymerase digests RNA primer and replaces it with DNA

7. DNA ligase joins the discontinuous of the lagging strand

8. enzymes catalyze the formation of sugar-phosphate bonds between sequential nucleotides on each resulting daughter strand

bacterial DNA replication

• bidirectional: two replication forks move in opposite directions away from the origin of replication

• since chromosome is circular → will meet at a single point → separated by topoisomerase

• each offspring cell receives one copy of the DNA molecule

• proof-reading capability of DNA polymerase: DNA evaluates whether the base pair is correct each time one is added

RNA

• single-strand of nucleotides

• 5-carbon ribose sugar

• contains uracil (U) instead of thymine (T)

ribosomal RNA (rRNA)

integral part of ribosomes

transfer RNA (tRNA)

transports amino acids during protein synthesis

messenger RNA (mRNA)

carries coded info from DNA to ribosomes

transcription in prokaryotes

• synthesis of a complementary RNA strand from a DNA template

• takes place in the cytoplasm of bacterial cell

• RNA polymerase synthesizes the RNA

steps for transcription in prokaryotes

1. initiation: transcription begins when RNA polymerase binds to the promoter sequence on DNA

2. elongation: transcription proceeds in the 5’ to 3’ direction of the growing DNA

   1. A on DNA- U in RNA

   2. G on DNA - C in RNA

   3. C on DNA - G in RNA

   4. T on DNA - A in RNA

3. site of synthesis moves along DNA

4. DNA that has been transcribed rewinds

5. termination: transcription stops when it reaches the terminator sequence on DNA

6. RNA and RNA polymerase → DNA helix reforms

codons

• groups of three mRNA nucleotides that specify a particular amino acid or stop signal

• 64 different codons

• 61 sense codons: encode 20 amino acids including the start codon

• 3 stop/nonsense codons: UAA, UAG, UGA

  • signal the end of synthesis → but don’t code for anything

• each amino acid can be coded for by several codons

process of translation

1. components needed to begin translation come together → 2 ribosomal subunits and mRNA

2. start codon enters the A site 

3. tRNA carries first amino acid with the anticodon that matches the start codon on mRNA → attaches to codon

4. first tRNA attached to codon moves to the P site → tRNA carrying second amino acid enters A site and binds to matching codon

5. first amino acid joins the second by a peptide bond

6. ribosome moves along mRNA until second tRNA is in the P site, next codon to be translated is brought into the A site, first tRNA is now in the E site

7. second amino acid joins to third by peptide bond and first tRNA is released from the E site

8. ribosome continuous to move along mRNA → codons from before are exposed → new ribosomes can come and use it to synthesize proteins

9. ribosome reaches stop codon → polypeptide is released and ribosome comes apart

translation can begin..

before transcription is complete

where do transcription and translation take place

cytoplasm of bacterial cell

where does transcription occur in eukaryotes

the nucleus

where does translation occur in eukaryotes

the cytoplasm

exons

regions of DNA that code for proteins

introns

regions of DNA that do not code for proteins

what happens after the RNA transcript is made

the RNA copies of introns are removed by small nuclear ribonucleoproteins (snRNPs) and splice exons together

constitutive genes

• expressed at a fixed rate

• do not appear to be regulated

• always “turned on”

genes that are expressed as needed

• inducible genes

• repressible genes

inducible operon

• default position is “off” → repressor bound to DNA operator region

• gene expression needs to be turned on 

• turns on when an inducer binds to the repressor and makes it inactive

repressible operon

• default position is “on”

• mediated by repressors to turn off the DNA and its expression → need to avoid making too much product

• corepressor (sometimes the product itself) binds to the allosteric site of the inactive repressor → makes it active

• repressors bind to operator region to block transcription from occurring

operon

• segment of DNA consisting of:

  • regulatory gene (I): codes for the repressor

  • promoter (P): segment of DNA where RNA polymerase initiates transcription of structural genes

  • operator (O): segment of DNA that controls transcription → traffic light for whether to start or stop transcription → where the repressor binds

  • structural genes: determine the structure of proteins

example of an inducible operon

the lac operon of E. coli

activity of the lac operon in the ABSENCE of lactose

1. I gene transcribed to be repressor mRNA → translates to repressor protein

2. repressor is active

3. RNA polymerase binds to promoter region

4. BUT active repressor binds to the operator region

5. repressor prevents RNA polymerase from moving forward to transcribe the structural genes

activity of the lac operon in the PRESENCE of lactose

1. I gene transcribed to be repressor mRNA → translated into active repressor protein

2. presence of lactose → molecule of it called allolactose acts as an inducer

3. inducer allolactose binds to repressor protein → inactivates it

4. operator region is free → goes ahead with transcription of structural genes

5. operon mRNA made → translated into enzymes

6. enzymes used for the catabolism of lactose into glucose → which enters glycolysis to make ATP

enzymes that metabolize glucose are

constitutive

what happens when there is no more glucose

1. cAMP accumulates

2. cAMP binds to the allosteric site of the catabolic activator protein (CAP)

3. CAP binds to lac promoter region → makes it easy for RNA polymerase to bind to promoter region

4. BUT transcription will occur under 3 conditions

   1. lactose must be present → both to break down after enzymes are produced and for a modified version (allolactose) to act as the inducer and turn off the repressor → transcription will not occur if repressor is still active

   2. glucose must be absent → using glucose is more efficient so if it is still present, the cell would prefer to use it

catabolite repression

inhibits cells from using carbon sources other than glucose

when glucose is present

• cAMP levels low

• CAP inactive (not bound to cAMP)

when glucose is absent

• cAMP levels high

• CAP active (bound to cAMP)

growth rate when glucose is the sole carbon source

grows faster than lactose

growth rate when the medium contains glucose and lactose

• glucose first consumed

• lag time occurs

  • intracellular cAMP increases

  • lac operon transcribed

  • lactose is transported into the cell

  • enzymes produced to break down lactose

• lactose turns into glucose → glucose enters cellular respiration to make ATP

activity of the trp operon with repressor off

1. I gene codes for inactive repressor protein

2. nothing is there to bind to it to make it active

3. RNA polymerase binds to promoter region → operator region goes ahead with transcription

4. operon mRNA translated → produces enzymes used to synthesize tryptophan

activity of the trp operon with repressor on

1. I gene codes for inactive repressor protein

2. tryptophan product from earlier cycles acts as a corepressor → binds to the repressor protein

3. repressor becomes active → binds to the operator region

4. RNA polymerase binds to promoter region → but cannot move forward since repressor is blocking the way

5. structural genes cannot be transcribed

epigenetic control

• adding a methyl group (-CH₃) to genes → turns them off

• methylated genes can be passed down to offspring

• not permanent → genes may be turned on in another generation

mutation

• a permanent change in the base sequence of DNA

• may cause a change in product encoded by the gene

• may be neutral, beneficial, or harmful

mutation rate

the probability that a gene will mutate when a cell divides

mutagens

• agents that cause mutations

• increase the rate of mutations 10 to 1000 times

• there are physical agents and chemical agents

spontaneous mutations

• occur in the absence of a mutagen

• result from replication errors

• spontaneous mutation rate is 1 in 10⁹ bps

base substitution

• aka point mutation

• change in one base in DNA

• mRNA will carry the incorrect base in that position → can cause the insertion of the wrong amino acid

• 3 types: silent, nonsense, and missense

silent mutation

• leads to a different codon that encodes the same amino acid

• results in a full-length protein with the same amino acid sequence

• no effect on function → redundancy

nonsense mutation

• leads to a stop (nonsense) codon

• results in a truncated protein

missense mutation

• leads to a codon that encodes a different amino acid

• results in a change in one amino acid in the protein sequence

• vary from minimal impact to significant loss of function

frameshift mutation

• insertion or deletion of one or more nucleotide pairs that are not multiple of 3

  • if multiple of 3 → results in one extra or one less amino acid → can’t tell where mutation occurred

• shifts the translational reading frame

• causes changes in many amino acids downstream from the site of the original mutation → different codons result

• usually loss of function

nitrous acid

• chemical mutagen that removes amino groups from DNA bases and causes mistakes in base pairing

• alters the amino group of adenine so it resembles guanine → this binds with cytosine instead of thymine

2-aminopurine (nucleoside analog)

• incorporated into DNA in place of adenine

• this analog can pair with cytosine

• so it resembles a G-C pair in the next DNA replication

  • DNA poly would pair the analog with another cytosine and the cytosine with guanine each replication

5-bromouracil (nucleoside analog)

• pairs with cytosine

• mistaken for thymine by cellular enzymes → buts this down instead of thymine

• first pairing from this → A to analog (instead of A-T

• next replication with the strand with analog in it → binds to C

• C will bind to G in the following replication

• after a few generations → G-C pair results

ionizing radiation

• causes electrons to pop out of their shells and bombard other molecules with them

• some molecules lose electrons and some gain them → ions

• the ions are reactive → oxidize nucleotides

• result: errors in DNA and breakage of deoxyribose-phosphate backbone

UV raditation

• causes pyrimidine dimers on the same DNA strand

• two adjacent thymine bases on the same strand become covalently bonded → causes a kink in the strand

• leads to mutations if not repaired → prevents proper replication/transcription → skin cancer

repairing a thymine dimer

• endonuclease cuts the DNA

• exonuclease removes damaged DNA

• DNA polymerase fills the gap by synthesizing new DNA → uses the intact strand as a template

• DNA ligase seals the remaining gap by joining the old and new DNA

photolyases

• light-repair enzymes

• uses light to separate thymine dimers

nucleotide excision repair

enzymes cut out incorrect bases and fill in correct bases

auxotroph

mutant that has a nutritional requirement absent in the parent

prototroph

wild type that does not have such nutritional requirement

positive (direct) selection

detects mutant cells because they grow or appear different than unmutated cells

• aka apply something in which the unmutated cells cannot survive

negative (indirect) selection

detects mutant cells that cannot grow or perform a certain function

replica plating steps

1. about 100 bacterial cells are inoculated onto an agar plate

2. sterile velvet is pressed on the grown colonies on the master plate

3. cells from each colony are transferred from the velvet to new plates

   1. one plate containing histidine

   2. one plate without histidine

4. comparing results

   1. any colony that grows on the medium with histidine but cannot synthesize its own histidine → cannot grow on the plate without histidine = auxotrophic

many mutagens are

carcinogens (cause cancer)

Ames test

• based on the observation that: exposure of already mutant bacteria to mutagenic substances may cause new mutations that reverse the effects of the original mutation

• uses a auxotroph of Salmonella that is unable to synthesize histidine → measures its reversion to a histidine-synthesizing bacterium after mutagen exposure

• rat liver extract is added → provides activation enzymes

• reversion rate indicates the degree to which a substance is mutagenic

vertical gene transfer

transfer of genes from an organism to its offspring

horizontal gene transfer

• transfer of genes between the cells of the same generation

• involve a donor cell that gives some of its DNA to a recipient cell

• major mechanisms: transformation, conjugation, and transduction

• plays a significant role in bacterial evolution and the spread of traits among bacterial populations

genetic recombination

• exchange of genes between two DNA molecules to form new combinations of genes

• horizontal gene transfer involves incorporation of the donor DNA into the recipient DNA → becomes recombinant

steps of horizontal gene transfer (crossing over)

1. DNA from one cell aligns with DNA in the recipient cell → nick is formed in donor DNA

2. DNA from the donor aligns with complementary base pairs in the recipient’s chromosome → can involve thousands of base pairs

3. RecA protein catalyzes the joining of the two strands

4. result: recipients chromosome contains new DNA, complementary base pairs will be joined by DNA polymerase and ligase, and donor DNA will be destroyed

transformation

genes transferred from one bacterium to another as “naked” DNA

transformation in bacteria

• demonstrated by Griffith’s experiment with smooth strains (S strain) and rough strains (R strain) of Streptococcus pneumoniae

• occurs when:

  • bacterial cells die and lyse → releases DNA

  • other bacteria may take up and incorporate fragments of that DNA into their chromosome by recombination

• occurs naturally among a few bacterial genera

Griffith’s experiment

• Types of strains

  • S strain (smooth): encapsulated and virulent (causes disease)

  • R strain (rough): nonencapsulated and nonvirulent (harmless)

• Live S strain = mouse dies

• Live R strain = mouse lives

• Heat-killed S strain = mouse lives

• Heat-killed S strain + live R strain = mouse dies

  • Conclusion: the dead S cells transformed the originally harmless R cells into virulent S cells = transformation

plasmids

• extra-chromosomal, self-replicating, circular pieces of DNA

• carry nonessential genes

• 1-5% the size of bacterial chromosome

• found primarily in bacteria

conjugative plasmids

• carry genes for sex pili and transfer of the plasmids

• F factor: conjugative plasmid that carries genes for sex pili and for the transfer of the plasmid to another cell

dissimilation plasmids

• encode enzymes for the catabolism of unusual compounds → break it down for energy

• allows them to survive in diverse/challenging environments

resistance factors (R factors)

encode for antibiotic resistance → spreads these genes to make more bacteria resistant

virulence plasmids

carry genes that code for toxins or bacteriocins

resistance (R) factors

• plasmids that encode antibiotic resistance → can be transferred horizontally and recombine

• resistance transfer factor (RTF): group of genes for replication and conjugation

• R determinant: resistance genes that code for enzymes that inactivate certain drugs/toxins

conjugation

• plasmids transferred from one bacterium to another through cell-to-cell contact

• occurs commonly among gram negative bacteria

• gram-negative bacteria: attachment and transfer is via sex pilus → encoded by conjugative plasmid

• gram-positive bacteria: a sticky substance that holds participating cells together

• used to map the location of genes on a chromosome