Bacterial genetics

1
Chapter 5 Bacterial genetics ?????? ? ? ?
2

• DNA The genetic material
• Bacterial genome (prokaryotes)
• chromosome, a single, double-stranded,
  circular DNA
• Extrachromosomal genetic elements
  plasmids, bacteriophages
• only one copy of chromosome (haploid)
• structure of the bacterial chromosome
  polyamines, spermine, spermidine
• Operons groups of one or more structural
  genes expressed from a particular
• promoters and ending at a
  transcriptional terminator
• - polycistronic, e.g, E. coli
  lac operon
• Replication of DNA
• Initiation of synthesis
• Initiated a specific sequence (at one fixed
  point)
• OriC
• 1700bps/sec, 40 min for entire genome copy
• DNA ???? ?? double helix ? replication fork ??
• ??, ? strand ? DNA
  polymerase ?

3

• Semiconservative replication
• one original strand (conserved) one
  newly-synthesized strand
• DNA ? 5-gt3 ?? ??
• replication fork ?? ??? daughter strand ?
  continuously ?? (leading)
• ?? ???
  discontinuously ?? (lagging)
• Lagging strand ??? DNA ???
• replication fork ? ????? ?????? ????
• RNA polymerase, RNA primer, DNA-dependent DNA
  polymerase, DNA ligase ? ?? ??
• Bidirectional replication
• replicative origin ?? initiation ?? ???
  ?????? ???? replication ? ??
• DNA ??? ??? ??? ? ??
• 1) unwinding enzymes
• 2) stabilizing proteins single stranded
  parental DNA ? rewinding ? ??? ??.
• 3) DNA polymerases
• Pol I role for maturation, Pol II
  unclear role, Pol III 3-gt5 exonuclease
  activity
• 4) RNA polymerase leading strand RNA
  primer ??
• Primase lagging strand ? RNA primer ?? ,
  
• insensitive to the RNA
  polymerase inhibitor rifampicin

4
The rate of DNA replication - Log phase ??
multiple replication fork ??? ??? ? ?? generation
time ? ??? ??? ??? ???? ?? ? ??.
5

• Transcriptional control
• Transcription
• 3 types of RNA rRNA, tRNA, mRNA
• ds DNA ? ?? strand ? template ? ?? RNA polymerase
  ? RNA ? ??
• RNA ? nucleotide A, C, G, U ? ????.
• Promoter site transcription ????? RNA
  polymerase ? binding
• Terminator site transcription end point
• 4) RNA ? 5-gt3 ?? ????.
• 5) Prokaryotes ????? transcription
• Eukaryotes ??? transcription (hnRNA) -gt RNA
  processing -gt mRNA -gt ???
• Operon ??? ??? structural gene ? ?? ????.
  ?????? ?? ???
• ????? ??? ?? ??? structural gene ??
  ?? mechanism ? ??
• ???? ??? ??.
• promoter, operator site ? ?? ??
  structural gene ?? transcription ?
• ????? ??? structural gene ?
  promoter ? operator site ? operon ?? ?

6
Regulation of gene expression - Regulating
protein synthesis at the gene level is energy
efficient
because proteins are only synthesized as they
are needed - Constitutive enzymes are always
present in a cell Repression and Induction 1)
Repression ? ??? ??? ????, ?? ??? ???? mechanism
Repression ? ?? ??? repressible enzyme ?
??? ?? - regulatory proteins
Repressor protein capable of binding to
specific sequences of DNA (Operator)
overlap with the promoter ?
turning off the gene or set of genes
Corepressor 2) ????? ??? ?? end-product ? ???? ?
end-product ? ??? ???? ??? ??? ????. 3)
Induction ?? ??? turn-on Inducer ? ?? ??
??? ?? ?? ??? ??? ? ???, ?? induction ?? ?
Effectors inducers-combine with repressors to
decrease binding affinity for DNA Activators
positive-acting proteins, e.g. CAP protein 4)
Induction ? ? E. coli ?? lactose ?? ??
b-galactosidase? ??? ????
lactose ? ??? ????.
7

• Operon model
• 1961, Jacob Monod
• Induction (inducer) ? repression (repressor)
• ? ?? ????? ??
• ??? glucose ? lactose ? ??? ?
• carbon? energy source ? glucose ? ??
• ???? ??? lactose operon ? ????
• ?? (catabolite repression)
• Glucose ???? energy (ATP) ?? ?
• cAMP ?? ?? ? catabolite repression
• - Inducer ? ?? ? (Allolactose, IPTG)
• active repressor ? operator ? ????
• transcription ??
• - Inducer ? ?? ?
• inducer ? repressor ? ???? inactive
• repressor ? ?? operator ? ???? ??
• - In poor medium
• cAMP ?? ?? ?

8

• Attenuation tryptophan operon (trp operon)
• mRNA ? ??? ?????? ???
• transcription dl ??? ??
• ????? ?? ???? ??? E. coli ?
• ???? ??? ??? ??? ??? ??
• ??? ???? (? Trp) ? ????
• tryptophan ? ???? ??? ??? ? ??
• ???? ?? ??? ?? ??? ??? ??
• Attenuator tryptophan ????
• transcription termination
  site
• mRNA ??? premature termination ?
• ???? ??? ????? attenuation
• - Tryptophan operon
• 2) ???? tryptophan ? ???? ??
• - attenuator site ?? transcription ?
• premature termination ? ???
• 3) ???? tryptophan ? ???? ??

9

• Mutation, Repair, and Recombination
• Mutation
• ???? ?? ? gene product (?? ?) ? ?? ? ??? ???? ?
  lethal
• ? ??? activity ? benefit
• DNA base sequence ? ??? mutation ?? ?
• Types of mutations
• Point mutation DNA one base pair ? ?? base
  pair ? ???? ??
• - Transition one purine ? another purine
• one pyrimidine ? another
  pyrimidine
• - Transversion Purine ?? Pyrimidine
• - Silent mutation base pair ? ???? ??????
  ??? ?? ??
• ?) codon? 3?? ??? mutation
  (degeneracy)
• - Missence mutation DNA ? ??? ?? ??? ?????
  ?? ?????? ??
• sickle cell
  anemia
• - Conservative mutation DNA ? ??? ?? ???
  ?????
• ?? ??????
  ????? ??? ??? ?? ?????? ??

10

• Mutagen
• - DNA ? permanent change ? ???? ? ?? ??
• - Spontaneous mutation ?? mutagen ??? ???? ??
• - Physical mutagens
• 1) heat deamination of nucleotides
• 2) ionizing radiation X-rays, g-rays.
• DNA ? ??? ? ?? ion ? free radical ??
  ? combine with DNA bases
• ? DNA ??, repair ? error ?? ?
  mutation
• Base substitution ?? sugar-phosphate
  backbone ? break
• 3) UV nonionizing ??? thymidine ?
  crosslinking ? thymidine dimer
• Chemical mutagens
• 1) Nucleotide-base analogues 5-bromouracil ?
  incorporation into DNA
• 
  ? base pairing with guanine ? mistake in
  replication
• 2) Frameshift mutagens intercalating agents,
  polycyclic, flat, molecules
• Ethidium
  bromide, acridine derivatives
• 3) DNA-reactive chemicals Chemicals that
  react with DNA

11

• Repair mechanisms of DNA
• Direct DNA repair
• Excision repair
• Recombinational or postreplication repair
• SOS response
• Error-prone repair

12
Gene exchange in prokaryotic cells

• Exchange of DNA between cells ? New strain
• 
  Advantage ?? Antibiotic resistance gene
• Plasmid
• Circular, double-stranded DNA ?? ??
• ?? ??? ????? ?? ???? ?? ???? ??? ???
• extrachromosomal element
• Antibiotic resistance, bacteriocins, toxins,
• virulence determinant..
• 11 1 several copies (up to 50 copies)
• Replicons Autonomously replication,
• Capable of self duplication
• DNA molecules possess their
• own origin of replication
• Episomes integrate into host chromosome
• ? E. coli F plasmid
• Transfer conjugation F plasmid
• transformation
• transduction

13

• Bacteriophages
• Bacterial viruses
• Survive outside of a host cell
• totally dependent for Metabolic precursors
• Ribosomes, Cellular organelles necessary
• for making proteins
• Lysogenic state integrate into host genome
• Lytic infection
• insertion Bacteriophage lamda
• Lytic phage T2, T7
• How phage chromosomes are inserted into
• bacterial chromosome
• Circularize DNA
• attach to E. coli chromosome
• (very homologous section)
• crossover break and rejoin.

14

• Transposon
• Jumping genes, mobile genetic elements
• ???? ?? ???? ?????? ??
• ??? ??? ?? ? ?? ?? DNA segment
• Insertion sequence (IS, simple transposon)
• carry no genetic information
• except for
  transposition
• Specific terminal DNA sequence
• - Inverted repeat 15-40 bases
• coding region for an enzyme (transposase)
• cutting and ligating DNA, recognition site
• Composite transposons (complex transposons)
• contain genetic information in addition to
• that needed for transposition
• TnA family
• transposase(tnpA)
• resolvase (tnpR)
• b-lactamase resistance to ampicillin
• resolution site (Res site)

15

• Mechanism of genetic transfer between cells
• - Exchange of genetic material between bacterial
  cells
• - Transformation ????? naked DNA ??? ????
• ? ?????? ?? ????? ????
  ??
• Streptococcus pneumoniae ?? ?? ??
• ? ?? ???????
• natural transformation ??
• - Haemophilus influenzae
• - Neisseria
• - Acinetobacter
• - Streptococcus
• - Bacillus
• Artificial transformation
• Competence
• end of logarithmic growth
• before stationary phase
• work only in complete replicon
• capable of self-duplication

16

• Conjugation
• ???? ???? contact
• Donor F cells plasmid F factor ? ??? ?? ??
• Recipient F- - F factor ? transfer ??.
• F factor ? ???? ??? ??? Hfr (high frequency
  recombination) ?? ?
• chromosomal DNA ??? transfer ?? ???
  ?? ?? ??? transfer
• 2 distinct sexes
• F(fertility) factor
• - sex chromosome
• - circular dsDNA molecule
• - one copy/cell
• - capable of transferring genes
• into female cells
• -1/3 of the F DNA
• consist of 19 transfer (tra) genes
• specifically involved in the transfer of
• genetic material
• responsible for synthesis of the
• sex specific F pili

17

• Recombination
• Genetic recombination ? ?? DNA ?? ??? ??? ???
  ???
• ???? ??? ???
  ???? ??
• - Homologous (legitimate) recombination
• - Nonhomologous (illegitimate) recombination
• Genetic engineering