Fundamentals ii bacterial genetics
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Fundamentals II: Bacterial Genetics. Janet Yother, Ph.D. Department of Microbiology [email protected] 4-9531. Learning Objectives. Bacterial transcription and translation Examples of transcriptional regulation Gene transfer mechanisms

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Fundamentals II: Bacterial Genetics

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Fundamentals ii bacterial genetics

Fundamentals II:Bacterial Genetics

Janet Yother, Ph.D.

Department of Microbiology

[email protected]

4-9531


Learning objectives

Learning Objectives

  • Bacterial transcription and translation

  • Examples of transcriptional regulation

  • Gene transfer mechanisms

  • Roles of mutation, gene transfer, and recombination in virulence and antibiotic resistance


Central dogma of molecular biology

transcription translation

DNA (m)RNA protein

reverse transcription

(some viruses)

replication

replication

Central Dogma of Molecular Biology


Fundamentals ii bacterial genetics

Cytoplasmic membrane

d.s.

circular

single

s.c.

RNA polymerase - recognizes specific sequences (promoters)

in DNA to initiate transcription

s.s.

linear

NH2-(aa)n-COOH

Replication -

DNA polymerase

and other enzymes

Ribosome - recognizes specific sequences

(Ribosome binding sites) in

mRNA to initiate translation;

catalyzes amino acid additions

transcription translation

DNA mRNA protein


Promoters and transcription initiation in bacteria

Promoters and Transcription Initiation in Bacteria

Molecular Genetics of Bacteria 2nd Ed, 2003


Transcription dna mrna

mRNA 3’ UCAGUCGUG 5’

mRNA 5’ AGUCAGCAC 3’

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/

chroms-genes-prots/transcription-translation.html

Transcription(DNA mRNA)

DNA 5’ AGTCAGCAC3’

3’ TCAGTCGTG5’

mRNA - synthesized 5’ to 3’

- complement of DNA (U instead of T)


Fundamentals ii bacterial genetics

Transcription can occur on either DNA strand - the one used depends on the presence of the proper signals

On average, 1 gene ~ 1 kb

Escherichia coli ~ 4500 kb

Streptococcus pneumoniae ~ 2300 kb


Transcription translation in bacteria

Cytoplasmic membrane

d.s.

circular

single

s.c

RNA polymerase - recognizes specific sequences (promoters)

in DNA to initiate transcription

s.s.

linear

Replication -

DNA polymerase

and other enzymes

Ribosome - recognizes specific sequences

(Ribosome binding sites) in

mRNA to initiate translation;

catalyzes amino acid additions

Transcription/Translation in Bacteria

transcription translation

DNA mRNA protein


Translation mrna polypeptide

Translation(mRNA polypeptide)

  • Initiation - 30S subunit of ribosome binds mRNA at specific site

  • 30S subunit binds 50S subunit 70S

  • Synthesis - tRNA anticodons pair with complementary codons in mRNA, add amino acid to growing chain


Translation initiation

3’ end of

16S rRNA

3’5’

A N

U N

UCCUCCA

5’-NNNNNNAGGAGGU-N5-10-AUG-NNNn-3’

mRNA

Shine-

Delgarno

sequence

Initiation

Codon

Translation Initiation

Ribosome (30S)

Ribosome Binding Site


Fundamentals ii bacterial genetics

aminoacyl-tRNA to

A-site of ribosome

peptidyl transfer

(peptide bind formation)

translocation

Molecular Biology of the Cell 4th Ed, 2002


Translation genetic code

Translation - Genetic Code

  • Essentially universal

  • Amino acid determined by mRNA codon (codon = 3 nucleotides; complement of anticodon in tRNA)

  • Translation start = AUG (Met); less often GUG

  • Translation stop = UAA, UAG, UGA

    • Exception: UGA in

      mycoplasma = Trp


Fundamentals ii bacterial genetics

ribosome

subunits

protein

Simultaneous translation of same mRNA by multiple ribosomes

http://www.sci.sdsu.edu/~smaloy/MicrobialGenetics/topics/chroms-genes-prots/transcription-translation.html


Coupled transcription translation

RNAP

DNA

mRNA

AUG

ribosome

growing polypeptide

Science 169: 392-395.

Coupled Transcription-Translation


Operons

Operons

Bacterial genes can be organized into operons - more than one gene transcribed

from a single promoter

Promoter = non-coding sequence

RBS

RBS

RBS

RBS

mRNA

monocistronic message

mRNA

polycistronic message

No introns in bacteria although there is non-coding sequence


Mechanisms of transcriptional regulation

Mechanisms of Transcriptional Regulation

  • Alternative Sigma Factors

  • Two component (signal transduction)

  • Quorum sensing


Alternative sigma factors

Alternative Sigma Factors

  • Bind RNA polymerase, allow recognition of alternative promoter sequences

  • s70 (70 kDa) = major sigma factor in E. coli

    • At least 8 alternative sigma factors

      • s54 – nitrogen limitation

      • s32 – heat shock

  • Bacillus – sporulation


Signal transduction

Signal Transduction

  • Two-component regulatory systems.

Input signal

Sensor autophosphorylates

(usually histidine kinase)

)

P

ATP

Interacts with and phosphorylates RR

Response regulator -

Mediates downstream effects

P

Signals include - temperature, O2, phosphate, sugar

Downstream effects include DNA binding and transcription alterations,

protein interactions


Quorum sensing

Quorum Sensing

  • Accumulation and detection of small molecule leads to transcription regulation

  • Gram-negative signal = acyl homoserine lactone

  • Gram-positive signal = oligopeptide

Gram-positive

Gram-negative

ABC-

transporter

Two-comp

Regulator


Mutations

Mutations

  • Any change in DNA sequence whether effect observable or not

  • Causes

    • spontaneous - errors in DNA replication. Arise at a low but constant and often detectable frequency (always occurring).

    • induced - radiation (X-ray, uv), chemicals. Increase frequency.


Classes and results of mutations i

Classes and Results of Mutations - I

  • Point Mutation

    • alteration of single nucleotide. Can have multiple point mutations.

  • Possible Results

    • samesense - codes for same amino acid. No effect (silent).

    • missense - codes for different amino acid. Protein function may/may not be altered.

    • nonsense - now codes for translation stop codon. Premature stop >> truncated product, function probably lost (depending on where stop occurs)


Classes and results of mutations ii

X

DNA

mRNA

RBS

RBS

Polar effect of insertion - multiple genes may be affected due to transcription from same promoter

Classes and Results of Mutations - II

  • Deletion - DNA lost. Function lost if most/all of gene deleted.

  • Insertion - new DNA has been added. Gene interrupted. Function usually lost.


Recombination homologous

Recombination - Homologous

  • Occurs between regions of DNA that are highly similar

  • Involves specific bacterial enzymes

  • RecA-mediated


Recombination non homologous

Recombination: Non-homologous

  • Occurs between DNAs without significant similarity. Best example, important in pathogens - transposons.

  • Transposons - mobile (transposable) genetic elements (Tn)

    • DNA sequences that can insert essentially at random into chromosome/plasmid (some have some site specificities)

    • result is an insertion mutation: disrupt function, polar (affect expression of downstream genes)

    • 2 to 50 kb

    • cannot replicate autonomously

    • encode functions for own transposition

    • often, encode antibiotic resistance (Amp, Km, e.g.), virulence factors.


Bacterial gene transfer mechanisms

Bacterial Gene Transfer Mechanisms


Extrachromosomal dna

Extrachromosomal DNA

  • Plasmids - Replicate in cytoplasm, independent of chromosome.

    • Usually circular (borrelia = linear)

    • Few to several hundred kb

    • Few to several hundred copies

    • Conjugative (F, R), antibiotic resistance, metabolic, virulence

  • Bacteriophage - virus; replicates in cytoplasm or integrates into chromosome

    • Seen with electron microscope

    • DNA or RNA; no metabolic apparatus

    • Specific phage infects specific bacterium(a)


Bacteriophage

Bacteriophage

  • virus; replicates in cytoplasm or integrates into chromosome

    • Seen with electron microscope

    • DNA or RNA (in phage head); no metabolic apparatus

    • Specific phage infects specific bacterium(a)

  • Types

    • Virulent - continually in lytic cycle, making phage; bacterial host usually killed

    • Temperate - may undergo lytic cycle OR lysogenic cycle (symbiotic with host; may encode virulence factors); >90% of known phages


Significance of bacteriophage

Significance of bacteriophage

  • Phage (lysogenic) conversion - observable effect of phage carried by bacterium. Medically important. Every bacterium may carry a phage.

    • Corynebacterium diptheriae - Gm + rod; diptheria toxin = phage-encoded

    • Clostridium botulinum - Gm + rod; botulism toxin = phage-encoded

  • Gene transfer (transduction)


Transduction

Transduction

  • Mediated by bacteriophage

  • Transduction = accidental packaging of bacterial DNA during lytic cycle, transfer to new host (transducing phages)

100s released


Conjugation

Conjugation

F-pilus

  • Mediated by F-factor or similar conjugative plasmids (in Gram-negatives)

  • F-factors can encode antibiotic resistance = R factor

  • F-factors replicate in cytoplasm and be transferred - - -

F ~ 100 kb

Donor F+ Recipient F- one strand of F transferred;

replicated in donor and recip

Both donor and recipient = F+


Conjugation1

Conjugation

F-pilus

  • OR F-factors can integrate into chromosome and transfer part of the chromosome

enters last enters first

Genes near oriT transferred

most frequently

F rarely transferred - recipient

does not become Hfr

  • Mediated by pheromones in Gram-positives


Transformation

Transformation

  • Uptake and integration into chromosome (usually) of free DNA (plasmids can also be transformed)

  • First demonstrated in Streptococcus pneumoniae (1928)


Transformation1

Transformation

  • Homologous DNA integrated (though non-homologous DNA may be taken up by some bacteria)

  • DNA from lysed bacteria or secretion

  • Highly regulated - uptake machinery may be expressed only when other like bacteria are present

  • Gm +: Streptococcus, Bacillus, Streptomyces

  • Gm -: Haemophilus, Pseudomonas, Neisseria


Roles of mutation recombination and gene transfer in virulence and antibiotic resistance

Roles of Mutation, Recombination, and Gene Transfer in Virulence and Antibiotic Resistance


Variation antigenic

Variation - Antigenic

  • Antigenic Variation (Microbial evasion)

    • Antigenic drift - slow accumulation of point or other “small” mutations. Alter specific protein at one or few antigenic epitopes (influenza virus)

    • Antigenic shift - major change. Results from recombination (new DNA from gene transfer; intracellular deletions, insertions)

    • Permanent change


Variation phase

Variation - Phase

  • Phase Variation (Microbial Variation)

    • Switching back and forth between expressing/not expressing

    • Involves recombination

    • Not permanent, can revert to original type

  • Advantages for Pathogen

    • Avoid antibody, avoid having antibody made to antigen

    • Express antigen only when important (attachment, e.g.)


Variation phase1

Variation - Phase

On/off of one antigen: E. coli pili involved in attachment. Inversion of promoter.

Expression of

alternative antigens

Neisseria gonorrhoeae pili


Genetics and antibiotic resistance

Genetics and Antibiotic Resistance


Antibiotics mechanisms of action differences between prokaryotes and eukaryotes

Antibiotics - Mechanisms of Action (Differences between Prokaryotes and Eukaryotes)

  • inhibit protein synthesis

    • bind ribosomal proteins, RNA polymerase

    • Aminoglycosides (kanamycin), tetracyclines, macrolides (erythromycin)

    • Rifampin (binds RNA polymerase)

  • inhibit DNA synthesis

    • bind enzymes/proteins involved in DNA replication

    • Fluoroquinolones (ciprofloxacin)

  • inhibit metabolic activity

    • bind enzymes

    • Sulfonamides-bactrim (inhibit tetrahydrofolate production)

  • Cell wall synthesis

    • Penicillins (block transpeptidation)

    • Vancomycin (blocks transglycosylation)


Antibiotics specificity for bacteria

Antibiotics - Specificity for Bacteria

  • Differences between bacterial (70S) and mammalian ribosomes (80S)

  • Analogous mammalian enzymes insensitive

  • Antibiotic doesn’t enter mammalian cells

  • Absence of peptidoglycan in mammalian cells


Fundamentals ii bacterial genetics

Antibiotic Mechanism

Inhibit DNA replication (gyrase)

Inhibit transcription (RNA polymerase)

Inhibit translation (ribosome)

Inhibit Cell Wall Synthesis

Bacterial Resistance

Altered gyrase doesn’t bind antibiotic (point mutations*)

Altered RNA polymerase (point mutations*)

Altered ribosome*; enzyme (plasmid-encoded) inactivates antibiotic; protein (plasmid-encoded) prevents antibiotic entry into cell

Altered cell wall synthesis proteins*; enzyme (plasmid-encoded) inactivates antibiotic

* chromosome-encoded


Development and spread of antibiotic resistance

Development and Spread of Antibiotic Resistance

  • Mutations (point, in chromosome)

  • Plasmids, transposons

  • Gene transfer

  • **Mutations arise at a low but constant frequency.

  • **Antibiotics SELECT FOR naturally-occurring resistant isolates.


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