Fundamentals II:Bacterial Genetics

Janet Yother, Ph.D.

Department of Microbiology

jyother@uab.edu

4-9531

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

DNA (m)RNA proteintranscription translation

reverse transcription (some viruses)replication replication

DNA mRNA proteintranscription translation

Replication - DNA polymeraseand other enzymes

d.s.circularsingles.c.

RNA polymerase - recognizes specific sequences (promoters) in DNA to initiate transcription

Cytoplasmic membrane

s.s.linear

Ribosome - recognizes specific sequences (Ribosome binding sites) in mRNA to initiate translation; catalyzes amino acid additions

NH2-(aa)n-COOH

Promoters and Transcription Initiation in Bacteria

Molecular Genetics of Bacteria 2nd Ed, 2003

Transcription(DNA mRNA)

mRNA - synthesized 5’ to 3’ - complement of DNA (U instead of T)

DNA 5’ AGTCAGCAC 3’ 3’ TCAGTCGTG 5’

mRNA 5’ AGUCAGCAC 3’

mRNA 3’ UCAGUCGUG 5’

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

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

On average, 1 gene ~ 1 kbEscherichia coli ~ 4500 kbStreptococcus pneumoniae ~ 2300 kb

DNA mRNA proteintranscription translation

Replication - DNA polymeraseand other enzymes

d.s.circularsingles.c

RNA polymerase - recognizes specific sequences (promoters) in DNA to initiate transcription

Cytoplasmic membrane

s.s.linear

Ribosome - recognizes specific sequences (Ribosome binding sites) in mRNA to initiate translation; catalyzes amino acid additions

Transcription/Translation in Bacteria

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’ 5’ A N U N

UCCUCCA5’-NNNNNNAGGAGGU-N5-10-AUG-NNNn-3’

3’ end of16S rRNA

mRNA

Shine-Delgarnosequence

InitiationCodon

Ribosome (30S)

Ribosome Binding Site

aminoacyl-tRNA to A-site of ribosome

peptidyl transfer(peptide bind formation)

translocation

Molecular Biology of the Cell 4th Ed, 2002

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

1st Second Position 3rd5 U C A G 3

UPhePheLeuLeu

SerSerSerSer

TyrTyrstopstop

CysCysstopTrp

UCAG

CLeuLeuLeuLeu

ProProProPro

HisHisGlnGln

ArgArgArgArg

UCAG

AIleIleIle

Met

ThrThrThrThr

AsnAsnLysLys

SerSerArgArg

UCAG

GValValValVal

AlaAlaAlaAla

AspAspGluGlu

GlyGlyGlyGly

UCAG

protein

ribosomesubunits

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

Simultaneous translation of same mRNA by multiple ribosomes

Coupled Transcription-Translation

Science 169: 392-395.

DNA

ribosome

growing polypeptide

AUG mRNA

RNAP

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

from a single promoter

Promoter = non-coding sequence

mRNApolycistronic message

mRNAmonocistronic message

RBS RBSRBSRBS

No introns in bacteria although there is non-coding sequence

Mechanisms of Transcriptional Regulation

Alternative Sigma Factors Two component (signal transduction) Quorum sensing

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

Two-component regulatory systems.

Input signalSensor autophosphorylates (usually histidine kinase) ) ATP

P Response regulator -Mediates downstream effects

Signals include - temperature, O2, phosphate, sugarDownstream effects include DNA binding and transcription alterations, protein interactions

P

Interacts with and phosphorylates RR

Quorum Sensing

Accumulation and detection of small molecule leads to transcription regulation

Gram-negative signal = acyl homoserine lactone Gram-positive signal = oligopeptide

Gram-negativeGram-positive

ABC-transporter

Two-compRegulator

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

• 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

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

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

RBS RBS

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

XDNA

mRNA

Recombination - Homologous

• Occurs between regions of DNA that are highly similar

• Involves specific bacterial enzymes

• RecA-mediated

A B C D E

a b c d e

recipient chromosome

incoming donor DNA

A b c d E

a B C D e

resulting recombinant chromosome

incoming DNA lost; not replicated

- results in replacement of portions of host DNA with portions of donor DNA - multiple crossovers can occur

A B C D E

a b c d e

- can result in insertion or deletion of recipient chromosomal DNA since not all DNA between recipient and donor need be homologous

A B C D E

a b X Y Z c d

A B C D E

a b c d

X

Y

Z

A b X Y Z c D E

insertion

A B C D E

a b d e

A B D E

C

A B D E

deletion

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

Mediated by Cell-cell contact

DNase

Transduction Bacteriophage No Resistant

Conjugation F-factor (Gm -)

Pheromones (Gm +)

Yes Resistant

Transformation Free DNA No Sensitive

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• 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

• 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 Mediated by bacteriophage Transduction = accidental packaging of bacterial DNA

during lytic cycle, transfer to new host (transducing phages)

1

2transc.

early mRNA

DNA

transl.early proteins

host DNA, RNA, protein syn. shut off host chromosome degraded

middle proteins late proteins, incl. heads, tails

DNA replication

3a

0 5 10 15 22

minutes after attachment

3b 3c 4 5 6

Lytic Cycle (T4)

100s released

F

chrom (4500 kb)

F FF

Conjugation 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

F-pilus

F

chrom (4500 kb)

F FFF

chrom (4500 kb)

F FF

F

chrom (4500 kb)

F FF

Donor F+ Recipient F- one strand of F transferred; replicated in donor and recip

Both donor and recipient = F+

Conjugation OR F-factors can integrate into chromosome and transfer

part of the chromosome

Mediated by pheromones in Gram-positives

F-pilus

tra

X

bacterial chromosome

F

IS3

bacterial chromosome

IS3

tra

oriT

F

cointegrate

oriTtra bacterial chromosome

F F

IS3oriT

enters last enters first

Genes near oriT transferred most frequentlyF rarely transferred - recipient does not become Hfr

Transformation Uptake and integration into chromosome (usually) of free

DNA (plasmids can also be transformed) First demonstrated in Streptococcus pneumoniae (1928)

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

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

• 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 - Phase

promoter pil

pilin expressed

promoter pilinversion of promoter region

no pilin expressed

Salmonella flagella

P H1 P H1 repressor H2

no H1

H2+

P H1 H1 repressor H2 P H1+

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

Expression of alternative antigens

Neisseria gonorrhoeae pili

p ilA

p ilB

no

p ro m o t ers

P ilB +

P

p ilA

p ilD

no

p ro m o t ers

P ilD +

P

rec o m b inat io n

ac ro s s t he

c hro m o s o m e

Genetics and Antibiotic Resistance

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

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

Analogous mammalian enzymes insensitive Antibiotic doesn’t enter mammalian cells Absence of peptidoglycan in mammalian

cells

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

• Mutations (point, in chromosome)• Plasmids, transposons• Gene transfer• **Mutations arise at a low but constant

frequency.• **Antibiotics SELECT FOR naturally-

occurring resistant isolates.