Fundamentals II: Bacterial Genetics Janet Yother, Ph.D. Department of Microbiology [email protected]...
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Transcript of Fundamentals II: Bacterial Genetics Janet Yother, Ph.D. Department of Microbiology [email protected]...
Fundamentals II:Bacterial Genetics
Janet Yother, Ph.D.
Department of Microbiology
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
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
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
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