Download Microbial Genetics

Microbiology: Microbial Genetics (Withey)
PROKARYOTIC GENOME:
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Genetic Composition of Prokaryotes:
Chromosome:
o Haploid and circular
o ~4,000,000 bp (~4,000 genes)

Typical Gene: 1000bp
o No nucleus, introns or histones

Still highly structured, using histone-like proteins for form a nucleoid
o Usually only have one chromosome
May also possess plasmids and bacteriophages
o Plasmid: circular, extrachromosomal elements
o Bacteriophage: bacterial viruses, integrated or autonomous
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Plasmids:
Replication: autonomously replicating DNA (have their own origin of replication; can replicate independent of
the chromosome)
Size: 5000-200,000 bps
Quantity: 1-500 per cell
Epsiome: a plasmid that can integrate into chromosome; some encode elements required for conjugation
Plasmid-Encoded Virulence Factors:
o Heat labile and heat stable toxins of E.coli
o Tetanus toxin of Clostridium tetani
o Anthrax toxin of Bacillus anthracis
o Shigella spp.’s ability to invade colonic epithelium
o Antibiotic resistance (in some circumstances)
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Episomes/Resistance Factors:
R Factors: conjugative episomes that encode antibiotic resistance
o Composed of 2 Subunits:

Resistance Transfer Factor (RTF): allows for autonomous replication and conjugal transfer

Resistance Determinant: composed of one or more transposons, which carry the antibiotic
resistance gene
 Tranposons mediate the formation/resolution of R factors
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Transposons (Tn):
Definition: a sequence of DNA that can “hop” from place to place
Composition: antibiotic resistance gene flanked by insertion sequences, which encode for transposon mobility
and allow for entry into host genome
Function: disseminate antibiotic resistance
o Carried on a conjugative episome
o Hop into chromosome (overcome host restriction barriers)
Complex Transposons: consists of drug resistance (and other genes) flanked by 2 different insertion sequences
Example of Resistance:
o Enterobacteriaceae have transferred ampicillin resistance to Haemophilus influenza and Neisseri
gonorrhoeae
o This concept of transferred resistance is the rationale behind using combinations of unrelated
antibiotics
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Bacteriophage: viruses that only infect bacteria
Two Types:
o Lytic Phages: infect, reproduce and kill bacteria by lysis
o Temperant Phages: integrate into chromosome to form lysogen or prophage
Many toxin/virulence factor genes are carried in phage genomes:
o Examples: Vibrio cholera, E.coli
Can be used as therapy to kill antibiotic resistant bacteria
o Phage specific for a bacterial species can be isolated in a few days (very quick)
o They are very specific for their host bacterial species (protect normal flora)
GENE TRANSFER:

Restriction/Modification:
Function: a sort of bacterial immune system (prevents incorporation of foreign DNA into the genome)
o Modification: species-specific methylation of certain DNA sequences
o
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Restriction: cleavage of unmethylated DNA at the same sequences by restriction enzymes (foreign
DNA will not be methylated)

Bacterial methylase recognizes hemi-methylated DNA after replication, and methylates the
other strand

If it remains only hemi-methylated (unmethylated on one strand), restriction enzymes will
recognize and cleave out
3 Bacterial Gene Transfer Methods:
1. Transformation:
Basics: uptake of DNA from extracellular milieu (species-specific, sequence specific or non-specific)
Steps:
o Naked DNA adsorbs to bacteria and enters cytoplasm

Competence: ability to accept DNA; mechanisms vary among bacteria

Transformation is DNase sensitive
o Incoming DNA must recombine with host chromosome using RecA enzyme

Any DNA may gain entry, however does not mean it will be incorporated

Incoming DNA subject to host restriction barriers (Restriction/Modification system)

Incoming DNA must have some sequence homology with the host DNA for RecA to function
2. Transduction:
Basics: transfer of genetic information by a bacteriophage (can be generalized or specialized)
Generalized Transduction: indiscriminate transfer of chromosomal sequences
o Phage “accidentally” packages host sequences into a pseudovirion (new phages made that have some
host DNA; see step 3 pg 7)
o Transducing phages released from host cell, and injects this host DNA into new recipient cell
o Original host DNA incorporated into recipient’s chromosome by recombination (requires RecA)
Specialized Transduction: transfer of specific chromosomal sequences; can only occur through lysogenic phages
o Bacteriophage integrates into host chromosome to form a lysogen/prophage

Integration is site-specific and reversible

Integrated prophage is lysogenic
o DNA damage induces excision of the bacteriophage, and pieces of the host chromosome are pulled out
with the phage

This combination of chromosome + phage DNA is then transferred to the next host, where it
is integrated into the chromosome
o Virulence Factors Controlled by Lysogenic (Specialized) Conversion:

Diphtheria toxin (Corynebacterium diphtheria)

Streptococcal pyrogenic exotoxin A (SPE A; S.pyogenes)

Shiga toxins (Enterohemorrhagic E.coli)

Botulinum toxin (Clostridium botulinum)

Cholera toxin (Vibrio cholera)
3.
-
Conjugation:
Basics: sex in bacteria; DNA transfer by cell –cell contact (can occur with both Gram (+) and Gram (-) bacteria)
Steps:
o Merozygote Formation:

Temporary partial diploid/merozygote: recipient carries 2 copies of transferred genes
 These extra incoming genes may or may not integrate into the recipient
chromosome
o Specialized episome required for conjugation

(EX) Conjugative F episome carried by E.coli, which encodes for:
 Sex pili for cell-cell contact and cytoplasmic fusion
 Conjugative transfer and the repression of transfer
 Surface exclusion that prevents F+ from being a recipient (need F- recipient)
GENETIC REGULATION IN PROKARYOTES:

General:
Gene expression may be regulated by:
o Transcriptional control (regulation of mRNA production; most common)
o Translational control
o Post-translational control


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Operon Structure:
Basics: functional transcription unit
Consists of:
o Promoter: a type of cis-acting regulatory region; DNA sequence recognized by RNA polymerase sigma
factor
o Single gene (monocistronic) or a series of genes (polycistronic) that are transcribed into one mRNA

Since only one mRNA produced, all genes are coordinately regulated

Often, genes in an operon encode products with related functions
o Regulatory Sequences:

Trans-acting sequences encode regulatory proteins that diffuse to site

Cis-acting sequences are binding sites for regulatory proteins
 Promoter
 Operator: near promoter; binds the repressor to modulate transcription
 Attenuator: mRNA secondary structure that modulates transcription
Regulon: a set of operons regulated by the same transcription factor
Regulation of the lac Operon:
General:
o Example of negative and positive regulation
Stuctural Genes:
o B-galactosidase (lacZ)
o Galactoside permease (lacY)
o Galactoside acetylase (lacA)
Regulatory Sequences:
o Promoter: cis-actng
o Operator (lacO): cis-acting
o Repressor (lacI): trans-acting

Binds operator and blocks transcription from promoter
o Inducer (allolactose): inactivates repressor (when lactose is present), allowing transcription to occur

IPTG is an artificial inducer
Role of cAMP:
o Glucose is the favored carbon source, but will use lactose when glucose levels are low
o cAMP levels increase as glucose levels decrease
o cAMP binding protein (CRP/CAP) is a DNA binding protein and positive regulator
o Basics: glucose decreases, cAMP levels increase, bind CRP, which binds DNA and activates transcription
Varying Activity of lac Operon:
o Glucose, no lactose: repressor bound, CRP not bound  very low transcription (never 0!!)
o No glucose, no lactose: repressor bound, CRP bound  low transcription
o Glucose, lactose: repressor not bound, CRP not bound  moderate transcription
o No glucose, lactose: represspr not bound, CRP bound  high transcription
Other Types of Regulation:
Transcription Level:
o Purely negative (repressor)
o Purely positive (activator)
o Negative and positive (like the lac operon)
Translation Level:
o Usually negative control

Prevent binding of ribosome to mRNA

Make mRNA unstable (sRNA, RNases)
Post-Translation:
o Protein level (proteolysis)
o Protein activity (binding of small molecules or other proteins)
GENETIC REGULATION OF VIRULENCE FACTORS:

General:
Virulence factor expression is usually highly regulated
If it is unregulated, often has deleterious effects on bacterial survival (especially if they also inhabit non-host
environments)
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Antigenic Variation:
Antigenic Phase Variation in Salmonella enteritidis:
o Basics: changes flagellar Ags to evade the immune system
o Details:

Flagellar type switch due to inversion of promoter region

Inversion process control by Hin protein (invertase), which causes site-specific
recombination

Promoter orientation governs the expression of H1 and H2 (whichever one is not being
expressed is repressed due to the transcription of a repressor gene upon flipping of the
promoter)
Neisseria gonorrheae Pilus Type Variation by Gene Conversion:
o Promoterless silent gene moved to expression site promoter
o Immune clearance followed by expression of new antigenic type
o Hinders the development of an effective vaccine for gonorrhea
Signal Transduction:
Basics: allows for global regulation of virulence factors
Details:
o Transmembrane sensor response to environmental conditions
o Signal transmitted from sensor to a regulator by protein kinase

Cytoplasmic regulator is a DNA binding protein

Regulator enhances or represses transcription of a gene
Examples:
o Vibrio cholera: cholera toxin and pilus production (regulation by cascade of transcription factors)
o Bordetella pertussis: pertussis toxin production (regulation by signal transduction- phosphorelay)
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BvgS becomes phosphorylated in the body at body temperature

Transfers phosphate to BvgA, which determines virulence gene expression
Pathogenicity Islands:
Basics: stretches of chromosome that encode virulence attributes (usually have a higher A/T content than the
rest of the genome)
o Clustered genes for adhesions and toxins
o Operons with common function
Pathogenicity Islands with Repetitive Terminal Sequences Indicating Transposition:
o Acquisition of pathogenicity island can render harmless bacteria pathogenic
o Removal of pathogenicity islands from chromosome often eliminated virulence (potentially used in
vaccine production)
Examples of Pathogenicity Islands:
o Diarrheagenic E.coli: clustered loci for adhesions and toxins
o Vibrio Pathogenicity Island: TCP and regulatory proteins encoded here