Withey: Bacterial Genetics Flashcards
Chromosome: Shape bps/gene #: Typical gene: Nucleus? Nucleoid def: Cs #:
Haploid and circular
~4,000,000 bp (~4,000 genes)
Typical Gene: 1000bp
No nucleus, introns or histones
Still highly structured, using histone-like proteins for form a nucleoid
Usually only have one chromosome
Plasmids and bacteriophages definition:
Plasmid: circular, extrachromosomal elements
Bacteriophage: bacterial viruses, integrated or autonomous
Plasmids:
Replication:
Size:
Quantity:
Episome:
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 (5):
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)
Episomes/Resistance Factors:
- R Factors:
R Factors: conjugative episomes that encode antibiotic resistance
R Factors
Composed of 2 Subunits:
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
- Transposons mediate the formation/resolution of R factors
Transposons (Tn)
Definition:
Composition
Definition: a sequence of DNA that can “hop” from place to place. An insertion sequence that has assimilated a drug-resistance gene.
Composition: antibiotic resistance gene flanked by insertion sequences, which encode for transposon mobility and allow for entry into host genome
Transposons (Tn)
Function:
Complex Transposons:
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
Transposons (Tn)
Example of Resistance:
Enterobacteriaceae have transferred ampicillin resistance to Haemophilus influenza and Neisseri gonorrhoeae
This concept of transferred resistance is the rationale behind using combinations of unrelated antibiotics
Bacteriophage definition:
Two types:
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
Examples of toxin/virulence factor genes that are carried in phage genomes:
How can bacteriophages be used as therapy?
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
- Phage specific for a bacterial species can be isolated in a few days (very quick)
- They are very specific for their host bacterial species (protect normal flora)
- No effect on eukaryotic cells
Gene transfer restriction/modification:
A. Bacterial “immune system”
B. Defends against foreign DNA
C. Modification is the species-specific methylation of certain DNA sequences
D. Restriction is cleavage of unmethylated DNA at the same sequences by restriction enzymes
- Properly modified DNA is protected from cleavage by restriction enzyme
Transformation
Basics:
Competence definition:
Transformation is sensitive to what?
- Transformation:
- Basics: uptake of DNA from extracellular milieu (species-specific, sequence specific or non-specific)
Naked DNA adsorbs to bacteria and enters cytoplasm
Competence: ability to accept DNA; mechanisms vary among bacteria
Transformation is DNase sensitive
Transformation
Incoming DNA must recombine with host chromosome using:
Entry vs incorporation:
Incoming DNA subject to:
What must the incoming DNA have for RecA to function?
Incoming DNA must recombine with host chromosome using RecA enzyme
Any DNA may gain entry, however this 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
Transduction definition:
Transfer of genetic information by bacteriophage (phage)
Phage can be lytic (produce more phage, kill host cell) or lysogenic (integrate into host chromosome, do not kill host cell)
Generalized Transduction:
What is a pseudovirion?
Transferred DNA must:
Generalized Transduction: indiscriminate transfer of chromosomal sequences
Phage “accidentally” packages host sequences in pseudovirion (new phages made that have some host DNA)
Transferred DNA must integrate into recipient chromosome (RecA)
Specialized Transduction:
Can only occur via:
Specialized Transduction: transfer of specific chromosomal sequences
Can only occur through lysogenic phages
Specialized Transduction
Steps:
Bacteriophage specifically integrates into host chromosome (to form prophage)
a) Integrated prophage is lysogenic
b) Integration is site-specific & reversible
DNA damage induces excision of the bacteriophage
a) Pieces of chromosome pulled out with phage
b) Chromosome + phage DNA transferred to next host
Virulence Factors Controlled by Lysogenic (Specialized) Conversion:
Corynebacterium diphtheria SPE A; S.pyogenes Enterohemorrhagic E.coli Clostridium botulinum Vibrio cholera
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)
Conjugation:
Basics:
Sex in bacteria; DNA transfer by cell –cell contact (can occur with both Gram (+) and Gram (-) bacteria)
Simple conjugation:
F copy number as a plasmid:
Hfr:
F has very low copy number as a plasmid
F can recombine onto the bacterial chromosome
-“Hfr” can then transfer whole chromosome
F has replication origins for:
What are tra elements required for?
F episome is transferred from:
When does F episome replicate?
F has replication origins for dsDNA (Plasmid) and ssDNA (for transfer)
Plasmid encodes tra elements required for episomal transfer
-Pili, replication enzymes
F episome is transferred from an F+ to F- only
F episome replicates upon transfer
Merozygote formation
- Partial diploid or merozygote: Recipient carries 2 copies of transferred genes
- Incoming gene may integrate into chromosome
Specialized episome required for conjugation
Plasmid:
Episome:
Specialized episome required for conjugation
- Plasmid: Extrachromosomal, autonomously replicating DNA
- Episome: Autonomous or integrated plasmid
F episome:
Encodes (3):
Conjugative episome carried by E. coli encoding:
- 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
Gene expression in prokaryotes may be regulated by (3):
What is the most common mechanism of regulation?
o Transcriptional control (regulation of mRNA production; most common)
o Translational control
o Post-translational control
Regulation of transcription
Operon Definition:
Consists of:
Functional transcription unit
It consists of a:
- Promoter
- A single gene (mono-cistronic) or series of genes (poly-cistronic) that is/are transcribed into one mRNA, and may include
- Regulatory elements
Promoter:
Promoter: a type of cis-acting regulatory region; DNA sequence recognized by RNA polymerase sigma factor
Regulatory Sequences:
Trans-acting vs cis-acting
3 types of cis-acting regulatory regions:
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:
Structural genes
• Regulation of the lac Operon:
General:
o Example of negative and positive regulation
Structural Genes:
o B-galactosidase (lacZ)
o Galactoside permease (lacY)
o Galactoside acetylase (lacA)
lac Operon:
If glucose is absent, what happens to cAMP?
cAMP increases
lac Operon:
Regulatory Sequences:
Promoter:
Operator:
Repressor:
Inducer:
Promoter: cis-actng
Operator (lacO): cis-acting
Repressor (lacI): trans-acting
- Binds operator and blocks transcription from promoter
Inducer (allolactose): inactivates repressor (when lactose is present), allowing transcription to occur
- IPTG is an artificial inducer
lac Operon:
Role of cAMP:
cAMP in comparison with glucose levels:
cAMP binding protein (CRP/CAP):
Basics:
Glucose is the favored carbon source, but will use lactose when glucose levels are low
cAMP levels increase as glucose levels decrease
cAMP binding protein (CRP/CAP) is a DNA binding protein and positive regulator
Basics: glucose decreases, cAMP levels increase, bind CRP, which binds DNA and activates transcription
lac Operon
Glucose, no lactose:
No glucose, no lactose:
Glucose, lactose:
No glucose, lactose:
Glucose, no lactose: repressor bound, CRP not bound –> very low transcription (never 0!!)
No glucose, no lactose: repressor bound, CRP bound –> low transcription
Glucose, lactose: repressor not bound, CRP not bound –> moderate transcription
No glucose, lactose: repressor not bound, CRP bound –> high transcription
lac Operon
Catabolite repression overrides other regulatory systems
- Highest levels of transcription require catabolite activator protein (CAP) + cAMP
- CAP {a.k.a. cAMP Receptor Protein (CRP)} is a DNA-binding protein
- CAP is a positive regulator- it activates transcription when [cAMP] is high
- High levels of glucose decrease [cAMP], lac operon has low transcription
- Low glucose permits CAP activation of the lac operon
- Catabolite repression system constitutes a Regulon (separate operons)
Other Types of Regulation:
Transcription Level:
o Purely negative (repressor)
o Purely positive (activator)
o Negative and positive (like the lac operon)
Other Types of Regulation:
Translation Level:
Usually negative control
- Prevent binding of ribosome to mRNA
- Make mRNA unstable (sRNA, RNases)
Other Types of Regulation:
Post-Translation:
Protein level (proteolysis)
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)
Antigenic phase variation in Salmonella enteritidis:
Flagella type switch due to:
Inversion process controlled by:
Promoter orientation governs expression of:
Antigenic phase variation in Salmonella enteritidis
- Flagella type switch due to inversion of promoter region
- Inversion process controlled by Hin protein (invertase) - Site-specific recombination
- Promoter orientation governs expression of rH1 and H2
Signal Transduction:
Basics:
Details:
Basics: allows for global regulation of virulence factors
Details:
Transmembrane sensor response to environmental conditions
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
Signal Transduction examples (2):
Vibrio cholera: cholera toxin and pilus production (regulation by cascade of transcription factors)
Bordetella pertussis: pertussis toxin production (regulation by signal transduction- phosphorelay)
- BvgS becomes phosphorylated in the body at body temperature
- Transfers phosphate to BvgA, which determines virulence gene expression
Pathogenicity Islands:
Basics:
Basics: stretches of chromosome that encode virulence attributes (usually have a higher A/T content than the rest of the genome)
- Clustered genes for adhesins and toxins
- Operons with common function
Pathogenicity Islands with Repetitive Terminal Sequences Indicating Transposition:
What can the acquisition of a pathogenicity island do?
What can removal of pathogenicity islands from chromosome do?
Acquisition of pathogenicity island can render harmless bacteria pathogenic
Removal of pathogenicity islands from chromosome often eliminated virulence (potentially used in vaccine production)
Examples of Pathogenicity Islands (2):
Diarrheagenic E.coli: clustered loci for adhesions and toxins
Vibrio Pathogenicity Island: TCP and regulatory proteins encoded here
