Bacterial Genetics Flashcards
How large is the typical gene in prokaryotes, and how large is the genome? Does it have introns?
Typical gene is about 1000 bases (333 amino acids).
Genome is 4000kb, meaning about 4k genes.
Haploid chromosomes with no introns or histones
What is a plasmid vs an episome? What is the significance of these?
Plasmid - autonomously replicating, circular DNA
Episome - plasmid that can integrate into chromosome, some of which can encode for conjugation “transfer genes”
Many virulence factors are plasmid-encoded
What is an R factor and their two subunits?
R(esistance) factor is a conjugative episome which encodes for Abx resistance
- RTF - resistance transfer factor, coding autonomous replication and conjugal transfer
- Resistance determinant - composed of 1+ transposons, which carries the antibiotic resistance gene
What is a complex transposon?
More than one insertion sequence (including transposase gene) flanking another gene, often including antibiotic resistance (in the case of R factor).
This is able to hop into chromosome and overcome the host restriction barrier
What is a lytic vs temperate phage?
Lytic - infects and reproduces via bacterial lysis
Temperate = lysogenic, can integrate into chromosome and only become lytic during stress. Includes many toxin genes
What is phage therapy?
Infection of bacterial pathogens which may be effective for those who have antibiotic resistance
What is the bacterial innate immune system?
Restriction / Modification - breaks probable viral (unmethylated at certain spots) DNA via enzymes. Methylation is species-specific, and sterically blocks digestion via restriction enzymes
What are the three bacterial gene transfer methods?
- Transformation
- Transduction
- Conjugation
What is “competence” in transformation?
Ability of bacteria to accept DNA, including naked DNA.
Not all external DNA is accepted via bacteria, and many species have specific sequence requirements to accept foreign DNA. It will also be subject to restriction enzyme degradation
What enzyme mediates integration of external DNA into host chromosome during transformation?
RecA, which requires some sequence homology for DNA to be integrated. Otherwise, the DNA will be destroyed.
What is generalized transduction?
As phage is packaging during bacterial infection, it “accidentally” packages host DNA into pseudovirion particle.
DNA integrates into recipient chromosome via RecA following infection via pseudophage
“head full” mechanism
Does generalized transduction happen via lytic or lysogenic phages? Specialized?
Generalized: Can happen via both types
Specialized: Only lysogenic phages (prophage)
What is specialized transduction?
Transfer of specific chromosomal sequences via prophage (i.e. lambda phage) exiting out of lysogenic state and carrying some of the host chromosome with it. This reintegrates into new recipient genome upon infection of the phage
What is lysogenic conversion, and what are some examples?
Change in bacterial phenotype after phage infection (bacterial virulence factors carried by specialized transduction)
Examples:
Diphtheria toxin, botulinum toxin, cholera toxin, exotoxin A (S. pyogenes), Shigatoxin
What is conjugation and what type of bacteria does it occur in?
DNA transfer via cell-cell contact (sex)
Occurs in gram positive or gram negative
What is required for conjugation and what is the E. coli example?
Specialized episome, including DNA encoding for sex pilus
E coli - F plasmid - F+ always transfers to F- via rolling replication. F+ is resistant to conjugation via another F+.
What is an operon?
Functional transcriptional unit, includes promoter and associated genes (can be mono or poly-cistronic).
All genes are coordinated regulated in case of polycistronic
What is a cis vs trans-acting regulatory sequence?
Trans (away) - encode regulatory proteins which diffuse to site
Cis - DNA binding sites which bind the regulatory proteins
What are three types of cis-acting regulatory regions?
- Promoter - DNA sequence recognized via RNA polymerase
- Operator - near promotor, binds repressor protein and reduces transcription when bound (i.e. lac repressor)
- Activator - near promotor, binds activator protein to increase transcription (i.e. CRP protein)
What is an attenuator?
mRNA secondary structure which modulates transcription (i.e. hairpin loop)
What are the 5 genes associated with the lac operon?
Structural
- LacZ - betagalactosidase
- LacY - galactoside permease
- LacA - galactoside acetylase
Regulatory
- LacI - codes repressor
- LacO - operator site which binds the lac repressor
What is the inducer of the lac operon?
Allolactose (indicates lactose presence), binds lac repressor to inactivate it, increasing transcription
What is CRP’s function in lac operon?
CRP binds cAMP and binds the activator sequence to increase transcription when glucose is low
When will you have low vs medium vs high levels of transcription
Low - glucose low but no lactose present (repressor bound, activator bound)
Medium - glucose high but lactose is present (repressor not bound, activator not bound)
High - glucose low and lactose present (repressor not bound, activator bound)
What are the levels of transcriptional regulation?
- Positive only - use of activator
- Negative only - use of repressor
- Combination - i.e. lac operon, uses both activator and repressor
What are some methods of translation regulation?
Usually negative control, prevent bindong of ribosome to mRNA (mRNA looping or silencing RNA), can also make RNA unstable via digestion via RNases
How can post-translational regulation be done?
Protein lysis or reduction of protein activity via small molecules / proteins
Why is virulence factor regulation highly regulated?
Virulence factors are costly and often only advantageous in specific host environments
How is flagella type expression controlled in Salmonella enteritidis (pathogen of raw eggs)?
Antigen variation via inversion of promoter direction, changing phase 1 to phase 2 flagella.
Process is dictated via Hin protein (H invertase) which does site-specific inversion / recombination
What is the general principle underlying virulence factor production in Vibrio cholerae?
Transmembrane sensor responds to environmental conditions, and signal is transduced via protein kinase
Cytoplasmic sensory is a DNA binding protein which can modify transcription of a gene
These genes use multiple sensors to respond to environmental cues (quorum sensing, presence of bile salts indicating entrance into GI tract, sensing of bicarbonate in intestinal lumen) to produce virulence factors
What is the mechanism of Pertussis toxin production activation in Bordetella pertussis?
BvgS - transmembrane sensory kinase, recognizes a change in temperature to 37 degrees Celsius (indicates entrance into human body)
BvgA - virulence Activator - amount phosphorylated by BvgS determines activity. Relative concentration determines if Pertussis toxin is synthesized
What is a pathogenicity island? Why are they clinically important?
Stretch of chromosome which encodes for virulence attributes, especially a number of operons with a common function helping bacterial survival in a host.
often code for adhesins and toxins
These sections can be removed from chromosome to eliminate virulence, so strains without them can be used in potential vaccines
What is a regulon?
Series of operons under control via one transcription factor
What DNA patterns do pathogenicity islands often contain?
Repetitive terminal sequences - indicating transposition
Also have higher AT concentration than normal, indicating it is foreign DNA (some bacteria can regulate these abnormal genes and prevent transcription)
