Withey: Bacterial Genetics Flashcards Preview

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Flashcards in Withey: Bacterial Genetics Deck (46):
1

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

2

Plasmids and bacteriophages definition:

Plasmid: circular, extrachromosomal elements

Bacteriophage: bacterial viruses, integrated or autonomous

3

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

4

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)

5

Episomes/Resistance Factors:
- R Factors:

R Factors: conjugative episomes that encode antibiotic resistance

6

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

7

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

8

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

9

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

10

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

11

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

12

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

13

Transformation
Basics:
Competence definition:
Transformation is sensitive to what?

1. 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

14

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

15

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)

16

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)

17

Specialized Transduction:

Can only occur via:

Specialized Transduction: transfer of specific chromosomal sequences

Can only occur through lysogenic phages

18

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

19

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)

20

Conjugation:
Basics:

Sex in bacteria; DNA transfer by cell –cell contact (can occur with both Gram (+) and Gram (-) bacteria)

21

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

22

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

23

Merozygote formation

1. Partial diploid or merozygote: Recipient carries 2 copies of transferred genes
2. Incoming gene may integrate into chromosome

24

Specialized episome required for conjugation
Plasmid:
Episome:

Specialized episome required for conjugation
1. Plasmid: Extrachromosomal, autonomously replicating DNA
2. Episome: Autonomous or integrated plasmid

25

F episome:

Encodes (3):

Conjugative episome carried by E. coli encoding:

1. Sex pili for cell-cell contact and cytoplasmic fusion
2. Conjugative transfer and the repression of transfer
3. Surface exclusion that prevents F+ from being a recipient

26

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

27

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

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Promoter:

Promoter: a type of cis-acting regulatory region; DNA sequence recognized by RNA polymerase sigma factor

29

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

30

Regulon

A set of operons regulated by the same transcription factor

31

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)

32

lac Operon:
If glucose is absent, what happens to cAMP?

cAMP increases

33

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

34

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

35

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

36

lac Operon

Catabolite repression overrides other regulatory systems

1. Highest levels of transcription require catabolite activator protein (CAP) + cAMP
2. CAP {a.k.a. cAMP Receptor Protein (CRP)} is a DNA-binding protein
3. CAP is a positive regulator- it activates transcription when [cAMP] is high
4. High levels of glucose decrease [cAMP], lac operon has low transcription
5. Low glucose permits CAP activation of the lac operon
6. Catabolite repression system constitutes a Regulon (separate operons)

37

Other Types of Regulation:

Transcription Level:

o Purely negative (repressor)
o Purely positive (activator)
o Negative and positive (like the lac operon)

38

Other Types of Regulation:

Translation Level:

Usually negative control
- Prevent binding of ribosome to mRNA
- Make mRNA unstable (sRNA, RNases)

39

Other Types of Regulation:

Post-Translation:

Protein level (proteolysis)

Protein activity (binding of small molecules or other proteins)

40

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)

41

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
1. Flagella type switch due to inversion of promoter region
2. Inversion process controlled by Hin protein (invertase) - Site-specific recombination
3. Promoter orientation governs expression of rH1 and H2

42

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

43

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

44

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

45

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)

46

Examples of Pathogenicity Islands (2):

Diarrheagenic E.coli: clustered loci for adhesions and toxins

Vibrio Pathogenicity Island: TCP and regulatory proteins encoded here