Bacterial Genetics

Question 1

Differences between bacteria and Eukaryotes Chromosomes:

• Compartmentalization:
• Plasmids:
• DNA:

Answer 1

Chromosomes: Bacteria have single chromosome that is circular < 5mb; Several to many linear chromosomes > 5mb

• Compart: Bacterial genome not compartmentalized
• Plasmids: Eukaryotes have no plasmids
• DNA: Chormosome condensed by supercoiling in bacteria; in Eukaryotes wrap around histones

Question 2

Mechanism of replication in bacteria

Answer 2

Single origin, second round of replication can start before cell divides

Replicated DNA partitioned into daughter cell - binary fission

Question 3

Differences in transcription/translation

Answer 3

Bacteria - Genes encoded with operons; no introns or exons; 1 RNA polymerase

Eukaryotes - No operons; introns and exons; 3 RNA polymerase

* in bacteria, transcription/translation coupled since no compartmentalization

Question 4

How do bacteria generate diversity

Answer 4

1. Replication errors - introduce mutations into gene
2. Exchanging genetic material

Question 5

How do replication errors occur in bacteria? What is the frequency?

Answer 5

• DNA polymerase misincorporates nucleotide and does not correct this mistake through proofreading activity
• Frequency is 1 mutation/300 chromosome replications = 10^(-6) mutations per genome per generation

Question 6

Horizontal Gene transfer (vs. vertical transmission in Eukaryotes)

Answer 6

There is no natural species boundary in prokaryotes like there is for Eukaryotes

• Bacteria can acquire genetic material from another organism

Question 7

Why/How does bacteria perform gene exchange?

Answer 7

• The species barrier in bacteria is less stringent than in Eukaryotes
• Only need a single organism in the population to survive
• Help outcompete or kill other organisms

Question 8

Exchangeable genetic elements

Answer 8

1. Plasmids
2. Insertion sequences/Transposons
3. Pathogenicity islands

Question 9

Of the exchangeable genetic elements, most concern involves genes encoding _____ ______ and _____ _______ ______

Answer 9

• Virulence factors
• Antibiotic resistance determinants

Question 10

Methods of exchange

Answer 10

1. Transformation
2. Transduction
3. Conjugation

Question 11

Plasmids

Size:

Exchange:

Answer 11

DNA molecules that replicate independently of bacterial chromosomes

Size: from 1500bp to 400,000bp

Exchange: transformation, conjugation, transductoin

Question 12

Single copy plasmids

Answer 12

F plasmids or episomes

Question 13

Insertion sequences/ transposons

Answer 13

• Mobile genetic elements
• linear DNA segment - often disrupt genes in recipient bacterium following transfer
• Cannot replicate on their own - often transferred on plasmid or by bacteriophage

Question 14

ITR

Answer 14

Inverted terminal repeats at the ends of insertion sequences and simple transposons

Question 15

Transposase

Answer 15

Enzyme that recognizes ITR and cuts the DNA allowing transposition of element from one location to another

Question 16

Difference between insertion sequences and composite transposons

Answer 16

Insertion sequence: Sequence with inverted repeats on each side

Composite transposons: Insertion sequence on each end with central region: ex. genes for antibiotic resistance - all transposed together

Question 17

Phase variation

Answer 17

variation of protein expression, frequently in an on-off fashion, within different parts of a bacterial population

Question 18

Phase variation in Escherichia coli that are responsible for UTIs (UPEC)

Answer 18

• Associated with site-specific inversion of an insertion sequence
• Spontaneous switch between non-fimbriated and fimbriated forms
• Fimbriae promote attachment to urinary tract epithelial cell surface

Question 19

Genomic pathogenicity islands

Answer 19

Segment of bacterial genome = carried on plasmid or by bacteriophage

Question 20

Distinguishing Genomic Pathogenicity Islands

Answer 20

• G+C content differs from majority of chromosome
• Can encode adherence factors, invasion genes, toxins etc.
• Vary in size from 10,000bp to 200,000bp
• Cannot replicate by iteself

Question 21

Transformation:

Conjugation:

Transduction:

Answer 21

Transformation: DNA taken up from environment

Conjugation: Direct cell-to-cell contact

Transduction: Mediated by bacteriophage

Question 22

Bacterial transformation

Answer 22

• Uptake of DNA from environment
• Occurs in gram + and gram - bateria
• DNA released following lysis of bacteria
• dsDNA bound then processed to ssDNA before internalization
• Homologous recombination

Question 23

Conjugation

Answer 23

• DNA passed unidirectionally from Donor to Recipient
• Requires physical contact
• Both gram + and gram -
• DNA transferred through mating bridge
• Transfers plasmids, transposons, and chromosomal DNA
• Donor retains copy of original genetic material

Question 24

Mechanism of conjugation

Answer 24

1. Generation of pilus
2. Pilus retraction to form mating bridge

Question 25

Transduction-transfer of DNA via bacteriophage (types of bacteriophage)

Answer 25

* Bacteriophages are obligate intracellular parasites * 3 main types: icosahedral head; icosahedral head with tail; filamentous * Single nucleic acid molecule * Nucleic acid protected by protein coat or capsid

Question 26

Lytic vs Lysogenic lifecycle

Answer 26

Lytic: Make new progeny phage = phage replicated independently of host genome Lysogenic: Vertical transmission of phage = phage genome integrated and replicated with bacterial genome \* temperate phage can switch between these lifecycles

Question 27

Mechanism of transduction

Answer 27

* During lytic replication, fragment of bacterial genome packaged into phage particle rather than phage genome = transducing phage * Upon infection of new bacterium, transducing phage releases fragment of bacterial genome into host * Recombination between introduced DNA and host DNA result in stable inheritance

Question 28

Cholera toxin phage (Ctx)

Answer 28

* Filamentous bacteriophage that infects *Vibrio cholera* * Comprised of core element and repetitive sequence * Ctx is 5B to 1a (5 binding subunits with 1 enzymatic subunit) * Phage morphogenesis and enterotoxic activity

Question 29

Mechanism of cholera toxin activity

Answer 29

* A-5B toxin → B subunit binds ganglioside GM1 * A subunit internalized and interacts with G proteins regulating adenylate cyclase * Induces conversion of ATP to cAMP which results in enhanced secretion of water and electrolytes (diarhhea)

Question 30

Shiga Toxin phage (Stx)

Answer 30

* Icosahedral head with short tail * Temperate bacteriophage: lytic and lysogenic * Shiga toxin is made by both Shigella dysenteriae and EHEC strains of E. coli * Induces severe diarrhea, hemorrhagic colitis, and hemolytic-uremic syndrome (HUS)

Question 31

Mechanisms of Shiga Toxin

Answer 31

* A-5B toxin * Mechanism identical between Shiga- and Shiga-like toxin * B subunit binds Gb3 glycolipid * A subunit is translocated into cytosol and modifies ribosome acceptor site * Blocks protein synthesis