Microbial Genetics

Question 1

Bacterial Chromosome

Answer 1

• Single, large circular DNA (~1 to 6 megabases)

- Contains all genes for survival

Question 2

Plasmid

Answer 2

• Small, circular DNA (~1.5 to 400 kilobases)
• Separate from bacterial chromosome
• Replicated and transferred to daughter cells
• Copy number varies from 1 to 100

Question 3

What is an episome?

Answer 3

A plasmid that has been integrated into the bacterial chromosome

Question 4

Importance of Horizontal Gene Transfer

Answer 4

Allows bacteria to develop abx resistance and produce new virulence factors more quickly compared to Vertical Gene Transfer and natural slow rate of mutations

Question 5

Homologous Recombination

Answer 5

Exchange of DNA between two DNA molecules based on homology (sequence similarity) resulting in the incorporated DNA into the chromosome being passed on to daughter cells and becoming a permanent part of the genome

***Gene MUST be incorporated into the host plasmid/DNA in order to be passed on

Question 6

Transformation (Definition and Process)

Answer 6

1. Uptake of free DNA FROM THE ENVIRONMENT
2. Process: DNA is released into environment (e.g. bacterial death), DNA is actively imported into the bacterial cell, homologous can occur if there is sequence similarity, donor DNA is passed to progeny

Question 7

Conjugation (Definition and Process)

Answer 7

1. Direct transfer of DNA from one bacterial cell to another via CELL CONTACT
2. Process:

Donor: carries Fertility Factor (F-Factor) which contains the tra operon (encodes sex pilus); carries oriT, where a single strand break occurs for transfer of DNA

Either Chromosomal DNA can be transferred (requires homologous recombination in order to be passed on to progeny) OR Plasmid DNA can be transferred

Question 8

Donors vs Recipients

Answer 8

F+ and Hfr (High frequency recombination; F factor integrated into bacterial chromosome) can both act as donors and mate with F- (No F factor)

Question 9

Conjugation: F+ x F-

Answer 9

• Transfer is unidirectional
• F- undergoes sex change to F+
• Only one strand is transferred
• No bacterial genes are transferred
• tra region comes in last and then replication machinery forms second strand of DNA

Question 10

Conjugation: Hfr x F-

Answer 10

• Hfr donates to F-
• Mating does not generally last long enough for tra gene to move in (so, F- DOES NOT undergo sex change)
• F- receives new bacteria genes

Question 11

Transduction (Definition and Phage Life Cycles)

Answer 11

1. Transfer of bacterial DNA via a bacteriophage (virus)
2. Phage Life cycles:
   - Lytic/Virulent phage –> lytic replication upon entry
   - Temperate phage –> LYSOGENY (integrated into chromsome); can excise and convert to lytic replication when bacteria is dying

Question 12

Generalized Transduction

Answer 12

• A lytic phage incorporates bacterial chromsomal DNA into its phage head
• When phage infects another bacteria, the DNA can be incorporated into its chromosome via homologous recombination or can be degraded and lost

Question 13

Specialized Transduction

Answer 13

• A lysogenic phage incorporates into the chromosome as a PROPHAGE
• Event (e.g. UV damage) leads to conversion to lytic cycle
• Upon excision from the genome, small portion of chromosome is packaged into phage head

***Cause many diseases including cholera, botulism, diphtheria, shigellosis, and hemorrhagic colitis

Question 14

What is the only type of DNA transfer in bacteria that uses FREE DNA?

Answer 14

Transformation

Question 15

Type of DNA in Generalized vs Specialized Transduction

Answer 15

Generalized: Any DNA

Specialized: Only genes adjacent to phage insertion

Question 16

Is homologous recombination required for: Transformation? Conjugation? Transduction?

Answer 16

Transformation: Yes

Conjugation: Hfr x F - (Yes) / F+ x F- (NO)

Transduction: Yes

Question 17

Why is homologous recombination not needed for F+ x F-?

Answer 17

Because the complete plasmid is transferred in this type, whereas, in Hfr type, only part of the plasmid is incorporated into the F plasmid and transferred (this is why there is no sex change with Hfr x F-)

Question 18

Intrinsic Antibiotic Resistance

Answer 18

• NOT transferrable (horizontally) between bacteria
• NOT increasing among bacterial populations
• Example: lack of antibiotic target (e.g. Mycoplasma lack cell wall/peptidoglycan)

Question 19

Chromosome-Mediated Antibiotic Resistance

Answer 19

Arises through 1) Random Mutation and 2) Acquisition of DNA through horizontal gene transfer and incorporation into the genome via homologous recombination

e.g. Ribosomal proteins, Penicillin-binding proteins, DNA gyrase

Question 20

Plasmid-Mediated Antibiotic Resistance

Answer 20

Genes encoding antibiotic resistance located on a plasmid

Commonly, plasmids carry genes that encode proteins that degrade or modify an abx or an efflux pump to pump the abx out of the cell

Question 21

Resistance Factor (R-Factor): Two components

Answer 21

1. Resistance Tranfer Factor (equivalent to F-factor, as it encodes all proteins needed for conjugation)
2. Resistance Determinant (gene(s) for drug resistance)

Question 22

Transposons

Answer 22

• Mobile DNA elements that transfer or copy themselves from one molecule of DNA to another
• Present in both Eukaryotic and Bacterial cells, as well as Viruses

Question 23

What does the simplest Transposon contain? (3 things)

Answer 23

1. Indirect repeat sequences on each end (recognized by transpoase, TnpA)
2. A single gene for transposase, TnpA, an enzyme required for transposon movement (i.e. “Jumping”)
3. Single or multiple antibiotic resistance genes

Question 24

Integrons and Antibiotic Resistance Gene Cassettes

Answer 24

Integrons are a diverse group of genetic elements which encode SITE SPECIFIC RECOMBINATION SYSTEMS that can capture antibiotic resistance gene cassettes and express them under the control of an integron promoter

-Often found with large mobile genetic elements like PLASMIDS or TRANSPOSONS, as well as on the CHROMOSOME

***Play important role in MULTI DRUG RESISTANCE BACTERIA

Question 25

Pathogenicity Island

Answer 25

Basically, a very large transposon

• Can transfer itself into a bacterial chromosome via horizontal gene transfer
• Often carry coordinately regulated VIRULENCE GENES surrounded by insertion sequences