OBJ - Gene Transfer Mechanism

Question 1

DNA recombination

Answer 1

Bacterium’s goal is to become 2 bacteria
-growth & reproduction

Only part of genome is transferred

If transferred DNA is not a replicon, it
requires recombination to be stably
inherited in recipient

Recombination – replacement of one
allele/piece of DNA with another

Physical breakage and rejoining of two
DNA molecules to form hybrid recombinant molecules

Enzymes mediate cutting/splicing/rejoining ends
i.e. endonucleases, exonucleases,
single-stranded DNA binding proteins,
DNA polymerases, and DNA ligases

Question 2

Transformation

Answer 2

-Simplest - think phagocytosis

• Uptake of naked DNA from lysis of other bacteria in neighborhood
• Fragments of DNA from medium & integrated into genome

Process is sensitive to DNAse and requires:
– Competence, i.e. the ability to take up DNA (not all bacteria are able to)
– required to be DOUBLE-stranded DNA substrate
– Recombination

• Induced in response to environmental conditions such as altered growth conditions, nutrient limitation, cell density or starvation

• Co-transformation of two genes implies linkage on the genetic map (close to each other in genome)

Question 3

Conjugation

Answer 3

Cell-to-cell genetic exchange via sex pilus

• Highly specific process
• Resistant to DNase
• Requires cell-to-cell contact
• Requires F Plasmid (for sex pilus)
    dsDNA with F pilus genes
• Integration into host chromosome to form 
Hfr (high frequency recombination)

1. Mating pair formation – pilus contact
2. Pilus retraction
3. DNA transfer
   a. Relaxase generates single strand nick at oriT
   b. Strand displacement and transfer into recipient; donor DNA replication
4. Strand invasion in recipient
5. Recombination

Question 4

Transduction

Answer 4

Mediated by bacteriophage

Types

• Generalized – error in DNA packaging (not host’s DNA)
• Specialized – aberrant excision of prophage

Question 5

Plasmids

Answer 5

Genetic loose change; mobile genetic elements that aren’t bound to chromosomes & add diversity

Replicon – autonomous replication
Non-essential but provide benefits to host
– Novel metabolic pathways
– Pathogenicity factors
           Toxins
           Adherence factors
           Antibiotic resistance

Question 6

Transposons (Tn)

Answer 6

“Jumping Genes”

CUT & paste with DNA (not conserved) or can be COPY & paste with DNA (conserved)

Does not require homology between donor site and recipient site

• Transposases = Requires specific enzymes
• Independent of the RecA protein

• Larger than IS (insertion sequence) elements - a bunch linked together
• Contain direct or inverted terminal repeats; some repeats are IS elements
• Encode transposase
• Recognizable phenotype unrelated to
transposition - drug resistant

Question 7

Horizontal gene transfer & Microbial Evolution

Answer 7

Bacteriophages that accidentally up take random fragments of host chromosomal instead of bacterial DNA are packaged into phage heads

Question 8

Replicon

Answer 8

DNA molecule or RNA molecule, or a region of DNA or RNA, that replicates from a single origin of replication

Question 9

Types of Recombination

Answer 9

Homologous

• Double stranded break
• Reciprocal exchange between donor and recipient DNA that are homologous in nucleotide sequences
• need similar/homologous sequences
• *Mediated by RecA-dependent

Site Specific
- Breakage and rejoining at specific sites of the donor and recipient DNA molecules

**Mediated by integrases - recognize those specific ends of DNA

Transposition
– one site-specific substrate (transposon ends) and one non-site specific substrate (target DNA); jumping gene

**Mediated by transposases

Question 10

Bacteriophages

Answer 10

Medically important:
• Lysogenic conversion (Bacteria acquiring new properties - MRSA)
• Detection as a diagnostic tool (being developed)
• Antimicrobial therapy
• Vaccine vectors

Properties of Bacteriophages:
• Small (filterable)
• Nucleic acid
       – ssDNA, dsDNA, ssRNA, dsRNA 
       – Unusual nucleic acids (hydroxymethyl C)   
          that act protectively
• Phage coat can be protein or membrane
• Contain no ribosomes or enzyme systems 
for protein synthesis
• Replicate by synthesis of separate 
components followed by assembly into 
mature phage particles
• No division by binary fission

Plaque Assay - black wholes in agar/bacteria are the bacteriophage

Question 11

Lytic & Lysogenic Cycles

Answer 11

LYTIC - infect reproduce & lyse
Adsorption:
Host range specificity - determined by phage attachment protein & receptor protein

Resistance = Absence of phage receptor

Assembly
• Assembly of phage components into
infectious particles

Release of phage progeny
– Lysis of host bacterium by lytic enzymes = Lysin, lysozyme
– Extrusion - Release through bacterium without cell lysis

LYSOGENIC
Lysogenic - hybernate & integrate DNA (chromosomal + bacterial)
-> then are induced to return to lytic cycle

The Prophage State

• Integrated in bacterial genome at a Fixed site or random sites
• Autonomously replicating plasmid

Repression
– Shutoff of phage genes involved in replication
– Only phage repressor gene is transcribed
– Repressor is a diffusible, cytoplasmic protein

Immunity
– Lysogens are “immune” to infection by “like” phages
– Repressor binds to operator sites on incoming phage and blocks initiation of lytic cycle
-represses phage itself & other phages from invading

Continuous expression of repressor protein

Lambda Lysogen -> switch to Lytic cycle