Chapter 9. Genetic Recombination II S. Gbadamosi

 

9.1 Bacterial Conjugation

Lederberg and Tatum defined conjugation as a process in which transfer of genetic material from one bacterial strain to another is dependent upon cellular contact between members of the two bacterial strains. 

9.1.1 The conjugation process

1. The principal mediator of the conjugal process is a plasmid. Plasmids are DNA molecules that replicate separately from the bacterial chromosome. Some are conjugative, i.e., able to effect their own transfer from one cell to another. Plasmids may autonomously replicate as an extrachromosomal element or become integrated with the bacterial chromosome.

2. Kinds of Plasmids:

F. factor - (Fertility factor) governs the inherited property of maleness in bacteria. Conjugation is a mechanism imposed on the bacteria cell by a plasmid. The transfer of the bacterial chromosome is a secondary consequence.

1. Cells harboring the F factor are called F+; cells lacking the fertility factor are called F- F+ x F- matings are the most fertile; F- x F- are infertile.

2. In a population of F+ cells, the integration of a F plasmid with the bacterial chromosome produces a Hfr (high frequency of recombination) cell. Ent factor - plasmid with coded information for enterotoxin. K88 factor - plasmid controlling surface capsular antigen synthesis Example: In an experimental study, a correlation was established between the severity of diarrhea in piglets and the presence of Ent and K88 plasmids.

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Enterotoxin Capsular Markers

Marker K88- K88+

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Ent- no effect Mild diarrhea

Ent+ no effect severe diarrhea 

Col factor - colicin control 

Hly factor - alpha hemolysin factor 

R factor - resistance to antibiotics 

SUMMARY

May be capable of self-transmission (conjugative) or incapable (nonconjugative). In general, plasmids in Gram + organisms are nonconjugative. This may be a reflection of a host property since upon introduction into G-bacteria can be conjugative. R factors of Gram + plasmids can be maintained and expressed as resistant characters in Gram - organisms. Plasmids vary in size from approximately 106 to 200 X 106 daltons of DNA. Many resistant plasmids are unstable and may be rapidly lost by most members of a bacterial population in the absence of antibiotics In a sense, plasmids are gratuitous in that they enhance the survival of the bacterial host under adverse conditions and may be neutral in a stable environment.

 

9.1.2 Stages in Bacterial Conjugation:

• 1. Effective Pair Formation: a process by which a specific bacterial pair establishes a cellular connection between donor (D) and recipient (R) such that the passage of genetic material can commence. This formation is mediated by F plasmid.
• 2. Preparation of the circular D chromosome for linear sequential transfer.
• 3. Polymerization and sequential synthesis of a complimentary DNA strand in the R cell.
• 4. Some requirement for homologous pairing of the new chromosome with a comparable portion of the old chromosome.
• 5. The F cell must pull in the D chromosome while continuing to replicate chromosomes to ensure its own genetic integrity.

   

  9.1.3 Recombinant DNA Technology

• 1. Recombinant DNA techniques utilize the study of gene structure through microbial genetics and DNA biochemistry.
• 2. The DNA of prokaryotes has been moved about for centuries by inherent exchange elements called transposons, plasmids and bacterial viruses.
• 3. Micromanipulation of DNA has made possible a series of enzymes that catalyze various reactions of DNA molecules. Restriction endonucleases have the ability to cut double-stranded DNA at particular mucleotide sequences:

Example: Enzyme Eco RI 5' XXXGAATTCXXXX-3' 3' XXXCTTAAGXXXX-5' 5' XXXG AATTCXXXX-3' 3' XXXCTTAA + GXXXX-5'

 

• 4. The staggered cuts made by these enzymes create 'sticky ends' which can be realigned in solution by their sequence complementarity. Two different DNA molecules can be covalently linked by first cutting with the same endonuclease and then splicing them together in the presence of DNA ligase.
• 5. Other enzymes such as DNA polymerase and reverse transcriptase are capable of synthesizing copies of DNA or RNA templates respectively.

Example: 5'-GCC.UUU.AUG.GAA.AUU---3' RNA (REVERSE TRANSCRIPTASE) 3'-CGG.AAA TAC.CTT.TAA--- 5'-GCC.TTT.ATG.GAA.ATT---- DNA

 

The DNA fragment inserted into plasmids can be natural or synthetic.

• 6. The initial DNA product resulting from the construction of a recombinant plasmid is never homogeneous. The desired DNA sequence is obtained by cloning. Cloning is the isolation and proliferation of individual, genetically unique cells thus providing a high-resolution separation method for DNA molecules.

  9.1.4 Biological Applications of Recombinant DNA Technology

• 1. Environmental Effects: potential use of plasmids in the degradation of aromatic hydrocarbons, pesticides, plastics, PCB, etc.
• 2. Infectious Diseases: the role of plasmids or transposons in the establishment of nosocomial infections, propagation of multiple drug resistant strains and transfer of toxigenicity.
• 3. Expression in mammalian cells: plasmid-induced synthesis of interferon and somatostatin.
• 4. Characterization of Gene Structure: immunoglobulins and hemoglobin

   

  References Microbiology (Pelozar et al) Microbiology (Davis et al) Recombinant DNA and Transposons (Science 209: 1319, 1370, 1980) Recombinant DNA (American Scientist 68: 664, 1980) Plasmids and Transposons (Stuttard/Rozee 1979).