Question

1.) Vibrio cholerae is a Gram-negative, curved-shaped bacterial pathogen which causes nausea, vomiting, diarrhea and muscle cramps after consuming fecal contaminated water. Cholera killed thousands of people during the 1800’s as people traveled West to colonize the United States and more recently in Haiti after a devastating earthquake in 2010. Vibrio’s ability to cause potentially deadly diarrhea is due to a toxin it secretes called Cholera Toxin (CT). Vibrio is thought to have gained the gene which codes for CT from a bacteriophage.

A.) Which form of horizontal gene transfer (conjugation, transduction OR transformation) allowed Vibrio to produce the cholera toxin? (1p)

B.) Briefly describe the steps involved during this particular form of horizontal gene transfer. (2pts)

C.) Research and briefly explain how the rapid dipstick test works for identification of V. cholerae. Is it biochemical, immunological or genetic identification? (2pts)

Answer 1

A) As it is said that Vibrio gained gene for CT toxin from a bacteriophage, the horizontal gene transfer should have been occured through transduction.

Because in transformation the competent bacteria takes free DNA released by other bacteria in the environment. In conjugation cell-to-cell contact takes place between two bacteria and conjugative plasmid is transferred through pili from donor cell to recipient cell. Only in transduction bacteriophage acts as a vector between donor and recipient organisms.

B) Steps involved in transduction

1. Bacteriophage attaches to host cell surface and releases its genetic material into the cell

2. Viral genetic material gets integrated to host DNA. From this stage replication takes place in two ways viz., lytic cycle and lysogenic cycle

Lytic cycle

• Phage DNA starts replicating and produces new copies of phage DNA and proteins
• Phage assembles and lyses the cell open
• The produced phage copies attack new bacterial hosts and continue the same process

Lysogenic cycle

• Phage DNA remains integrated with the host DNA
• Phage DNA undergoes bacterial reproduction as the lysoenic bacterium reproduces replicating the prophage at each cell division
• At any stage of lysogenic cycle prophage lyses open the host bacterium to move out and enter the lytic cycle.

During this life cycle of bacteriophage the horizontal gene transfer of bacterial genes is mediated in two ways viz., Generalized transductiion and specialized transduction

Generalized transduction:

In this the defective virus particles randomly incorporate host DNA fragments and transfer it to recipient cell on infection. But its efficiency is very low.

Specialized transduction:

In this the DNA of temperate virus excises incorrectly and the host genes which are close to the integration point of virus are transduced.

C) Rapid Dipstick test for V.cholerae

Since Vibrio cholerae is a gram negative bacteria, its cell surface contains Lipopolysaccharides (LPS). Rapid Dipstick Test works with the monoclonal antiboies specific for cell surface LPS for different V.cholerae serogroups. Gold particles are used for the detectiomn of LPS and quantification of LPS is done by laterla flow immunochromatographic tests using specific monoclonal antibodies.

It is a immunological detection.