Background: Genomic islands (GIs) are discrete segments of mobile DNA with defined
boundaries according to recent patents, acquired in the bacterial genome from another organism by horizontal
gene transfer during the course of evolution. GIs contribute significantly to virulence, disease
development, antimicrobial resistance and metabolic process.
Objective: The present study focuses on the development of a vector based genetic tool carrying selectable
and counter-selectable markers, in order to flag the GIs in the bacterial chromosome and monitor
their stability under in vitro and in vivo conditions.
Method: We engineered suicide vectors, pSB40 and pSB41, carrying single or tandem copies of chloramphenicol
acetyltransferase (cat) and levansucrase (sacB) alleles, respectively. The sacB-cat allele in
both the vectors is flanked by several restriction sites. To test the suitability of sacB-cat allele for monitoring
GI loss, we introduced the allele in the Vibrio Pathogenicity Island-1 (VPI-1) in Vibrio cholerae
Results: The V. cholerae strain carrying sacB-cat allele in VPI-1 element showed resistance to chloramphenicol
and sensitivity to sucrose at optimal growth conditions. Loss of VPI-1 element from the V.
cholerae genome was simply monitored by growing the cells on selection agar plates supplemented
with sucrose. Our results showed that the genetic tool we developed is suitable for monitoring GI stability
in the bacterial genome.
Conclusion: The present study indicates that pSB40 and pSB41are efficient and sensitive genetic tool
that can be used for reverse genetics experiments and monitoring stability of mobile genetic elements in
the bacterial genome.