April 16, 2010
Identifying essential genes of Acinetobacter baumannii using an antisense expression strategy
MBRS-RISE MS to PhD Scholar
Department of Biological Sciences California State University, Los Angeles
Background: A. baumannii is a Gram negative, MDR soil bacterium that is responsible for responsible for a variety of nosocomial infections. Essential proteins, which are encoded by essential genes, are absolutely required for bacterial growth and survival. Consequently, the products of essential genes identified in pathogenic bacteria can be used as targets for the discovery of novel antibiotics. One important strategy for identifying bacterial essential genes is regulated antisense expression. An antisense expression strategy enables the down-regulation of gene expression at the post-transcriptional level using an inducible promoter and a plasmid vector containing an internal fragment of a gene in the antisense orientation. If the gene is essential, robust synthesis of the antisense RNA will inhibit cell growth thus confirming the essentiality of the gene by binding to the cognate mRNA to render the mRNA nonfunctional largely by increasing its degradation as well as inhibiting translation by hybridizing to sequences flanking the ribosome binding site (RBS) and the mRNAÂs start codon. Methods: Origin of replication (ori) and kanamycin resistance marker (kan) were individually PCR amplified using pWH1266 and pET-30 Ek/LIC, respectively. These amplified fragments were fused through overlap extension PCR. This fragment was blunt-ligated into pHN678 to yield expression vector pHN678-ok. E. coli DH5? cells were transformed with the ligation reaction. Plasmid DNA extraction of positive clones was conducted followed by agarose gel electrophoresis to confirm the presence of the fused fragment. Positive cloneÂs plasmid DNA was used to transform A. baumannii ATCC 17978 cells. A reporter gene, xylE, which encodes catechol 2,3Âdioxygenase, was cloned into linearized pHN678-ok. Quantitative spectrophotometric analysis of the regulation of expression of the reporter gene under the control of the shuttle vectorÂs PA1-03/04 promoter was conducted using A. baumannii cells. Primers were designed to amplify gene fragments of fabI, murA, and murG that contained sequences flanking the targetÂs mRNA RBS and start codon. The amplified fragments contained restriction enzyme sites that enabled ligation into pHN678-ok in the antisense orientation. Results: The fragment containing ori and kan was cloned into pHN678-ok. The shuttle vector replicated successfully in A. baumannii, as made evident by gel electrophoresis of plasmid DNA digestion reactions. The promoterÂs profile was quantitatively analyzed in a cell-based assay using xylE as a reporter gene. Using varying IPTG concentrations, we were able to successfully obtain the promoter activity profile of pHN678-ok in A. baumannii. Conclusions: Establishment of an inducible vector system is the initial step in the search for essential genes using an antisense expression strategy. IPTG will be used to control the expression of antisense gene fragments. We are confident that pHN678-ok, as an inducible vector, will serve as a valuable molecular tool in the search for A. baumanniiÂs essential genes. Acknowledgements: Funding for this project has been partially provided by NIH RIMI (P20MD001824) and NIH MBRS-SCORE grants (S06GM008101) to H. H. Xu.