Construction Of A Pseudomonas Aeruginosa Random Promoter Library And Its Application

Pseudomonas aeruginosa is a gram-negative opportunistic pathogen that is a major infectious agent in hospitals. It can cause fatal acute and chronic infections. One of the most important characteristics of P. aeruginosa is its intrinsic resistance to many antibiotics. It is important to study the pathogenesis and antibiotic resistance of P. aeruginosa.Iron is an essential nutrientional element to virtually all bacteria, and pathogens require iron when causing infection. Screen of the iron-responsive genes of P. aeruginosa at the genomic scale helps our systemically understanding of iron metabolism, iron regulation pathways and the changes of gene expression after P. aeruginosa enters the host.Antibiotics are bioactive compounds, which are recently recognized as not only being antagonist agents but also intermicrobial signaling agents capable of modulating many bacterial genes. They have been shown to possess biological activities other than inhibition; under the subinhibitory concentration they are able to regulate the expression of virulence factors, induce antibiotic resistance of pathogens, and affect the interactions among pathogens and between pathogens and hosts. So, it is important to study regulation of gene expression by antibiotics in order to understand the biological functions of antibiotic and reveal gene regulation pathways.pMS402 that contains a promoterless luminescence reporter, LuxCDABE, was used as the vector to construct a P.aeruginosa random promoter library with a size of about 5700 clones. It enables real-time, in vivo high-throughput gene expression profiling at genomic scale. Using this random promoter library we screened for iron-responsive genes in the P. aeruginosa genome. Two previously reported iron-regulated genes were identified, and ten new iron-regulated genes were uncovered. The genes encoding dihydrolipoamide acetyltransferase, phosphogluconate dehydratase and Fe (III) dicitrate transporter were found to be iron-regulated together with four function-unknown genes and three putative protein encoding genes. These results provide a basis for elucidating the complex iron regulation network in P. aeruginosa and help our understanding of the roles of iron in bacterial physiology and pathogenesis. At subinhibitory concentrations of carbenicillin, polymyxin and ciprofloxacin, part of the random promoter library of P. aeruginosa were screened. Genes encoding acyl-CoA dehydrogenase and lysophosphatidic acid acyltransferase (LptA) were found to be active by ciprofloxacin; one function-unkown gene was found to be inhibited by carbenicillin. These results provide new information in the regulation of subinhibitory concentrations of antibiotics in P. aeruginosa. The random promoter library system also offers a useful tool in bacterial genomic studies.