g. polA, holE, holB, holC, dnaG, dnaJ, dnaK, rpoC, infC, and ftsYEX) were Tn inserted in previous investigations (Additional file 4: Table S4) [9, 10, 23] and, for this reason, P. aeruginosa alleles were not included in the Database of Essential Genes (DEG) [20]. SAR302503 in vivo Some disadvantages of this kind of approach could be overcome by using growth-conditional mutagenesis. To generate growth-conditional phenotypes, we decided to use the antisense-mediated STA-9090 in vitro strategy established previously in S. aureus[13, 14]. This technique is not affected by some of the bias linked to transposon mutagenesis mentioned above. However, it can present limitations in the multi-step process of antisense

libraries preparation such as the blunt-end cloning of mechanically sheared DNA fragments, library clones carrying multigenic inserts, the reintroduction efficiency of libraries into the original host. In addition, the efficiency of antisense inhibition, supposed to affect gene translatability and/or mRNA stability, can be gene-dependent and also differential

for distinct DNA fragments belonging to the same gene. We report here, for the first time, successful application of regulated antisense RNA technology to discover novel essential Entinostat ic50 functions in P. aeruginosa. To also screen for low expressed essential genes, we added a preliminary shotgun library construction in E. coli to the previous strategy, followed by mating transfer to P. aeruginosa. The subset of growth-impairing fragments that targeted single loci (Table 1) directly defined 28 “essential-for-growth” genes. Only five of these genes were “classical” essential genes involved in DNA replication, transcription, and translation. The remaining 23 genes are suggested to take part in disparate cellular functions,

including protein secretion, biosynthesis of cofactors, prosthetic groups, and carriers, energy metabolism, central intermediary metabolism, else transport of small molecules, translation, post-translational modification, non-ribosomal peptide synthesis, lipopolysaccharide synthesis/modification, and transcriptional regulation. Finally, some of the gene products described in Table 1 were annotated as “hypothetical” proteins. We suggest that these proteins may be involved in unexplored essential functions, either as stand-alone proteins or connected to classical housekeeping processes. This is the case for the inner membrane protein TgpA (PA2873; Table 1) [21], which was found in our antisense screenings and was previously reported as hypothetical, whose transglutaminase activity associated with the periplasmic domain might be either linked to cell wall metabolism or be involved in unknown key functions of protein maturation, secretion, and/or modification. Only two of the 23 non-classical essential genes, PA4669 (ipk) and PA3820 (secF), were already indicated as essential in P. aeruginosa[9, 20]. For the remaining 21 genes, no evidence for essentiality has been reported previously in P. aeruginosa[20].