Seven of these genes were indicated previously as essential in P. aeruginosa[9, 20]. The 25 genes were annotated as involved in multiple cellular functions: lipid A biosynthesis (lpxA, lpxB; lpxD, fabZ) [31], amino acid GDC-0973 mouse biosynthesis and metabolism (glyA3, proC, hom, lysC, ldh), DNA replication and recombination (dnaX, recB, recR), transport of small molecules (potD, mgtA, fadL, fepG, pstC),

biosynthesis of cofactors, prosthetic groups and carriers (folD), translation and post-translational modification (tufB), nucleotide biosynthesis (purL), protein secretion (secE), tRNA modification (gcp) [32], central intermediary metabolism (glpK), and energy metabolism (fdx2). Other genes present in the multigenic inserts might be essential, but their identification would require further analysis via subcloning and/or conditional mutagenesis. Sepantronium price Interestingly, four multigenic inserts contained genes belonging to a single operon (Table 2), a feature that suggests a functional association. One such gene, proC, codes for pyrroline-5-carboxylate reductase [33] and was reported as essential in E. coli, Mycobacterium tuberculosis and Acinetobacter baylyi[20]. Other gene products

of these operons are annotated as hypothetical click here proteins. Therefore, we suggest that these operonic genes might be involved in novel essential pathways. Overall, they are well-conserved in the sequenced Pseudomonas species (Additional file 5: Table S5). Exceptions are PA1088-1089-1090 which appear restricted to few Pseudomonas species and not conserved in all

sequenced P. aeruginosa strains. Finally, one operonic growth-impairing insert included PA1001-1002 (phnAB) implicated in the biosynthesis of pyocyanin. Previous reports on P. aeruginosa PAO1 phnA and PA14 phnAB function [34, 35] did not mention growth defects associated to deletion of these genes. As in the case of PA1709 (popD), discrepancies between our results and previous works could be attributable to differences in experimental conditions. Conclusions Taken together, our results show the feasibility of antisense technology in P. aeruginosa Tolmetin for identifying novel essential genes. Because of its supposed inefficiency [16], this approach has been neglected in Gram-negative bacteria for several years, and was only recently recovered in E. coli[17]. By comparison with this previous work, the results presented here strongly suggest that our modification of the antisense strategy could broaden the class variety of the identified essential genes. We expect that our methodology could be well suited for antisense-mediated searches of essential genes in other Gram-negative bacterial species. Methods Bacterial strains, plasmids, and growth conditions Bacterial strains and plasmids used in this study are listed in Additional file 6: Table S1. Bacteria were grown at 37°C in Luria-Bertani (LB) broth, or in M9 minimal medium supplemented with 0.2% citrate (M9-citrate).