The discovery of novel essential genes or pathways that have not yet been targeted by clinical antibiotics can underlie the development of alternative effective antibacterials to overcome the extant mechanisms of resistance. In P. aeruginosa, a genome-wide assessment of essential genes has been performed previously by constructing an ordered, nonredundant random transposon (Tn) insertion library [9, 10, 23]. An approach of this kind has proven invaluable in studying bacterial genomes and in detecting novel essential genes. However, there can be some degree of imprecision in tagging for essentiality owing to Tn insertions into possible permissive site(s) of essential genes. For example, “classical” essential genes involved in DNA replication, transcription, translation, and cell division (e.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) . 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 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 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, 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) , 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. We propose these genes as novel essential genes in P. aeruginosa. PA2951 (etfA), PA3687 (ppc), PA3758 (nagA), PA1183 (dctA), and PA1805 (ppiD) are homologous to genes previously shown to be essential in a limited number of bacterial species . Interestingly, for the remaining 16 genes, no homologs have been reported as essential in other bacteria . Among these, PA1709 (popD), coding for a subunit of the PopB/D translocon complex of the type III secretion-translocation system (TTSS), is implicated in effector translocation across the host plasma membrane. Previous reports on P. aeruginosa PopD function [24–26] did not mention growth defects associated to deletion of popD gene. Therefore, the growth-impairing effects of S5A10 insert corresponding to PA1709 (Table 1) did not seem to match the PopD role characterized so far. These discrepancies could be due to differences in experimental conditions between our study and earlier works.
We evaluated the set of 21 novel candidate essential genes for degree of conservation in Pseudomonas species according to the computationally-based analysis of orthologs of the Pseudomonas Genome Database  (Additional file 5: Table S5). Interestingly, they are well-conserved in the sequenced Pseudomonas species, with the exceptions of PA5548 and PA1709 (popD) that are unique in P. aeruginosa. However, PA5548 and PA1709 (popD) orthologs can be found in other bacterial species. Remarkably, 17 of 21 novel essential candidates are conserved in all twelve sequenced P. aeruginosa genomes (Additional file 5: Table S5). Instead, PA2220 (oprR), PA5264, PA1709 (popD) and PA3687 (ppc) are present in 3, 8, 9 and 10 of the sequenced genomes, respectively. Essential genes that are not fully conserved in all strains of a bacterial species can occur infrequently. As an example, the Escherichia coli genes ytfI, ypjF, ymfJ, ymfI and ymcD, coding for hypothetical proteins, were reported as essential in the K12-MG1655 strain [28, 29] and are conserved in only a limited number of the sequenced E. coli genomes .
Moreover, we compared the novel essential candidates with a panel of “classical” essential genes that were not included in the Database of Essential Genes (DEG)  because of the occurence of Tn insertions in previous screenings in P. aeruginosa[9, 10, 23]. The Tn insertion patterns of the novel essential candidates (i.e. number of insertions and insertion site(s)- terminal vs internal; Additional file 5: Table S5) were similar to those of “classical” essential genes (Additional file 4: Table S4).
This study also identified growth-impairing inserts carrying multiple genes. Because of their multigenic composition, the tagging of genes in these constructs for essentiality is not as direct as for single locus inserts (see above). However, among the multigenic inserts, we identified sequences corresponding to 25 genes with homologs already annotated as essential in other bacterial species  (Additional file 3: Table S3). 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) , amino acid 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) , 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.
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  and was reported as essential in E. coli, Mycobacterium tuberculosis and Acinetobacter baylyi. Other gene products of these operons are annotated as hypothetical 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.