However, conditional TM can also be affected by systematic biases, see more deriving, for example, from transposon tools endowed with outward-facing promoters that are not strictly regulated in non-inducing conditions, resulting in a basal level of promoter expression. In fact, promoter leakage under non-inducing conditions would not completely switch off the gene downstream of the insertion site, significantly increasing the false-negative identification rate. The TM tools applicable for use with P. aeruginosa[12] are based on elements used for tightly regulated gene expression in E. coli, and are expected to not be completely PX-478 purchase switched off in non-inducing

conditions when used “out-of-context”. For these reasons, we set out to screen novel essential genes of P. aeruginosa using a method other than TM. To this end, we selected shotgun antisense RNA identification of essential genes, a technique that was developed a decade ago in Staphylococcus aureus[13, 14]. This technique originally only showed limited success in Gram-negative bacteria [15, 16], but

has recently been used effectively in E. coli[17]. In this approach, essential genes are identified after shotgun-cloned genomic fragments are conditionally expressed. The fragments are screened to identify those whose expression impairs growth [18]. The genes targeted by antisense RNA are identified by DNA sequencing of the growth-impairing fragments. This study shows for the first time the until feasibility of the antisense technology VX-809 order in P. aeruginosa for identifying novel essential genes. Moreover, we included some modifications to the original strategy that could have broadened the functional class variety of the identified essential genes in respect to a recent report in E. coli[17]. Results Ad hoc procedure to screen for essential P. aeruginosa genes by antisense RNA effects According to the scheme for antisense-mediated identification of essential genes established in S. aureus[13, 14], the shotgun genomic libraries generated in vitro are directly introduced into the original host

by transformation, and selected in permissive conditions, i.e., with the promoter vector in an off state, to allow the clones carrying inserts targeting essential genes to survive. However, basal vector promoter activity could be sufficient to elicit silencing effects against genes transcribed at low levels. This effect may introduce a bias in the subsequent conditional screening, favoring the identification of highly transcribed essential genes (e.g., tRNAs, tRNA synthetases, ribosomal proteins, translation factors, components of the transcription machinery). Cells transformed using constructs targeting essential genes expressed at low levels will fail to form a colony in the permissive conditions.