Influence of a putative ECF sigma factor on expression of the major outer membrane protein, OprF, in Pseudomonas aeruginosa and Pseudomonas fluorescens

The gene encoding OprF, a major outer membrane protein in Pseudomonas speci es (formerly known as type 1 pseudomonads), was thought to be constitutivel y transcribed from a single sigma 70 promoter immediately upstream of the g ene, We now report the identification of a no,el putative ECF (extracytopla smic function) sigma factor gene, sigX, located immediately upstream of opr F in both Pseudomonas aeruginosa PAO1 and Pseudomonas fluorescens OE 28.3 a nd show that disruption of this gene significantly reduces OprF expression. In P, aeruginosa, Northern analysis demonstrated that this reduction was a result of an effect on transcription of monocistronic oprF combined with a polar effect due to termination of a transcript containing sigX and oprF, Comparison of sigX-disrupted and wild-type cell transcripts by primer exten sion indicated that monocistronic transcription of oprF occurs from two ove rlapping promoters, one that is SigX-dependent and resembles ECF sigma fact or promoters in its minus-35 region and another promoter that is independen t of SigX and is analogous to the sigma 70-type promoter previously reporte d. Complementation of the P, aeruginosa sigX-disrupted mutant with plasmid- encoded OprF did not resolve the phenotypes associated with this mutant, wh ich included a markedly reduced logarithmic-phase growth rate in rich mediu m (compared to that in minimal medium), further reduction of the growth rat e in a low-osmolarity environment, secretion of an unidentified pigment, an d increased sensitivity to the antibiotic imipenem, This indicates that Sig X is involved in the regulation of other genes in P, aeruginosa. Disruption of the sigX gene in P. fluorescens also had an effect on the logarithmic-p hase growth rate in rich medium. A conserved sigX gene was also identified in a Pseudomonas syringae isolate and six P, aeruginosa clinical isolates. Collectively, these data indicate that an ECT; sigma factor plays a role in the regulation and expression of OprF and also affects other genes.