Pseudomonas aeruginosa fur overlaps with a gene encoding a novel outer membrane lipoprotein, OmlA

A novel outer membrane lipoprotein in Pseudomonas aeruginosa is encoded by the omlA gene, which was identified immediately upstream of the fur (ferric uptake regulator) gene. The omlA and fur genes were divergently transcribe d and had overlapping promoter regions, The proximal fur P2 promoter and th e omlA promoter shared a 5-bp DNA motif for their -10 promoter elements. Th e distal fur P1 promoter was located within the omlA coding sequence, and t he omlA and fur T1 mRNAs overlapped by 154 nucleotides, Optimal expression of both fur and omlA required roughly 200 bp of DNA upstream of the promote r regions, suggesting the presence of cia-acting transcriptional activation elements located within the omlA and fur genes, respectively. The levels o f Fur and OmlA proteins had no influence on omlA or fur expression, excludi ng any trans-acting cross-regulation between fur and omlA. Expression of om lA was constitutive regardless of growth phase, oxygen tension, iron concen tration, pH, and temperature. OmlA contained a signal sequence typical of b acterial lipoproteins, with a cysteine as a putative cleavage and lipid att achment site. Inhibition of signal peptidase II by globomycin resulted in f ailure to process OmlA, thus giving strong evidence that OmlA is a lipoprot ein. Cell fractionation followed by Western blot analysis indicated that al l OmlA protein is localized in the outer membrane. Mature OmlA was an acidi c (pI = 4.5) protein of 17.3 kDa and had close to 40% amino acid sequence i dentity to SmpA (small protein A) of Escherichia call, Vibrio cholerae, and Haemophilus influenzae, a protein of unknown function. All P. aeruginosa s trains tested as well as Pseudomonas fluorescens were found to produce OmlA , A mutant strain with impaired production of OmlA but no change in the exp ression of the overlapping fur gene was constructed. The omlA mutant was hy persusceptible to anionic detergents such as sodium dodecyl sulfate and deo xycholate, and it showed increased susceptibility to various antibiotics, i ncluding nalidixic acid, rifampin, novobiocin, and chloramphenicol. A struc tural role of OmlA in maintaining the cell envelope integrity is proposed.