<i>Pseudomonas aeruginosa fur</i> Overlaps with a Gene Encoding a Novel Outer Membrane Lipoprotein, OmlA

<jats:title>ABSTRACT</jats:title> <jats:p> A novel outer membrane lipoprotein in <jats:italic>Pseudomonas aeruginosa</jats:italic> is encoded by the <jats:italic>omlA</jats:italic> gene, which was identified immediately upstream of the <jats:italic>fur</jats:italic> (ferric uptake regulator) gene. The <jats:italic>omlA</jats:italic> and <jats:italic>fur</jats:italic> genes were divergently transcribed and had overlapping promoter regions. The proximal <jats:italic>fur</jats:italic> P2 promoter and the <jats:italic>omlA</jats:italic> promoter shared a 5-bp DNA motif for their −10 promoter elements. The distal <jats:italic>fur</jats:italic> P1 promoter was located within the <jats:italic>omlA</jats:italic> coding sequence, and the <jats:italic>omlA</jats:italic> and <jats:italic>fur</jats:italic> T1 mRNAs overlapped by 154 nucleotides. Optimal expression of both <jats:italic>fur</jats:italic> and <jats:italic>omlA</jats:italic> required roughly 200 bp of DNA upstream of the promoter regions, suggesting the presence of <jats:italic>cis</jats:italic> -acting transcriptional activation elements located within the <jats:italic>omlA</jats:italic> and <jats:italic>fur</jats:italic> genes, respectively. The levels of Fur and OmlA proteins had no influence on <jats:italic>omlA</jats:italic> or <jats:italic>fur</jats:italic> expression, excluding any <jats:italic>trans</jats:italic> -acting cross-regulation between <jats:italic>fur</jats:italic> and <jats:italic>omlA</jats:italic> . Expression of <jats:italic>omlA</jats:italic> was constitutive regardless of growth phase, oxygen tension, iron concentration, pH, and temperature. OmlA contained a signal sequence typical of bacterial lipoproteins, with a cysteine as a putative cleavage and lipid attachment site. Inhibition of signal peptidase II by globomycin resulted in failure to process OmlA, thus giving strong evidence that OmlA is a lipoprotein. Cell fractionation followed by Western blot analysis indicated that all OmlA protein is localized in the outer membrane. Mature OmlA was an acidic (pI = 4.5) protein of 17.3 kDa and had close to 40% amino acid sequence identity to SmpA (small protein A) of <jats:italic>Escherichia coli</jats:italic> , <jats:italic>Vibrio cholerae</jats:italic> , and <jats:italic>Haemophilus influenzae</jats:italic> , a protein of unknown function. All <jats:italic>P. aeruginosa</jats:italic> strains tested as well as <jats:italic>Pseudomonas fluorescens</jats:italic> were found to produce OmlA. A mutant strain with impaired production of OmlA but no change in the expression of the overlapping <jats:italic>fur</jats:italic> gene was constructed. The <jats:italic>omlA</jats:italic> mutant was hypersusceptible to anionic detergents such as sodium dodecyl sulfate and deoxycholate, and it showed increased susceptibility to various antibiotics, including nalidixic acid, rifampin, novobiocin, and chloramphenicol. A structural role of OmlA in maintaining the cell envelope integrity is proposed. </jats:p>