Identification of oprG, a gene encoding a major outer membrane protein of Pseudomonas aeruginosa.

Sir, Pseudomonas aeruginosa expresses between five and nine major outer membrane proteins (OMPs), depending on the conditions of growth; the genes encoding all but one of these, OprG, have been identified. OprG has an apparent molecular weight of 25,000 when outer membranes are heated to 88°C for 10 min in solubilization buffer before being subjected to SDS–PAGE and 19,000 when they are not pre-heated. Its appearance in the outer membrane is highly dependent on growth conditions. In particular, Yates et al. observed a direct relationship between the iron concentration in the medium and expression of OprG and suggested that this OMP is involved in low-affinity iron uptake. Other conditions, including growth into the stationary phase, higher growth temperatures, Mg deficiency, certain lipopolysaccharide alterations and the presence of certain carbon sources, also result in the expression of varying concentrations of OprG. Such apparently broad regulation of this OMP has frustrated efforts to assign a function to it. However, in addition to the potential role of OprG in iron uptake, two groups of investigators have suggested that it is involved in quinolone uptake/ susceptibility. In view of this uncertainty, we undertook to identify the oprG gene. Outer membranes were prepared according to the method described by Hancock & Carey from a 500 mL overnight culture of P. aeruginosa IA1 in L-broth (1% Bacto-Tryptone, 0.5% yeast extract; Difco Chemical Co., St Louis, MO, USA). The OMPs were electroblotted on to an Immobilon P polyvinylidinedifluoride membrane (Millipore, Bedford, MA, USA) which was then dried and stained. The OprG band was excised from the blot and subjected to N-terminal amino acid sequencing by Dr P. Williams (Department of Pharmaceutical Sciences, Univers i t y of Nottingham, Nottingham, UK). The procedure