MOLECULAR AND IMMUNOLOGICAL CHARACTERIZATION OF OPRL, THE 18 KDA OUTER-MEMBRANE PEPTIDOGLYCAN-ASSOCIATED LIPOPROTEIN (PAL) OF PSEUDOMONAS-AERUGINOSA

Immunological screening of a Pseudomonas aeruginosa cosmid library led to the identification of clones producing an 18 kDa outer-membrane pr otein. This protein reacted in Western blots with a polyclonal antiser um against outer-membrane proteins of P. aeruginosa and with a monoclo nal antibody (MA1-6) specific for OprL, the peptidoglycan-associated o uter-membrane lipoprotein (PAL). Sequencing of pOML7, a subclone expre ssing oprL, revealed an ORF of 504 bp encoding a polypeptide with a ty pical lipoprotein signal recognition sequence. Another ORF was found u pstream of oprL, with homology to the TolB protein of Escherichia coli and Haemophilus influenzae. Downstream of oprL, a second ORF, of 321 bp, was found (orf2), encoding a protein with a signal peptide and wit h no homology with proteins of known biological function. After the st op codon of orf2, a rho-independent terminator sequence was detected w hich is part of the P. aeruginosa PAO1 insertion element 1S222. OprL s howed homologies with all known PALs from Gram-negative bacteria, espe cially in the C-terminal part. mAb MA1-6 reacted with P. aeruginosa ce lls in immunofluorescence, and with E. coli cells expressing oprL, whi ch had an abnormal, elongated morphology, an indication that productio n of the protein perturbed the division process.