Cloning and analysis of the gene for the major outer membrane lipoprotein from Pseudomonas aeruginosa

Abstract

The gene for the Pseudomonas aeruginosa outer membrane lipoprotein I was isolated from a genomic library in the phage lambda EMBL3 vector and subsequently subcloned in the low copy-number, wide host-range plasmid vector, pKT240. The cloned gene was highly expressed, resulting in the production of a low molecular-weight protein (8 kD) that was found to be associated with the outer membrane. Sequence analysis showed an open reading frame of 83 amino acids with a putative N-terminal hydrophobic signal peptide of 19 residues immediately followed by the lipoprotein consensus sequence, GLY-CYS-SER-SER (residues 19-22). The predicted amino acid composition of the mature polypeptide and that of the purified lipoprotein I of P. aeruginosa (Mizuno and Kageyama, 1979) were identical. In contrast with other Gram-negative outer membrane lipoproteins, conformation predictions suggested that the mature protein was a single alpha helix.

Specificity of the Pseudomonas aeruginosa PA01 lipoprotein I gene as a DNA probe and PCR target region within the Pseudomonadaceae

“…Oligonucleotides derived from the recently published sequence of the oprl gene from a clinical isolate of P. aeruginosa (Cornelis et al, 1989) were used to identify the appropriate M13 clones and also to sequence the gene. The sequencing reactions were performed as described in the protocol for the T7 Sequencing Kit (Pharmacia) with [aJSS]dATP.…”

The Pseudomonas aeruginosa oxidative stress regulator OxyR influences production of pyocyanin and rhamnolipids: protective role of pyocyanin

“…For all primer sets, the following cycling parameters were used: 94 uC for 3 min followed by 40 cycles of 94 uC for 60 s, 55 uC for 45 s and 72 uC for 60 s, followed by 72 uC for 7 min. oprI (housekeeping gene control, outer-membrane lipoprotein precursor) was used to normalize gene expression (Cornelis et al, 1989). Amplification products were electrophoresed on 0.8 % agarose gels.…”

Use of Siderophores to Type Pseudomonads: The Three Pseudomonas Aeruginosa Pyoverdine Systems

“…However, three PVDdeficient strains, A l l , A13 and D, showed no incorporation with any of the three (ferri)pyoverdines. Their correct designation as P. aeruginosa species was checked by the accurate PCR-probing technique based on the detection of a 249 bp fragment of oprl coding for the outer-membrane lipoprotein of P. aeruginosa strains (Cornelis et al, 1989b;De Vos et al, 1993). The sequence of the corresponding PCR-amplified fragments from strains A l l , A13 and D showed a perfect identity with the P. aeruginosa ATCC 15692 sequence (data not shown).…”