Conservation of the gene for outer membrane protein OprF in the family Pseudomonadaceae: sequence of the Pseudomonas syringae oprF gene

Abstract

The conservation of the oprF gene for the major outer membrane protein OprF was determined by restriction mapping and Southern blot hybridization with the Pseudomonas aeruginosa oprF gene as a probe. The restriction map was highly conserved among 16 of the 17 serotype type strains and 42 clinical isolates of P. aeruginosa. Only the serotype 12 isolate and one clinical isolate showed small differences in restriction pattern. Southern probing of PstI chromosomal digests of 14 species from the family Pseudomonadaceae revealed that only the nine members of rRNA homology group I hybridized with the oprF gene. To reveal the actual extent of homology, the oprF gene and its product were characterized in Pseudomonas syringae. Nine strains of P. syringae from seven different pathovars hybridized with the P. aeruginosa gene to produce five different but related restriction maps. All produced an OprF protein in their outer membranes with the same apparent molecular weight as that of P. aeruginosa OprF. In each case the protein reacted with monoclonal antibody MA4-10 and was similarly heat and 2-mercaptoethanol modifiable. The purified OprF protein of the type strain P. syringae pv. syringae ATCC 19310 reconstituted small channels in lipid bilayer membranes. The oprF gene from this latter strain was cloned and sequenced. Despite the low level of DNA hybridization between P. aeruginosa and P. syringae DNA, the OprF gene was highly conserved between the species with 72% DNA sequence identity and 68% amino acid sequence identity overall. The carboxy terminus-encoding region of P. syringae oprF showed 85 and 33% identity, respectively, with the same regions of the P. aeruginosa oprF and Escherichia coli ompA genes.

Molecular characterization of the major outermembrane protein OprF from plant root-colonizing Pseudomonas fluorescens

“…This is not unexpected since earlier reports based on cross-reactivity of antibodies (Mutharia & Hancock, 1985) and on DNA hybridization (Ullstrom et al, 1991) indicated the occurrence of oprF-like genes and their products in several species of this group. OprF as a reference, only residues differing in the other proteins are shown.…”

“…Fig. 3 shows that the signal peptides of the different OprF proteins were highly similar and strongly resembled the corresponding part of the unprocessed OprF from P. syringae (Ullstrom et al, 1991). For the mature OprFs, the seven sequences showed 83.3 YO identity over 275 residues, and 27 of the 46 non-conserved residues contained at least one substitution with a dissimilar residue.…”

Phenotypic expression of the Pseudomonas syringae pv. syringae 61 hrp/hrm gene cluster in Escherichia coli MC4100 requires a functional porin

“…syringae 61 hrplhrm genes were expressed in E. coli at a level below the sensitivity of the assay, similar analyses were performed comparing the wild-type strain with one of the P. syringae pv. syringae 61 (24) have shown that inactivation of a single hrp gene is sufficient to block production of the HR-eliciting factor or activity. As before, no specific epitopes were associated with expression of the hrplhrm genes.…”

“…Cell collapse was evident within 18 h in leaf tissue infiltrated with P. syringae pv. syringae 61 and E. coli MC4100(pHIR11).…”

“…However, caution must be exercised in extending this conclusion to P. syningae. P. syningae porins have not been fully characterized, although some biochemical and immunological similarities have been noted (23,61). We are currently investigating the role of P. syningae porins in pathogenicity and HR elicitation.…”