The major outer membrane protein of Haemophilus ducreyi consists of two OmpA homologs

Abstract

The major outer membrane protein (MOMP) of Haemophilus ducreyi is an OmpA homolog that migrates on sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels as three species with apparent molecular weights ranging from 37,000 to 43,000. Monoclonal antibodies directed against this macromolecule were used to identify recombinant clones containing fragments of the gene encoding this protein. Nucleotide sequence analysis of these fragments confirmed that the MOMP encoded by the intact gene (momp) was a member of the OmpA family of outer membrane proteins. Construction of an isogenic H. ducreyi mutant unable to express the MOMP led to the discovery of a second outer membrane protein which migrated at the same rate on SDS-PAGE gels as the MOMP. N-terminal amino acid sequence analysis of this second protein revealed that its N terminus was nearly identical to that of the MOMP and also had homology with members of the OmpA family. Nucleotide sequence analysis of the region downstream from the momp gene revealed the presence of a partial open reading frame encoding a predicted OmpA-like protein. A modification of anchored PCR technology was used to obtain the nucleotide sequence of this downstream gene which was shown to encode a second OmpA homolog (OmpA2). The N-terminal amino acid sequence of OmpA2 was identical to that of the OmpA-like protein detected in the momp mutant. The H. ducreyi MOMP and OmpA2 proteins, which comigrated on SDS-PAGE gels and which were encoded by the tandem arranged momp and ompA2 genes, were 72% identical.

Evaluation of an Isogenic Major Outer Membrane Protein-Deficient Mutant in the Human Model of Haemophilus ducreyi Infection

“…The plasmid pMOMP1.2, which contains a promoterless momp ORF, was described previously (17). Robert S. Munson, Jr. (Ohio State University, Columbus), kindly provided pRSM1515, which contains the ⍀-Km2 cassette.…”

“…H. ducreyi expresses 2 OmpA homologs, designated MOMP (major outer membrane protein) and OmpA2. Depending on gel conditions, both MOMP and OmpA2 may migrate as three bands in sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) with apparent molecular masses of 37 to 39 kDa and a heat-modifiable 43-kDa species (17,30). MOMP is estimated to be four to five times more abundant in the outer membrane than OmpA2.…”

“…Our laboratories had constructed an H. ducreyi MOMPdeficient mutant (35000.60) by insertion of a chloramphenicol acetyltransferase gene (cat) cassette into the momp open reading frame (ORF) in the same orientation as the momp and ompA2 promoters (17). In Western blotting, monoclonal antibody (MAb) 2C7, which binds to both MOMP and OmpA2, reacts strongly with the 43-kDa band in 35000.60.…”

“…MOMP is estimated to be four to five times more abundant in the outer membrane than OmpA2. The genes encoding MOMP and OmpA2 are separated by 285 bp on the chromosome and share 74% identity (17,30). Their tandem arrangement on the chromosome may be due to a gene duplication event.…”

The Pseudomonas aeruginosa tonB gene encodes a novel TonB protein

“…While the tonB gene is not linked to exbB and exbD homologues in most of the bacteria where they have been identified, the three genes appear to form an operon in P.psrtida and H. injueqae (Bitter e t al., 1993;Jarosik et al, 1994). P. aeruginosa expresses a number of outer-membrane proteins with homology to TonB-dependent receptors, including receptors for the ferric complexes of the siderophores pyoverdine (Poole e t al., 1993c), pyochelin (Heinrichs et al, 1991 ;Ankenbauer & Quan, 1994) and enterobactin (Dean & Poole, 1993a).…”