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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 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.

SUBMITTER: Ullstrom CA

PROVIDER: S-EPMC207070 | biostudies-other | 1991 Jan

REPOSITORIES: biostudies-other

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

Ullstrom C A CA Siehnel R R Woodruff W W Steinbach S S Hancock R E RE

Journal of bacteriology 19910101 2

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 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 Ps ...[more]

PMID: 1898935

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Project description:The major surface protein (Msp) of Treponema denticola has been implicated as a mediator of the interaction between the spirochete and the gingival epithelium in periodontal diseases. Previous studies showed that the Msp of T. denticola ATCC 35405 had porin activity, depolarized epithelial cell membranes, bound to extracellular matrix components of epithelial cells, and formed a regular hexagonal surface array in the treponemal outer membrane. The gene encoding Msp in ATCC 35405 was recently cloned, sequenced, and expressed in Escherichia coli (J. C. Fenno, K.-H. Müller, and B. C. McBride, J. Bacteriol. 178:2489-2496, 1996). In the present study, we identified genes encoding Msp-like proteins in several oral spirochetes. A prominent heat-modifiable Msp-like protein having an apparent molecular mass of between 43 and 64 kDa was present in all oral spirochete strains tested. Antibodies raised against the ATCC 35405 Msp reacted strongly with the Msp proteins of T. denticola ATCC 35404 and T. vincentii, reacted very weakly with the Msp protein of T. denticola ATCC 33520, and did not react with T. denticola OTK, T. socranskii, and T. pectinovorum. The msp loci of the T. denticola strains and T. vincentii were identified in analyses using PCR with oligonucleotide primers derived from the DNA sequence flanking msp in ATCC 35405. Southern blot analysis showed at least three groups of related msp DNA sequences. Comparison of DNA sequences of the 5' and 3' ends of the msp genes showed high sequence homology in the flanking regions and signal peptide coding regions, while the homologies between regions encoding the mature peptide were as low as 50%. The entire msp DNA sequences of T. denticola ATCC 33520 and OTK were determined, and the deduced Msp amino acid sequences were compared to the sequence of the previously reported Msp of ATCC 35405. The results show that the msp locus is conserved in oral treponemes but that there are significant differences between the mature Msp peptides of different strains. Further studies of the antigenic domains, functional domains, and physical structures of Msp proteins, based on these results, will enhance understanding of the role of Msp in the cytopathology associated with oral spirochetes.

Project description:The pPT23A family of plasmids appears to be indigenous to the plant pathogen Pseudomonas syringae and these plasmids are widely distributed and widely transferred among pathovars of P. syringae and related species. pPT23A-family plasmids (PFPs) are sources of accessory genes for their hosts that can include genes important for virulence and epiphytic colonization of plant leaf surfaces. The occurrence of repeated sequences including duplicated insertion sequences on PFPs has made obtaining closed plasmid genome sequences difficult. Therefore, our objective was to obtain complete genome sequences from PFPs from divergent P. syringae pathovars and also from strains of P. syringae pv. syringae isolated from different hosts.The eight plasmids sequenced ranged in length from 61.6 to 73.8 kb and encoded from 65 to 83 annotated orfs. Virulence genes including type III secretion system effectors were encoded on two plasmids, and one of these, pPt0893-29 from P. syringae pv. tabaci, encoded a wide variety of putative virulence determinants. The PFPs from P. syringae pv. syringae mostly encoded genes of importance to ecological fitness including the rulAB determinant conferring tolerance to ultraviolet radiation. Heavy metal resistance genes encoding resistance to copper and arsenic were also present in a few plasmids. The discovery of part of the chromosomal genomic island GI6 from P. syringae pv. syringae B728a in two PFPs from two P. syringae pv. syringae hosts is further evidence of past intergenetic transfers between plasmid and chromosomal DNA. Phylogenetic analyses also revealed new subgroups of the pPT23A plasmid family and confirmed that plasmid phylogeny is incongruent with P. syringae pathovar or host of isolation. In addition, conserved genes among seven sequenced plasmids within the same phylogenetic group were limited to plasmid-specific functions including maintenance and transfer functions.Our sequence analysis further revealed that PFPs from P. syringae encode suites of accessory genes that are selected at species (universal distribution), pathovar (interpathovar distribution), and population levels (intrapathovar distribution). The conservation of type IV secretion systems encoding conjugation functions also presumably contributes to the distribution of these plasmids within P. syringae populations.

Dataset Information

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

Publications

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

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