Identification by Subtractive Hybridization of a Novel Insertion Sequence Specific for Virulent Strains ofPorphyromonas gingivalis

ABSTRACT Subtractive hybridization was employed to isolate specific genes from virulent Porphyromonas gingivalis strains that are possibly related to abscess formation. The genomic DNA from the virulent strain P. gingivalis W83 was subtracted with DNA from the avirulent strain ATCC 33277. Three clones unique to strain W83 were isolated and sequenced. The cloned DNA fragments were 885, 369, and 132 bp and had slight homology with only Bacillus stearothermophilus IS5377, which is a putative transposase. The regions flanking the cloned DNA fragments were isolated and sequenced, and the gene structure around the clones was revealed. These three clones were located side-by-side in a gene reported as an outer membrane protein. The three clones interrupt the open reading frame of the outer membrane protein gene. This inserted DNA, consisting of three isolated clones, was designated IS1598, which was 1,396 bp (i.e., a 1,158-bp open reading frame) in length and was flanked by 16-bp terminal inverted repeats and a 9-bp duplicated target sequence. IS1598 was detected inP. gingivalis W83, W50, and FDC 381 by Southern hybridization. All three P. gingivalis strains have been shown to possess abscess-forming ability in animal models. However, IS1598 was not detected in avirulent strains of P. gingivalis, including ATCC 33277. The IS1598 may interrupt the synthesis of the outer membrane protein, resulting in changes in the structure of the bacterial outer membrane. The IS1598 isolated in this study is a novel insertion element which might be a specific marker for virulent P. gingivalisstrains.

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The 120 kilodalton outer membrane protein (rOmp B) of Rickettsia rickettsii is encoded by an unusually long open reading frame: evidence for protein processing from a large precursor

Molecular Microbiology ◽

10.1111/j.1365-2958.1991.tb02082.x ◽

1991 ◽

Vol 5 (10) ◽

pp. 2361-2370 ◽

Cited By ~ 48

Author(s):

R. D. Gilmore ◽

W. Cieplak ◽

P. F. Policastro ◽

T. Hackstadt

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Protein Processing ◽

Open Reading Frame ◽

Reading Frame ◽

Rickettsia Rickettsii

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Characterization of Outer Membrane Proteins in Chlamydia trachomatis LGV Serovar L2

Journal of Bacteriology ◽

10.1128/jb.183.8.2686-2690.2001 ◽

2001 ◽

Vol 183 (8) ◽

pp. 2686-2690 ◽

Cited By ~ 38

Author(s):

Regina J. Tanzer ◽

Thomas P. Hatch

Keyword(s):

Chlamydia Trachomatis ◽

Membrane Proteins ◽

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Outer Membrane Proteins ◽

Major Outer Membrane Protein ◽

Developmental Cycle ◽

Reading Frame

ABSTRACT We used a photoactivatable, lipophilic reagent, 3′-(trifluoromethyl)-3-(m-[125I]iodophenyl)diazirine, to label proteins in the outer membrane of elementary bodies ofChlamydia trachomatis LGV serovar L2 and mass spectrometry to identify the labeled proteins. The identified proteins were polymorphic outer membrane proteins E, G, and H, which were made late in the developmental cycle, the major outer membrane protein, and a mixture of 46-kDa proteins consisting of the open reading frame 623 protein and possibly a modified form of the major outer membrane protein.

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HmbR, a Hemoglobin-Binding Outer Membrane Protein of Neisseria meningitidis, Undergoes Phase Variation

Journal of Bacteriology ◽

10.1128/jb.181.7.2067-2074.1999 ◽

1999 ◽

Vol 181 (7) ◽

pp. 2067-2074 ◽

Cited By ~ 70

Author(s):

Anthony R. Richardson ◽

Igor Stojiljkovic

Keyword(s):

Membrane Protein ◽

Neisseria Meningitidis ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Phase Variation ◽

Membrane Receptors ◽

Phenotypic Switch ◽

Reading Frame ◽

Single Colony ◽

Hemoglobin Binding

ABSTRACT Neisseria meningitidis uses hemoglobin (Hb) as an iron source via two TonB-dependent outer membrane receptors, HmbR and HpuB. Analysis of 25 epidemiologically unrelated clinical isolates from serogroups A, B, C, and Y revealed that 64% strains possessed both Hb receptor genes. Examination of the hmbR expression pattern in strains in which the hpuB gene was genetically inactivated revealed two distinct Hb utilization phenotypes. Five strains retained the ability to grow as a confluent lawn, while seven grew only as single colonies around Hb discs. The single-colony phenotype observed for some hpuB mutants is suggestive of phase variation of hmbR. The length of the poly(G) tract starting at position +1164 of hmbR absolutely correlated with the two Hb utilization phenotypes. All five strains that grew as confluent lawns around Hb discs possessed either 9 or 12 consecutive G residues. All seven strains that grew as single colonies around Hb discs had poly(G) tracts of a length other than 9 or 12. These single-colony variants that arose around the Hb discs had poly(G) tracts with either 9 or 12 consecutive G residues restoring thehmbR reading frame. Inactivation of hmbR in these strains resulted in a loss of Hb utilization, demonstrating that the change in the hmbR gene was responsible for the phenotypic switch. The switching rates from hmbR phase off to phase on were ∼5 × 10−4 in four serogroup C strains, 2 × 10−2 in the serogroup A isolate, and 7 × 10−6 in the serogroup B isolate.

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The LspB Protein Is Involved in the Secretion of the LspA1 and LspA2 Proteins by Haemophilus ducreyi

Infection and Immunity ◽

10.1128/iai.72.4.1874-1884.2004 ◽

2004 ◽

Vol 72 (4) ◽

pp. 1874-1884 ◽

Cited By ~ 25

Author(s):

Christine K. Ward ◽

Jason R. Mock ◽

Eric J. Hansen

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Culture Supernatant ◽

Cell Envelope ◽

Rabbit Model ◽

Haemophilus Ducreyi ◽

Pcr Analysis ◽

Supernatant Fluid ◽

Reading Frame

ABSTRACT The LspA1 and LspA2 proteins of Haemophilus ducreyi 35000 are two very large macromolecules that can be detected in concentrated culture supernatant fluid. Both of these proteins exhibit homology with the N-terminal region of the Bordetella pertussis filamentous hemagglutinin (FHA), which is involved in secretion of the latter macromolecule. The lspA2 open reading frame is flanked upstream by a gene, lspB, that encodes a predicted protein with homology to the B. pertussis FhaC outer membrane protein that is involved in secretion of FHA across the outer membrane. The H. ducreyi lspB gene encodes a protein with a predicted molecular mass of 66,573 Da. Reverse transcription-PCR analysis suggested that the lspB gene was transcribed together with the lspA2 gene on a single mRNA transcript. Polyclonal H. ducreyi LspB antiserum reacted with a 64-kDa antigen present in the Sarkosyl-insoluble cell envelope fraction of H. ducreyi 35000, which indicated that the LspB protein is likely an outer membrane protein. Concentrated culture supernatant fluids from H. ducreyi lspB and lspA1 lspB mutants did not contain detectable LspA1 and detectable LspA2, respectively. However, complementation of the lspB mutant with the wild-type lspB gene on a plasmid restored LspB protein expression and resulted in release of detectable amounts of the LspA1 protein into culture supernatant fluid. When evaluated in the temperature-dependent rabbit model of infection, the lspB mutant was attenuated in the ability to cause lesions and was never recovered in a viable form from lesions. These results indicated that the H. ducreyi LspB protein is involved in secretion of the LspA1 and LspA2 proteins across the outer membrane.

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

Infection and Immunity ◽

10.1128/iai.68.5.2602-2607.2000 ◽

2000 ◽

Vol 68 (5) ◽

pp. 2602-2607 ◽

Cited By ~ 22

Author(s):

Robert E. Throm ◽

Jaffar A. Al-Tawfiq ◽

Kate R. Fortney ◽

Barry P. Katz ◽

Antoinette F. Hood ◽

...

Keyword(s):

Membrane Protein ◽

Outer Membrane ◽

Outer Membrane Protein ◽

Transcriptional Start Site ◽

Haemophilus Ducreyi ◽

Human Model ◽

Deficient Mutant ◽

Double Blind ◽

Reading Frame ◽

Porin Protein

ABSTRACT Haemophilus ducreyi expresses 2 OmpA homologs, designated MOMP and OmpA2, whose genes are arranged in tandem on the chromosome. Northern blot analysis indicated that momp andompA2 are transcribed independently. Sequences of themomp open reading frame (ORF) lacking the transcriptional start site were amplified by PCR, and an Ω-Km2 cassette was ligated into the ORF. A plasmid containing this construction was electroporated into H. ducreyi 35000HP, and an isogenic MOMP-deficient mutant (35000HP-SMS2) was generated by allele exchange. In Southern blotting, 35000HP-SMS2 contained one copy of the Ω-Km2 cassette inmomp. 35000HP and 35000HP-SMS2 had similar outer membrane protein (OMP) and lipooligosaccharide profiles and growth rates except for up-regulation of a putative porin protein in the mutant. Five subjects were inoculated with three doses of live 35000HP-SMS2 on one arm and two doses of live 35000HP and one dose of a heat-killed control on the other arm in a double-blind escalating dose-response trial. Pustules developed at 7 of 10 sites inoculated with 35000HP and at 6 of 15 sites inoculated with 35000HP-SMS2 (P = 0.14). 35000HP and 35000HP-SMS2 were recovered at similar rates from daily surface cultures and semiquantitative cultures. The data suggest that expression of MOMP is not required for pustule formation by H. ducreyi in the human model of infection.

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