Cloning and nucleotide sequence of a gene (ompS) encoding the major outer membrane protein of Legionella pneumophila

Hoffman, Paul S.; Ripley, Murray; Weeratna, Risini D.

doi:10.1128/jb.174.3.914-920.1992

Cited by 64 publications

(48 citation statements)

References 45 publications

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“…We have used an oligonucleotide probe (24b) to clone the structural gene encoding the 28-and 31-kDa proteins (18 (2). We are presently exploring this possibility for L. pneumophila, since recent evidence indicates that the 60-kDa (Hsp6O) chaperone protein is associated with the cell surface (16,30).…”

Section: Discussionmentioning

confidence: 99%

Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein

Hoffman

Butler

1992

J Bacteriol

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The major outer membrane protein of LegioneHla pneumophila exhibits an apparent molcular mass of 100 kDa. Previous studies revealed the oligomer to be composed of 28-and 31-kDa subunits; the latter subunit is covalently bound to peptidoglycan. These proteins exhibit cross-reactivity with polyclonal anti-31-kDa protein serum. In this study, we present evidence to confirm that the 31-kDa subunit is a 28-kDa subunit containing a bound fragment of peptidoglycan. Peptide maps of purified proteins were generated following cyanogen bromide cleavage or proteolysis with staphylococcal V8 protease. A comparison of the banding patterns resulting from sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) revealed a common pattern. Selected peptide fragments were sequenced on a gas phase microsequencer, and the sequence was compared with the sequence obtained for the 28-kDa protein. While the amino terminus of the 31-kDa protein was blocked, peptide fragments generated by cyanogen bromide treatment exhibited a sequence identical to that of the amino terminus of the 28-kDa protein, but beginning at amino acid four (glycine), which is preceded by methionine at the third position. This sequence, (Gly-Thr-Met)-Gly-Pro-Val-Trp-ThrP Gly-Asn .... confirms that these proteins have a common amino terminus. An oligonucleotide synthesized from the codons of the common N-terminal amino acid sequence was used to establish by Southern and Northern (RNA) blot analyses that a single gene coded for both proteins. With regard to the putative porin stucture, we have identified two major bands at 70 kDa and at approximately 120 kDa by nonreducing SDS-PAGE. The former may represent the typical trimeric motif, while the latter may represent either a double trimer or an aggregate. Analysis of these two forms by two-dimensional SDS-PAGE (first dimension, nonreducing; second dimension, reducing) established that both were composed of 31-and 28-kDa subunits cross-linked via interchain disulfide bonds. These studies confirm that the novel L. pneumophila major outer membrane protein is covalently bound to peptidoglycan via a modified 28-kDa subunit (31-kDa anchor protein) and cross-linked to other 28-kDa subunits via interchain disulfide bonds.

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Section: Discussionmentioning

confidence: 99%

Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein

Hoffman

Butler

1992

J Bacteriol

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“…Primer extension was performed as described previously (Hoffman et al, 1992) by hybridizing a 59-end-labelled oligonucleotide primer TERZ2R (see Table 1) to P. mirabilis total RNA. End-labelling of TERZ2R was done using T4 polynucleotide kinase with subsequent purification using a Sep-Pack C 18 cartridge (Millipore).…”

Section: Methodsmentioning

confidence: 99%

“…Total RNA was extracted by the hot SDS/acid phenol method (Hoffman et al, 1992) from P. mirabilis strains grown in the presence or absence of K 2 TeO 3 or following treatments with hydrogen peroxide or methyl viologen. Northern blot hybridization assays were optimized as generally described (Kroczek & Siebert, 1990).…”

Section: Methodsmentioning

confidence: 99%

An inducible tellurite-resistance operon in Proteus mirabilis

et al. 2003

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Tellurite resistance (Te r ) is widespread in nature and it is shown here that the natural resistance of Proteus mirabilis to tellurite is due to a chromosomally located orthologue of plasmid-borne ter genes found in enteric bacteria. The P. mirabilis ter locus (terZABCDE) was identified in a screen of Tn5lacZ-generated mutants of which one contained an insertion in terC. The P. mirabilis terC mutant displayed increased susceptibility to tellurite (Te s ) and complementation with terC carried on a multicopy plasmid restored high-level Te r . Primer extension analysis revealed a single transcriptional start site upstream of terZ, but only with RNA harvested from bacteria grown in the presence of tellurite. Northern blotting and reverse transcriptase-PCR (RT-PCR) analyses confirmed that the ter operon was inducible by tellurite and to a lesser extent by oxidative stress inducers such as hydrogen peroxide and methyl viologen (paraquat). Direct and inverted repeat sequences were identified in the ter promoter region as well as motifs upstream of the 235 hexamer that resembled OxyR-binding sequences. Finally, the 390 bp intergenic promoter region located between orf3 and terZ showed no DNA sequence identity with any other published ter sequences, whereas terZABCDE genes exhibited 73-85 % DNA sequence identity. The ter operon was present in all clinical isolates of P. mirabilis and Proteus vulgaris tested and is inferred for Morganella and Providencia spp. based on screening for high level Te r and preliminary PCR analysis. Thus, a chromosomally located inducible tellurite resistance operon appears to be a common feature of the genus Proteus.

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“…For PCR assay, we use the gene of the major outer membrane protein (ompS), which encodes for a surface protein composed of two subunits (28 and 31 kDa). 16,17 It was shown that the ompS DNA sequence is highly conserved among the different serogroups of L. pneumophila. This makes the gene a perfect target for detection of this pathogen.…”

Section: Introductionmentioning

confidence: 99%

Automated Immunomagnetic Processing and Separation of Legionella Pneumophila with Manual Detection by Sandwich ELISA and PCR Amplification of the ompS Gene

Reidt¹,

Geisberger²,

Heller³

et al. 2011

JALA: Journal of the Association for Laboratory Automation

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T he culture-independent and automated detection of bacteria in the environment is a scientific and technological challenge. For detection alone, a number of sensitive methods are known (e.g., PCR, enzyme-linked immunosorbent assay [ELISA], fluorescent in situ hybridization) but a major problem remaining is the enrichment and separation of the bacteria that usually occur at low concentrations. Here, we present an automated capturing and separation system, which can easily be combined with one of the sensitive detection techniques.We have developed a method for enrichment and detection of Legionella pneumophila in liquid media. Concentrated microorganisms were either detected by PCR or by sandwich ELISA. The limit of detection with the immunological assay was about 750 bacteria. Using PCR, the equivalent of about 2000 genomes could be detected.The assays were then transferred to a laboratory prototype for automated processing. It was possible to automatically enrich L. pneumophila by immunomagnetic separation (IMS), and again, the bacteria were detected by sandwich ELISA and PCR amplification of the ompS gene. As a novel aspect, ompS gene was used for the first time as a target for the detection of L. pneumophila on magnetic beads.The aim of this work was to develop an automated procedure and a device for IMS of bacteria. With Legionella as a model organism, we could show that such a novel fully automated system can be an alternative to timeconsuming conventional cultivation methods for detecting bacteria or other microorganisms.

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Cloning and nucleotide sequence of a gene (ompS) encoding the major outer membrane protein of Legionella pneumophila

Cited by 64 publications

References 45 publications

Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein

Molecular characterization of the 28- and 31-kilodalton subunits of the Legionella pneumophila major outer membrane protein

An inducible tellurite-resistance operon in Proteus mirabilis

Automated Immunomagnetic Processing and Separation of Legionella Pneumophila with Manual Detection by Sandwich ELISA and PCR Amplification of the ompS Gene

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