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Cloning and nucleotide sequence of a gene (ompS) encoding the major outer membrane protein of Legionella pneumophila
P S Hoffman1, M Ripley, R Weeratna
1Department of Microbiology, Dalhousie University, Halifax, Nova Scotia, Canada.
Abstract:
The major outer membrane protein of Legionella pneumophila is composed of 28- and 31-kDa subunits cross-linked by interchain disulfide bonds. The oligomer is covalently anchored to the underlying peptidoglycan via the 31-kDa subunit. We have cloned the structural gene ompS encoding both proteins. Oligonucleotide probes synthesized from the codons of the N-terminal amino acid sequence of purified 28- and 31-kDa subunits were used to identify cloned sequences. A 2.9-kb HindIII fragment cloned into pBluescript (clone H151) contained the ompS gene. Nucleotide sequence analysis revealed an open reading frame of 891 bp encoding a polypeptide of 297 amino acids. A leader sequence of 21 amino acids was identified, and the mature protein contained 276 amino acids. The deduced amino acid sequence of OmpS matched the experimentally determined amino acid sequence (32 amino acids), with the exception of two cysteine residues. The deduced amino acid sequence was rich in glycine and aromatic amino acids and contained four cysteine residues, two in the amino terminus and two in the carboxy region. Primer extension analysis (total RNA from L. pneumophila) identified the transcription start at 96 to 98 bp upstream of the translation start, but no Escherichia coli-like promoter sequences were evident. While an mRNA transcript from clone H151 was detected, no cross-reactive protein was detected by immunoblotting with either monoclonal or polyclonal antibody. Attempts to subclone the gene in the absence of the putative promoter region (i.e., under the control of the lac promoter) proved unsuccessful, possibly because of overproduction lethality in E. coli. The ompS DNA sequence was highly conserved among the serogroups of L. pneumophila, and related species also exhibited homology in Southern blot analysis at a moderately high stringency. Evidence is presented to suggest that this gene may be environmentally regulated in L. pneumophila.
Insights
The ompS gene, encoding the major outer membrane protein of Legionella pneumophila, was cloned and sequenced. This protein is crucial for bacterial structure and may be environmentally regulated.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Chemistry
Background:
- Legionella pneumophila possesses a major outer membrane protein complex essential for its structure.
- This protein complex consists of 28- and 31-kDa subunits linked by disulfide bonds and anchored to peptidoglycan.
Purpose of the Study:
- To clone and characterize the structural gene (ompS) for the major outer membrane protein of Legionella pneumophila.
- To analyze the nucleotide sequence and deduced amino acid sequence of the ompS gene and its encoded protein.
- To investigate the expression and regulation of the ompS gene.
Main Methods:
- Gene cloning using oligonucleotide probes derived from N-terminal amino acid sequences.
- Nucleotide sequence analysis of the cloned ompS gene.
- Primer extension analysis to identify transcription start sites.
- Southern blot analysis to assess gene conservation.
Main Results:
- The ompS gene was successfully cloned on a 2.9-kb HindIII fragment (clone H151).
- Sequence analysis revealed an 891 bp open reading frame encoding a 297-amino acid polypeptide with a 21-amino acid leader sequence.
- The deduced amino acid sequence matched experimental data, revealing a protein rich in glycine and aromatic amino acids, with four cysteine residues.
- Transcription start sites were identified, but no typical E. coli-like promoter sequences were found.
- mRNA transcripts were detected, but protein expression was not confirmed via immunoblotting, possibly due to overproduction lethality in E. coli.
- The ompS DNA sequence demonstrated high conservation across L. pneumophila serogroups and homology in related species.
Conclusions:
- The ompS gene has been cloned and sequenced, providing insights into the structure of Legionella pneumophila's major outer membrane protein.
- The gene sequence is highly conserved, suggesting functional importance across different strains and related species.
- Evidence suggests that the ompS gene may be subject to environmental regulation in Legionella pneumophila.
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