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Published on: January 4, 2017
Cloning and DNA sequence of the omc gene encoding the outer membrane protein-macromolecular complex from Neisseria
W M Tsai1, S H Larsen, C E Wilde
1Department of Microbiology and Immunology, Indiana University School of Medicine, Indianapolis 46223.
Abstract:
The omc gene, encoding the outer membrane protein-macromolecular complex (OMP-MC), was cloned in two pieces from Neisseria gonorrhoeae 2686. The 5' fragment of the omc gene included a promoter sequence, as indicated by its unregulated expression in Escherichia coli. Attempts to reconstruct an intact omc gene were unsuccessful, suggesting that expression of the complete OMP-MC protein was toxic to E. coli. Complete sequence determination revealed a coding sequence of 2,133 nucleotides; the deduced amino acid sequence indicated a mature protein of 687 amino acids with an NH2-terminal signal peptide of 24 amino acids. Analysis of the deduced amino acid sequence revealed that the NH2-terminal half of OMP-MC is generally hydrophilic, while the COOH-terminal portion contains alternating hydrophobic and hydrophilic regions. Serological analyses demonstrated that the NH2-terminal portion of OMP-MC is exposed on the gonococcal surface and the COOH-terminal portion is membrane associated.
Insights
The Neisseria gonorrhoeae omc gene, encoding the outer membrane protein-macromolecular complex (OMP-MC), was cloned. Reconstructing the full OMP-MC gene in E. coli was unsuccessful, indicating potential toxicity of the complete protein.
Area of Science:
- Microbiology
- Molecular Biology
- Protein Biochemistry
Background:
- Neisseria gonorrhoeae is a significant human pathogen.
- Outer membrane proteins (OMPs) play crucial roles in bacterial structure and function.
- The omc gene encodes a novel outer membrane protein-macromolecular complex (OMP-MC).
Purpose of the Study:
- To clone and characterize the omc gene from Neisseria gonorrhoeae.
- To investigate the expression and structural properties of the OMP-MC protein.
- To determine the localization of OMP-MC domains within the bacterial cell.
Main Methods:
- Gene cloning of the omc gene in two fragments from Neisseria gonorrhoeae.
- Expression analysis in Escherichia coli to identify promoter activity.
- Complete nucleotide sequence determination and amino acid sequence analysis.
- Serological analyses to determine protein localization.
Main Results:
- The 5' fragment of the omc gene contained a promoter sequence, driving unregulated expression in E. coli.
- Attempts to express the full-length OMP-MC protein in E. coli failed, suggesting toxicity.
- The deduced amino acid sequence revealed a mature protein of 687 amino acids with distinct hydrophilic and hydrophobic regions.
- Serological data indicated the N-terminal portion is surface-exposed, while the C-terminal portion is membrane-associated.
Conclusions:
- The omc gene was successfully cloned and sequenced, providing insights into OMP-MC structure.
- The complete OMP-MC protein may exhibit toxicity to host cells, impacting its expression.
- The differential localization of OMP-MC domains suggests specific functional roles in the bacterial outer membrane.
