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Expression of a gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv.

ABSTRACT: An open reading frame in the genomic database of Mycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for a presumed signal sequence was well expressed, but the protein had no pore-forming activity in the liposome swelling assay. However, the intact protein, OmpATb, behaved as a porin of low specific activity, with a pore diameter of 1.4 to 1.8 nm, and was also active in planar lipid bilayers, showing a single-channel conductance of 700 pS. The protein had a molecular mass of about 38 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis. A polyclonal rabbit antiserum raised to the truncated protein recognized a protein of similar molecular mass in detergent extracts of broken M. tuberculosis cells. Reverse transcription-PCR confirmed that the gene for OmpATb was expressed in M. tuberculosis cells growing in culture. Comparison of the purified protein with that in the detergent-extracted preparation using liposomes and planar lipid bilayers showed that the two materials had similar pore-forming properties. OmpATb is different from either of the mycobacterial porins described so far. This is the first report of a porin-like molecule from M. tuberculosis; the porin is likely to be important in controlling the access of hydrophilic molecules to the bacterial cell.

SUBMITTER: Senaratne RH

PROVIDER: S-EPMC107320 | biostudies-literature | 1998 Jul

REPOSITORIES: biostudies-literature

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Expression of a gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv.

Senaratne R H RH Mobasheri H H Papavinasasundaram K G KG Jenner P P Lea E J EJ Draper P P

Journal of bacteriology 19980701 14

An open reading frame in the genomic database of Mycobacterium tuberculosis H37Rv was identified as having homology with an outer membrane protein. We found that the gene specified a protein belonging to the OmpA family, which includes some porins of gram-negative organisms. The gene was amplified by PCR and cloned into Escherichia coli. Overexpression of the gene was toxic to the host, but limited amounts could be purified from cells before growth ceased. A truncated gene devoid of the code for ...[more]

PMID: 9657995

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Project description:BackgroundMycobacterium tuberculosis (M.tb) is the causative agent of tuberculosis, killing ~1.7 million people annually. The remarkable capacity of this pathogen to escape the host immune system for decades and then to cause active tuberculosis disease, makes M.tb a successful pathogen. Currently available anti-mycobacterial therapy has poor compliance due to requirement of prolonged treatment resulting in accelerated emergence of drug resistant strains. Hence, there is an urgent need to identify new chemical entities with novel mechanism of action and potent activity against the drug resistant strains.ResultsThis study describes novel computational models developed for predicting inhibitors against both replicative and non-replicative phase of drug-tolerant M.tb under carbon starvation stage. These models were trained on highly diverse dataset of 2135 compounds using four classes of binary fingerprint namely PubChem, MACCS, EState, SubStructure. We achieved the best performance Matthews correlation coefficient (MCC) of 0.45 using the model based on MACCS fingerprints for replicative phase inhibitor dataset. In case of non-replicative phase, Hybrid model based on PubChem, MACCS, EState, SubStructure fingerprints performed better with maximum MCC value of 0.28. In this study, we have shown that molecular weight, polar surface area and rotatable bond count of inhibitors (replicating and non-replicating phase) are significantly different from non-inhibitors. The fragment analysis suggests that substructures like hetero_N_nonbasic, heterocyclic, carboxylic_ester, and hetero_N_basic_no_H are predominant in replicating phase inhibitors while hetero_O, ketone, secondary_mixed_amine are preferred in the non-replicative phase inhibitors. It was observed that nitro, alkyne, and enamine are important for the molecules inhibiting bacilli residing in both the phases. In this study, we introduced a new algorithm based on Matthews correlation coefficient called MCCA for feature selection and found that this algorithm is better or comparable to frequency based approach.ConclusionIn this study, we have developed computational models to predict phase specific inhibitors against drug resistant strains of M.tb grown under carbon starvation. Based on simple molecular properties, we have derived some rules, which would be useful in robust identification of tuberculosis inhibitors. Based on these observations, we have developed a webserver for predicting inhibitors against drug tolerant M.tb H37Rv available at http://crdd.osdd.net/oscadd/mdri/.

Dataset Information

Expression of a gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv.

Publications

Expression of a gene for a porin-like protein of the OmpA family from Mycobacterium tuberculosis H37Rv.

Similar Datasets

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