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Expression of the major porin gene mspA is regulated in Mycobacterium smegmatis.

ABSTRACT: MspA is the major porin of Mycobacterium smegmatis and is important for diffusion of small and hydrophilic solutes across its unique outer membrane. The start point of transcription of the mspA gene was mapped by primer extension and S1 nuclease experiments. The main promoter driving transcription of mspA was identified by single point mutations in lacZ fusions and resembled sigma(A) promoters of M. smegmatis. However, a 500-bp upstream fragment including P(mspA) in a transcriptional fusion with lacZ yielded only low beta-galactosidase activity, whereas activity increased 12-fold with a 700-bp fragment. Activation of P(mspA) by the 200-bp element was almost eliminated by increasing the distance by 14 bp, indicating binding of an activator protein. The chromosomal mspA transcript had a size of 900 bases and was very stable with a half-life of 6 minutes, whereas the stabilities of episomal mspA transcripts with three other 5' untranslated region (UTRs) were three- to sixfold reduced, indicating a stabilizing role of the native 5' UTR of mspA. Northern blot experiments revealed that the amount of mspA mRNA was increased under nitrogen limitation but reduced under carbon and phosphate limitation at 42 degrees C in stationary phase in the presence of 0.5 M sodium chloride, 18 mM hydrogen peroxide, and 10% ethanol and at acidic pH. These results show for the first time that M. smegmatis regulates porin gene expression to optimize uptake of certain nutrients and to protect itself from toxic solutes.

SUBMITTER: Hillmann D

PROVIDER: S-EPMC1797333 | biostudies-literature | 2007 Feb

REPOSITORIES: biostudies-literature

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Expression of the major porin gene mspA is regulated in Mycobacterium smegmatis.

Hillmann Dietmar D Eschenbacher Iris I Thiel Anja A Niederweis Michael M

Journal of bacteriology 20061201 3

MspA is the major porin of Mycobacterium smegmatis and is important for diffusion of small and hydrophilic solutes across its unique outer membrane. The start point of transcription of the mspA gene was mapped by primer extension and S1 nuclease experiments. The main promoter driving transcription of mspA was identified by single point mutations in lacZ fusions and resembled sigma(A) promoters of M. smegmatis. However, a 500-bp upstream fragment including P(mspA) in a transcriptional fusion with ...[more]

PMID: 17142388

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Project description:The recalcitrance of mycobacteria to antibiotic therapy is in part due to its ability to build proteins into a multi-layer cell wall. Proper synthesis of both cell wall constituents and associated proteins is crucial to maintaining cell integrity, and intimately tied to antibiotic susceptibility. How mycobacteria properly synthesize the membrane-associated proteome, however, remains poorly understood. Recently, we found that loss of lepA in Mycobacterium smegmatis (Msm) altered tolerance to rifampin, a drug that targets a non-ribosomal cellular process. LepA is a ribosome-associated GTPase found in bacteria, mitochondria, and chloroplasts, yet its physiological contribution to cellular processes is not clear. To uncover the determinants of LepA-mediated drug tolerance, we characterized the whole-cell proteomes and transcriptomes of a lepA deletion mutant relative to strains with lepA We find that LepA is important for the steady-state abundance of a number of membrane-associated proteins, including an outer membrane porin, MspA, which is integral to nutrient uptake and drug susceptibility. Loss of LepA leads to a decreased amount of porin in the membrane which leads to the drug tolerance phenotype of the lepA mutant. In mycobacteria, the translation factor LepA modulates mycobacterial membrane homeostasis, which in turn affects antibiotic tolerance.ImportanceThe mycobacterial cell wall is a promising target for new antibiotics due to the abundance of important membrane-associated proteins. Defining mechanisms of synthesis of the membrane proteome will be critical to uncovering and validating drug targets. We found that LepA, a universally conserved translation factor, controls the synthesis of a number of major membrane proteins in M. smegmatis LepA primarily controls synthesis of the major porin MspA. Loss of LepA results in decreased permeability through the loss of this porin, including permeability to antibiotics like rifampin and vancomycin. In mycobacteria, regulation from the ribosome is critical for the maintenance of membrane homeostasis and, importantly, antibiotic susceptibility.

Dataset Information

Expression of the major porin gene mspA is regulated in Mycobacterium smegmatis.

Publications

Expression of the major porin gene mspA is regulated in Mycobacterium smegmatis.

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