Expression of the major porin gene mspA is regulated in Mycobacterium smegmatis.
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.
Hillmann
Dietmar
D
Department of Microbiology, University of Alabama at Birmingham, 609 Bevill Biomedical Research Building, 845 19th Street South, Birmingham, AL 35294, USA.
Eschenbacher
Iris
I
Thiel
Anja
A
Niederweis
Michael
M
D024506:5' Flanking Region; D001483:Base Sequence; D015152:Blotting, Northern; D000431:Ethanol / Q000494:pharmacology; D020869:Gene Expression Profiling; D015964:Gene Expression Regulation, Bacterial / Q000187:drug effects; D005947:Glucose / Q000494:pharmacology; D006863:Hydrogen-Ion Concentration; D008958:Models, Molecular; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000254:growth & development; D018272:Porins / Q000235:genetics*; D011401:Promoter Regions, Genetic; D020871:RNA Stability; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D013696:Temperature; D024363:Transcription Initiation Site; D014158:Transcription, Genetic / Q000187:drug effects
D018272:Porins; D012333:RNA, Messenger; C121838:mspA protein, Mycobacterium smegmatis; D000431:Ethanol; D005947:Glucose