sigA is an essential gene in Mycobacterium smegmatis.
sigA encodes a sigma factor of the sigma70 family, sigmaA, that is found in all mycobacterial species. As sigmaA shows high similarity to the primary sigma factor in Streptomyces coelicolor, it was postulated that sigmaA has the same role in mycobacteria. However, a point mutation in sigA, resulting in the replacement of arginine 522 by histidine, was found responsible for the attenuated virulence of the Mycobacterium bovis strain ATCC 35721. This raised the possibility that sigmaA was an alternative sigma factor specifically required for virulence gene expression. In this work, we show that sigA can not be disrupted in Mycobacterium smegmatis unless an extra copy of the gene is provided at another chromosomal site, which demonstrates that sigA is essential. To characterize the pattern of sigA expression during exponential and stationary phase in M. smegmatis, we measured the beta-galactosidase activity in a strain carrying a sigA-lacZ transcriptional fusion and monitored sigmaA levels using Western blotting. Our results indicate that sigA is expressed throughout the growth of the culture. The essential character of sigA and its pattern of expression corroborate the hypothesis that sigA codes for the primary sigma factor in M. smegmatis and, most likely, in all mycobacteria.
Gomez
M
M
Department of Microbiology, Public Health Research Institute, New York, NY 10016, USA.
Doukhan
L
L
Nair
G
G
Smith
I
I
D001483:Base Sequence; D012321:DNA-Directed RNA Polymerases / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000235:genetics* / Q000254:growth & development; D011401:Promoter Regions, Genetic; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D012689:Sequence Homology, Nucleic Acid; D012808:Sigma Factor / Q000235:genetics*; D001616:beta-Galactosidase / Q000235:genetics / Q000378:metabolism
D011993:Recombinant Fusion Proteins; D012808:Sigma Factor; C081775:RNA polymerase sigma 70; D012321:DNA-Directed RNA Polymerases; D001616:beta-Galactosidase