Identification and characterization of dps promoter of Mycobacterium smegmatis: Promoter recognition by stress specific ECF sigma factors ^H and ^F

Molecular Biophysics Unit, Indian Institute of Science, Bangalore-560012, INDIA

^* To whom correspondence should be addressed. Email: dipankar{at}mbu.iisc.ernet.in.

	Abstract

Survival of a bacterium under depleted oxygen or nutrient supply is important for its long term persistence inside the host under stressful conditions. We have studied a gene dps from Mycobacterium smegmatis, encoding a protein Dps (DNA binding Protein from Starved cells), which gets over expressed under oxidative and nutritional stresses and provides bimodal protection to the bacterial DNA. Characterization of the dps promoter in vivo, hence, becomes important. We had cloned upstream 1 kb putative promoter region of the dps gene of M. smegmatis in an E. coli-Mycobacterium shuttle vector pSD5B, immediate upstream of the lacZ gene. Promoter activity was assayed in vivo both in solid medium as well as in liquid cultures by quantitative -galactosidase activity measurements. To characterize the minimal promoter region, a 200 bp fragment from this whole 1 kb sequence was further cloned in the same vector and in similar way -galactosidase activity was quantitated. Primer extension analysis was performed to obtain the +1 transcription start site of the gene. Point base mutations were inserted in the putative promoter sequences at -10 and -20 region and the promoter sequence was confirmed. The promoter was not recognized by purified M. smegmatis core RNA polymerase reconstituted with purified M. tuberculosis ^A or ^B during multiple and single round in vitro transcription assays. Promoter specific in vivo pull-down assay with immobilized 1 kb DNA fragment containing dps promoter established that ECF sigma factors were associated with this starvation inducible promoter. Single round transcription at dps promoter further supported that only core RNA polymerase reconstituted with ^F or ^H can generate proper transcripts.