Identification and semi-quantitative analysis of Mycobacterium tuberculosis H37Rv ftsZ gene-specific promoter activity-containing regions.
The cytokinetic protein FtsZ plays a pivotal role in regulation of cell division in bacteria. Multiple promoters regulate transcription of the ftsZ gene in Escherichia coli, Streptomyces and Bacillus species. In order to identify promoter activity-containing regions of the ftsZ gene of Mycobacterium tuberculosis H37Rv (MtftsZ) in vivo, different regions upstream of MtftsZ, namely, the ftsQ-ftsZ intergenic region, the ftsQ open reading frame (ORF), and different regions of ftsQ ORF, were analyzed in a gfp reporter plasmid in Mycobacterium smegmatis mc(2)155 cells. Flow cytometric analysis of mid-logarithmic M. smegmatis mc(2)155 cells containing these transcription fusion constructs revealed GFP expression in the cells harboring the ftsQ-ftsZ intergenic region (172 bp), the entire ftsQ ORF (945 bp), and 5' 467-bp and 3' 217-bp regions of ftsQ ORF. RT-PCR analyses on RNA from M. smegmatis mc(2)155 cells, transformed with the entire ftsQ ORF-ftsQ-ftsZ intergenic region-containing construct, as well as on RNA from M. tuberculosis, confirmed that the regions identified indeed elicit promoter activity. Semi-quantitative RT-PCR analyses of gfp transcripts driven by cloned MtftsZ promoter regions in M. smegmatis cells showed threefold higher promoter activity from ftsQ ORF than from the ftsQ-ftsZ intergenic region. Expression from the individual 5' and 3' regions of ftsQ ORF was almost equivalent to that from the ftsQ-ftsZ intergenic region. RT-PCR analyses on RNA from M. tuberculosis quantitatively confirmed these promoter activities. Thus, at least three independent regions in the immediate upstream sequence of MtftsZ contain promoter activity, with the major contribution coming from ftsQ ORF.
Roy
Sougata
S
Department of Microbiology and Cell Biology, Indian Institute of Science, Bangalore 560012, India.
Mir
Mushtaq Ahmad
MA
Anand
Syam Prasad
SP
Niederweis
Michael
M
Ajitkumar
Parthasarathi
P
D001426:Bacterial Proteins / Q000235:genetics*; D001483:Base Sequence; D003001:Cloning, Molecular; D003598:Cytoskeletal Proteins / Q000235:genetics*; D004269:DNA, Bacterial / Q000737:chemistry / Q000235:genetics; D005434:Flow Cytometry; D015964:Gene Expression Regulation, Bacterial; D049452:Green Fluorescent Proteins; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000235:genetics / Q000502:physiology; D009169:Mycobacterium tuberculosis / Q000235:genetics*; D011401:Promoter Regions, Genetic; D012329:RNA, Bacterial / Q000737:chemistry / Q000235:genetics; D011994:Recombinant Proteins; D020133:Reverse Transcriptase Polymerase Chain Reaction; D014158:Transcription, Genetic / Q000502:physiology
D001426:Bacterial Proteins; D003598:Cytoskeletal Proteins; D004269:DNA, Bacterial; C071525:FtsZ protein, Bacteria; D012329:RNA, Bacterial; D011994:Recombinant Proteins; D049452:Green Fluorescent Proteins