The Mycobacterium tuberculosis katG promoter region contains a novel upstream activator.
An Escherichia coli-mycobacterial shuttle vector, pJCluc, containing a luciferase reporter gene, was constructed and used to analyse the Mycobacterium tuberculosis katG promoter. A 1.9 kb region immediately upstream of katG promoted expression of the luciferase gene in E. coli and Mycobacterium smegmatis. A smaller promoter fragment (559 bp) promoted expression with equal efficiency, and was used in all further studies. Two transcription start sites were mapped by primer extension analysis to 47 and 56 bp upstream of the GTG initiation codon. Putative promoters associated with these show similarity to previously identified mycobacterial promoters. Deletions in the promoter fragment, introduced with BAL-31 nuclease and restriction endonucleases, revealed that a region between 559 and 448 bp upstream of the translation initiation codon, designated the upstream activator region (UAR), is essential for promoter activity in E. coli, and is required for optimal activity in M. smegmatis. The katG UAR was also able to increase expression from the Mycobacterium paratuberculosis P(AN) promoter 15-fold in E. coli and 12-fold in M. smegmatis. An alternative promoter is active in deletion constructs in which either the UAR or the katG promoters identified here are absent. Expression from the katG promoter peaks during late exponential phase, and declines during stationary phase. The promoter is induced by ascorbic acid, and is repressed by oxygen limitation and growth at elevated temperatures. The promoter constructs exhibited similar activities in Mycobacterium bovis BCG as they did in M. smegmatis.
Mulder
Michelle A
MA
Department of Medical Microbiology, University of Cape Town and Groote Schuur Hospital, Medical School, Observatory, 7925 Cape Town, South Africa1.
Zappe
Harold
H
Department of Medical Microbiology, University of Cape Town and Groote Schuur Hospital, Medical School, Observatory, 7925 Cape Town, South Africa1.
Steyn
Lafras M
LM
Department of Medical Microbiology, University of Cape Town and Groote Schuur Hospital, Medical School, Observatory, 7925 Cape Town, South Africa1.
D001426:Bacterial Proteins; D001483:Base Sequence; D017931:DNA Primers; D004269:DNA, Bacterial / Q000032:analysis; D004926:Escherichia coli / Q000235:genetics; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D005822:Genetic Vectors; D008969:Molecular Sequence Data; D020102:Mycobacterium smegmatis / Q000201:enzymology / Q000235:genetics / Q000254:growth & development; D009169:Mycobacterium tuberculosis / Q000201:enzymology / Q000235:genetics* / Q000254:growth & development; D010544:Peroxidases / Q000235:genetics* / Q000378:metabolism*; D016133:Polymerase Chain Reaction / Q000379:methods; D011401:Promoter Regions, Genetic / Q000235:genetics*; D012329:RNA, Bacterial / Q000032:analysis / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014158:Transcription, Genetic; D014169:Transformation, Bacterial
D001426:Bacterial Proteins; D017931:DNA Primers; D004269:DNA, Bacterial; D012329:RNA, Bacterial; D010544:Peroxidases; C076136:catalase HPI