Mycobacterium tuberculosis FurA autoregulates its own expression

The furA-katG region of Mycobacterium tuberculosis, encoding a Fur-like protein and the catalase-peroxidase, is highly conserved among mycobacteria. Both genes are induced upon oxidative stress. In this work we analyzed the M. tuberculosis furA promoter region. DNA fragments were cloned upstream of the luciferase reporter gene, and promoter activity in Mycobacterium smegmatis was measured in both the presence and absence of oxidative stress. The shortest fragment containing an inducible promoter extends 45 bp upstream of furA. In this region, -35 and -10 promoter consensus sequences can be identified, as well as a 23-bp AT-rich sequence that is conserved in the nonpathogenic but closely related M. smegmatis. M. tuberculosis FurA was purified and found to bind upstream of furA by gel shift analysis. A ca. 30-bp DNA sequence, centered on the AT-rich region, was essential for FurA binding and protected by FurA in footprinting analysis. Peroxide treatment of FurA abolished DNA binding. Three different AT-rich sequences mutagenized by site-directed mutagenesis were constructed. In each mutant, both M. tuberculosis FurA binding in vitro and pfurA regulation upon oxidative-stress in M. smegmatis were abolished. Thus, pfurA is an oxidative stress-responsive promoter controlled by the FurA protein.     

A new gene, sigG, encoding a putative alternative sigma factor of Streptomyces coelicolor A3(2)

An oligonucleotide probe encoding a peptide motif conserved in all sigma factors was used to isolate a new gene, sigG, from a Streptomyces coelicolor A3(2) genomic library. The deduced protein of 263 amino acids with an M(r) of 29,422 showed the greatest similarity to the previously identified sporulation sigma factor (sigma F) of Streptomyces coelicolor, and general stress response sigma factor (sigma B) of Bacillus subtilis, mostly in domains suggested to be involved in recognition of -10 and -35 promoter regions. Southern-blot hybridization with DNA from several Streptomyces spp. revealed the presence of a similar gene in all strains tested. Disruption of the S. coelicolor sigG gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin.     

Stress-response sigma factor σΗ is essential for morphological differentiation of Streptomyces coelicolor A3(2)

We previously cloned the sigH gene encoding a stress-response sigma factor sigma(H) in Streptomyces coelicolor A3(2), located in an operon with the gene encoding proposed anti-sigma factor UshX. To clarify the in vivo function of sigma(H), a stable null mutant of sigH was prepared by homologous recombination. This mutation appeared to have no obvious effect on vegetative growth, but dramatically affected morphological differentiation. Microscopy showed that the sigH mutant produced undifferentiated hyphae with rare spore chains, giving the colony a pale gray color compared to the dark gray wild-type spores. The sigH mutation partially affected growth under conditions of high osmolarity. Expression of the sigH operon was investigated in the S. coelicolor sigH mutant. Out of four promoters directing expression of the sigH operon, the sigH-P2 promoter--the only promoter preferentially induced by salt-stress conditions--was inactive in the sigH mutant. The results indicated that the sigH-P2 promoter is dependent (directly or indirectly) upon sigma(H) and that the operon is autocatalytically activated. We propose that in S. coelicolor sigma(H) has a dual role, regulating the osmotic response and morphological differentiation.