SummaryThe master regulator for entry into sporulation in Bacillus subtilis is the DNA-binding protein Spo0A, which has been found to influence, directly or indirectly, the expression of over 500 genes during the early stages of development. To search on a genomewide basis for genes under the direct control of Spo0A, we used chromatin immunoprecipitation in combination with gene microarray analysis to identify regions of the chromosome at which an activated form of Spo0A binds in vivo . This information in combination with transcriptional profiling using gene microarrays, gel electrophoretic mobility shift assays, using the DNA-binding domain of Spo0A, and bioinformatics enabled us to assign 103 genes to the Spo0A regulon in addition to 18 previously known members. Thus, in total, 121 genes, which are organized as 30 single-gene units and 24 operons, are likely to be under the direct control of Spo0A. Forty of these genes are under the positive control of Spo0A, and 81 are under its negative control. Among newly identified members of the regulon with transcription that was stimulated by Spo0A are genes for metabolic enzymes and genes for efflux pumps. Among members with transcription that was inhibited by Spo0A are genes encoding components of the DNA replication machinery and genes that govern flagellum biosynthesis and chemotaxis. Also included in the regulon are many (25) genes with products that are direct or indirect regulators of gene transcription. Spo0A is a master regulator for sporulation, but many of its effects on the global pattern of gene transcription are likely to be mediated indirectly by regulatory genes under its control.
Additional targets of CodY, a GTP-activated repressor of early stationary-phase genes in Bacillus subtilis, were identified by combining chromatin immunoprecipitation, DNA microarray hybridization, and gel mobility shift assays. The direct targets of CodY newly identified by this approach included regulatory genes for sporulation, genes that are likely to encode transporters for amino acids and sugars, and the genes for biosynthesis of branched-chain amino acids.Bacteria have evolved a variety of mechanisms to accommodate gene expression to changes in nutritional availability. Some of these mechanisms are specific to a particular gene or operon. In other cases, regulatory proteins control large groups of genes of related function, such as the nitrogen metabolism genes regulated by the Ntr system in enteric bacteria (43) and by TnrA in Bacillus subtilis (17) and the carbon metabolism genes regulated by CcpA in gram-positive bacteria (13) and catabolic gene activator protein-cyclic AMP complex in gram-negative bacteria (59). Even broader forms of regulation are mediated by the leucine-responsive protein (Lrp) of gram-negative bacteria and the sigma-B protein of B. subtilis. Lrp and sigma-B control the transcription of operons that have diverse functions but have a common need to be expressed under a particular set of environmental conditions (50, 54). Lrp regulates the biosynthesis of leucine, isoleucine, valine, serine, glycine, and glutamate; the degradation of serine and threonine; transport of peptides, amino acids, and sugars; and production of fimbriae in response to the availability of leucine and serine (50). Sigma-B activates transcription of a host of genes when cells are exposed to excessive heat, ethanol, salt, or acid (54). Sigma-B responds through a complex, multibranched signal transduction pathway.The B. subtilis CodY protein also has broad effects on gene expression. CodY is a GTP-binding repressor of several genes that are normally quiescent when cells are growing in a rich medium (57). A high concentration of GTP activates CodY as a repressor (57). When the growth rate of B. subtilis slows down because of limitation of the carbon or nitrogen or phosphorus source, the GTP level drops (39, 40), CodY loses repressing activity, and targets of CodY repression are transcribed. The known targets of CodY in B. subtilis include the genes that encode transport systems for dipeptides (dpp) (65) and ␥-aminobutyrate (gabP) (16); catabolic pathways for acetate (acsA) (S. H. Fisher, personal communication), urea (ureABC) (71), histidine (hut) (18), arginine (rocABC and roc-DEF) (B. Belitsky, personal communication), and branchedchain keto acids (the bkd operon) (12); an enzyme of surfactin synthesis (srfAA) (63); the transcription factor for DNA uptake genes (comK) (63); a ComA aspartyl phosphate phosphatase and its inhibitor (rapC-phrC) (37); motility and chemotaxis (hag, fla/che) (45; F. Bergara, C. Ibarra, J. Iwamasa, R. Aguilera, and L. M. Màrquez-Magaña, submitted for publication); and aconitase (citB) (3...
SummaryIn the Gram-positive, antibiotic-producing bacterium Streptomyces coelicolor A3(2), the thiol-disulphide status of the hyphae is controlled by a novel regulatory system consisting of a sigma factor, s R , and its cognate anti-sigma factor, RsrA. Oxidative stress induces intramolecular disulphide bond formation in RsrA, which causes it to lose affinity for s R , thereby releasing s R to activate transcription of the thioredoxin operon, trxBA. Here, we exploit a preliminary consensus sequence for s R target promoters to identify 27 new s R target genes and operons, thereby defining the global response to disulphide stress in this organism. Target genes related to thiol metabolism encode a second thioredoxin (TrxC), a glutaredoxin-like protein and enzymes involved in the biosynthesis of the low-molecular-weight thiol-containing compounds cysteine and molybdopterin. In addition, the level of the major actinomycete thiol buffer, mycothiol, was fourfold lower in a sigR null mutant, although no candidate mycothiol biosynthetic genes were identified among the s R targets. Three s R target genes encode ribosome-associated products (ribosomal subunit L31, ppGpp synthetase and tmRNA), suggesting that the translational machinery is modified by disulphide stress. The product of another s R target gene was found to be a novel RNA polymerase-associated protein, RbpA, suggesting that the transcriptional machinery may also be modified in response to disulphide stress. We present DNA sequence evidence that many of the targets identified in S. coelicolor are also under the control of the s R homologue in the actinomycete pathogen Mycobacterium tuberculosis.
SummaryMycolic acids are very long-chain fatty acids representing essential components of the mycobacterial cell wall. Considering their importance, characterization of key enzymes participating in mycolic acid biosynthesis not only allows an understanding of their role in the physiology of mycobacteria, but also might lead to the identification of new drug targets. Mycolates are synthesized by at least two discrete elongation systems, the type I and type II fatty acid synthases (FAS-I and FAS-II respectively). Among the FAS-II components, the condensing enzymes that catalyse the formation of carbon-carbon bonds have received considerable interest. Four condensases participate in initiation (mtFabH), elongation (KasA and KasB) and termination (Pks13) steps, leading to full-length mycolates. We present the recent biochemical and structural data for these important enzymes. Special emphasis is given to their role in growth, intracellular survival, biofilm formation, as well as in the physiopathology of tuberculosis. Recent studies demonstrated that phosphorylation of these enzymes by mycobacterial kinases affects their activities. We propose here a model in which kinases that sense environmental changes can phosphorylate the condensing enzymes, thus representing a novel mechanism of regulating mycolic acid biosynthesis. Finally, we discuss the attractiveness of these enzymes as valid targets for future antituberculosis drug development.
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