Spore formation by Clostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficileassociated disease. Spores are resistant to heat, radiation, chemicals, and antibiotics, making a contaminated environment difficult to clean. To cause disease, however, spores must germinate and grow out as vegetative cells. The germination of C. difficile spores has not been examined in detail. In an effort to understand the germination of C. difficile spores, we characterized the response of C. difficile spores to bile. We found that cholate derivatives and the amino acid glycine act as cogerminants. Deoxycholate, a metabolite of cholate produced by the normal intestinal flora, also induced germination of C. difficile spores but prevented the growth of vegetative C. difficile. A model of resistance to C. difficile colonization mediated by the normal bacterial flora is proposed.

We have determined the nucleotide sequence of two Bacillus subtilis promoters (veg and tms) that are utilized by the principal form of B. subtilis RNA polymerase found in vegetative cells (sigma 55-RNA polymerase) and have compared our sequences to those of several previously reported Bacillus promoters. Hexanucleotide sequences centered approximately 35 (the "--35" region) and 10 (the "--10" region) base pairs upstream from the veg and tms transcription starting points (and separated by 17 base pairs) corresponded closely to the consensus hexanucleotides (TTGACA and TATAAT) attributed to Escherichia coli promoters. Conformity to the preferred --35 and --10 sequences may not be sufficient to promote efficient utilization by B. subtilis RNA polymerase, however, since three promoters (veg, tms and E. coli tac) that conform to these sequences and that are utilized efficiently by E. coli RNA polymerase were used with highly varied efficiencies by B. subtilis RNA polymerase. We have also analyzed mRNA sequences in DNA located downstream from eight B. subtilis chromosomal and phage promoters for nucleotide sequences that might signal the initiation of translation. In accordance with the rules of McLaughlin, Murray and Rabinowitz (1981), we observe mRNA nucleotide sequences with extensive complementarity to the 3' terminal region of B. subtilis 16S rRNA, followed by an initiation codon and an open reading frame.

CodY, a highly conserved protein in the low G + C, gram-positive bacteria, regulates the expression of many Bacillus subtilis genes that are induced as cells make the transition from rapid exponential growth to stationary phase and sporulation. This transition has been associated with a transient drop in the intracellular pool of GTP. Many stationary-phase genes are also induced during exponential-growth phase by treatment of cells with decoyinine, a GMP synthetase inhibitor. The effect of decoyinine on an early-stationary-phase gene is shown here to be mediated through CodY and to reflect a reduction in guanine nucleotide accumulation. CodY proved to bind GTP in vitro. Moreover, CodY-mediated repression of target promoters was dependent on a high concentration of GTP, comparable to that found in rapidly growing exponential-phase cells. Because a codY-null mutant was able to sporulate under conditions of nutrient excess, CodY also appears to be a critical factor that normally prevents sporulation under such conditions. Thus, B. subtilis CodY is a novel GTP-binding protein that senses the intracellular GTP concentration as an indicator of nutritional conditions and regulates the transcription of early-stationary-phase and sporulation genes, allowing the cell to adapt to nutrient limitation. Our understanding of the relationship between environmental signals and global changes in gene expression is limited by the difficulty in identifying intracellular signaling molecules that interact with key regulatory proteins. This gap is particularly apparent for cases of general nutrient limitation. When Bacillus subtilis cells encounter nutrient limitation and enter stationary phase, a variety of adaptive processes-such as genetic competence, secretion of macromolecule-degrading enzymes, import of secondary nutrients, activation of metabolic pathways, chemotaxis and motility, production of antibiotics, and sporulation-are initiated (Sonenshein 1989). A network of global regulatory proteins modulates the cell's response and regulates the choice between adaptation to poor growth conditions and sporulation (Sonenshein 1989(Sonenshein , 2000Burkholder and Grossman 2000), but the specific signals to which these regulators respond have remained a mystery.Many B. subtilis genes that are expressed early in stationary phase are repressed by CodY (Table 1). Preliminary results indicate that CodY also contributes to regulation of at least two genes (citB, spo0A) whose products are necessary for sporulation (M. Ratnayake- Lecamwasam and A.L. Sonenshein, unpubl. Bolotin et al. 1999).CodY was first identified as a repressor of the B. subtilis dipeptide transport (dpp) operon and was found to be active when cells are grown with an excess of glucose or Casamino acids (CAA) as reported by Slack et al. (1995). During vegetative growth, the dpp operon is also directly repressed by AbrB, a second global regulator of earlystationary-phase genes Strauch 1993;Serror and Sonenshein 1996b), but the repressive effects of nutrient excess are mediat...

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...

SummaryThe Clostridium difficile toxA and toxB genes, encoding cytotoxic and enterotoxic proteins responsible for antibiotic-associated colitis and pseudomembranous colitis, were shown to be transcribed both from genespecific promoters and from promoters of upstream genes. However, the gene-specific transcripts represented the majority of tox gene mRNAs. The 5Ј ends of these mRNAs were shown to correspond to DNA sequences that had promoter activity when fused to the Escherichia coli ␤-glucuronidase (gusA) gene and introduced into C. perfringens. The appearance of tox mRNA in C. difficile was repressed during exponential growth phase but increased substantially as cells entered stationary phase. When glucose or other rapidly metabolizable sugars were present in the medium, the stationary phase-associated induction was inhibited, indicating that the toxin genes are subject to a form of catabolite repression. This glucose effect was general to many toxinogenic strains having varying levels of toxin production.

The remarkable ability of bacteria to adapt efficiently to a wide range of nutritional environments reflects their use of overlapping regulatory systems that link gene expression to intracellular pools of a small number of key metabolites. By integrating the activities of global regulators, such as CcpA, CodY and TnrA, Bacillus subtilis manages traffic through two metabolic intersections that determine the flow of carbon and nitrogen to and from crucial metabolites, such as pyruvate, 2-oxoglutarate and glutamate. Here, the latest knowledge on the control of these key intersections in B. subtilis is reviewed.

More than 200 direct CodY target genes in Staphylococcus aureus were identified by genome-wide analysis of in vitro DNA binding. This analysis, which was confirmed for some genes by DNase I footprinting assays, revealed that CodY is a direct regulator of numerous transcription units associated with amino acid biosynthesis, transport of macromolecules, and virulence. The virulence genes regulated by CodY fell into three groups. One group was dependent on the Agr system for its expression; these genes were indirectly regulated by CodY through its repression of the agr locus. A second group was regulated directly by CodY. The third group, which includes genes for alpha-toxin and capsule synthesis, was regulated by CodY in two ways, i.e., by direct repression and by repression of the agr locus. Since S. aureus CodY was activated in vitro by the branched chain amino acids and GTP, CodY appears to link changes in intracellular metabolite pools with the induction of numerous adaptive responses, including virulence.Bacterial survival depends upon the ability to sense and respond to environmental stresses, such as changes in temperature, pH, osmolarity, cell population density, and nutrient availability. Staphylococcus aureus has a well-characterized ability to survive when faced with suboptimal conditions, highlighted by the ability of S. aureus to persist in mammalian hosts both as a commensal and as a pathogen. Many regulators of S. aureus virulence gene expression have been characterized (6). With the exception of the stress-dependent activation of B and the link between CcpA (catabolite control protein A) and select virulence factor expression (46), however, the specific mechanisms of virulence regulation in response to changes in nutrient availability are largely unknown. The best-characterized regulator of S. aureus virulence in response to environmental changes is the Agr (accessory gene regulator) system. This system, encoded at the agr locus, includes a quorumsensing mechanism that activates a two-component system that controls synthesis of a regulatory RNA, RNAIII (for a review, see reference 33).CodY, a highly conserved regulatory protein of stationaryphase adaptation in low-GϩC Gram-positive bacteria, is emerging as a regulator of virulence in S. aureus (28,38,47) as well as in other Gram-positive pathogens (4,13,19,20,(28)(29)(30). First discovered in two nonpathogenic species, Bacillus subtilis and Lactococcus lactis, CodY senses nutrient availability by direct interaction with metabolite effectors. CodY homologs define a unique, winged helix-turn-helix-containing family of transcription factors. For B. subtilis CodY, as well as for CodY proteins from Clostridium difficile, Listeria monocytogenes, and Bacillus cereus, the effectors are GTP and the branched chain amino acids (BCAAs; isoleucine, leucine, and valine) (4,13,20,31,39,43). GTP and the BCAAs increase synergistically the affinity of CodY for its DNA target sites (17, 50). CodY proteins from L. lactis and Streptococcus pneumoniae, however, respond ...

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