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In several Gram-positive bacterial species, the global transcriptional regulatory protein CodY adjusts the expression of many metabolic genes, apparently in response to changes in the pools of specific metabolites, i.e., the branched-chain amino acids (BCAAs) isoleucine, leucine, and valine (ILV) and the nucleoside triphosphate GTP. CodY not only responds to these metabolites as measured in vitro but also regulates the genes that direct their synthesis. We have constructed a set of strains lacking binding sites for the CodY protein in c/s at loci coding for the ILV biosynthetic machinery, effectively overexpressing these genes in an attempt to modulate the ILV input signal to CodY. Metabolite analyses of strains derepressed for genes needed for ILV synthesis revealed more than a 6-fold increase in the valine pool and a 2-fold increase in the isoleucine and leucine pools. Accumulation of the branched-chain amino acids was accompanied by a 24-fold induction of the bkd operon (required for branched-chain fatty acid synthesis) and 6-fold hyperrepression of the CodY-regulated yhdG and yufN genes, demonstrating that CodY perceives intracellular fluctuations in at least one if its input signals. We conclude that changes in the rate of endogenous ILV synthesis serve as an important signal for CodY-mediated gene regulation. doi: 10.1128/JB.00937-10

CodY, a global regulatory protein that monitors the nutrient sufficiency of the environment by responding to the intracellular levels of GTP and the branched-chain amino acids, was previously shown to be a potent repressor of toxin gene expression in Clostridium difficile during growth in rich medium. In the intestinal tract, such derepression of toxin synthesis would lead to destruction of epithelial cells and the liberation of potential nutrients for the bacterium. CodY is likely to play an important role in regulating overall cellular physiology as well. In this study, DNA microarray analysis and affinity purification of CodY-DNA complexes were used to identify and distinguish the direct and indirect effects of CodY on global gene transcription. A codY null mutation resulted in >4-fold overexpression of 146 genes (organized in 82 apparent transcription units) and underexpression of 19 genes. In addition to the toxin genes, genes for amino acid biosynthesis, nutrient transport, fermentation pathways, membrane components, and surface proteins were overexpressed in the codY mutant. Genome-wide analysis identified more than 350 CodY binding regions, many of which are likely to correspond to sites of direct CodY-mediated regulation. About 60% of the CodY-repressed transcription units were associated with binding regions. Several of these genes were confirmed to be direct targets of CodY by gel mobility shift and DNase I footprinting assays. doi: 10.1128/JB.00341-10

To cause disease, Clostridium difficile spores must germinate in the host gastrointestinal tract. Germination is initiated upon exposure to glycine and certain bile acids, e.g., taurocholate. Chenodeoxycholate, another bile acid, inhibits taurocholate-mediated germination. By applying Michaelis-Menten kinetic analysis to C. difficile spore germination, we found that chenodeoxycholate is a competitive inhibitor of taurocholate-mediated germination and appears to interact with the spores with greater apparent affinity than does taurocholate. We also report that several analogs of chenodeoxycholate are even more effective inhibitors. Some of these compounds resist 7[alpha]-dehydroxylation by Clostridium scindens, a core member of the normal human colonic microbiota, suggesting that they are more stable than chenodeoxycholate in the colonic environment. doi: 10.1128/JB.00610-10

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. doi: 10.1128/JB.00220-10

Bacillus subtilis CodY protein is a DNA-binding global transcriptional regulator that responds to branched-chain amino acids (isoleucine, leucine, and valine) and GTP. Crystal structure studies have shown that the N-terminal region of the protein includes a GAF domain that contains a hydrophobic pocket within which isoleucine and valine bind. This region is well conserved in CodY homologs. Site-directed mutagenesis was employed to understand the roles of some of the residues in the GAF domain and hydrophobic pocket in interaction with isoleucine and GTP. The F40A, F71E, and F98A forms of CodY were inactive in vivo. They were activatable by GTP but to a much lesser extent by branched-chain amino acids in vitro. The CodY mutant R61A retained partial repression of target promoters in vivo and was able to respond to GTP in vitro but also responded poorly to branched-chain amino acids in vitro unless GTP was simultaneously present. Thus, the GAF domain includes residues essential for full activation of CodY by branched-chain amino acids, but these residues are not critical for activation by GTP. Binding studies with branched-chain amino acids and their analogs revealed that an amino group at position 2 and a methyl group at position 3 of valine are critical components of the recognition of the amino acids by CodY. doi: 10.1128/JB.00818-09

Citrate synthase, the first and rate-limiting enzyme of the tricarboxylic acid branch of the Krebs cycle, was shown to be required for de novo synthesis of glutamate and glutamine in Listeria monocytogenes. The citrate synthase (citZ) gene was found to be part of a complex operon with the upstream genes lmo1569 and lmo1568. The downstream isocitrate dehydrogenase (citC) gene appears to be part of the same operon as well. Two promoters were shown to drive citZ expression, a distal promoter located upstream of lmo1569 and a proximal promoter located upstream of the lmo1568 gene. Transcription of citZ from both promoters was regulated by CcpC by interaction with a single site; assays of transcription in vivo and assays of CcpC binding in vitro revealed that CcpC interacts with and represses the proximal promoter that drives expression of the lmo1568, citZ, and citC genes and, by binding to the same site, prevents read-through transcription from the distal, lmo1569 promoter. Expression of the lmo1568 operon was not affected by the carbon source but was repressed during growth in complex medium by addition of glutamine.

Clostridium thermocellum is an anaerobic thermophilic bacterium that grows efficiently on cellulosic biomass. This bacterium produces and secretes a highly active multienzyme complex, the cellulosome, that mediates the cell attachment to and hydrolysis of the crystalline cellulosic substrate. C. thermocellum can efficiently utilize only [beta]-1,3 and [beta]-1,4 glucans and prefers long celiodextrins. Since the bacterium can also produce ethanol, it is considered an attractive candidate for a consolidated fermentation process in which cellulose hydrolysis and ethanol fermentation occur in a single process. In this study, we have identified and characterized five sugar ABC transporter systems in C. thermocellum. The putative transporters were identified by sequence homology of the putative solute-binding lipoprotein to known sugar-binding proteins. Each of these systems is transcribed from a gene cluster, which includes an extracellular solute-binding protein, one or two integral membrane proteins, and, in most cases, an ATP-binding protein. The genes of the five solute-binding proteins were cloned, fused to His tags, overexpressed, and purified, and their abilities to interact with different sugars was examined by isothermal titration calorimetry. Three of the sugar-binding lipoproteins (CbpB to -D) interacted with different lengths of cellodextrins ([G.sub.2] to [G.sub.5]), with disassociation constants in the micromolar range. One protein, CbpA, binds only cellotriose ([G.sub.3]), while another protein, Lbp (laminaribiose- binding protein) interacts with laminaribiose. The sugar specificity of the different binding lipoproteins is consistent with the observed substrate preference of C. thermocellum, in which cellodextrins ([G.sub.3] to [G.sub.5]) are assimilated faster than cellobiose.

Spore formation by Ciostridium difficile is a significant obstacle to overcoming hospital-acquired C. difficile-associated 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.

CodY is a global regulatory protein that was first discovered in Bacillus subtilis, where it couples gene expression to changes in the pools of critical metabolites through its activation by GTP and branched-chain amino acids. Homologs of CodY can be found encoded in the genomes of nearly all low-G+C gram-positive bacteria, including Staphylococcus aureus. The introduction of a codY-null mutation into two S. aureus clinical isolates, SA564 and UAMS-1, through allelic replacement, resulted in the overexpression of several virulence genes. The mutant strains had higher levels of hemolytic activity toward rabbit erythrocytes in their culture fluid, produced more polysaccharide intercellular adhesin (PIA), and formed more robust biofilms than did their isogenic parent strains. These phenotypes were associated with derepressed levels of RNA for the hemolytic alpha-toxin (h/a), the accessory gene regulator (agr) (RNAII and RNAIII/hid), and the operon responsible for the production of PIA (icaADBC). These data suggest that CodY represses, either directly or indirectly, the synthesis of a number of virulence factors of S. aureus.

CodY is a global transcriptional regulator that is known to control directly the expression of at least two dozen operons in Bacillus subtilis, but the rules that govern the binding of CodY to its target DNA have been unclear. Using DNase I footprinting experiments, we identified CodY-binding sites upstream of the B. subtilis ylmA and yurP genes. The protected regions overlapped versions of a previously proposed CodY-binding consensus motif, AATTTTCWGAAAATT. Multiple single mutations were introduced into the CodY-binding sites of the ylmA, yurP, dppA, and ilvB genes. The mutations affected both the affinity of CodY for its binding sites in vitro and the expression in vivo of lacZ fusions that carry these mutations in their promoter regions. Our results show that versions of the AATTTTCWGAAAATT motif, first identified for Lactococcus lactis CodY, with up to five mismatches play an important role in the interaction of B. subtilis CodY with DNA.

Many of the adaptive mechanisms that allow Bacillus subtilis to adjust to changes in nutrient availability are controiled by CodY. Binding of CodY to its target genes is stimulated by interaction with its effectors, GTP and the branched-chain amino acids (BCAAs). Upon nutrient limitation, intracellular pools of these effectors are depleted and CodY can no Ionger repress genes required for adaptation. In vitro studies reported here explored in more detail the interaction of CodY with GTP. DNase I footprinting experiments indicated that CodY has an atfinity for GTP in the millimolar range. Further, CodY was shown to interact specifically with GTP and dGTP; no other naturally occurring nucleotides that were tested, including ppGpp and pppGpp, resulted in DNA protection. Two nonhydrolyzable anaiogs of GTP were fully able to activate CodY binding to target DNA, demonslrating that GTP hydrolysis is not necessary for CodY-dependent regulation. GTP and the BCAAs were shown to act additively to increase the affinity of CodY for DNA; increased protection was observed in DNase I footprinting experiments when both effectors were present, compared to either effector alone, and in in vitro transcription reactions, transcriptional repression by CodY was stronger in the presence of both GTP and BCAAs than of BCAAs alone. Thus, interaction of CodY with GTP is specific and results in increased attinity for its target genes. This increase in aflinity is independent of GTP hydrolysis and is augmented in the presence of BCAAs.

Expression of yeast mitochondrial aconitase (Aco1) in a Bacillus subtilis aconitase null mutant restored aconitase activity and glutamate prototrophy but only partially restored sporulation. Late sporulation gene expression in the Aco1-expressing strain was delayed.

Bacillus subtilis aconitase, encoded by the citB gene, is homologous to the bifunctional eukaryotic protein IRP-1 (iron regulatory protein 1). Like IRP-1, B. subtilis aconitase is both an enzyme and an RNA binding protein. In an attempt to separate the two activities of aconitase, the C-terminal region of the B. subtilis citB gene product was mutagenized. The resulting strain had high catalytic activity but was defective in sporulation. The defect was at a late stage of sporulation, specifically affecting expression of [[sigma].sup.K]-dependent genes, many of which are important for spore coat assembly and require transcriptional activation by GerE. Accumulation of gerE mRNA and GerE protein was delayed in the aconitase mutant strain. Pure B. subtilis aconitase bound to the 3' untranslated region of gerE mRNA in in vitro gel mobility shift assays, strongly suggesting that aconitase RNA binding activity may stabilize gerE mRNA in order to allow efficient GerE synthesis and proper timing of spore coat assembly.

In Bacillus subtilis, the catabolite control protein C (CcpC) plays a critical role in regulating the genes encoding the enzymes of the tricarboxylic acid branch of the Krebs citric acid cycle. A gene encoding a potential CcpC homolog and two potential target genes were identified in the Listeria monocytogenes genome. In vitro gel mobility shift assays and DNase I footprinting experiments showed that L. monocytogenes CcpC (CCp[C.sub.Lm]) interacts with the promoter regions of citBLm (the gene that is likely to encode aconitase) and lmo0847 (encoding a possible glutamine transporter) and that citrate is a specific inhibitor of this interaction. To study in vivo promoter activity, a new lacZ reporter system was developed. This system allows stable integration into the chromosome of a promoter region transcriptionally fused to a promoterless lacZ gene at a nonessential, ectopic locus. Analysis of strains carrying a cit[B.sub.LB]-lacZ or lmo0847-lacZ fusion revealed that CCp[C.sub.Lm] represses cit[B.sub.Lm] and lmo0847 in media containing an excess of glucose and glutamine. In addition, regulation of CCp[C.sub.Lm] expression in rich medium was growth phase dependent; during exponential growth phase, expression was very low even in the absence of CCp[C.sub.Lm], but a higher level of CCp[C.sub.Lm] expression was induced in stationary phase, suggesting the involvement of another, as yet unidentified regulatory factor.

Clostridium difficile is the primary causative agent of antibiotic-associated diarrheal disease. To facilitate molecular genetic analysis of gene expression in this organism, methods were developed to study transcriptional regulation in vitro and in vivo. That is, C. difficile RNA polymerase was partially purified and shown to bind to and initiate transcription in vitro from bona fide C. difficile promoters for rRNA and glutamate dehydrogenase genes. In addition, primer extension analyses and a [beta]-glucuronidase reporter system were used to quantitate transcription from these promoters in vivo. With these tools in hand, it is now possible to characterize the behavior of any C. difficile gene in vivo and to study the regulation of its expression in detail.

Bacillus subtilis CodY protein is the best-studied member of a novel family of global transcriptional regulators found ubiquitously in low-G+C gram-positive bacteria. As for many DNA-binding proteins, CodY appears to have a helix-turn-helix (HTH) motif thought to be critical for interaction with DNA. This putative HTH motif was found to be highly conserved in the CodY homologs. Site-directed mutagenesis was used to identify amino acids within this motif that are important for DNA recognition and binding. The effects of each mutation on DNA binding in vitro and on the regulation of transcription in vivo from two target promoters were tested. Each of the mutations had similar effects on binding to the two promoters in vitro, but some mutations had differential effects in vivo.

The Bacillus subtilis rocG gene, encoding catabolic glutamate dehydrogenase, was found to be subject to direct CcpA-dependent glucose repression. The effect of CcpA required the presence of both the HPr and Crh proteins. The primary CcpA binding site was identified by mutational analysis and DNase I footprinting. In the absence of inducers of the Roc pathway, rocG was still expressed at a low level due to readthrough transcription. CcpA-dependent repression of rocG readthrough transcription proved to contribute to the slow growth rate of B. subtilis cells in glucose-glutamate medium. Increased readthrough expression of rocG was shown to be partially responsible for the growth defect of ccpA strains in glucose-ammonium medium.

The Bacillus subtilis gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression and is repressed by the global regulatory protein TnrA. The factor that controls TnrA activity, a complex of glutamine synthetase and a feedback inhibitor, such as glutamine, is known, but the signal for modulation of GltC activity has remained elusive. GltC-dependent gltAB expression was drastically reduced when cells were grown in media containing arginine or ornithine or proline, all of which are inducers and substrates of the Roc catabolic pathway. Analysis of gltAB expression in mutants with various defects in the Roc pathway indicated that rocG-encoded glutamate dehydrogenase was required for such repression, suggesting that the substrates or products of this enzyme are the real electors of GltC. Given that RocG is an enzyme of glutamate catabolism, the main regulatory role of GltC may be prevention of a futile cycle of glutamate synthesis and degradation in the presence of arginine-related amino acids or proline. In addition, high activity of glutamate dehydrogenase was incompatible with activity of TnrA.

Research has been conducted on sigma factor Clostridium difficile TxeR for toxin gene expression. The role of environmental conditions in tox gene expression and the possibility that these conditions control TxeR synthesis and regulate tox genes indirectly have been tested, and the results are reported.

Research discusses the genetic basis for the origin of nitrogen-containing groups in bacteria. Results demonstrate that glutamate or glutamine are the key compounds involved whose synthesis is mediated by glutamine synthetase and glutamine synthase. The enzyme synthesis is regulated by gltAB operon and TnrA regulator.

Research was conducted to understand the role of the two-component ResD-ResE signal transduction system in the respiratory metabolism of Bacillus subtilis. Results show glycolytic intermediate 1,3-diphosphoglycerate activate ResD independent of ResE as revealed by an ResE mutant strain.

Research was conducted to examine the role and regulation of Bacillus subtilis glutamate dehydrogenase (GlutDH) genes. The objective was to analyze the involvement of the two genes in utilization of amino acids of the glutamate family. Results provide evidence that the rocG gene encoded themajor catabolic GlutDH while gudB seemed to encode an intrinsically inactive GlutDH. Active catabolic GlutDH were generated by spontaneous mutations in gudB.

Research was conducted to examine a citB null mutant of Bacillus subtilis. Plasmid DNA was determined from Escherichia coli while heat resistance was measured after the end of the exponential phase for all cultures. Results indicated that the null mutant was characterized by a severe early defect in sporulation when prepared in nutrient broth sporulation medium. They also suggested that the relationship between citrate accumulation and the early block in sporulation may be related to the capability of citrate to chelate divalent cations.

The lrp-like gene of Bacillus subtilis that participates in branched-chain amino acid transport was examined. It was discovered that azlB is the first gene of an operon involved in branched-chain amino acid transport. Furthermore, the B. subtilis lrp-like gene is identical to the azlB. The azlB gene does not play any role in the glutamate synthase regulation. Five new B. subtilis gene products belonging to the AsnC-lrp family of transcription were determined.

A bacillus subtilis mutant with the deleted gene citC encoding isocitrate dehydrogenase has educed sporulation ability. The expression of lacZ fusions to sporulation gene promoters showed that in the citC mutant development may be blocked between stage 0 and stage II. Cell morphology analysis by thin-section electron microscopy and immunofluerescence microscopy showed the formation of axial chromosomal filaments and rings of FtsZ protein.

The genetic characteristics and biochemical function of purified GltR proteins from Bacillus subtilis cells were analyzed to determine the mechanisms that mediated the genetic transcription of the LysR gene. The Bacillus subtilis LysR transcriptional regulators such as GltR and GltR24 bonded in different ways to the gltR and gltC-gltA regulatory regions. Furthermore, a mutation in the gltR gene induced the activation of gltAB by GltR in the absence of the GltC transcriptional regulator.

The CodY gene is required for the effect of Casamino acids on competence. When the growth medium contains Casamino acids, the acquisition of genetic competence by Bacillus subtilis is repressed. Such repression is exerted at the level of expression from the promoters of the competence-regulatory genes srfA and comK and is relieved in strains carrying a null mutation in the codY gene.

Bacillus subtilis GlnR was purified to apparent homogeneity and demonstrated to interfere with transcription by binding to four tandem segments of DNA that overlap the glnRA promoter site. The sites of binding correlate well with the results of mutagenesis experiments that define the glnRA operator site and with in vivo determinations of the GlnR-DNA contact points.

A gene located downstream of the genes coding for isocitrate dehydrogenase and citrate synthase in Bacillus subtilis was found to encode malate dehydrogenase in the bacterium. The gene, designated citH, was initially discovered as an open reading frame consisting of 312 codons. Results revealed that in mutants where the citH gene was disrupted, sporulation required higher aspartate levels to progress.

Mutant forms of GltC in Bacillus subtilis may cause an increase in gltA expression by two times in wild-type bacteria and by 80 times in bacteria with promoter mutations. Researchers analyzed the effects of three gltC mutations on gltA expression, which is regulated by GltC at its promoter. The mutations also caused gltA expression to require less of a signal from a nitrogen source.

The gltA and gltC genes, involved in the production of glutamate synthase in Bacillus subtilis, may be regulated by two consensus binding sites, Box I and Box II. Box I is involved in activation of gltA and autorepression of gltC. Box II is necessary for gltA activation. The location of the Boxes and the effects of inserting helical twists between them are discussed.

Analysis of the modulation of transcription of the citrate synthase genes citZ and citA in Bacillus subtilis reveals that after induction of the drug decoyinine into the bacterial cells, the citZ mRNA level was five times higher than the citA mRNA level. The citA gene transcription level is six times less with glucose as carbon source than with succinate as carbon source. Glutamate and glucose caused a 10-fold reduction and glucose alone causes a 2-fold reduction in the transcription of citZ gene. CitR protein from LysR family inhibits citA but not citZ gene expression.

Genetic complementation of the citrate synthase mutant strain W620 of Escherichia coli and PCR-based probing of the chromosomal DNA of Bacillus subtilis help isolate two different citrate synthase genes citZ and citA from B. subtilis. Null mutations in the citA gene have no influence on sporulation or citrate synthase enzyme functions, while citZ gene mutations induce severe abnormalities in sporulation and reduce citrate synthase enzyme functions. The citA gene was present in a monocistronic transcriptional unit while the citZ gene was present in a tricistronic cluster, coexisting with citH and citC genes.

The sporulation and transcription of some metabolic genes are suppressed by glucose and amino acids but the molecular mechanism by which nutrients influence these metabolic activities remain unidentified. An attempt was made to identify the additional regulators of metabolic activities by seeking mutations that would facilitate the transcription of dciA in cells ordinarily exposed to low concentrations of nutrients. These mutations were identified as Cmlr colonies by employing the dciAp-cat-lacZ fusion construction. Some of these mutations were identified near the dciA promoter region while others were found near the chromosomes.

The control of early sporulation gene expression by the Bacillus subtilis gsiA operon was reported. It was shown that the product of the spo0A gene is necessary to turnoff gsiA transcription, while the product of another locus, gsiC, is necessary to turnoff expression of gsiA. Overexpression of gsiA causes reduced expression of the spoIIA, spoIIG and spoIID genes. Moreover, it was also shown that gsiC82 is an allele of the kinA gene, which encodes the histidine protein kinase of a two-component signal transduction system.

The structure and regulation of Bacillus subtilis glucose starvation-inducible genes gsiA and gsiB were characterized. Both genes are repressed during rapid exponential growth and are induced in response to glucose starvation and other forms of nutritional limitation. Induction of gsiA expression is dependent on the ComP-ComA signal transduction system. Furthermore, it was proposed that gsiA and gsiB are members of an adaptive pathway of genes whose products are involved in nonsporulation responses to nutrient limitation.

The RNA polymerase factor sigma-E directs the transcription of sporulation genes spoIID and spoIIID as well as other Bacillus subtilis cloned genes. The primary translation product of sigE is pro-sigma-E, the inactive form of sigma-E, which is later processed to its active form by the removal of 19 N-terminal amino acids. A mutational screening isolated mutants in the precursor region of pro-sigma-E, resulting in high level expression of spoIID. The region involved in the inactivation of pro-sigma-E are the amino acids 1 to 16.

Some cholate derivatives that are normal components of bile can act with glycine to induce the germination of Clostridium difficile spores, but at least one bile component, chenodeoxycholate, does not induce germination. Here we show that chenodeoxycholate inhibits the germination of C. difficile spores in response to cholate and taurocholate.

Purification and characterization of the major citrate synthase of Bacillus subtilis (CS-II) have identified a protein corresponding to the product of the citZ gene. Analysis of the physical and kinetic properties showed that CS-II has high homology to the citrate synthase enzymes from B. megaterium and from eukaryotic cells, but is significantly different to enzymes from gram-negative bacteria.