Effects of surotomycin on Clostridium difficile viability and toxin production in vitro.

The increasing incidence and severity of infection by Clostridium difficile have stimulated attempts to develop new antimicrobial therapies. We report here the relative abilities of two antibiotics (metronidazole and vancomycin) in current use for treating C. difficile infection and of a third antimicrobial, surotomycin, to kill C. difficile cells at various stages of development and to inhibit the production of the toxin proteins that are the major virulence factors. The results indicate that none of the drugs affects the viability of spores at 8× MIC or 80× MIC and that all of the drugs kill exponential-phase cells when provided at 8× MIC. In contrast, none of the drugs killed stationary-phase cells or inhibited toxin production when provided at 8× MIC and neither vancomycin nor metronidazole killed stationary-phase cells when provided at 80× MIC. Surotomycin, on the other hand, did kill stationary-phase cells when provided at 80× MIC but did so without inducing lysis.

Identification of CodY targets in Bacillus anthracis by genome-wide in vitro binding analysis.

In Gram-positive bacteria, CodY is an important regulator of genes whose expression changes under conditions of nutrient limitation. Bacillus anthracis CodY represses or activates directly or indirectly approximately 500 genes. Affinity purification of CodY-DNA complexes was used to identify the direct targets of CodY. Of the 389 DNA binding sites that were copurified with CodY, 132 sites were in or near the regulatory regions governing the expression of 197 CodY-controlled genes, indicating that CodY controls many other genes indirectly. CodY-binding specificity was verified using electrophoretic mobility shift and DNase I footprinting assays for three CodY targets. Analysis of the bound sequences led to the identification of a B. anthracis CodY-binding consensus motif that was found in 366 of the 389 affinity-purified DNA regions. Regulation of the expression of the two genes directly controlled by CodY, sap and eag, encoding the two surface layer (S-layer) proteins, was analyzed further by monitoring the expression of transcriptional lacZ reporter fusions in parental and codY mutant strains. CodY proved to be a direct repressor of both sap and eag expression. Since the expression of the S-layer genes is under the control of both CodY and PagR (a regulator that responds to bicarbonate), their expression levels respond to both metabolic and environmental cues.

Multiple genes for the last step of proline biosynthesis in Bacillus subtilis.

The complete Bacillus subtilis genome contains four genes (proG, proH, proI, and comER) with the potential to encode Delta(1)-pyrroline-5-carboxylate reductase, a proline biosynthetic enzyme. Simultaneous defects in three of these genes (proG, proH, and proI) were required to confer proline auxotrophy, indicating that the products of these genes are mostly interchangeable with respect to the last step in proline biosynthesis.

Bacillus subtilis CodY represses early-stationary-phase genes by sensing GTP levels.

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.

Control of sporulation initiation in Bacillus subtilis.

Recent work has provided new insights into the mechanisms by which Bacillus subtilis responds to signals that reflect high population density and nutritional limitation, the mechanisms that regulate activation of the key transcription factor Spo0A, and the physical basis for critical aspects of the Spo0A phosphorelay.

Role of TnrA in nitrogen source-dependent repression of Bacillus subtilis glutamate synthase gene expression.

Synthesis of glutamate, the cell's major donor of nitrogen groups and principal anion, occupies a significant fraction of bacterial metabolism. In Bacillus subtilis, the gltAB operon, encoding glutamate synthase, requires a specific positive regulator, GltC, for its expression. In addition, the gltAB operon was shown to be repressed by TnrA, a regulator of several other genes of nitrogen metabolism and active under conditions of ammonium (nitrogen) limitation. TnrA was found to bind directly to a site immediately downstream of the gltAB promoter. As is true for other genes, the activity of TnrA at the gltAB promoter was antagonized by glutamine synthetase under certain growth conditions.

CcpC, a novel regulator of the LysR family required for glucose repression of the citB gene in Bacillus subtilis.

Synergistic carbon catabolite repression of the Bacillus subtilis aconitase (citB) gene by glucose and a source of 2-ketoglutarate is dependent on DNA sequences located upstream of the gene. Mutations in a dyad symmetry element centered at position -66 and in a repeat of the downstream arm of the dyad symmetry at position -27 cause derepressed citB expression. In this work, a protein able to bind to a DNA fragment containing these elements was purified and identified. This protein, named CcpC (Catabolite control protein C), shares sequence similarity with members of the LysR family of transcriptional regulators. In addition to binding to the citB promoter, CcpC bound to the promoter of the citZ gene, which encodes the cell's major citrate synthase and is subject to carbon catabolite repression. In a ccpC null mutant, expression of both citB and citZ was derepressed in glucose-glutamine minimal medium, indicating that CcpC is a negative regulator of citB and citZ gene expression. DNase I footprinting experiments showed that CcpC binds to two sites within the citB promoter region, corresponding to the dyad symmetry and -27 elements. In the presence of citrate, a putative inducer, only the dyad symmetry element was fully protected by CcpC. When the dyad symmetry element was mutated, CcpC was no longer able to bind to either the dyad symmetry or -27 elements. Repression of citB and citZ gene expression during anaerobiosis also proved to be mediated by CcpC.

A mutation in the 3-phosphoglycerate kinase gene allows anaerobic growth of Bacillus subtilis in the absence of ResE kinase.

The Bacillus subtilis ResD-ResE two-component signal transduction system is essential for aerobic and anaerobic respiration. A spontaneous suppressor mutant that expresses ResD-controlled genes and grows anaerobically in the absence of the ResE histidine kinase was isolated. In addition, aerobic expression of ResD-controlled genes in the suppressed strain was constitutive and occurred at a much higher level than that observed in the wild-type strain. The suppressing mutation, which mapped to pgk, the gene encoding 3-phosphoglycerate kinase, failed to suppress a resD mutation, suggesting that the suppressing mutation creates a pathway for phosphorylation of the response regulator, ResD, which is independent of the cognate sensor kinase, ResE. The pgk-1 mutant exhibited very low but measurable 3-phosphoglycerate kinase activity compared to the wild-type strain. The results suggest that accumulation of a glycolytic intermediate, probably 1, 3-diphosphoglycerate, is responsible for the observed effect of the pgk-1 mutation on anaerobiosis of resE mutant cells.

An enhancer element located downstream of the major glutamate dehydrogenase gene of Bacillus subtilis.

The rocG gene of Bacillus subtilis, encoding a catabolic glutamate dehydrogenase, is transcribed by SigL (sigma(54))-containing RNA polymerase and requires for its expression RocR, a member of the NtrC/NifA family of proteins that bind to enhancer-like elements, called upstream activating sequences (UAS). Unlike the case for other sigma(54)-dependent genes, rocG has no UAS; instead, its expression depends on a sequence located 1.5 kilobases downstream of the rocG promoter, beyond the end of the rocG coding region. The same sequence also serves as the UAS for the downstream rocABC operon and can activate rocG if moved upstream of its promoter. Furthermore, the activating sequence can be moved as far as 15 kilobases downstream of the rocG promoter and still retain partial activity.

Bacillus subtilis aconitase is an RNA-binding protein.

The aconitase protein of Bacillus subtilis was able to bind specifically to sequences resembling the iron response elements (IREs) found in eukaryotic mRNAs. The sequences bound include the rabbit ferritin IRE and IRE-like sequences in the B. subtilis operons that encode the major cytochrome oxidase and an iron uptake system. IRE binding activity was affected by the availability of iron both in vivo and in vitro. In eukaryotic cells, aconitase-like proteins regulate translation and stability of iron metabolism mRNAs in response to iron availability. A mutant strain of B. subtilis that produces an enzymatically inactive aconitase that was still able to bind RNA sporulated 40x more efficiently than did an aconitase null mutant, suggesting that a nonenzymatic activity of aconitase is important for sporulation. The results support the idea that bacterial aconitases, like their eukaryotic homologs, are bifunctional proteins, showing aconitase activity in the presence of iron and RNA binding activity when cells are iron-deprived.

The glucuronic acid utilization gene cluster from Bacillus stearothermophilus T-6.

A lambda-EMBL3 genomic library of Bacillus stearothermophilus T-6 was screened for hemicellulolytic activities, and five independent clones exhibiting beta-xylosidase activity were isolated. The clones overlap each other and together represent a 23.5-kb chromosomal segment. The segment contains a cluster of xylan utilization genes, which are organized in at least three transcriptional units. These include the gene for the extracellular xylanase, xylanase T-6; part of an operon coding for an intracellular xylanase and a beta-xylosidase; and a putative 15.5-kb-long transcriptional unit, consisting of 12 genes involved in the utilization of alpha-D-glucuronic acid (GlcUA). The first four genes in the potential GlcUA operon (orf1, -2, -3, and -4) code for a putative sugar transport system with characteristic components of the binding-protein-dependent transport systems. The most likely natural substrate for this transport system is aldotetraouronic acid [2-O-alpha-(4-O-methyl-alpha-D-glucuronosyl)-xylotriose] (MeGlcUAXyl3). The following two genes code for an intracellular alpha-glucuronidase (aguA) and a beta-xylosidase (xynB). Five more genes (kdgK, kdgA, uxaC, uxuA, and uxuB) encode proteins that are homologous to enzymes involved in galacturonate and glucuronate catabolism. The gene cluster also includes a potential regulatory gene, uxuR, the product of which resembles repressors of the GntR family. The apparent transcriptional start point of the cluster was determined by primer extension analysis and is located 349 bp from the initial ATG codon. The potential operator site is a perfect 12-bp inverted repeat located downstream from the promoter between nucleotides +170 and +181. Gel retardation assays indicated that UxuR binds specifically to this sequence and that this binding is efficiently prevented in vitro by MeGlcUAXyl3, the most likely molecular inducer.

Metabolic imbalance and sporulation in an isocitrate dehydrogenase mutant of Bacillus subtilis.

A Bacillus subtilis mutant with a deletion in the citC gene, encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, exhibited reduced growth yield in broth medium and had greatly reduced ability to sporulate compared to the wild type due to a block at stage I, i.e., a failure to form the polar division septum. In early stationary phase, mutant cells accumulated intracellular and extracellular concentrations of citrate and isocitrate that were at least 15-fold higher than in wild-type cells. The growth and sporulation defects of the mutant could be partially bypassed by deletion of the major citrate synthase gene (citZ), by raising the pH of the medium, or by supplementation of the medium with certain divalent cations, suggesting that abnormal accumulation of citrate affects survival of stationary-phase cells and sporulation by lowering extracellular pH and chelating metal ions. While these genetic and environmental alterations were not sufficient to allow the majority of the mutant cell population to pass the stage I block (lack of asymmetric septum formation), introduction of the sof-1 mutant form of the Spo0A transcription factor, when coupled with a reduction in citrate synthesis, restored sporulation gene expression and spore formation nearly to wild-type levels. Thus, the primary factor inhibiting sporulation in a citC mutant is abnormally high accumulation of citrate, but relief of this metabolic defect is not by itself sufficient to restore competence for sporulation.

Role of SpoVG in asymmetric septation in Bacillus subtilis.

Deletion of the citC gene, coding for isocitrate dehydrogenase, arrests sporulation of Bacillus subtilis at stage I after bipolar localization of the cell division protein FtsZ but before formation of the asymmetric septum. A spontaneous extragenic suppressor mutation that overcame the stage I block was found to map within the spoVG gene. The suppressing mutation and other spoVG loss-of-function mutations enabled citC mutant cells to form asymmetric septa and to activate the forespore-specific sigma factor sigmaF. However, little induction of mother cell-specific, sigmaE-dependent sporulation genes was observed in a citC spoVG double mutant, indicating that there is an additional defect(s) in compartmentalized gene expression in the citC mutant. These other defects could be partially overcome by reducing the synthesis of citrate, by buffering the medium, or by adding excess MnCl2. Overexpression of the spoVG gene in wild-type cells significantly delayed sigmaF activation. Increased expression and stability of SpoVG in citC mutant cells may contribute to the citC mutant phenotype. Inactivation of the spoVG gene caused a population of otherwise wild-type cells to produce a small number of minicells during growth and caused sporulating cells to complete asymmetric septation more rapidly than normal. Unlike the case for inactivation of the cell division inhibitor gene minD, many of these minicells contained DNA and appeared only when the primary sporulation signal transduction pathway, the Spo0A phosphorelay, was active. These results suggest that SpoVG interferes with or is a negative regulator of the pathway leading to asymmetric septation.

Role and regulation of Bacillus subtilis glutamate dehydrogenase genes.

The complete Bacillus subtilis genome contains two genes with the potential to encode glutamate dehydrogenase (GlutDH) enzymes. Mutations in these genes were constructed and characterized. The rocG gene proved to encode a major GlutDH whose synthesis was induced in media containing arginine or ornithine or, to a lesser degree, proline and was repressed by glucose. A rocG null mutant was impaired in utilization of arginine, ornithine, and proline as nitrogen or carbon sources. The gudB gene was expressed under all growth conditions tested but codes for a GlutDH that seemed to be intrinsically inactive. Spontaneous mutations in gudB that removed a 9-bp direct repeat within the wild-type gudB sequence activated the GudB protein and allowed more-efficient utilization of amino acids of the glutamate family.

Anaerobic regulation of Bacillus subtilis Krebs cycle genes.

Krebs cycle enzyme activity in Bacillus subtilis was examined under aerobic and anaerobic conditions. Citrate synthase and aconitase activities in cells grown anaerobically in the presence of nitrate were reduced by as much as 10- and 30-fold, respectively, from levels observed under aerobic culture conditions. The maximum level of isocitrate dehydrogenase activity during anaerobic growth was only twofold lower than that in aerobic cultures. These reductions in activity under conditions of anaerobiosis were found to be primarily the result of reduced Krebs cycle gene transcription. This repression was not dependent on either the fnr or resDE gene products, which have been shown to regulate expression of other B. subtilis genes in response to anaerobic conditions. Additionally, catabolite control proteins CcpA and CcpB were not responsible for the repression. A dyad symmetry element located between positions -73 and -59 relative to the transcription start site of the aconitase gene (citB) promoter was previously shown to be a target of catabolite repression and the binding site for a putative negative regulator during aerobic growth. The deletion of the upstream arm of the dyad symmetry region abolished the citB repression observed during anaerobic growth. Furthermore, neither citZ or citB was repressed in an anaerobically grown citB mutant, an effect that was very likely the result of citrate accumulation. These results suggest that catabolite repression and anaerobic repression of citZ and citB are regulated by a common mechanism that does not involve CcpA, CcpB, Fnr, or ResDE.

Regulated transcription of Clostridium difficile toxin genes.

The 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 gene-specific promoters and from promoters of upstream genes. However, the gene-specific transcripts represented the majority of tox gene mRNAs. The 5' ends of these mRNAs were shown to correspond to DNA sequences that had promoter activity when fused to the Escherichia coli beta-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.

A null mutation in the Bacillus subtilis aconitase gene causes a block in Spo0A-phosphate-dependent gene expression.

The citB gene of Bacillus subtilis encodes aconitase, the enzyme of the Krebs citric acid cycle, which is responsible for the interconversion of citrate and isocitrate. A B. subtilis strain with an insertion mutation in the citB gene was devoid of aconitase activity and aconitase protein, required glutamate for growth in minimal medium, and was unable to sporulate efficiently in nutrient broth sporulation medium. Mutant cells failed to form the asymmetric septum characteristic of sporulating cells and were defective in transcription of the earliest-expressed spo genes, that is, the genes dependent on the Spo0A phosphorelay. However, this early block in sporulation was partially overcome when cells of the citB mutant were induced to sporulate by resuspension in a poor medium. Accumulation of citrate in the mutant cells or in their culture fluid may be responsible for the early block, possibly because citrate can chelate divalent cations needed for the activity of the phosphorelay.

An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport.

The azlB locus of Bacillus subtilis was defined previously by a mutation conferring resistance to a leucine analog, 4-azaleucine (J. B. Ward, Jr., and S. A. Zahler, J. Bacteriol. 116:727-735, 1973). In this report, azlB is shown to be the first gene of an operon apparently involved in branched-chain amino acid transport. The product of the azlB gene is an Lrp-like protein that negatively regulates expression of the azlBCDEF operon. Resistance to 4-azaleucine in azlB mutants is due to overproduction of AzlC and AzlD, two novel hydrophobic proteins.

Deletion of the Bacillus subtilis isocitrate dehydrogenase gene causes a block at stage I of sporulation.

A Bacillus subtilis mutant with a deletion of citC, the gene encoding isocitrate dehydrogenase, the third enzyme of the tricarboxylic acid branch of the Krebs cycle, had a greatly reduced ability to sporulate. Analysis of expression of lacZ fusions to various sporulation gene promoters revealed that in the citC mutant development is probably blocked between stage 0 and stage II. That is, genes expressed very early in sporulation, under the direct control of the Spo0A transcription factor, were induced normally in the citC mutant. However, genes expressed after asymmetric septation (stage II) in wild-type cells were not induced in the citC mutant. Analysis of cell morphology by thin-section electron microscopy and immunofluorescence microscopy showed that the mutant formed axial chromosomal filaments and accumulated rings of FtsZ protein at potential polar division sites but failed to form asymmetric division septa, indicating that sporulation is blocked at stage I. The growth and sporulation defects of the B. subtilis citC mutant were fully overcome by introduction and expression of the Escherichia coli icd gene, encoding an isocitrate dehydrogenase similar to the enzyme from B. subtilis.