The dipeptide D-Ala-D-Ala is an essential component of peptidoglycan and the target of vancomycin. Most Clostridioides difficile strains possess the vanG operon responsible for the synthesis of D-Ala-D-Ser, which can replace D-Ala-D-Ala in peptidoglycan. The C. difficile vanG operon is regulated by a two-component system, VanRS, but is not induced sufficiently by vancomycin to confer resistance to this antibiotic. Surprisingly, in the absence of the VanS histidine kinase (HK), the vanG operon is still induced by vancomycin and also by another antibiotic, ramoplanin, in a VanR-dependent manner. This suggested the cross-regulation of VanR by another HK or kinases that are activated in the presence of certain lipid II-targeting antibiotics. We identified these HKs as CD35990 and CD22880. However, mutations in either or both HKs did not affect the regulation of the vanG operon in wild-type cells suggesting that intact VanS prevents the cross-activation of VanR by non-cognate HKs. Overproduction of VanR in the absence of VanS, CD35990, and CD22880 led to high expression of the vanG operon indicating that VanR can potentially utilize at least one more phosphate donor for its activation. Candidate targets of CD35990- and CD22880-mediated regulation in the presence of vancomycin or ramoplanin were identified by RNA-Seq.

A Clostridioides difficile strain deficient in the ddl gene is unable to synthesize the dipeptide D‐Ala‐D‐Ala, an essential component of peptidoglycan and the target of vancomycin. We isolated spontaneous suppressors of a ∆ ddl mutation that allowed cell growth in the absence of D‐Ala‐D‐Ala. The mutations caused constitutive or partly constitutive expression of the vancomycin‐inducible vanG operon responsible for the synthesis of D‐Ala‐D‐Ser, which can replace D‐Ala‐D‐Ala in peptidoglycan. The mutations mapped to the vanS or vanR genes, which regulate expression of the vanG operon. The constitutive level of vanG expression was about 10‐fold above that obtained by vancomycin induction. The incorporation of D‐Ala‐D‐Ser into peptidoglycan due to high expression of the vanG operon conferred only low‐level resistance to vancomycin, but VanG was found to synthesize D‐Ala‐D‐Ala in addition to D‐Ala‐D‐Ser. However, the same, low resistance to vancomycin was also observed in cells completely unable to synthesize D‐Ala‐D‐Ala and grown in the presence of D‐Ala‐D‐Ser. D‐Ala‐D‐Ala presence was required for efficient vancomycin induction of the vanG operon showing that vancomycin is not by itself able to activate VanS. D‐Ala‐D‐Ser, similar to D‐Ala‐D‐Ala, served as an anti‐activator of DdlR, the positive regulator of the ddl gene, thereby coupling vanG and ddl expression.

CodY is a global transcriptional regulator that controls, directly or indirectly, the expression of dozens of genes and operons in Listeria monocytogenes. We used in vitro DNA affinity purification combined with massively parallel sequencing (IDAP-Seq) to identify genome-wide L. monocytogenes chromosomal DNA regions that CodY binds in vitro. The total number of CodY-binding regions exceeded 2,000, but they varied significantly in their strengths of binding at different CodY concentrations. The 388 strongest CodY-binding regions were chosen for further analysis. A strand-specific analysis of the data allowed pinpointing CodY-binding sites at close to single-nucleotide resolution. Gel shift and DNase I footprinting assays confirmed the presence and locations of several CodY-binding sites. Surprisingly, most of the sites were located within genes' coding regions. The binding site within the beginning of the coding sequence of the prfA gene, which encodes the master regulator of virulence genes, has been previously implicated in regulation of prfA, but this site was weaker in vitro than hundreds of other sites. The L. monocytogenes CodY protein was functionally similar to Bacillus subtilis CodY when expressed in B. subtilis cells. Based on the sequences of the CodY-binding sites, a model of CodY interaction with DNA is proposed.

L. monocytogenes is a widespread foodborne pathogen. Nitrogen-containing compounds, such as the glutamate-containing tripeptide, glutathione, and glutamine, have been shown to be important for expression of L. monocytogenes virulence genes. In this work, we showed that a transcriptional regulator, GlnR, controls expression of critical listerial genes of nitrogen metabolism that are involved in ammonium uptake and biosynthesis of glutamine and glutamate. A different mode of GlnR-mediated regulation was found for each of these three pathways.

D-Ala-D-Ala ligase, encoded by ddl genes, is responsible for the synthesis of a dipeptide, D-Ala-D-Ala, an essential precursor of bacterial peptidoglycan. In Clostridioides difficile, the single ddl gene is located upstream of the ddlR gene, which encodes a putative transcriptional regulator. Using mutational and transcriptional analysis and DNA-binding assays, DdlR was found to be a direct activator of the ddl ddlR operon. DdlR is a member of the MocR/GabR-type proteins that have aminotransferase-like, pyridoxal 5 '-phosphate-binding domains. A DdlR mutation that prevented covalent binding of pyridoxal 5 '-phosphate abolished the ability of DdlR to activate transcription. Addition of D-Ala-D-Ala to the medium inactivated DdlR, reducing dipeptide biosynthesis. In contrast, D-Ala-D-Ala limitation caused a dramatic increase in expression from the ddl promoter. Though uncommon for transcription regulators, C. difficile DdlR is essential, as the ddlR null mutant cells could not grow even in complex laboratory media in the absence of D-Ala-D-Ala. A dyad symmetry sequence, which is located immediately upstream of the -35 region of the ddl promoter, serves as an important element of the DdlR-binding site. This sequence is conserved upstream of putative DdlR targets in other bacteria of classes Clostridia and Bacilli, indicating a similar mode of regulation of these genes.

Toxin synthesis and endospore formation are two of the most critical factors that determine the outcome of infection by Clostridioides difficile. The two major toxins, TcdA and TcdB, are the principal factors causing damage to the host. Spores are the infectious form of C. difficile, permit survival of the bacterium during antibiotic treatment and are the predominant cell form that leads to recurrent infection. Toxin production and sporulation have their own specific mechanisms of regulation, but they share negative regulation by the global regulatory protein CodY. Determining the extent of such regulation and its detailed mechanism is important for understanding the linkage between two apparently independent biological phenomena and raises the possibility of creating new ways of limiting infection. The work described here shows that a codY null mutant of a hypervirulent (ribotype 027) strain is even more virulent than its parent in a mouse model of infection and that the mutant expresses most sporulation genes prematurely during exponential growth phase. Moreover, examining the expression patterns of mutants producing CodY proteins with different levels of residual activity revealed that expression of the toxin genes is dependent on total CodY inactivation, whereas most sporulation genes are turned on when CodY activity is only partially diminished. These results suggest that, in wild-type cells undergoing nutrient limitation, sporulation genes can be turned on before the toxin genes.

The Bacillus subtilis protein regulator of the gabTD operon and its own gene (GabR) is a transcriptional activator that regulates transcription of γ-aminobutyric acid aminotransferase (GABA-AT; GabT) upon interactions with pyridoxal-5'-phosphate (PLP) and GABA, and thereby promotes the biosynthesis of glutamate from GABA. We show here that the external aldimine formed between PLP and GABA is apparently responsible for triggering the GabR-mediated transcription activation. Details of the "active site" in the structure of the GabR effector-binding/oligomerization (Eb/O) domain suggest that binding a monocarboxylic γ-amino acid such as GABA should be preferred over dicarboxylic acid ligands. A reactive GABA analog, (S)-4-amino-5-fluoropentanoic acid (AFPA), was used as a molecular probe to examine the reactivity of PLP in both GabR and a homologous aspartate aminotransferase (Asp-AT) from Escherichia coli as a control. A comparison between the structures of the Eb/O-PLP-AFPA complex and Asp-AT-PLP-AFPA complex revealed that GabR is incapable of facilitating further steps of the transamination reaction after the formation of the external aldimine. Results of in vitro and in vivo assays using full-length GabR support the conclusion that AFPA is an agonistic ligand capable of triggering GabR-mediated transcription activation via formation of an external aldimine with PLP.

CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Grampositive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (c (G) under bar MP-stimulated phosphodiesterases, (a) under bar denylate cyclases, (F) under bar hlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn alpha-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodYIle derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19-36-bp operator fragments are consistent with isoleucine dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed.

ABSTRACT AprE and NprE are two major extracellular proteases in Bacillus subtilis whose expression is directly regulated by several pleiotropic transcriptional factors, including AbrB, DegU, ScoC, and SinR. In cells growing in a rich, complex medium, the aprE and nprE genes are strongly expressed only during the post-exponential growth phase; mutations in genes encoding the known regulators affect the level of post-exponential-phase gene expression but do not permit high-level expression during the exponential growth phase. Using DNA-binding assays and expression and mutational analyses, we have shown that the genes for both exoproteases are also under strong, direct, negative control by the global transcriptional regulator CodY. However, because CodY also represses scoC , little or no derepression of aprE and nprE was seen in a codY null mutant due to overexpression of scoC . Thus, CodY is also an indirect positive regulator of these genes by limiting the synthesis of a second repressor. In addition, in cells growing under conditions that activate CodY, a scoC null mutation had little effect on aprE or nprE expression; full effects of scoC or codY null mutations could be seen only in the absence of the other regulator. However, even the codY scoC double mutant did not show high levels of aprE and nprE gene expression during exponential growth phase in a rich, complex medium. Only a third mutation, in abrB , allowed such expression. Thus, three repressors can contribute to reducing exoprotease gene expression during growth in the presence of excess nutrients. IMPORTANCE The major Bacillus subtilis exoproteases, AprE and NprE, are important metabolic enzymes whose genes are subject to complex regulation by multiple transcription factors. We show here that expression of the aprE and nprE genes is also controlled, both directly and indirectly, by CodY, a global transcriptional regulator that responds to the intracellular pools of amino acids. Direct CodY-mediated repression explains a long-standing puzzle, that is, why exoproteases are not produced when cells are growing exponentially in a medium containing abundant quantities of proteins or their degradation products. Indirect regulation of aprE and nprE through CodY-mediated repression of the scoC gene, encoding another pleiotropic repressor, serves to maintain a significant level of repression of exoprotease genes when CodY loses activity.

CodY and ScoC are Bacillus subtilis transcriptional regulators that control the expression of dozens of genes and operons. Using scoC-lacZ fusions and DNA-binding experiments, we show here that scoC is directly repressed by CodY. This effect creates multiple forms of cascade regulation. For instance, expression of the dtpT gene, which is directly and negatively controlled by ScoC and encodes a putative oligopeptide permease, was activated indirectly by CodY due to CodY-mediated repression of scoC. The opp operon, which encodes an oligopeptide permease that is essential for sporulation and genetic competence development, proved to be a direct target of repression by both ScoC and CodY but was not significantly affected in codY or scoC single mutants. The combined actions of CodY and ScoC maintain opp repression when either one of the regulators loses activity but limit the level of repression to that provided by one of the regulators acting alone. Under conditions of nitrogen limitation, repression by ScoC of dtpT and opp was partly prevented by TnrA. Thus, the functioning of ScoC is determined by other transcription factors via modulation of its expression or DNA binding.

ABSTRACT CodY is a global transcriptional regulator in low-G+C Gram-positive bacteria that is responsive to GTP and branched-chain amino acids. By interacting with its two cofactors, it is able to sense the nutritional and energetic status of the cell and respond by regulating expression of adaptive genetic programs. In Bacillus subtilis , more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. In this study, we demonstrated that expression of two extracellular proteases, Vpr and Mpr, is negatively controlled by CodY. By gel mobility shift and DNase I footprinting assays, we showed that CodY binds to the regulatory regions of both genes, in the vicinity of their transcription start points. The mpr gene is also characterized by the presence of a second, higher-affinity CodY-binding site located at the beginning of its coding sequence. Using strains carrying vpr - or mpr-lacZ transcriptional fusions in which CodY-binding sites were mutated, we demonstrated that repression of both protease genes is due to the direct effect by CodY and that the mpr internal site is required for regulation. The vpr promoter is a rare example of a sigma H-dependent promoter that is regulated by CodY. In a codY null mutant, Vpr became one of the more abundant proteins of the B. subtilis exoproteome. IMPORTANCE CodY is a global transcriptional regulator of metabolism and virulence in low-G+C Gram-positive bacteria. In B. subtilis , more than 200 genes, including those for peptide transporters, intracellular proteolytic enzymes, and amino acid degradative pathways, are controlled by CodY. However, no role for B. subtilis CodY in regulating expression of extracellular proteases has been established to date. In this work, we demonstrate that by binding to the regulatory regions of the corresponding genes, B. subtilis CodY negatively controls expression of Vpr and Mpr, two extracellular proteases. Thus, in B. subtilis , CodY can now be seen to regulate the entire protein utilization pathway.

ABSTRACT CodY is a branched-chain amino acid-responsive transcriptional regulator that controls the expression of several dozen transcription units in Bacillus subtilis . The presence of isoleucine, valine, and leucine in the growth medium is essential for achieving high activity of CodY and for efficient regulation of the target genes. We identified three permeases—BcaP, BraB, and BrnQ—that are responsible for the bulk of isoleucine and valine uptake and are also involved in leucine uptake. At least one more permease is capable of efficient leucine uptake, as well as low-affinity transport of isoleucine and valine. The lack of the first three permeases strongly reduced activity of CodY in an amino acid-containing growth medium. BcaP appears to be the most efficient isoleucine and valine permease responsible for their utilization as nitrogen sources. The previously described strong CodY-mediated repression of BcaP provides a mechanism for fine-tuning CodY activity by reducing the availability of amino acids and for delaying the utilization of isoleucine and valine as nitrogen and carbon sources under conditions of nutrient excess. IMPORTANCE Bacillus subtilis CodY is a global transcriptional regulator that is activated by branched-chain amino acids (BCAA). Since the level of BCAA achieved by intracellular synthesis is insufficient to fully activate CodY, transport of BCAA from the environment is critical for CodY activation, but the permeases needed for such activation have not been previously identified. This study identifies three such permeases, reports their amino acid transport specificity, and reveals their impact on CodY activation.

The global transcriptional regulator, CodY, binds strongly to the regulatory region of the braB gene, which encodes a Bacillus subtilis branched-chain amino acid (BCAA) permease. However, under conditions that maximize CodY activity, braB expression was similar in wild-type and codY null mutant cells. Nonetheless, expression from the braB promoter was significantly elevated in cells containing partially active mutant versions of CodY or in wild-type cells under growth conditions leading to intermediate levels of CodY activity. This novel pattern of regulation was shown to be due to two opposing mechanisms, negative and positive, by which CodY affects braB expression. A strong CodY-binding site located downstream of the transcription start point conferred negative regulation by direct interaction with CodY. Additionally, sequences upstream and downstream of the promoter were required for repression by a second pleiotropic B. subtilis regulator, ScoC, whose own expression is repressed by CodY. ScoC-mediated repression of braB in codY null mutants cells was as efficient as direct, CodY-mediated repression in wild-type cells under conditions of high CodY activity. However, under conditions of reduced CodY activity, CodY-mediated repression was relieved to a greater extent than ScoC-mediated repression was increased, leading to elevated braB expression. We conclude that restricting increased expression of braB to conditions of moderate nutrient limitation is the raison d’être of the feed-forward regulatory loop formed by CodY and ScoC at the braB promoter. The increase in BraB expression only at intermediate activities of CodY may facilitate the uptake of BCAA when they are not in excess but prevent unneeded BraB synthesis when other BCAA transporters are active.

Listeria monocytogenes PdxR is a member of the poorly characterized but widespread group of MocR/GabR-type chimeric bacterial proteins that have DNA-binding and aminotransferase-like domains. Using mutational analysis, real-time RT-PCR, transcriptional fusions, gel-shift assays, DNase I footprinting, and in vitro transcription, it was shown that PdxR is a direct activator of the pdxST operon, transcribed divergently from pdxR and responsible for the de novo synthesis of pyridoxal 5'-phosphate (PLP), the major active form of vitamin B6 . PLP acts as an anti-activator of PdxR and is the only effector required to reduce the activity of PdxR. PdxR is also a negative autoregulator, and its ability to repress is increased by PLP. A dyad-symmetry sequence, which overlaps the -35 region of the pdxS promoter and lies downstream of the pdxR transcription start point, serves as an important element of the PdxR binding site. Unexpectedly, some mutations in this activator binding site, disrupting the dyad-symmetry element, caused constitutive, B6 -independent expression from the pdxS promoter. The data suggest that PdxR-like proteins, for which PLP plays just a signalling role, form a separate functional group among the MocR/GabR-type proteins.

SummaryMetabolic adaptations are critical to the ability of bacterial pathogens to grow within host cells and are normally preceded by sensing of host‐specific metabolic signals, which in turn can influence the pathogen's virulence state. Previously, we reported that the intracellular bacterial pathogen Listeria monocytogenes responds to low availability of branched‐chain amino acids (BCAAs) within mammalian cells by up‐regulating both BCAA biosynthesis and virulence genes. The induction of virulence genes required the BCAA‐responsive transcription regulator, CodY, but the molecular mechanism governing this mode of regulation was unclear. In this report, we demonstrate that CodY directly binds the coding sequence of the L. monocytogenes master virulence activator gene, prfA, 15 nt downstream of its start codon, and that this binding results in up‐regulation of prfA transcription specifically under low concentrations of BCAA. Mutating this site abolished CodY binding and reduced prfA transcription in macrophages, and attenuated bacterial virulence in mice. Notably, the mutated binding site did not alter prfA transcription or PrfA activity under other conditions that are known to activate PrfA, such as during growth in the presence of glucose‐1‐phosphate. This study highlights the tight crosstalk between L. monocytogenes metabolism and virulence, while revealing novel features of CodY‐mediated regulation.

This chapter provides an overview on biosynthesis of amino acids. Glutamine and glutamate occupy a special place in nitrogen metabolism because virtually all nitrogen-containing groups of biological molecules are derived from these two amino acids, glutamate being the major donor of nitrogen. The major pathway of glutamate synthesis in many bacteria is catalyzed by glutamate synthase (GOGAT or GltAB) through reductive transamidation of a-ketoglutarate. Glutamate can also be generated through degradation of several amino acids (glutamine, arginine, proline, aspartate, ?-aminobutyrate, histidine). The major regulator appears to be the leucine-responsive protein, Lrp, a global transcriptional regulator, that carries out many of its functions in response to leucine or alanine availability. thrBC and thrD gene of unknown function is cotranscribed with the pheA gene and was suggested to be involved in phenylalanine synthesis. Some of the polyamine synthesis genes are organized in two operons, speAB and speED. In Bacillus subtilis, proline is synthesized from glutamate in three enzymatic steps. proA mutants, defective in γ- glutamyl phosphate reductase, are auxotrophic for proline, indicating that this is the only important pathway of de novo proline biosynthesis. The complex regulation of the pro genes in B. subtilis stands in sharp contrast with the constitutive expression of the proBA and proC genes in Escherichia coli. The proBA-dependent pathway of proline synthesis, similar to that of B. subtilis, is also found in S. bovis, Streptococcus thermophilus, Listeria monocytogenes, and, as deduced by genome analysis, in most other low-G+C gram-positive bacteria.

Significance GabR is a member of the MocR/GabR subfamily of the GntR family of bacterial transcription regulators. It regulates the metabolism of γ-aminobutyric acid, an important nitrogen and carbon source in many bacteria. The crystal structures reported here show that this protein has evolved from the fusion of a type I aminotransferase and a winged helix-turn-helix DNA-binding protein to form a chimeric protein that adopts a dimeric head-to-tail configuration. The pyridoxal 5′-phosphate–binding regulatory domain of GabR is therefore an example of a coenzyme playing a role in transcription regulation rather than in enzymatic catalysis. Our structural and biochemical studies lay the mechanistic foundation for understanding the regulatory functions of the MocR/GabR subfamily of transcription regulators.

The CodY protein is a global transcriptional regulator that controls, directly or indirectly, expression of more than 100 genes and operons in Bacillus subtilis. We used in vitro DNA affinity purification combined with massively parallel sequencing, to identify B. subtilis chromosomal DNA fragments that bind CodY in vitro. A nonstandard strand-specific analysis of the data allowed us to pinpoint CodY-binding sites at single-nucleotide resolution. By comparing the extent of binding at decreasing CodY concentrations, we were able to classify binding regions according to their relative strengths and construct a subset of the 323 strongest CodY-binding regions that included sites associated with nearly all genes reported to be direct CodY targets. Many of the identified sites were located within coding regions. At such sites within the ispA, rapA, and rapE genes CodY-dependent repression was demonstrated using lacZ fusions and mutational analysis.

Sublingual (SL) and intranasal (IN) administration of a Bacillus subtilis-based tetanus vaccine was tested in piglets, which more closely mimic the human immune system than mice. Piglets were immunized by the SL, IN or oral routes with vaccine expressing tetanus toxin fragment C, or commercial tetanus vaccine given by intramuscular injection as a control. Tetanus toxoid specific ELISA and passive neutralization tests were used to measure IgG and IgA levels in serum and mucosal secretions, and assess protective serum antibodies, respectively. The nature of the immune response was explored by MHC Class II, TGF-beta 1 expression, and ELISA assays for multiple cytokines. SL or IN immunization of piglets induced neutralizing tetanus toxoid specific serum antibody and local salivary and vaginal IgA responses. Standard tetanus vaccine resulted in systemic antibodies, whereas oral administration of the Bacillus-based vaccine was ineffective. Further analyses indicated a balanced Th1/Th2 response to SL or IN immunization.Conclusion: This study demonstrates for the first time that SL or IN administration is effective for inducing both systemic and mucosal responses in a piglet model, indicating that SL or IN delivery of a B. subtilis-based tetanus vaccine can be a simple, non-invasive, low cost strategy to induce immunity to tetanus. (c) 2011 Published by Elsevier Masson SAS on behalf of Institut Pasteur.

Proline is an efficient source of both carbon and nitrogen for many bacterial species. In Bacillus subtilis, the proline utilization pathway, encoded by the putBCP operon, is inducible by proline. Here, we show that this induction is mediated by PutR, a proline-responsive transcriptional activator of the PucR family. When other amino acids are present in the medium, proline utilization is prioritized through transient repression by CodY, a global transcriptional regulator in Gram-positive bacteria that responds to amino acid availability. CodY-mediated repression of the putBCP operon has two novel features. First, repression requires the cooperative binding of CodY to at least two adjacent motifs. Second, though CodY binds to the region that overlaps the putB promoter, repression is due to displacement of PutR rather than competition with RNA polymerase.