Structure of the Branched Chain Amino Acid and GTP Sensing Global Regulator, CodY, from Bacillus subtilis

CodY is a branched-chain amino acid (BCAA) and GTP sensor, and a global regulator of transcription in low G + C Gram-positive 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. CodY consists of a GAF 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 reorganised 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 CodY-Ile 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 base-pair operator fragment 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

PLP and GABA Trigger GabR-mediated Transcription Regulation in Bacillus Subtilis via External Aldimine Formation

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

Impact of CodY protein on metabolism, sporulation and virulence in Clostridioides difficile ribotype 027

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

Modulation of Activity of Bacillus subtilis Regulatory Proteins GltC and TnrA by Glutamate Dehydrogenase

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 effectors 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

CodY-Mediated Regulation of Guanosine Uptake in Bacillus subtilis ▿

CodY is a global transcriptional regulator known to control expression of more than 100 genes and operons in Bacillus subtilis. Some of the most strongly repressed targets of CodY, the nupNOPQ (formerly, yufNOPQ) genes, were found to encode a guanosine transporter. Using DNase I footprinting experiments, we identified two high-affinity CodY-binding sites in the regulatory region of the nupN gene. The two sites are located 50 bp upstream and 163 bp downstream of the transcription start site. The downstream site was responsible for 6- to 8-fold nupN repression in the absence of the upstream site. When the upstream site was intact, however, only a minor contribution of the downstream site to nupN regulation could be detected under the conditions tested. Both sites contained 15-bp CodY-binding motifs with two mismatches each with respect to the consensus sequence, AATTTTCWGTTTTAA. However, the experimentally determined binding sites included additional sequences flanking the 15-bp CodY-binding motifs. An additional version of the 15-bp CodY-binding motif, with 5 mismatches with respect to the consensus but essential for efficient regulation by CodY, was found within the upstream site. The presence of multiple 15-bp motifs may be a common feature of CodY-binding sites

Contributions of Multiple Binding Sites and Effector-Independent Binding to CodY-Mediated Regulation in Bacillus subtilis▿ †

CodY is a branched-chain amino acid-responsive transcriptional regulator that controls, directly or indirectly, the expression of more than 100 genes and operons in Bacillus subtilis. Using DNase I footprinting and gel-shift experiments, we identified two CodY-binding regions upstream of a B. subtilis gene (bcaP, previously known as yhdG) that encodes a transporter of branched-chain amino acids. Mutational analysis revealed that both CodY-binding regions contribute to repression in vivo and do so independently of each other. Thus, a single CodY-binding site is apparently sufficient for substantial CodY-dependent regulation. By analyzing affinities of wild-type and mutant CodY-binding sites for CodY and their regulation by wild-type CodY and forms of CodY with various levels of activation by branched-chain amino acids, we concluded that unliganded CodY cannot repress transcription in vivo and that the level of endogenously produced effectors is sufficient for CodY-mediated regulation of promoters with stronger sites. Because the sites with higher affinity apparently respond to lower concentrations of CodY effectors and saturate faster as the concentrations of effectors increase, having two sites of binding with different affinities for CodY permits a promoter to respond to a wider range of intracellular concentrations of effectors