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.

A widespread family of serine/threonine protein phosphatases shares a common regulatory switch with proteasomal proteases.

PP2C phosphatases control biological processes including stress responses, development, and cell division in all kingdoms of life. Diverse regulatory domains adapt PP2C phosphatases to specific functions, but how these domains control phosphatase activity was unknown. We present structures representing active and inactive states of the PP2C phosphatase SpoIIE from .

Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, from .

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. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear.

Human Lin28 Forms a High-Affinity 1:1 Complex with the 106~363 Cluster miRNA miR-363.

Lin28A is a post-transcriptional regulator of gene expression that interacts with and negatively regulates the biogenesis of let-7 family miRNAs. Recent data suggested that Lin28A also binds the putative tumor suppressor miR-363, a member of the 106~363 cluster of miRNAs. Affinity for this miRNA and the stoichiometry of the protein-RNA complex are unknown.

12-Fold symmetry of the putative portal protein from the Thermus thermophilus bacteriophage G20C determined by X-ray analysis.

In tailed bacteriophages and several animal viruses, the portal protein forms the gateway through which viral DNA is translocated into the head structure during viral particle assembly. In the mature virion the portal protein exists as a dodecamer, while recombinant portal proteins from several phages, including SPP1 and CNPH82, have been shown to form 13-subunit assemblies. A putative portal protein from the thermostable bacteriophage G20C has been cloned, overexpressed and purified.

The putative small terminase from the thermophilic dsDNA bacteriophage G20C is a nine-subunit oligomer.

The assembly of double-stranded DNA bacteriophages is dependent on a small terminase protein that normally plays two important roles. Firstly, the small terminase protein specifically recognizes viral DNA and recruits the large terminase protein, which makes the initial cut in the dsDNA. Secondly, once the complex of the small terminase, the large terminase and the DNA has docked to the portal protein, and DNA translocation into a preformed empty procapsid has begun, the small terminase modulates the ATPase activity of the large terminase.

Structure of components of an intercellular channel complex in sporulating Bacillus subtilis.

Following asymmetric cell division during spore formation in Bacillus subtilis, a forespore expressed membrane protein SpoIIQ, interacts across an intercellular space with a mother cell-expressed membrane protein, SpoIIIAH. Their interaction can serve as a molecular "ratchet" contributing to the migration of the mother cell membrane around that of the forespore in a phagocytosis-like process termed engulfment. Upon completion of engulfment, SpoIIQ and SpoIIIAH are integral components of a recently proposed intercellular channel allowing passage from the mother cell into the forespore of factors required for late gene expression in this compartment.

Structure of the phosphatase domain of the cell fate determinant SpoIIE from Bacillus subtilis.

Sporulation in Bacillus subtilis begins with an asymmetric cell division producing two genetically identical cells with different fates. SpoIIE is a membrane protein that localizes to the polar cell division sites where it causes FtsZ to relocate from mid-cell to form polar Z-rings. Following polar septation, SpoIIE establishes compartment-specific gene expression in the smaller forespore cell by dephosphorylating the anti-sigma factor antagonist SpoIIAA, leading to the release of the RNA polymerase sigma factor σ(F) from an inhibitory complex with the anti-sigma factor SpoIIAB.

Structure and organisation of SinR, the master regulator of biofilm formation in Bacillus subtilis.

sinR encodes a tetrameric repressor of genes required for biofilm formation in Bacillus subtilis. sinI, which is transcribed under Spo0A control, encodes a dimeric protein that binds to SinR to form a SinR-SinI heterodimer in which the DNA-binding functions of SinR are abrogated and repression of biofilm genes is relieved. The heterodimer-forming surface comprises residues conserved between SinR and SinI.

Compensating stereochemical changes allow murein tripeptide to be accommodated in a conventional peptide-binding protein.

The oligopeptide permease (Opp) of Escherichia coli is an ATP-binding cassette transporter that uses the substrate-binding protein (SBP) OppA to bind peptides and deliver them to the membrane components (OppBCDF) for transport. OppA binds conventional peptides 2-5 residues in length regardless of their sequence, but does not facilitate transport of the cell wall component murein tripeptide (Mtp, L-Ala-γ-D-Glu-meso-Dap), which contains a D-amino acid and a γ-peptide linkage. Instead, MppA, a homologous substrate-binding protein, forms a functional transporter with OppBCDF for uptake of this unusual tripeptide.

The structure and interactions of SpoIISA and SpoIISB, a toxin-antitoxin system in Bacillus subtilis.

Spore formation in Bacillus subtilis begins with an asymmetric cell division, following which differential gene expression is established by alternative compartment-specific RNA polymerase σ factors. The spoIISAB operon of B. subtilis was identified as a locus whose mutation leads to increased activity of the first sporulation-specific sigma factor, σ(F).

Expression of soluble, active fragments of the morphogenetic protein SpoIIE from Bacillus subtilis using a library-based construct screen.

SpoIIE is a dual function protein that plays important roles during sporulation in Bacillus subtilis. It binds to the tubulin-like protein FtsZ causing the cell division septum to relocate from mid-cell to the cell pole, and it dephosphorylates SpoIIAA phosphate leading to establishment of differential gene expression in the two compartments following the asymmetric septation. Its 872 residue polypeptide contains a multiple-membrane spanning sequence at the N-terminus and a PP2C phosphatase domain at the C-terminus.

Structure of the catalytic domain of the human mitochondrial Lon protease: proposed relation of oligomer formation and activity.

ATP-dependent proteases are crucial for cellular homeostasis. By degrading short-lived regulatory proteins, they play an important role in the control of many cellular pathways and, through the degradation of abnormally misfolded proteins, protect the cell from a buildup of aggregates. Disruption or disregulation of mammalian mitochondrial Lon protease leads to severe changes in the cell, linked with carcinogenesis, apoptosis, and necrosis.

Genetic and biochemical analysis of the interaction of Bacillus subtilis CodY with branched-chain amino acids.

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.

Structural rearrangement accompanying ligand binding in the GAF domain of CodY from Bacillus subtilis.

The GAF domain is a simple module widespread in proteins of diverse function, including cell signalling proteins and transcription factors. Its structure, typically spanning 150 residues, has three tiers: a basal layer of two or more alpha-helices, a middle layer of beta-pleated sheet and a top layer formed by segments of the polypeptide that connect strands of the beta-sheet. In structures of GAF domains in complex with their effectors, these polypeptide segments envelop the ligand, enclosing it in a cavity whose base is formed by the beta-sheet, such that ligand binding and release must be accompanied by conformational rearrangements of the distal portion of the structure.

The structure of Rph, an exoribonuclease from Bacillus anthracis, at 1.7 A resolution.

Maturation of tRNA precursors into functional tRNA molecules requires trimming of the primary transcript at both the 5' and 3' ends. Cleavage of nucleotides from the 3' stem of tRNA precursors, releasing nucleotide diphosphates, is accomplished in Bacillus by a phosphate-dependent exoribonuclease, Rph. The crystal structure of this enzyme from B.

Functional role for a conserved aspartate in the Spo0E signature motif involved in the dephosphorylation of the Bacillus subtilis sporulation regulator Spo0A.

Sporulation is a complex developmental system characterizing Gram-positive bacteria of the genus Bacillus and Clostridium. In Bacillus subtilis the phosphorelay signal transduction system regulates the initiation of sporulation by integrating a myriad of positive and negative signals through the action of histidine sensor kinases and aspartyl phosphate phosphatases. The Spo0E family of phosphatases dephosphorylates the Spo0A response regulator and transcription factor of the phosphorelay.

Structures of two superoxide dismutases from Bacillus anthracis reveal a novel active centre.

The BA4499 and BA5696 genes of Bacillus anthracis encode proteins homologous to manganese superoxide dismutase, suggesting that this organism has an expanded repertoire of antioxidant proteins. Differences in metal specificity and quaternary structure between the dismutases of prokaryotes and higher eukaryotes may be exploited in the development of therapeutic antibacterial compounds. Here, the crystal structure of two Mn superoxide dismutases from B.

Structure of purine nucleoside phosphorylase (DeoD) from Bacillus anthracis.

Protein structures from the causative agent of anthrax (Bacillus anthracis) are being determined as part of a structural genomics programme. Amongst initial candidates for crystallographic analysis are enzymes involved in nucleotide biosynthesis, since these are recognized as potential targets in antibacterial therapy. Purine nucleoside phosphorylase is a key enzyme in the purine-salvage pathway.

The structure of CodY, a GTP- and isoleucine-responsive regulator of stationary phase and virulence in gram-positive bacteria.

CodY is a global regulator of transcription in gram-positive bacteria. It represses during growth genes required for adaptation to nutrient limitation, including virulence genes in some human pathogens. CodY activity is regulated by GTP and branched chain amino acids, metabolites whose intracellular concentrations drop as cells enter stationary phase.

An ATP-binding cassette-type cysteine transporter in Campylobacter jejuni inferred from the structure of an extracytoplasmic solute receptor protein.

Campylobacter jejuni is a Gram-negative food-borne pathogen associated with gastroenteritis in humans as well as cases of the autoimmune disease Guillain-Barré syndrome. C. jejuni is asaccharolytic because it lacks an active glycolytic pathway for the use of sugars as a carbon source.

The structure of the oligopeptide-binding protein, AppA, from Bacillus subtilis in complex with a nonapeptide.

Besides their role as a source of amino acids for Bacillus subtilis, exogenous peptides play important roles in the signalling pathways leading to the development of competence and sporulation. B.subtilis has three peptide transport systems all belonging to the ATP-binding cassette family, a dipeptide permease (Dpp) and two oligopeptide permeases (Opp and App) with overlapping specificity.

The crystal structure of YloQ, a circularly permuted GTPase essential for Bacillus subtilis viability.

yloQ is one of 11 essential genes in Bacillus subtilis with unknown roles in the physiology of the cell. It encodes a polypeptide of 298 residues with motifs characteristic of GTPases. As a contribution to elucidating its indispensable cellular function, we have solved the crystal structure of YloQ to 1.