Recent Advances in Peptidoglycan Synthesis and Regulation in Bacteria.

Bacteria must synthesize their cell wall and membrane during their cell cycle, with peptidoglycan being the primary component of the cell wall in most bacteria. Peptidoglycan is a three-dimensional polymer that enables bacteria to resist cytoplasmic osmotic pressure, maintain their cell shape and protect themselves from environmental threats. Numerous antibiotics that are currently used target enzymes involved in the synthesis of the cell wall, particularly peptidoglycan synthases. In this review, we highlight recent progress in our understanding of peptidoglycan synthesis, remodeling, repair, and regulation in two model bacteria: the Gram-negative Escherichia coli and the Gram-positive Bacillus subtilis. By summarizing the latest findings in this field, we hope to provide a comprehensive overview of peptidoglycan biology, which is critical for our understanding of bacterial adaptation and antibiotic resistance.

Metabolic Control of Cell Elongation and Cell Division in Bacillus subtilis.

To survive and adapt to changing nutritional conditions, bacteria must rapidly modulate cell cycle processes, such as doubling time or cell size. Recent data have revealed that cellular metabolism is a central regulator of bacterial cell cycle. Indeed, proteins that can sense precursors or metabolites or enzymes, in addition to their enzymatic activities involved in metabolism, were shown to directly control cell cycle processes in response to changes in nutrient levels. Here we focus on cell elongation and cell division in the Gram-positive rod-shaped bacterium Bacillus subtilis and we report evidences linking these two cellular processes to environmental nutritional availability and thus metabolic cellular status.

Uridine diphosphate N-acetylglucosamine orchestrates the interaction of GlmR with either YvcJ or GlmS in Bacillus subtilis.

In bacteria, glucosamine-6-phosphate (GlcN6P) synthase, GlmS, is an enzyme required for the synthesis of Uridine diphosphate N-acetylglucosamine (UDP-GlcNAc), a precursor of peptidoglycan. In Bacillus subtilis, an UDP-GlcNAc binding protein, GlmR (formerly YvcK), essential for growth on non-glycolytic carbon sources, has been proposed to stimulate GlmS activity; this activation could be antagonized by UDP-GlcNAc. Using purified proteins, we demonstrate that GlmR directly stimulates GlmS activity and the presence of UDP-GlcNAc (at concentrations above 0.1 mM) prevents this regulation. We also showed that YvcJ, whose gene is associated with yvcK (glmR), interacts with GlmR in an UDP-GlcNAc dependent manner. Strains producing GlmR variants unable to interact with YvcJ show decreased transformation efficiency similar to that of a yvcJ null mutant. We therefore propose that, depending on the intracellular concentration of UDP-GlcNAc, GlmR interacts with either YvcJ or GlmS. When UDP-GlcNAc concentration is high, this UDP-sugar binds to YvcJ and to GlmR, blocking the stimulation of GlmS activity and driving the interaction between GlmR and YvcJ to probably regulate the cellular role of the latter. When the UDP-GlcNAc level is low, GlmR does not interact with YvcJ and thus does not regulate its cellular role but interacts with GlmS to stimulate its activity.

YvcK, a protein required for cell wall integrity and optimal carbon source utilization, binds uridine diphosphate-sugars.

In Bacillus subtilis, Listeria monocytogenes and in two Mycobacteria, it was previously shown that yvcK is a gene required for normal cell shape, for optimal carbon source utilization and for virulence of pathogenic bacteria. Here we report that the B. subtilis protein YvcK binds to Uridine diphosphate-sugars like Uridine diphosphate-Glucose (UDP-Glc) and Uridine diphosphate-N-acetylglucosamine (UDP-GlcNAc) in vitro. Using the crystal structure of Bacillus halodurans YvcK, we identified residues involved in this interaction. We tested the effect of point mutations affecting the ability of YvcK to bind UDP-sugars on B. subtilis physiology and on cell size. Indeed, it was shown that UDP-Glc serves as a metabolic signal to regulate B. subtilis cell size. Interestingly, we observed that, whereas a yvcK deletion results in the formation of unusually large cells, inactivation of YvcK UDP-sugar binding site does not affect cell length. However, these point mutations result in an increased sensitivity to bacitracin, an antibiotic which targets peptidoglycan synthesis. We thus propose that UDP-GlcNAc, a precursor of peptidoglycan, could be a good physiological ligand candidate of YvcK.

Impact of Serine/Threonine Protein Kinases on the Regulation of Sporulation in Bacillus subtilis.

Bacteria possess many kinases that catalyze phosphorylation of proteins on diverse amino acids including arginine, cysteine, histidine, aspartate, serine, threonine, and tyrosine. These protein kinases regulate different physiological processes in response to environmental modifications. For example, in response to nutritional stresses, the Gram-positive bacterium Bacillus subtilis can differentiate into an endospore; the initiation of sporulation is controlled by the master regulator Spo0A, which is activated by phosphorylation. Spo0A phosphorylation is carried out by a multi-component phosphorelay system. These phosphorylation events on histidine and aspartate residues are labile, highly dynamic and permit a temporal control of the sporulation initiation decision. More recently, another kind of phosphorylation, more stable yet still dynamic, on serine or threonine residues, was proposed to play a role in spore maintenance and spore revival. Kinases that perform these phosphorylation events mainly belong to the Hanks family and could regulate spore dormancy and spore germination. The aim of this mini review is to focus on the regulation of sporulation in B. subtilis by these serine and threonine phosphorylation events and the kinases catalyzing them.

Phosphorylation of the cell division protein GpsB regulates PrkC kinase activity through a negative feedback loop in Bacillus subtilis.

Although many membrane Ser/Thr-kinases with PASTA motifs have been shown to control bacterial cell division and morphogenesis, inactivation of the Ser/Thr-kinase PrkC does not impact Bacillus subtilis cell division. In this study, we show that PrkC localizes at the division septum. In addition, three proteins involved in cell division/elongation, GpsB, DivIVA and EzrA are required for stimulating PrkC activity in vivo. We show that GpsB interacts with the catalytic subunit of PrkC that, in turn, phosphorylates GpsB. These observations are not made with DivIVA and EzrA. Consistent with the phosphorylated residue previously detected for GpsB in a high-throughput phosphoproteomic analysis of B. subtilis, we show that threonine 75 is the single PrkC-mediated phosphorylation site in GpsB. Importantly, the substitution of this threonine by a phospho-mimetic residue induces a loss of PrkC kinase activity in vivo and a reduced growth under high salt conditions as observed for gpsB and prkC null mutants. Conversely, substitution of threonine 75 by a phospho-ablative residue does not induce such growth and PrkC kinase activity defects. Altogether, these data show that proteins of the divisome control PrkC activity and thereby phosphorylation of PrkC substrates through a negative feedback loop in B. subtilis.

PrkC-mediated phosphorylation of overexpressed YvcK protein regulates PBP1 protein localization in Bacillus subtilis mreB mutant cells.

The YvcK protein has been shown to be necessary for growth under gluconeogenic conditions in Bacillus subtilis. Amazingly, its overproduction rescues growth and morphology defects of the actin-like protein MreB deletion mutant by restoration of PBP1 localization. In this work, we observed that YvcK was phosphorylated at Thr-304 by the protein kinase PrkC and that phosphorylated YvcK was dephosphorylated by the cognate phosphatase PrpC. We show that neither substitution of this threonine with a constitutively phosphorylated mimicking glutamic acid residue or a phosphorylation-dead mimicking alanine residue nor deletion of prkC or prpC altered the ability of B. subtilis to grow under gluconeogenic conditions. However, we observed that a prpC mutant and a yvcK mutant were more sensitive to bacitracin compared with the WT strain. In addition, the bacitracin sensitivity of strains in which YvcK Thr-304 was replaced with either an alanine or a glutamic acid residue was also affected. We also analyzed rescue of the mreB mutant strain by overproduction of YvcK in which the phosphorylation site was substituted. We show that YvcK T304A overproduction did not rescue the mreB mutant aberrant morphology due to PBP1 mislocalization. The same observation was made in an mreB prkC double mutant overproducing YvcK. Altogether, these data show that YvcK may have two distinct functions: 1) in carbon source utilization independent of its phosphorylation level and 2) in cell wall biosynthesis and morphogenesis through its phosphorylation state.

Strategies to adapt cellular processes to nutrient availability in bacteria.

Bacteria are able to adapt to nutrient availability in the environment. For example, when nutritional conditions are not favorable, bacterial size can be reduced and duplication time can be significantly extended in comparison to rich growth conditions. These observations suggest that essential cellular processes like cell division, morphogenesis and chromosome dynamics are highly coordinated with central metabolism to ensure the production of fit progeny. The aim of this review is to provide an overview extending from physiological observations done more than fifty years ago to recent discoveries showing strategies to control essential functions in relation with metabolism in the model bacterium Bacillus subtilis.

The YvcK protein is required for morphogenesis via localization of PBP1 under gluconeogenic growth conditions in Bacillus subtilis.

The YvcK protein was previously shown to be dispensable when B. subtilis cells are grown on glycolytic carbon sources but essential for growth and normal shape on gluconeogenic carbon sources. Here, we report that YvcK is localized as a helical-like pattern in the cell. This localization seems independent of the actin-like protein, MreB. A YvcK overproduction restores a normal morphology in an mreB mutant strain when bacteria are grown on PAB medium. Reciprocally, an additional copy of mreB restores a normal growth and morphology in a yvcK mutant strain when bacteria are grown on a gluconeogenic carbon source like gluconate. Furthermore, as already observed for the mreB mutant, the deletion of the gene encoding the penicillin-binding protein PBP1 restores growth and normal shape of a yvcK mutant on gluconeogenic carbon sources. The PBP1 is delocalized in an mreB mutant grown in the absence of magnesium and in a yvcK mutant grown on gluconate medium. Interestingly, its proper localization can be rescued by YvcK overproduction. Therefore, in gluconeogenic growth conditions, YvcK is required for the correct localization of PBP1 and hence for displaying a normal rod shape.

Characterization of YvcJ, a conserved P-loop-containing protein, and its implication in competence in Bacillus subtilis.

The uncharacterized protein family UPF0042 of the Swiss-Prot database is predicted to be a member of the conserved group of bacterium-specific P-loop-containing proteins. Here we show that two of its members, YvcJ from Bacillus subtilis and YhbJ, its homologue from Escherichia coli, indeed bind and hydrolyze nucleotides. The cellular function of yvcJ was then addressed. In contrast to results recently obtained for E. coli, which indicated that yhbJ mutants strongly overproduced glucosamine-6-phosphate synthase (GlmS), comparison of the wild type with the yvcJ mutant of B. subtilis showed that GlmS expression was quite similar in the two strains. However, in mutants defective in yvcJ, the transformation efficiency and the fraction of cells that expressed competence were reduced. Furthermore, our data show that YvcJ positively controls the expression of late competence genes. The overexpression of comK or comS compensates for the decrease in competence of the yvcJ mutant. Our results show that even if YvcJ and YhbJ belong to the same family of P-loop-containing proteins, the deletion of corresponding genes has different consequences in B. subtilis and in E. coli.

YvcK of Bacillus subtilis is required for a normal cell shape and for growth on Krebs cycle intermediates and substrates of the pentose phosphate pathway.

The HPr-like protein Crh has so far been detected only in the bacillus group of bacteria. In Bacillus subtilis, its gene is part of an operon composed of six ORFs, three of which exhibit strong similarity to genes of unknown function present in many bacteria. The promoter of the operon was determined and found to be constitutively active. A deletion analysis revealed that gene yvcK, encoded by this operon, is essential for growth on Krebs cycle intermediates and on carbon sources metabolized via the pentose phosphate pathway. In addition, cells lacking YvcK acquired media-dependent filamentous or L-shape-like aberrant morphologies. The presence of high magnesium concentrations restored normal growth and cell morphology. Furthermore, suppressor mutants cured from these growth defects appeared spontaneously with a high frequency. Such suppressing mutations were identified in a transposon mutagenesis screen and found to reside in seven different loci. Two of them mapped in genes of central carbon metabolism, including zwf, which encodes glucose-6-phosphate dehydrogenase and cggR, the product of which regulates the synthesis of glyceraldehyde-3-phosphate dehydrogenase. All these results suggest that YvcK has an important role in carbon metabolism, probably in gluconeogenesis required for the synthesis of cell wall precursor molecules. Interestingly, the Escherichia coli homologous protein, YbhK, can substitute for YvcK in B. subtilis, suggesting that the two proteins have been functionally conserved in these different bacteria.

IMGT unique numbering for immunoglobulin and T cell receptor constant domains and Ig superfamily C-like domains.

IMGT, the international ImMunoGeneTics information system (http://imgt.cines.fr) provides a common access to expertly annotated data on the genome, proteome, genetics and structure of immunoglobulins (IG), T cell receptors (TR), major histocompatibility complex (MHC), and related proteins of the immune system (RPI) of human and other vertebrates. The NUMEROTATION concept of IMGT-ONTOLOGY has allowed to define a unique numbering for the variable domains (V-DOMAINs) and for the V-LIKE-DOMAINs. In this paper, this standardized characterization is extended to the constant domains (C-DOMAINs), and to the C-LIKE-DOMAINs, leading, for the first time, to their standardized description of mutations, allelic polymorphisms, two-dimensional (2D) representations and tridimensional (3D) structures. The IMGT unique numbering is, therefore, highly valuable for the comparative, structural or evolutionary studies of the immunoglobulin superfamily (IgSF) domains, V-DOMAINs and C-DOMAINs of IG and TR in vertebrates, and V-LIKE-DOMAINs and C-LIKE-DOMAINs of proteins other than IG and TR, in any species.

Fully human IgG and IgM antibodies directed against the carcinoembryonic antigen (CEA) Gold 4 epitope and designed for radioimmunotherapy (RIT) of colorectal cancers.

BACKGROUND: Human monoclonal antibodies (MAbs) are needed for colon cancer radioimmunotherapy (RIT) to allow for repeated injections. Carcinoembryonic antigen (CEA) being the reference antigen for immunotargeting of these tumors, we developed human anti-CEA MAbs. METHODS: XenoMouse-G2 animals were immunized with CEA. Among all the antibodies produced, two of them, VG-IgG2kappa and VG-IgM, were selected for characterization in vitro in comparison with the human-mouse chimeric anti-CEA MAb X4 using flow cytometry, surface plasmon resonance, and binding to radiolabeled soluble CEA and in vivo in human colon carcinoma LS174T bearing nude mice. RESULTS: Flow cytometry analysis demonstrated binding of MAbs on CEA-expressing cells without any binding on NCA-expressing human granulocytes. In a competitive binding assay using five reference MAbs, directed against the five Gold CEA epitopes, VG-IgG2kappa and VG-IgM were shown to be directed against the Gold 4 epitope. The affinities of purified VG-IgG2kappa and VG-IgM were determined to be 0.19 +/- 0.06 x 10(8) M(-1) and 1.30 +/- 0.06 x 10(8) M(-1), respectively, as compared with 0.61 +/- 0.05 x 10(8) M(-1) for the reference MAb X4. In a soluble phase assay, the binding capacities of VG-IgG2kappa and VG-IgM to soluble CEA were clearly lower than that of the control chimeric MAb X4. A human MAb concentration of about 10(-7) M was needed to precipitate approximatively 1 ng 125I-rhCEA as compared with 10(-9) M for MAb X4, suggesting a preferential binding of the human MAbs to solid phase CEA. In vivo, 24 h post-injection, 125I-VG-IgG2kappa demonstrated a high tumor uptake (25.4 +/- 7.3%ID/g), close to that of 131I-X4 (21.7 +/- 7.2%ID/g). At 72 h post-injection, 125I-VG-IgG2kappa was still concentrated in the tumor (28.4 +/- 11.0%ID/g) whereas the tumor concentration of 131I-X4 was significantly reduced (12.5 +/- 4.8%ID/g). At no time after injection was there any accumulation of the radiolabeled MAbs in normal tissues. A pertinent analysis of VG-IgM biodistribution was not possible in this mouse model in which IgM displays a very short half-life due to poly-Ig receptor expression in the liver. CONCLUSION: Our human anti-CEA IgG2kappa is a promising candidate for radioimmunotherapy in intact form, as F(ab')2 fragments, or as a bispecific antibody.

IMGT unique numbering for immunoglobulin and T cell receptor variable domains and Ig superfamily V-like domains.

IMGT, the international ImMunoGeneTics database (http://imgt.cines.fr) is a high quality integrated information system specializing in immunoglobulins (IG), T cell receptors (TR) and major histocompatibility complex (MHC) of human and other vertebrates. IMGT provides a common access to expertly annotated data on the genome, proteome, genetics and structure of the IG and TR, based on the IMGT Scientific chart and IMGT-ONTOLOGY. The IMGT unique numbering defined for the IG and TR variable regions and domains of all jawed vertebrates has allowed a redefinition of the limits of the framework (FR-IMGT) and complementarity determining regions (CDR-IMGT), leading, for the first time, to a standardized description of mutations, allelic polymorphisms, 2D representations (Colliers de Perles) and 3D structures, whatever the antigen receptor, the chain type, or the species. The IMGT numbering has been extended to the V-like domain and is, therefore, highly valuable for comparative analysis and evolution studies of proteins belonging to the IG superfamily.