Search

International audience; During the past decade, Chromosome Conformation Capture (3C/Hi-C)-based methods have been used to probe the 3D structure and organization of bacterial genomes, revealing fundamental aspects of chromosome dynamics. However, the current protocols are expensive, inefficient, and limited in their resolution. Here we present a simple, cost-effective Hi-C approach that is readily applicable to a range of Gram-positive and Gram-negative bacteria.

Phylogenomic analysis supports a diderm ancestor of the Firmicutes and points to an early origin of two-membraned cells in Bacteria and the derived nature of the Gram-positive envelope following multiple outer membrane losses. The transition between cell envelopes with one membrane (Gram-positive or monoderm) and those with two membranes (Gram-negative or diderm) is a fundamental open question in the evolution of Bacteria. Evidence of the presence of two independent diderm lineages, the Halanaerobiales and the Negativicutes, within the classically monoderm Firmicutes has blurred the monoderm/diderm divide and specifically anticipated that other members with an outer membrane (OM) might exist in this phylum. Here, by screening 1,639 genomes of uncultured Firmicutes for signatures of an OM, we highlight a third and deep branching diderm clade, the Limnochordia, strengthening the hypothesis of a diderm ancestor and the occurrence of independent transitions leading to the monoderm phenotype. Phyletic patterns of over 176,000 protein families constituting the Firmicutes pan-proteome identify those that strongly correlate with the diderm phenotype and suggest the existence of new potential players in OM biogenesis. In contrast, we find practically no largely conserved core of monoderms, a fact possibly linked to different ways of adapting to repeated OM losses. Phylogenetic analysis of a concatenation of main OM components totalling nearly 2,000 amino acid positions illustrates the common origin and vertical evolution of most diderm bacterial envelopes. Finally, mapping the presence/absence of OM markers onto the tree of Bacteria shows the overwhelming presence of diderm phyla and the non-monophyly of monoderm ones, pointing to an early origin of two-membraned cells and the derived nature of the Gram-positive envelope following multiple OM losses.

International audience; The safe use and design of nanoparticles (NPs) ask for a comprehensive interpretation of their potentially adverse effects on (micro)organisms. In this respect, the prior assessment of the interactions experienced by NPs in the vicinity of - and in contact with - complex biological surfaces is mandatory. It requires the development of suitable techniques for deciphering the processes that govern nano-bio interactions when a single organism is exposed to an extremely low dose of NPs. Here, we used atomic force spectroscopy (AFM)-based force measurements to investigate at the nanoscale the interactions between carboxylate-terminated polyamidoamine (PAMAM) nanodendrimers (radius ca. 4.5 nm) and two bacteria with very distinct surface properties, Escherichia coli and Lactococcus lactis. The zwitterionic nanodendrimers exhibit a negative peripheral surface charge and/or a positive intraparticulate core depending on the solution pH and salt concentration. Following an original strategy according to which a single dendrimer NP is grafted at the very apex of the AFM tip, the density and localization of NP binding sites are probed at the surface of E. coli and L. lactis mutants expressing different cell surface structures (presence/absence of the O-antigen of the lipopolysaccharides (LPS) or of a polysaccharide pellicle). In line with electrokinetic analysis, AFM force measurements evidence that adhesion of NPs onto pellicle-decorated L. lactis is governed by their underlying electrostatic interactions as controlled by the pH-dependent charge of the peripheral and internal NP components, and the negatively-charged cell surface. In contrast, the presence of the O-antigen on E. coli systematically suppresses the adhesion of nanodendrimers onto cells, may the apparent NP surface charge be determined by the peripheral carboxylate groups or by the internal amine functions. Altogether, this work highlights the differentiated roles played by surface polysaccharides in mediating NP attachment to Gram-positive and Gram-negative bacteria. It further demonstrates that the assessment of NP bioadhesion features requires a critical analysis of the electrostatic contributions stemming from the various structures composing the stratified cell envelope, and those originating from the bulk and surface NP components. The joint use of electrokinetics and AFM provides a valuable option for rapidly addressing the binding propensity of NPs to microorganisms, as urgently needed in NP risk assessments.

International audience; Bacterial surface proteins constitute an amazing repertoire of molecules with important functions such as adherence, invasion, signalling and interaction with the host immune system or environment. In Gram-positive bacteria, many surface proteins of the "LPxTG" family are anchored to the peptidoglycan (PG) by an enzyme named sortase. While this anchoring mechanism has been clearly deciphered, less is known about the spatial organization of cell wall-anchored proteins in the bacterial envelope. Here, we review the question of the precise spatial and temporal positioning of LPxTG proteins in subcellular domains in spherical and ellipsoid bacteria (Staphylococcus aureus, Streptococcus pyogenes, Streptococcus agalactiae and Enterococcus faecalis) and in the rod-shaped bacterium Listeria monocytogenes. Deposition at specific sites of the cell wall is a dynamic process tightly connected to cell division, secretion, cell morphogenesis and levels of gene expression. Studying spatial occupancy of these cell wall-anchored proteins not only provides information on PG dynamics in responses to environmental changes, but also suggests that pathogenic bacteria control the distribution of virulence factors at specific sites of the surface, including pole, septa or lateral sites, during the infectious process.

International audience; Methanolic extracts prepared from the leaves, twigs and the roots of Vismia laurentii De Wild as well as nine compounds isolated from these crude extracts, were tested for their antimicrobial activity against Gram-positive bacteria (six species), Gram-negative bacteria (12 species) and two Candida species using disc diffusion and well micro-dilution methods. The disc diffusion assay indicated that the crude extract was active against all the pathogens tested, whereas isolated compounds showed selective activities. The degree of sensitivity of the test organisms to purified compounds varied from 25 to 90%. Fridelin (8) was found to be the most active compound, while Bivismiaquinone (3) was the least active. The lowest minimum inhibition concentration (MIC) values as obtained by the micro-dilution assays were 19.53 and 1.22 microg/ml for the crude extracts and purified compounds, respectively. The lowest value for the purified compounds (1.22 microg/ml) was obtained with O(1)-demethyl-3',4'-deoxypsorospermin-3',4'-diol (6) on Candida gabrata and Bacillus subtilis; 1,8-dihydroxy-6-methoxy-3-methylanthraquinone (5) on Bacillus subtilis and 6-deoxyisojacareubin (7) on Bacillus stearothermophilus. These results provide promising baseline information for the potential use of these crude extracts as well as some of the isolated compounds in the treatment of bacterial and fungal infections.

International audience; In the last decade, pili, which are encoded within pathogenicity islands, have been found in many Gram-positive bacteria, including the major streptococcal and enterococcal pathogens. These long proteinaceous polymers extending from the bacterial surface are constituted of covalently linked pilin subunits, which play major roles in adhesion and host colonization. They are also involved in biofilm formation, a characteristic life-style of the bacteria constituting the oral flora. Pili are highly immunogenic structures that are under the selective pressure of host immune responses. Indeed, pilus expression was found to be heterogeneous in several bacteria with the co-existence of two subpopulations expressing various levels of pili. The molecular mechanisms underlying this complex regulation are poorly characterized except for Streptococcus pneumoniae. In this review, we will discuss the roles of Gram-positive bacteria pili in adhesion to host extracellular matrix proteins, tissue tropism, biofilm formation, modulation of innate immune responses and their contribution to virulence, and in a second part the regulation of their expression. This overview should help to understand the rise of pili as an intensive field of investigation and pinpoints the areas that need further study.

Contains fulltext : 96277.pdf (Publisher’s version ) (Open Access) BACKGROUND: Two NF-kappaB signaling pathways, Toll and immune deficiency (imd), are required for survival to bacterial infections in Drosophila. In response to septic injury, these pathways mediate rapid transcriptional activation of distinct sets of effector molecules, including antimicrobial peptides, which are important components of a humoral defense response. However, it is less clear to what extent macrophage-like hemocytes contribute to host defense. METHODOLOGY/PRINCIPAL FINDINGS: In order to dissect the relative importance of humoral and cellular defenses after septic injury with three different gram-positive bacteria (Micrococcus luteus, Enterococcus faecalis, Staphylococcus aureus), we used latex bead pre-injection to ablate macrophage function in flies wildtype or mutant for various Toll and imd pathway components. We found that in all three infection models a compromised phagocytic system impaired fly survival--independently of concomitant Toll or imd pathway activation. Our data failed to confirm a role of the PGRP-SA and GNBP1 Pattern Recognition Receptors for phagocytosis of S. aureus. The Drosophila scavenger receptor Eater mediates the phagocytosis by hemocytes or S2 cells of E. faecalis and S. aureus, but not of M. luteus. In the case of M. luteus and E. faecalis, but not S. aureus, decreased survival due to defective phagocytosis could be compensated for by genetically enhancing the humoral immune response. CONCLUSIONS/SIGNIFICANCE: Our results underscore the fundamental importance of both cellular and humoral mechanisms in Drosophila immunity and shed light on the balance between these two arms of host defense depending on the invading pathogen.

A novel strictly anaerobic, moderately halophilic and mesophilic bacterium, designated strain SOL3f37T, was isolated from a hydrocarbon-polluted soil surrounding a deep petroleum environment located in south Tunisia. Cells of strain SOL3f37T stained Gram-positive and were motile, straight and spore-forming. Strain SOL3f37T had a typical Gram-positive-type cell-wall structure, unlike the thick, multilayered cell wall of its closest relative Clostridiisalibacter paucivorans. The major fatty acids were iso-C15 : 0 (41 %), iso-C14 : 0 3-OH and/or iso-C15 : 0 dimethyl acetal (21.6 %), iso-C13 : 0 (4.4 %), anteiso-C15 : 0 (3.9 %) and iso-C15 : 1 (2.8 %). Strain SOL3f37T grew between 20 and 48 °C (optimum 40 °C) and at pH 6.2–8.1 (optimum pH 6.9). Strain SOL3f37T required at least 0.5 g NaCl l−1 and grew in the presence of NaCl concentrations up to 150 g l−1 (optimum 40 g l−1). Yeast extract (2 g l−1) was required for degradation of pyruvate, fumarate, fructose, glucose and mannitol. Also, strain SOL3f37T grew heterotrophically on yeast extract, peptone and bio-Trypticase, but was unable to grow on Casamino acids. Sulfate, thiosulfate, sulfite, elemental sulfur, fumarate, nitrate and nitrite were not reduced. The DNA G+C content was 30.7 mol%. Phylogenetic analysis based on 16S rRNA gene sequences revealed that strain SOL3f37T was a member of the family Clostridiaceae in the order Clostridiales; strain SOL3f37T was related to members of various genera of the family Clostridiaceae. It exhibited highest 16S rRNA gene sequence similarity (93.4 %) with Clostridiisalibacter paucivorans 37HS60T, 91.8 % with Thermohalobacter berrensis CTT3T and 91.7 % with Caloranaerobacter azorensis MV1087T. On the basis of genotypic, phenotypic and phylogenetic data, it is suggested that strain SOL3f37T represents a novel species in a new genus. The name Sporosalibacterium faouarense gen. nov., sp. nov. is proposed, with SOL3f37T (=DSM 21485T =JCM 15487T) as the type strain of Sporosalibacterium faouarense.

International audience; The serine-rich repeat family of fimbriae play important roles in the pathogenesis of streptococci and staphylococci. Despite recent attention, their finer structural details and precise adhesion mechanisms have yet to be determined. Fap1 (Fimbriae-associated protein 1) is the major structural subunit of serine-rich repeat fimbriae from Streptococcus parasanguinis and plays an essential role in fimbrial biogenesis, adhesion, and the early stages of dental plaque formation. Combining multidisciplinary, high resolution structural studies with biological assays, we provide new structural insight into adhesion by Fap1. We propose a model in which the serine-rich repeats of Fap1 subunits form an extended structure that projects the N-terminal globular domains away from the bacterial surface for adhesion to the salivary pellicle. We also uncover a novel pH-dependent conformational change that modulates adhesion and likely plays a role in survival in acidic environments.

International audience; The WalK/WalR (YycG/YycF) two-component system, which is essential for cell viability, is highly conserved and specific to low-GC percentage of Gram-positive bacteria, making it an attractive target for novel antimicrobial compounds. Recent work has shown that WalK/WalR exerts an effect as a master regulatory system in controlling and coordinating cell wall metabolism with cell division in Bacillus subtilis and Staphylococcus aureus. In this paper, we develop a high-throughput screening system for WalR inhibitors and identify two novel inhibitors targeting the WalR response regulator (RR): walrycin A (4-methoxy-1-naphthol) and walrycin B (1,6-dimethyl-3-[4-(trifluoromethyl)phenyl]pyrimido[5,4-e][1,2,4]triazine-5,7-dione). Addition of these compounds simultaneously affects the expression of WalR regulon genes, leading to phenotypes consistent with those of cells starved for the WalK/WalR system and having a bactericidal effect. B. subtilis cells form extremely long aseptate filaments and S. aureus cells form large aggregates under these conditions. These results show that walrycins A and B are the first antibacterial agents targeting WalR in B. subtilis and S. aureus.

International audience; Most secretion pathways in bacteria and eukaryotic cells are challenged by the requirement for their substrate proteins to mature after they traverse a membrane barrier and enter a reactive oxidizing environment. For Gram-positive bacteria, the mechanisms that protect their exported proteins from misoxidation during their post-translocation maturation are poorly understood. To address this, we separated numerous bacterial species according to their tolerance for oxygen and divided their proteomes based on the predicted subcellular localization of their proteins. We then applied a previously established computational approach that utilizes cysteine incorporation patterns in proteins as an indicator of enzymatic systems that may exist in each species. The Sec-dependent exported proteins from aerobic Gram-positive Actinobacteria were found to encode cysteines in an even-biased pattern indicative of a functional disulfide bond formation system. In contrast, aerobic Gram-positive Firmicutes favor the exclusion of cysteines from both their cytoplasmic proteins and their substantially longer exported proteins. Supporting these findings, we show that Firmicutes, but not Actinobacteria, tolerate growth in reductant. We further demonstrate that the actinobacterium Corynebacterium glutamicum possesses disulfide-bonded proteins and two dimeric Dsb-like enzymes that can efficiently catalyze the formation of disulfide bonds. Our results suggest that cysteine exclusion is an important adaptive strategy against the challenges presented by oxidative environments.

The dramatic rise of antibiotic-resistant bacteria over the past two decades has stressed the need for completely novel classes of antibacterial agents. Accordingly, recent advances in the study of prokaryotic transcription open new opportunities for such molecules. This paper reports the structure-activity relationships of a series of phenyl-furanyl-rhodanines (PFRs) as antibacterial inhibitors of RNA polymerase (RNAP). The molecules have been evaluated for their ability to inhibit transcription and affect growth of bacteria living in suspension or in a biofilm and for their propensity to interact with serum albumin, a critical parameter for antibacterial drug discovery. The most active of these molecules inhibit Escherichia coli RNAP transcription at concentrations of/=10 microM and have promising activities against various Gram-positive pathogens including Staphylococcus epidermidis biofilms, a major cause of nosocomial infection.

International audience; Over the last 10 years, DNA sequences of more than 600 bacterial species have been deposited in databases and are now available to search any gene, motif, or regulatory sequence of interest. Although genome data are instrumental in phylogenetic analysis and in silico design of metabolic and regulatory networks, only a very small fraction of the information has been experimentally validated. A striking example is lipoproteins predicted from genome sequences. Despite the predominance of this class of surface proteins in bacteria (up to 0.5 to 8% of the proteome), very few of these proteins have been identified as lipoproteins by biochemical methods (19). In this issue of the Journal of Bacteriology, Baumgärtner et al. (2) report a systematic analysis of lipoproteins of Listeria monocytogenes, a facultative gram-positive intracellular bacterial pathogen that causes severe infections (listeriosis) in both human and animals. These authors used an L. monocytogenes mutant defective in lipoprotein diacylglyceryl transferase (Lgt), an enzyme involved in lipoprotein processing. Three aspects of their study should be highlighted: (i) new findings concerning the roles of Lgt and lipoprotein-specific signal peptidase II (Lsp) during lipoprotein processing (22); (ii) the identification of 26 of the 68 lipoproteins predicted in the initial annotation of the L. monocytogenes strain EGD-e genome (7); and (iii) experimental evidence that a few of these lipoproteins are regulated by PrfA, the master virulence regulator of L. monocytogenes (8). Below, we discuss the significance of these findings separately.

International audience; A phytochemical investigation of the constituents of the roots of Vismia laurentii has resulted in the isolation of two xanthone derivatives named laurentixanthone A (1) (6-hydroxy-3,3-dimethyl-11-(3-methylbut-2-enyl)pyrano[2,3-c]xanthen-7(3H)-one) and laurentixanthone B (2) (1-hydroxy-5,6,7,8-tetramethoxyxanthone), along with 11 known compounds: 1,7-dihydroxyxanthone, vismiaquinone, vismiaquinone B, bivismiaquinone, 3-geranyloxy-6-methyl-1,8-dihydroxyanthraquinone, O(1)-demethyl-3',4'-deoxypsorospermin-3',4'-diol, 6-deoxyisojacareubin, 1,8-dihydroxy-6-methoxy-3-methylanthraquinone, kaempferol, friedelin and stigmasterol. The structures of compounds were established by means of spectroscopic methods. Furthermore, the compounds were screened for antimicrobial activities in vitro.

The complement system is a major humoral portion of the innate immune system, playing a significant role in host defence against microorganisms. The biological importance of this system is underlined by the fact that at least three different pathways for its activation exist, the classical, the MBL and the alternative pathway. To elucidate the involvement of the classical and/or the MBL pathway during bacterial septicemia, 32 patients with gram-positive and 30 patients with gram-negative bacterial infections were investigated. In patients with gram-positive bacteria, a significant consumption of C1q (p=0.005) but not of mannose-binding lectin (MBL) (p=0.2) was found during the acute phase of infection. In contrast, in patients with gram-negative bacterial infections, a significant reduction of MBL (p=0.002) and only a moderate, less significant reduction of C1q (p=0.03) were observed. As a model for the binding of MBL to gram-negative bacteria, Salmonella strains with defined mutations in their lipopolysaccharide (LPS) structure were used. The comparison of the binding of MBL to these Salmonella strains with that of the corresponding isolated LPS forms bound to microtiter plates revealed a similar binding pattern, supporting the interpretation that LPS on the surface of gram-negative bacteria is the major acceptor molecule for MBL on these bacteria, which according to our results obviously also takes place during gram-negative bacterial septicaemia. Furthermore, we were able to demonstrate that MBL bound to LPS was able to initiate activation of the complement cascade as measured by the occurrence of the cleavage product C4c.

International audience; Bacterial surface proteins constitute a diverse group of molecules with important functions, such as adherence, invasion, signaling and interaction with the host immune system or the environment. In Gram-positive bacteria, many surface proteins are anchored to the cell wall envelope by an enzyme named sortase, which recognizes a conserved carboxylic sorting motif. The sequence of the prototype staphylococcal SrtA has been widely used to identify homologs in bacterial genomes, revealing a profusion of sortases in almost all Gram-positive bacteria, often with more than one sortase-like protein per genome [M.J. Pallen, A.C. Lam, M. Antonio, K. Dunbar, Trends Microbiol. 9 (2001) 97-102]. In light of increasing reports on the identification and/or characterization of paralogous sortase genes, a classification of sortases now appears necessary. This report provides an analysis of sixty-one sortases from complete Gram-positive genomes, and suggests the existence of four structural groups of sortases. We propose the classification of sortases into 4 classes designated A, B, C and D. This classification should help to discriminate between sortases in the future.

Equipe Osmorégulation chez les Bactéries (OB) devenue équipe Dualité et Universalité de l'Adaptation Lors d'un Stress (DUALS); International audience; Glycine betaine (GB) analogues were obtained using solid phase organic synthesis and assayed for their toxic activity against 15 Gram positive and Gram negative bacteria. Four benzyl derivatives of GB were selected to determine their effect on bacterial growth. Bacteriostatic and lethal effects were observed for compound 1 and compound 2, respectively. The importation of the two GB analogues into bacterial cells appeared strictly dependent on the presence of the powerful betaine membrane osmoporters; their capacity to be amassed intracellularly at molar levels from extremely dilute solutions might constitute a basis to design a new class of antimicrobial agents.

International audience; clpP and clpC of Bacillus subtillis encode subunits of the Clp ATP-dependent protease and are required for stress survival, including growth at high temperature. They play essential roles in stationary phase adaptive responses such as the competence and sporulation developmental pathways, and belong to the so-called class III group of heat shock genes, whose mode of regulation is unknown and whose expression is induced by heat shock or general stress conditions. The product of ctsR, the first gene of the clpC operon, has now been shown to act as a repressor of both clpP and clpC, as well as clpE, which encodes a novel member of the Hsp100 Clp ATPase family. The CtsR protein was purified and shown to bind specifically to the promoter regions of all three clp genes. Random mutagenesis, DNasel footprinting and DNA sequence deletions and comparisons were used to define a consensus CtsR recognition sequence as a directly repeated heptad upstream from the three clp genes. This target sequence was also found upstream from clp and other heat shock genes of several Gram-positive bacteria, including Listeria monocytogenes, Streptococcus salivarius, S. pneumoniae, S. pyogenes, S. thermophilus, Enterococcus faecalis, Staphylococcus aureus, Leuconostoc oenos, Lactobacillus sake, Lactococcus lactis and Clostridium acetobutylicum. CtsR homologues were also identified in several of these bacteria, indicating that heat shock regulation by CtsR is highly conserved in Gram-positive bacteria.