Biosynthèse des glucanes périplasmiques osmorégulés chez Escherichia colis (analyse fonctionnelle des protéines MdoG et MdoH et caractérisation de deux nouvelles activités)

Les glucanes périplasmiques osmorégulés (OPG) sont présents chez toutes les Protéobactéries testées, où ils peuvent représenter jusqu'à 5% du poids sec de la bactérie. Leur biosynthèse est d'autant plus importante que l'osmolarité du milieu est faible. Les OPG d'E. coli sont formés d'une chaîne linéaire de résidus de glucose liés en b-1,2 et ramifiés par des résidus de glucose liés en b-1,6. Ces OPG peuvent être substitués par des résidus de phosphoglycérol, de succinate et de phosphoéthanolamine. Un opéron de deux gènes (mdoGH) soumis à une régulation osmotique est nécessaire à la biosynthèse du squelette glucosidique. Trois types de mutagenèse (dirigée, ou par greffage d'épitope aléatoire ou localisée) ont été effectués sur cet opéron dans le but d'établir une topologie fonctionnelle de ces deux protéines. Ces mutagenèses ont révélé la présence dans MdoH de deux domaines cytoplasmiques actifs, l'importance d'une boucle périplasmique et la stricte nécessité de 3 résidus d'acide aspartique, conservés dans les glycosyltransférase de la famille 2, Cette approche a aussi révélé l'importance des 80 derniers acides aminés de la protéine MdoG dans sa fonction. Un gène mdoD, paralogue du gène mdoG, est impliqué dans la biosynthèse du squelette glucosidique mais n'est pas indispensable. L'expression du gène mdoD, étudiée grâce à des fusions traductionnelles, est maintenue à un niveau de base en phase exponentielle et augmentée lors de l'entrée en phase stationnaire et de l'adaptation à une haute osmolarité. L'expression du gène mdoD dépend des facteurs de transcription s70 et sS et d'un régulateur inconnu. Le produit du gène mdoB responsable de l'activité phosphoglycérol-transférase II périplasmique. Nos études indiquent que le transfert du succinate s'effectue simultanément à la biosynthèse du squelette glucosidique et de manière plus tardive et séquentielle pour les résidus de phosphoglycérol.LILLE1-BU (590092102) / SudocSudocFranceF

Timing and Localization of Rhamnolipid Synthesis Gene Expression in Pseudomonas aeruginosa Biofilms

Pseudomonas aeruginosa biofilms can develop mushroom-like structures with stalks and caps consisting of discrete subpopulations of cells. Self-produced rhamnolipid surfactants have been shown to be important in development of the mushroom-like structures. The quorum-sensing-controlled rhlAB operon is required for rhamnolipid synthesis. We have introduced an rhlA-gfp fusion into a neutral site in the P. aeruginosa genome to study rhlAB promoter activity in rhamnolipid-producing biofilms. Expression of the rhlA-gfp fusion in biofilms requires the quorum-sensing signal butanoyl-homoserine lactone, but other factors are also required for expression. Early in biofilm development rhlA-gfp expression is low, even in the presence of added butanoyl-homoserine lactone. Expression of the fusion becomes apparent after microcolonies with a depth of >20 μm have formed and, as shown by differential labeling with rfp or fluorescent dyes, rhlA-gfp is preferentially expressed in the stalks rather than the caps of mature mushrooms. The rhlA-gfp expression pattern is not greatly influenced by addition of butanoyl-homoserine lactone to the biofilm growth medium. We propose that rhamnolipid synthesis occurs in biofilms after stalks have formed but prior to capping in the mushroom-like structures. The differential expression of rhlAB may play a role in the development of normal biofilm architecture

Identification of mdoD, an mdoG Paralog Which Encodes a Twin-Arginine-Dependent Periplasmic Protein That Controls Osmoregulated Periplasmic Glucan Backbone Structures

Osmoregulated periplasmic glucans (OPGs) of Escherichia coli are anionic and highly branched oligosaccharides that accumulate in the periplasmic space in response to low osmolarity of the medium. The glucan length, ranging from 5 to 12 glucose residues, is under strict control. Two genes that form an operon, mdoGH, govern glucose backbone synthesis. The new gene mdoD, which appears to be a paralog of mdoG, was characterized in this study. Cassette inactivation of mdoD resulted in production of OPGs with a higher degree of polymerization, indicating that OpgD, the mdoD product (according to the new nomenclature), controls the glucose backbone structures. OpgD secretion depends on the Tat secretory pathway. Orthologs of the mdoG and mdoD genes are found in various proteobacteria. Most of the OpgD orthologs exhibit a Tat-dependent secretion signal, while most of the OpgG orthologs are Sec dependent

Selective adhesion of Bacillus cereus spores on heterogeneously wetted silicon nanowires

ISI Document Delivery No.: 556ZPTimes Cited: 1Cited Reference Count: 33Galopin, Elisabeth Piret, Gaelle Szunerits, Sabine Lequette, Yannick Faille, Christine Boukherroub, RabahCentre National de la Recherche Scientifique (CNRS); Nord-Pas-de Calais; Agence Nationale de la Recherche[ANR-07-PNRA-009-01 InterSpore]We gratefully acknowledge the Centre National de la Recherche Scientifique (CNRS) and the Nord-Pas-de Calais region for support. We are grateful to G. Ronse, A. Ronse. and M. Clarisse from INRA for technical assistance provided for biological sample preparation. This work has been partially financed by the Agence Nationale de la Recherche under the Programme National de Recherche en Alimentation et Nutrition Humaine, project ANR-07-PNRA-009-01 InterSpore.Amer chemical socWashingtonBoukherroub, R (reprint author), CNRS, IRI, USR 3078, Parc Haute Borne,50 Ave Halley,BP 70478, F-59658 Villeneuve Dascq, [email protected] audienceThe article reports on the selective adhesion of Bacillus cereus spores oil patterned and heterogeneously wetted superhydrophobic silicon nanowires surfaces. Superhydrophilic patterns on superhydrophobic silicon nanowire (SiNW) surfaces were prepared by a standard optical lithography technique. Exposure of the patterned surface to a suspension of B. cercus spores in water led to their specific adsorption in superhydrophobic areas. Comparable results were obtained on a patterned hydrophobic/hydrophilic flat silicon (Si) surface even though at higher concentration of spores wits observed on the hydrophobic areas, its compared to the superhydrophobic regions of the SiNW substrate, The surfaces were characterized using scanning electron microscopy (SEM), fluorescence spectroscopy, and contact angle measurements

A Distinct QscR Regulon in the Pseudomonas aeruginosa Quorum-Sensing Circuit

The opportunistic pathogen Pseudomonas aeruginosa possesses two complete acyl-homoserine lactone (acyl-HSL) signaling systems. One system consists of LasI and LasR, which generate a 3-oxododecanoyl-homoserine lactone signal and respond to that signal, respectively. The other system is RhlI and RhlR, which generate butanoyl-homoserine lactone and respond to butanoyl-homoserine lactone, respectively. These quorum-sensing systems control hundreds of genes. There is also an orphan LasR-RhlR homolog, QscR, for which there is no cognate acyl-HSL synthetic enzyme. We previously reported that a qscR mutant is hypervirulent and showed that QscR transiently represses a few quorum-sensing-controlled genes. To better understand the role of QscR in P. aeruginosa gene regulation and to better understand the relationship between QscR, LasR, and RhlR control of gene expression, we used transcription profiling to identify a QscR-dependent regulon. Our analysis revealed that QscR activates some genes and represses others. Some of the repressed genes are not regulated by the LasR-I or RhlR-I systems, while others are. The LasI-generated 3-oxododecanoyl-homoserine lactone serves as a signal molecule for QscR. Thus, QscR appears to be an integral component of the P. aeruginosa quorum-sensing circuitry. QscR uses the LasI-generated acyl-homoserine lactone signal and controls a specific regulon that overlaps with the already overlapping LasR- and RhlR-dependent regulons

Viability and surface properties of spores subjected to a cleaning-in-place procedure: consequences on their ability to contaminate surfaces of equipment

International audienceThis study was designed to evaluate how conditions encountered by spores during cleaning-in-place (CIP) procedures affected their surface properties, their viability and ability to contaminate materials. Spores from five Bacillus cereus strains were treated with NaOH at high temperature. Results revealed that high temperatures (exceeding 60 degrees C) and NaOH concentrations (over 0.5%) were required to significantly decrease spore viability (3-5 log decrease). In these conditions, modifications were also clearly observed by microscopy to various surface structures of spores (appendages, exosporium, and especially to the hair-like nap) but also to their coat. Therefore, the ability of culturable spores to adhere decreased for the majority of strains tested. We then demonstrated that spores in suspension in NaOH could adhere to surfaces of a CIP rig and that the contamination level was controlled by flow pattern. Consequently, re-adhesion along the processing line might occur during CIP procedures and this phenomenon must be taken into account when defining cleaning strategies

Glycosylation of BclA glycoprotein from Bacillus cereus and Bacillus anthracis exosporium Is domain-specific

The spores of the Bacillus cereus group (B. cereus, Bacillus anthracis, and Bacillus thuringiensis) are surrounded by a paracrystalline flexible yet resistant layer called exosporium that plays a major role in spore adhesion and virulence. The major constituent of its hairlike surface, the trimerized glycoprotein BclA, is attached to the basal layer through an N-terminal domain. It is then followed by a repetitive collagen-like neck bearing a globular head (C-terminal domain) that promotes glycoprotein trimerization. The collagen-like region of B. anthracisis known to be densely substituted by unusual O-glycans that may be used for developing species-specific diagnostics of B. anthracis spores and thus targeted therapeutic interventions. In the present study, we have explored the species and domain specificity of BclA glycosylation within the B. cereus group. First, we have established that the collagen-like regions of both B. anthracis and B. cereus are similarly substituted by short O-glycans that bear the species-specific deoxyhexose residues anthrose and the newly observed cereose, respectively. Second we have discovered that the C-terminal globular domains of BclA from both species are substituted by polysaccharide-like O-linked glycans whose structures are also species-specific. The presence of large carbohydrate polymers covering the surface of Bacillus spores may have a profound impact on the way that spores regulate their interactions with biotic and abiotic surfaces and represents potential new diagnostic targets