The Pyrimidine Nucleotide Biosynthetic Pathway Modulates Production of Biofilm Determinants in Escherichia coli

Bacteria are often found in multicellular communities known as biofilms, which constitute a resistance form against environmental stresses. Extracellular adhesion and cell aggregation factors, responsible for bacterial biofilm formation and maintenance, are tightly regulated in response to physiological and environmental cues. We show that, in Escherichia coli, inactivation of genes belonging to the de novo uridine monophosphate (UMP) biosynthetic pathway impairs production of curli fibers and cellulose, important components of the bacterial biofilm matrix, by inhibiting transcription of the csgDEFG operon, thus preventing production of the biofilm master regulator CsgD protein. Supplementing growth media with exogenous uracil, which can be converted to UMP through the pyrimidine nucleotide salvage pathway, restores csgDEFG transcription and curli production. In addition, however, exogenous uracil triggers cellulose production, particularly in strains defective in either carB or pyrB genes, which encode enzymes catalyzing the first steps of de novo UMP biosynthesis. Our results indicate the existence of tight and complex links between pyrimidine metabolism and curli/cellulose production: transcription of the csgDEFG operon responds to pyrimidine nucleotide availability, while cellulose production is triggered by exogenous uracil in the absence of active de novo UMP biosynthesis. We speculate that perturbations in the UMP biosynthetic pathways allow the bacterial cell to sense signals such as starvation, nucleic acids degradation, and availability of exogenous pyrimidines, and to adapt the production of the extracellular matrix to the changing environmental conditions

A colanic acid operon deletion mutation enhances induction of early antibody responses by live attenuated salmonella vaccine strains

Colanic acid (CA) is a common exopolysaccharide produced by many genera in the Enterobacteriaceae. It is critical for biofilm formation on HEp-2 cells and on chicken intestinal tissue by Salmonella. In this study, we generated different CA synthesis gene mutants and evaluated the immune responses induced by these mutants. One of these mutations, Δ(wza-wcaM)8, which deleted the whole operon for CA synthesis, was introduced into two Salmonella vaccine strains attenuated by auxotrophic traits or by the regulated delayed attenuation strategy (RDAS). The mice immunized with the auxotrophic Salmonella vaccine strain with the deletion mutation Δ(wza-wcaM)8 developed higher vaginal IgA titers against the heterologous protective antigen and higher levels of antigen-specific IgA secretion cells in lungs. In Salmonella vaccine strains with RDAS, the strain with the Δ(wza-wcaM)8 mutation resulted in higher levels of protective antigen production during in vitro growth. Mice immunized with this strain developed higher serum IgG and mucosal IgA antibody responses at 2 weeks. This strain also resulted in better gamma interferon (IFN-γ) responses than the strain without this deletion at doses of 10(8) and 10(9) CFU. Thus, the mutation Δ(wza-wcaM)8 will be included in various recombinant attenuated Salmonella vaccine (RASV) strains with RDAS derived from Salmonella enterica serovar Paratyphi A and Salmonella enterica serovar Typhi to induce protective immunity against bacterial pathogens

Salmonella Biofilm Formation on Aspergillus niger Involves Cellulose – Chitin Interactions

Salmonella cycles between host and nonhost environments, where it can become an active member of complex microbial communities. The role of fungi in the environmental adaptation of enteric pathogens remains relatively unexplored. We have discovered that S. enterica Typhimurium rapidly attaches to and forms biofilms on the hyphae of the common fungus, Aspergillus niger. Several Salmonella enterica serovars displayed a similar interaction, whereas other bacterial species were unable to bind to the fungus. Bacterial attachment to chitin, a major constituent of fungal cell walls, mirrored this specificity. Pre-incubation of S. Typhimurium with N-acetylglucosamine, the monomeric component of chitin, reduced binding to chitin beads by as much as 727-fold and inhibited attachment to A. niger hyphae considerably. A cellulose-deficient mutant of S. Typhimurium failed to attach to chitin beads and to the fungus. Complementation of this mutant with the cellulose operon restored binding to chitin beads to 79% of that of the parental strain and allowed for attachment and biofilm formation on A. niger, indicating that cellulose is involved in bacterial attachment to the fungus via the chitin component of its cell wall. In contrast to cellulose, S. Typhimurium curli fimbriae were not required for attachment and biofilm development on the hyphae but were critical for its stability. Our results suggest that cellulose–chitin interactions are required for the production of mixed Salmonella-A. niger biofilms, and support the hypothesis that encounters with chitinaceous alternate hosts may contribute to the ecological success of human pathogens

The Spatial Architecture of Bacillus subtilis Biofilms Deciphered Using a Surface-Associated Model and In Situ Imaging

The formation of multicellular communities known as biofilms is the part of bacterial life cycle in which bacteria display cooperative behaviour and differentiated phenotypes leading to specific functions. Bacillus subtilis is a Gram-positive bacterium that has served for a decade as a model to study the molecular pathways that control biofilm formation. Most of the data on B. subtilis biofilms have come from studies on the formation of pellicles at the air-liquid interface, or on the complex macrocolonies that develop on semi-solid nutritive agar. Here, using confocal laser scanning microcopy, we show that B. subtilis strains of different origins are capable of forming biofilms on immersed surfaces with dramatically protruding “beanstalk-like” structures with certain strains. Indeed, these structures can reach a height of more than 300 µm with one undomesticated strain from a medical environment. Using 14 GFP-labeled mutants previously described as affecting pellicle or complex colony formation, we have identified four genes whose inactivation significantly impeded immersed biofilm development, and one mutation triggering hyperbiofilm formation. We also identified mutations causing the three-dimensional architecture of the biofilm to be altered. Taken together, our results reveal that B. subtilis is able to form specific biofilm features on immersed surfaces, and that the development of these multicellular surface-associated communities involves regulation pathways that are common to those governing the formation of pellicle and/or complex colonies, and also some specific mechanisms. Finally, we propose the submerged surface-associated biofilm as another relevant model for the study of B. subtilis multicellular communities

A Global Metabolic Shift Is Linked to Salmonella Multicellular Development

Bacteria can elaborate complex patterns of development that are dictated by temporally ordered patterns of gene expression, typically under the control of a master regulatory pathway. For some processes, such as biofilm development, regulators that initiate the process have been identified but subsequent phenotypic changes such as stress tolerance do not seem to be under the control of these same regulators. A hallmark feature of biofilms is growth within a self-produced extracellular matrix. In this study we used metabolomics to compare Salmonella cells in rdar colony biofilms to isogenic csgD deletion mutants that do not produce an extracellular matrix. The two populations show distinct metabolite profiles. Even though CsgD controls only extracellular matrix production, metabolite signatures associated with cellular adaptations associated with stress tolerances were present in the wild type but not the mutant cells. To further explore these differences we examine the temporal gene expression of genes implicated in biofilm development and stress adaptations. In wild type cells, genes involved in a metabolic shift to gluconeogenesis and various stress-resistance pathways exhibited an ordered expression profile timed with multicellular development even though they are not CsgD regulated. In csgD mutant cells, the ordered expression was lost. We conclude that the induction of these pathways results from production of, and growth within, a self produced matrix rather than elaboration of a defined genetic program. These results predict that common physiological properties of biofilms are induced independently of regulatory pathways that initiate biofilm formation

Comparison of the virulence of exopolysaccharide-producing Prevotella intermedia to exopolysaccharide non-producing periodontopathic organisms

<p>Abstract</p> <p>Background</p> <p>Evidence in the literature suggests that exopolysaccharides (EPS) produced by bacterial cells are essential for the expression of virulence in these organisms. Secreted EPSs form the framework in which microbial biofilms are built.</p> <p>Methods</p> <p>This study evaluates the role of EPS in <it>Prevotella intermedia </it>for the expression of virulence. This evaluation was accomplished by comparing EPS-producing <it>P. intermedia </it>strains 17 and OD1-16 with non-producing <it>P. intermedia </it>ATCC 25611 and <it>Porphyromonas gingivalis </it>strains ATCC 33277, 381 and W83 for their ability to induce abscess formation in mice and evade phagocytosis.</p> <p>Results</p> <p>EPS-producing <it>P. intermedia </it>strains 17 and OD1-16 induced highly noticeable abscess lesions in mice at 10⁷colony-forming units (CFU). In comparison, <it>P. intermedia </it>ATCC 25611 and <it>P. gingivalis </it>ATCC 33277, 381 and W83, which all lacked the ability to produce viscous materials, required 100-fold more bacteria (10⁹CFU) in order to induce detectable abscess lesions in mice. Regarding antiphagocytic activity, <it>P. intermedia </it>strains 17 and OD1-16 were rarely internalized by human polymorphonuclear leukocytes, but other strains were readily engulfed and detected in the phagosomes of these phagocytes.</p> <p>Conclusions</p> <p>These results demonstrate that the production of EPS by <it>P. intermedia </it>strains 17 and OD1-16 could contribute to the pathogenicity of this organism by conferring their ability to evade the host's innate defence response.</p

Extracellular DNA Chelates Cations and Induces Antibiotic Resistance in Pseudomonas aeruginosa Biofilms

Biofilms are surface-adhered bacterial communities encased in an extracellular matrix composed of DNA, bacterial polysaccharides and proteins, which are up to 1000-fold more antibiotic resistant than planktonic cultures. To date, extracellular DNA has been shown to function as a structural support to maintain Pseudomonas aeruginosa biofilm architecture. Here we show that DNA is a multifaceted component of P. aeruginosa biofilms. At physiologically relevant concentrations, extracellular DNA has antimicrobial activity, causing cell lysis by chelating cations that stabilize lipopolysaccharide (LPS) and the outer membrane (OM). DNA-mediated killing occurred within minutes, as a result of perturbation of both the outer and inner membrane (IM) and the release of cytoplasmic contents, including genomic DNA. Sub-inhibitory concentrations of DNA created a cation-limited environment that resulted in induction of the PhoPQ- and PmrAB-regulated cationic antimicrobial peptide resistance operon PA3552–PA3559 in P. aeruginosa. Furthermore, DNA-induced expression of this operon resulted in up to 2560-fold increased resistance to cationic antimicrobial peptides and 640-fold increased resistance to aminoglycosides, but had no effect on β-lactam and fluoroquinolone resistance. Thus, the presence of extracellular DNA in the biofilm matrix contributes to cation gradients, genomic DNA release and inducible antibiotic resistance. DNA-rich environments, including biofilms and other infection sites like the CF lung, are likely the in vivo environments where extracellular pathogens such as P. aeruginosa encounter cation limitation

Acquisition of phosphorus and nitrogen in the rhizosphere and plant growth promotion by microorganisms

Copyright © 2009, Springer Science+Business Media B.V.The rhizosphere is a complex environment where roots interact with physical, chemical and biological properties of soil. Structural and functional characteristics of roots contribute to rhizosphere processes and both have significant influence on the capacity of roots to acquire nutrients. Roots also interact extensively with soil microorganisms which further impact on plant nutrition either directly, by influencing nutrient availability and uptake, or indirectly through plant (root) growth promotion. In this paper, features of the rhizosphere that are important for nutrient acquisition from soil are reviewed, with specific emphasis on the characteristics of roots that influence the availability and uptake of phosphorus and nitrogen. The interaction of roots with soil microorganisms, in particular with mycorrhizal fungi and non-symbiotic plant growth promoting rhizobacteria, is also considered in relation to nutrient availability and through the mechanisms that are associated with plant growth promotionAlan E. Richardson, José-Miguel Barea, Ann M. McNeill and Claire Prigent-Combare

The Pseudomonas secondary metabolite 2, 4-diacetylphloroglucinol is a signal inducing rhizoplane expression of Azospirillum genes involved in plant-growth promotion

International audienceDuring evolution, plants have become associated with guilds of plant-growth-promoting rhizobacteria (PGPR), which raises the possibility that individual PGPR populations may have developed mechanisms to cointeract with one another on plant roots. We hypothesize that this has resulted in signaling phenomena between different types of PGPR colonizing the same roots. Here, the objective was to determine whether the Pseudomonas secondary metabolite 2,4-diacetylphloroglucinol (DAPG) can act as a signal on Azospirillum PGPR and enhance the phytostimulation effects of the latter. On roots, the DAPG-producing Pseudomonas fluorescens F113 strain but not its phl-negative mutant enhanced the phytostimulatory effect of Azospirillum brasilense Sp245-Rif on wheat. Accordingly, DAPG enhanced Sp245-Rif traits involved in root colonization (cell motility, biofilm formation, and poly-beta-hydroxybutyrate production) and phytostimulation (auxin production). A differential fluorescence induction promoter-trapping approach based on flow cytometry was then used to identify Sp245-Rif genes upregulated by DAPG. DAPG enhanced expression of a wide range of Sp245-Rif genes, including genes involved in phytostimulation. Four of them (i.e., ppdC, flgE, nirK, and nifX-nifB) tended to be upregulated on roots in the presence of P fluorescens F113 compared with its phl-negative mutant. Our results indicate that DAPG can act as a signal by which some beneficial pseudomonads may stimulate plant-beneficial activities of Azospirillum PGPR