Allelic replacement of the streptococcal cysteine protease SpeB in a Δsrv mutant background restores biofilm formation

<p>Abstract</p> <p>Background</p> <p>Group A <it>Streptococcus </it>(GAS) is a Gram-positive human pathogen that is capable of causing a wide spectrum of human disease. Thus, the organism has evolved to colonize a number of physiologically distinct host sites. One such mechanism to aid colonization is the formation of a biofilm. We have recently shown that inactivation of the streptococcal regulator of virulence (Srv), results in a mutant strain exhibiting a significant reduction in biofilm formation. Unlike the parental strain (MGAS5005), the streptococcal cysteine protease (SpeB) is constitutively produced by the <it>srv </it>mutant (MGAS5005Δ<it>srv</it>) suggesting Srv contributes to the control of SpeB production. Given that SpeB is a potent protease, we hypothesized that the biofilm deficient phenotype of the <it>srv </it>mutant was due to the constitutive production of SpeB. In support of this hypothesis, we have previously demonstrated that treating cultures with E64, a commercially available chemical inhibitor of cysteine proteases, restored the ability of MGAS5005Δ<it>srv </it>to form biofilms. Still, it was unclear if the loss of biofilm formation by MGAS5005Δ<it>srv </it>was due only to the constitutive production of SpeB or to other changes inherent in the <it>srv </it>mutant strain. To address this question, we constructed a Δ<it>srv</it>Δ<it>speB </it>double mutant through allelic replacement (MGAS5005Δ<it>srv</it>Δ<it>speB</it>) and tested its ability to form biofilms <it>in vitro</it>.</p> <p>Findings</p> <p>Allelic replacement of <it>speB </it>in the <it>srv </it>mutant background restored the ability of this strain to form biofilms under static and continuous flow conditions. Furthermore, addition of purified SpeB to actively growing wild-type cultures significantly inhibited biofilm formation.</p> <p>Conclusions</p> <p>The constitutive production of SpeB by the <it>srv </it>mutant strain is responsible for the significant reduction of biofilm formation previously observed. The double mutant supports a model by which Srv contributes to biofilm formation and/or dispersal through regulation of <it>speB</it>/SpeB.</p

BiofOmics: A Web Platform for the Systematic and Standardized Collection of High-Throughput Biofilm Data

Background: Consortia of microorganisms, commonly known as biofilms, are attracting much attention from the scientific community due to their impact in human activity. As biofilm research grows to be a data-intensive discipline, the need for suitable bioinformatics approaches becomes compelling to manage and validate individual experiments, and also execute inter-laboratory large-scale comparisons. However, biofilm data is widespread across ad hoc, non-standardized individual files and, thus, data interchange among researchers, or any attempt of cross-laboratory experimentation or analysis, is hardly possible or even attempted. Methodology/Principal findings This paper presents BiofOmics, the first publicly accessible Web platform specialized in the management and analysis of data derived from biofilm high-throughput studies. The aim is to promote data interchange across laboratories, implementing collaborative experiments, and enable the development of bioinformatics tools in support of the processing and analysis of the increasing volumes of experimental biofilm data that are being generated. BiofOmics data deposition facility enforces data structuring and standardization, supported by controlled vocabulary. Researchers are responsible for the description of the experiments, their results and conclusions. BiofOmics curators interact with submitters only to enforce data structuring and the use of controlled vocabulary. Then, BiofOmics search facility makes publicly available the profile and data associated with a submitted study so that any researcher can profit from these standardization efforts to compare similar studies, generate new hypotheses to be tested or even extend the conditions experimented in the study. Significance BiofOmics novelty lays on its support to standardized data deposition, the availability of computerizable data files and the free-of-charge dissemination of biofilm studies across the community. Hopefully, this will open promising research possibilities, namely: the comparison of results between different laboratories, the reproducibility of methods within and between laboratories, and the development of guidelines and standardized protocols for biofilm formation devices and analytical methods.The financial support from the Institute of Biotechnology and Bioengineering - Center of Biological Engineering (IBB-CEB), Fundacao para a Ciencia e Tecnologia (FCT) and European Community fund FEDER (Program COMPETE), project PTDC/SAU-ESA/646091/2006/FCOMP-01-0124-FEDER-007480 and PhD grant of Idalina Machado (SFRH/BD/31065/2006) are gratefully acknowledged. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript

Unusual trivial trauma may end with extrusion of a well-functioning penile prosthesis: a case report

<p>Abstract</p> <p>Background</p> <p>Diabetes mellitus (DM) is the most common indication for insertion of a penile prosthesis and is a risk factor for infection of such prostheses.</p> <p>Case presentation</p> <p>Two patients presented with infected prostheses following unusual trivial penile trauma. Both patients underwent exploration and removal of the prostheses with uneventful recovery.</p> <p>Conclusion</p> <p>Appropriate sizing of the prosthesis should be taken into account to ensure good concealment and avoid easy exposure of the penis to unexpected trauma. Use of the newly designed antibiotic-coated prostheses appears preferable. As soon as signs of prosthesis infection appeared, extrusion of the device should be expedited.</p

Self-Organization, Layered Structure, and Aggregation Enhance Persistence of a Synthetic Biofilm Consortium

Microbial consortia constitute a majority of the earth’s biomass, but little is known about how these cooperating communities persist despite competition among community members. Theory suggests that non-random spatial structures contribute to the persistence of mixed communities; when particular structures form, they may provide associated community members with a growth advantage over unassociated members. If true, this has implications for the rise and persistence of multi-cellular organisms. However, this theory is difficult to study because we rarely observe initial instances of non-random physical structure in natural populations. Using two engineered strains of Escherichia coli that constitute a synthetic symbiotic microbial consortium, we fortuitously observed such spatial self-organization. This consortium forms a biofilm and, after several days, adopts a defined layered structure that is associated with two unexpected, measurable growth advantages. First, the consortium cannot successfully colonize a new, downstream environment until it selforganizes in the initial environment; in other words, the structure enhances the ability of the consortium to survive environmental disruptions. Second, when the layered structure forms in downstream environments the consortium accumulates significantly more biomass than it did in the initial environment; in other words, the structure enhances the global productivity of the consortium. We also observed that the layered structure only assembles in downstream environments that are colonized by aggregates from a previous, structured community. These results demonstrate roles for self-organization and aggregation in persistence of multi-cellular communities, and also illustrate a role for the techniques of synthetic biology in elucidating fundamental biological principles

Gac two-component system in Pseudomonas syringae pv. tabaci is required for virulence but not for hypersensitive reaction

Pseudomonas syringae pv. tabaci 6605 causes wildfire disease on host tobacco plants. To investigate the regulatory mechanism of the expression of virulence, Gac two-Component system-defective mutants, Delta gacA and Delta gacS, and a double mutant, Delta gacA Delta gacS, were generated. These mutants produced smaller amounts of N-acyl homoserine lactones required for quorum sensing, had lost swarming motility, and had reduced expression of virulence-related hrp genes and the algT gene required for exopolysaccharide production. The ability of the mutants to cause disease symptoms in their host tobacco plant was remarkably reduced, while they retained the ability to induce hypersensitive reaction (HR) in the nonhost plants. These results indicated that the Gac two-component system of P. syringae pv. tabaci 6605 is indispensable for virulence on the host plant, but not for HR induction in the nonhost plants.</p

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

The Pseudomonas aeruginosa Chemotaxis Methyltransferase CheR1 Impacts on Bacterial Surface Sampling

The characterization of factors contributing to the formation and development of surface-associated bacterial communities known as biofilms has become an area of intense interest since biofilms have a major impact on human health, the environment and industry. Various studies have demonstrated that motility, including swimming, swarming and twitching, seems to play an important role in the surface colonization and establishment of structured biofilms. Thereby, the impact of chemotaxis on biofilm formation has been less intensively studied. Pseudomonas aeruginosa has a very complex chemosensory system with two Che systems implicated in flagella-mediated motility. In this study, we demonstrate that the chemotaxis protein CheR1 is a methyltransferase that binds S-adenosylmethionine and transfers a methyl group from this methyl donor to the chemoreceptor PctA, an activity which can be stimulated by the attractant serine but not by glutamine. We furthermore demonstrate that CheR1 does not only play a role in flagella-mediated chemotaxis but that its activity is essential for the formation and maintenance of bacterial biofilm structures. We propose a model in which motility and chemotaxis impact on initial attachment processes, dispersion and reattachment and increase the efficiency and frequency of surface sampling in P. aeruginosa

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