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      A Novel Signaling Network Essential for Regulating Pseudomonas aeruginosa Biofilm Development

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      PLoS Pathogens
      Public Library of Science

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          Abstract

          The important human pathogen Pseudomonas aeruginosa has been linked to numerous biofilm-related chronic infections. Here, we demonstrate that biofilm formation following the transition to the surface attached lifestyle is regulated by three previously undescribed two-component systems: BfiSR (PA4196-4197) harboring an RpoD-like domain, an OmpR-like BfmSR (PA4101-4102), and MifSR (PA5511-5512) belonging to the family of NtrC-like transcriptional regulators. These two-component systems become sequentially phosphorylated during biofilm formation. Inactivation of bfiS, bfmR, and mifR arrested biofilm formation at the transition to the irreversible attachment, maturation-1 and -2 stages, respectively, as indicated by analyses of biofilm architecture, and protein and phosphoprotein patterns. Moreover, discontinuation of bfiS, bfmR, and mifR expression in established biofilms resulted in the collapse of biofilms to an earlier developmental stage, indicating a requirement for these regulatory systems for the development and maintenance of normal biofilm architecture. Interestingly, inactivation did not affect planktonic growth, motility, polysaccharide production, or initial attachment. Further, we demonstrate the interdependency of this two-component systems network with GacS (PA0928), which was found to play a dual role in biofilm formation. This work describes a novel signal transduction network regulating committed biofilm developmental steps following attachment, in which phosphorelays and two sigma factor-dependent response regulators appear to be key components of the regulatory machinery that coordinates gene expression during P. aeruginosa biofilm development in response to environmental cues.

          Author Summary

          Biofilms are complex communities of microorganisms encased in a matrix and attached to surfaces. It is well recognized that biofilm cells differ from their free swimming counterparts with respect to gene expression, protein production, and resistance to antibiotics and the human immune system. However, little is known about the underlying regulatory events that lead to the formation of biofilms, the primary cause of many chronic and persistent human infections. By mapping the phosphoproteome over the course of P. aeruginosa biofilm development, we identified three novel two-component regulatory systems that were required for the development and maturation of P. aeruginosa biofilms. Activation (phosphorylation) of these three regulatory systems occurred in a sequential manner and inactivation arrested biofilm formation at three distinct developmental stages. Discontinuation of bfiS, bfmR, or mifR expression after biofilms had already matured resulted in disaggregation/collapse of biofilms. Furthermore, this regulatory cascade appears to be linked via BfiS-dependent GacS-phosphorylation to the previously identified LadS/RetS/GacAS/RsmA network that reciprocally regulates virulence and surface attachment. Our data thus indicate the existence of a previously unidentified regulatory program of biofilm development once P. aeruginosa cells have committed to a surface associated lifestyle, and may provide new targets for controlling the programmed differentiation process of biofilm formation.

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          Most cited references52

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          Flagellar and twitching motility are necessary for Pseudomonas aeruginosa biofilm development.

          The formation of complex bacterial communities known as biofilms begins with the interaction of planktonic cells with a surface in response to appropriate environmental signals. We report the isolation and characterization of mutants of Pseudomonas aeruginosa PA14 defective in the initiation of biofilm formation on an abiotic surface, polyvinylchloride (PVC) plastic. These mutants are designated surface attachment defective (sad ). Two classes of sad mutants were analysed: (i) mutants defective in flagellar-mediated motility and (ii) mutants defective in biogenesis of the polar-localized type IV pili. We followed the development of the biofilm formed by the wild type over 8 h using phase-contrast microscopy. The wild-type strain first formed a monolayer of cells on the abiotic surface, followed by the appearance of microcolonies that were dispersed throughout the monolayer of cells. Using time-lapse microscopy, we present evidence that microcolonies form by aggregation of cells present in the monolayer. As observed with the wild type, strains with mutations in genes required for the synthesis of type IV pili formed a monolayer of cells on the PVC plastic. However, in contrast to the wild-type strain, the type IV pili mutants did not develop microcolonies over the course of the experiments, suggesting that these structures play an important role in microcolony formation. Very few cells of a non-motile strain (carrying a mutation in flgK) attached to PVC even after 8 h of incubation, suggesting a role for flagella and/or motility in the initial cell-to-surface interactions. The phenotype of these mutants thus allows us to initiate the dissection of the developmental pathway leading to biofilm formation.
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            Quantification of biofilm structures by the novel computer program COMSTAT.

            The structural organization of four microbial communities was analysed by a novel computer program, COMSTAT, which comprises ten features for quantifying three-dimensional biofilm image stacks. Monospecies biofilms of each of the four bacteria, Pseudomonas: putida, P. aureofaciens, P. fluorescens and P. aeruginosa, tagged with the green fluorescent protein (GFP) were grown in flow chambers with a defined minimal medium as substrate. Analysis by the COMSTAT program of four variables describing biofilm structure - mean thickness, roughness, substratum coverage and surface to volume ratio - showed that the four Pseudomonas: strains represent different modes of biofilm growth. P. putida had a unique developmental pattern starting with single cells on the substratum growing into micro-colonies, which were eventually succeeded by long filaments and elongated cell clusters. P. aeruginosa colonized the entire substratum, and formed flat, uniform biofilms. P. aureofaciens resembled P. aeruginosa, but had a stronger tendency to form micro-colonies. Finally, the biofilm structures of P. fluorescens had a phenotype intermediate between those of P. putida and P. aureofaciens. Analysis of biofilms of P. aureofaciens growing on 0.03 mM, 0.1 mM or 0.5 mM citrate minimal media showed that mean biofilm thickness increased with increasing citrate concentration. Moreover, biofilm roughness increased with lower citrate concentrations, whereas surface to volume ratio increased with higher citrate concentrations.
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              Microbial pathogenesis in cystic fibrosis: mucoid Pseudomonas aeruginosa and Burkholderia cepacia.

              Respiratory infections with Pseudomonas aeruginosa and Burkholderia cepacia play a major role in the pathogenesis of cystic fibrosis (CF). This review summarizes the latest advances in understanding host-pathogen interactions in CF with an emphasis on the role and control of conversion to mucoidy in P. aeruginosa, a phenomenon epitomizing the adaptation of this opportunistic pathogen to the chronic chourse of infection in CF, and on the innate resistance to antibiotics of B. cepacia, person-to-person spread, and sometimes rapidly fatal disease caused by this organism. While understanding the mechanism of conversion to mucoidy in P. aeruginosa has progressed to the point where this phenomenon has evolved into a model system for studying bacterial stress response in microbial pathogenesis, the more recent challenge with B. cepacia, which has emerged as a potent bona fide CF pathogen, is discussed in the context of clinical issues, taxonomy, transmission, and potential modes of pathogenicity.
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                Author and article information

                Contributors
                Role: Editor
                Journal
                PLoS Pathog
                plos
                plospath
                PLoS Pathogens
                Public Library of Science (San Francisco, USA )
                1553-7366
                1553-7374
                November 2009
                November 2009
                20 November 2009
                : 5
                : 11
                : e1000668
                Affiliations
                [1]Department of Biological Sciences, Binghamton University, Binghamton, New York, United States of America
                University of California San Francisco, United States of America
                Author notes

                Conceived and designed the experiments: OEP KS. Performed the experiments: OEP KS. Analyzed the data: OEP KS. Contributed reagents/materials/analysis tools: KS. Wrote the paper: OEP KS.

                Article
                09-PLPA-RA-1434R2
                10.1371/journal.ppat.1000668
                2774163
                19936057
                78cde735-9246-4f2d-9183-52096c4d0062
                Petrova, Sauer. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
                History
                : 18 August 2009
                : 27 October 2009
                Page count
                Pages: 16
                Categories
                Research Article
                Biochemistry/Cell Signaling and Trafficking Structures
                Genetics and Genomics/Gene Function
                Infectious Diseases/Bacterial Infections
                Microbiology/Medical Microbiology
                Microbiology/Microbial Growth and Development
                Microbiology/Microbial Physiology and Metabolism
                Microbiology/Plant-Biotic Interactions

                Infectious disease & Microbiology
                Infectious disease & Microbiology

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