scholarly journals Identification of a Diguanylate Cyclase That Facilitates Biofilm Formation on Electrodes by Shewanella oneidensis MR-1

2021 ◽  
Vol 87 (9) ◽  
Author(s):  
Akiho Matsumoto ◽  
Ryota Koga ◽  
Robert A. Kanaly ◽  
Atsushi Kouzuma ◽  
Kazuya Watanabe
Keyword(s):  
Microbial Fuel Cells ◽  
Biofilm Formation ◽  
Shewanella Oneidensis ◽  
Electric Energy ◽  
Diguanylate Cyclase ◽  
Flow Conditions ◽  
Content Type ◽  
Electrochemically Active ◽  
Medium Flow

ABSTRACT In many bacteria, cyclic diguanosine monophosphate (c-di-GMP), synthesized by diguanylate cyclase (DGC), serves as a second messenger involved in the regulation of biofilm formation. Although studies have suggested that c-di-GMP also regulates the formation of electrochemically active biofilms (EABFs) by Shewanella oneidensis MR-1, DGCs involved in this process remained to be identified. Here, we report that the SO_1646 gene, hereafter named dgcS, is upregulated under medium flow conditions in electrochemical flow cells (EFCs), and its product (DgcS) functions as a major DGC in MR-1. In vitro assays demonstrated that purified DgcS catalyzed the synthesis of c-di-GMP from GTP. Comparisons of intracellular c-di-GMP levels in the wild-type strain and a dgcS deletion mutant (ΔdgcS mutant) showed that production of c-di-GMP was markedly reduced in the ΔdgcS mutant when cells were grown in batch cultures and on electrodes in EFCs. Cultivation of the ΔdgcS mutant in EFCs also revealed that the loss of DgcS resulted in impaired biofilm formation and decreased current generation. These findings demonstrate that MR-1 uses DgcS to synthesize c-di-GMP under medium flow conditions, thereby activating biofilm formation on electrodes. IMPORTANCE Bioelectrochemical systems (BESs) have attracted wide attention owing to their utility in sustainable biotechnology processes, such as microbial fuel cells and electrofermentation systems. In BESs, electrochemically active bacteria (EAB) form biofilms on electrode surfaces, thereby serving as effective catalysts for the interconversion between chemical and electric energy. It is therefore important to understand mechanisms for the formation of biofilm by EAB grown on electrodes. Here, we show that a model EAB, S. oneidensis MR-1, expresses DgcS as a major DGC, thereby activating the formation of biofilms on electrodes via c-di-GMP-dependent signal transduction cascades. The findings presented herein provide the molecular basis for improving electrochemical interactions between EAB and electrodes in BESs. The results also offer molecular insights into how Shewanella regulates biofilm formation on solid surfaces in the natural environment.

scholarly journals Structures, Compositions, and Activities of Live Shewanella Biofilms Formed on Graphite Electrodes in Electrochemical Flow Cells

2017 ◽  
Vol 83 (17) ◽  
Author(s):  
Miho Kitayama ◽  
Ryota Koga ◽  
Takuya Kasai ◽  
Atsushi Kouzuma ◽  
Kazuya Watanabe
Keyword(s):  
Wastewater Treatment ◽  
Water Flow ◽  
Biofilm Formation ◽  
Shewanella Oneidensis ◽  
Flow Conditions ◽  
Current Generation ◽  
Content Type ◽  
Flow Cells ◽  
Electrode Potentials ◽  
Over Time

ABSTRACT An electrochemical flow cell equipped with a graphite working electrode (WE) at the bottom was inoculated with Shewanella oneidensis MR-1 expressing an anaerobic fluorescent protein, and biofilm formation on the WE was observed over time during current generation at WE potentials of +0.4 and 0 V (versus standard hydrogen electrodes), under electrolyte-flow conditions. Electrochemical analyses suggested the presence of unique electron-transfer mechanisms in the +0.4-V biofilm. Microscopic analyses revealed that, in contrast to aerobic biofilms, current-generating biofilm (at +0.4 V) was thin and flat (∼10 μm in thickness), and cells were evenly and densely distributed in the biofilm. In contrast, cells were unevenly distributed in biofilm formed at 0 V. In situ fluorescence staining and biofilm recovery experiments showed that the amounts of extracellular polysaccharides (EPSs) in the +0.4-V biofilm were much smaller than those in the aerobic and 0-V biofilms, suggesting that Shewanella cells suppress the production of EPSs at +0.4 V under flow conditions. We suggest that Shewanella cells perceive electrode potentials and modulate the structure and composition of biofilms to efficiently transfer electrons to electrodes. IMPORTANCE A promising application of microbial fuel cells (MFCs) is to save energy in wastewater treatment. Since current is generated in these MFCs by biofilm microbes under horizontal flows of wastewater, it is important to understand the mechanisms for biofilm formation and current generation under water-flow conditions. Although massive work has been done to analyze the molecular mechanisms for current generation by model exoelectrogenic bacteria, such as Shewanella oneidensis, limited information is available regarding the formation of current-generating biofilms over time under water-flow conditions. The present study developed electrochemical flow cells and used them to examine the electrochemical and structural features of current-generating biofilms under water-flow conditions. We show unique features of mature biofilms actively generating current, creating opportunities to search for as-yet-undiscovered current-generating mechanisms in Shewanella biofilms. Furthermore, information provided in the present study is useful for researchers attempting to develop anode architectures suitable for wastewater treatment MFCs.

Molecular mechanisms regulating the catabolic and electrochemical activities of Shewanella oneidensis MR-1

2021 ◽  
Author(s):  
Atsushi Kouzuma
Keyword(s):  
Microbial Fuel Cells ◽  
Biofilm Formation ◽  
Molecular Mechanisms ◽  
Current Knowledge ◽  
Shewanella Oneidensis ◽  
Extracellular Electron Transfer ◽  
Cellular Functions ◽  
Regulatory Systems ◽  
Electrochemically Active ◽  
Recent Developments

Abstract Electrochemically active bacteria (EAB) interact electrochemically with electrodes via extracellular electron transfer (EET) pathways. These bacteria have attracted significant attention due to their utility in environment-friendly bioelectrochemical systems (BESs), e.g. microbial fuel cells and electrofermentation systems. The electrochemical activity of EAB is dependent on their carbon catabolism and respiration; thus, understanding how these processes are regulated will provide insights into the development of a more efficient BES. The process of biofilm formation by EAB on BES electrodes is also important for electric current generation, because it facilitates physical and electrochemical interactions between EAB cells and electrodes. This article summarizes the current knowledge on EET-related metabolic and cellular functions of a model EAB, Shewanella oneidensis MR-1, focusing specifically on regulatory systems for carbon catabolism, EET pathways, and biofilm formation. Based on recent developments, the author also discusses potential uses of engineered S. oneidensis strains for various biotechnological applications.

scholarly journals In VitroAnalyses of the Effects of Heparin and Parabens on Candida albicans Biofilms and Planktonic Cells

2011 ◽  
Vol 56 (1) ◽  
pp. 148-153 ◽  
Author(s):  
Marisa H. Miceli ◽  
Stella M. Bernardo ◽  
T. S. Neil Ku ◽  
Carla Walraven ◽  
Samuel A. Lee
Keyword(s):  
Candida Albicans ◽  
Metabolic Activity ◽  
Biofilm Formation ◽  
High Dose ◽  
Dependent Manner ◽  
Planktonic Cells ◽  
Content Type ◽  
Heparin Sodium ◽  
The Individual

ABSTRACTInfections and thromboses are the most common complications associated with central venous catheters. Suggested strategies for prevention and management of these complications include the use of heparin-coated catheters, heparin locks, and antimicrobial lock therapy. However, the effects of heparin onCandida albicansbiofilms and planktonic cells have not been previously studied. Therefore, we sought to determine thein vitroeffect of a heparin sodium preparation (HP) on biofilms and planktonic cells ofC. albicans. Because HP contains two preservatives, methyl paraben (MP) and propyl paraben (PP), these compounds and heparin sodium without preservatives (Pure-H) were also tested individually. The metabolic activity of the mature biofilm after treatment was assessed using XTT [2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-carboxanilide] reduction and microscopy. Pure-H, MP, and PP caused up to 75, 85, and 60% reductions of metabolic activity of the mature preformedC. albicansbiofilms, respectively. Maximal efficacy against the mature biofilm was observed with HP (up to 90%) compared to the individual compounds (P< 0.0001). Pure-H, MP, and PP each inhibitedC. albicansbiofilm formation up to 90%. A complete inhibition of biofilm formation was observed with HP at 5,000 U/ml and higher. When tested against planktonic cells, each compound inhibited growth in a dose-dependent manner. These data indicated that HP, MP, PP, and Pure-H havein vitroantifungal activity againstC. albicansmature biofilms, formation of biofilms, and planktonic cells. Investigation of high-dose heparin-based strategies (e.g., heparin locks) in combination with traditional antifungal agents for the treatment and/or prevention ofC. albicansbiofilms is warranted.

scholarly journals A Single B-Repeat of Staphylococcus epidermidis Accumulation-Associated Protein Induces Protective Immune Responses in an Experimental Biomaterial-Associated Infection Mouse Model

2014 ◽  
Vol 21 (9) ◽  
pp. 1206-1214 ◽  
Author(s):  
Lin Yan ◽  
Lei Zhang ◽  
Hongyan Ma ◽  
David Chiu ◽  
James D. Bryers
Keyword(s):  
Staphylococcus Epidermidis ◽  
Biofilm Formation ◽  
Porous Scaffold ◽  
The United States ◽  
Protein Vaccine ◽  
Weight Changes ◽  
Biofilm Inhibition ◽  
Content Type ◽  
Accumulation Associated Protein

ABSTRACTNosocomial infections are the fourth leading cause of morbidity and mortality in the United States, resulting in 2 million infections and ∼100,000 deaths each year. More than 60% of these infections are associated with some type of biomedical device.Staphylococcus epidermidisis a commensal bacterium of the human skin and is the most common nosocomial pathogen infecting implanted medical devices, especially those in the cardiovasculature.S. epidermidisantibiotic resistance and biofilm formation on inert surfaces make these infections hard to treat. Accumulation-associated protein (Aap), a cell wall-anchored protein ofS. epidermidis, is considered one of the most important proteins involved in the formation ofS. epidermidisbiofilm. A small recombinant protein vaccine comprising a single B-repeat domain (Brpt1.0) ofS. epidermidisRP62A Aap was developed, and the vaccine's efficacy was evaluatedin vitrowith a biofilm inhibition assay andin vivoin a murine model of biomaterial-associated infection. A high IgG antibody response againstS. epidermidisRP62A was detected in the sera of the mice after two subcutaneous immunizations with Brpt1.0 coadministered with Freund's adjuvant. Sera from Brpt1.0-immunized mice inhibitedin vitroS. epidermidisRP62A biofilm formation in a dose-dependent pattern. After receiving two immunizations, each mouse was surgically implanted with a porous scaffold disk containing 5 × 106CFU ofS. epidermidisRP62A. Weight changes, inflammatory markers, and histological assay results after challenge withS. epidermidisindicated that the mice immunized with Brpt1.0 exhibited significantly higher resistance toS. epidermidisRP62A implant infection than the control mice. Day 8 postchallenge, there was a significantly lower number of bacteria in scaffold sections and surrounding tissues and a lower residual inflammatory response to the infected scaffold disks for the Brpt1.0-immunized mice than for of the ovalbumin (Ova)-immunized mice.

scholarly journals Abrogation of Neuraminidase Reduces Biofilm Formation, Capsule Biosynthesis, and Virulence of Porphyromonas gingivalis

2011 ◽  
Vol 80 (1) ◽  
pp. 3-13 ◽  
Author(s):  
Chen Li ◽  
Kurniyati ◽  
Bo Hu ◽  
Jiang Bian ◽  
Jianlan Sun ◽  
...  
Keyword(s):  
Porphyromonas Gingivalis ◽  
Biofilm Formation ◽  
Adult Population ◽  
Transcriptional Analysis ◽  
Wild Type ◽  
Content Type ◽  
Multiple Organs ◽  
Biochemical Analyses

ABSTRACTThe oral bacteriumPorphyromonas gingivalisis a key etiological agent of human periodontitis, a prevalent chronic disease that affects up to 80% of the adult population worldwide.P. gingivalisexhibits neuraminidase activity. However, the enzyme responsible for this activity, its biochemical features, and its role in the physiology and virulence ofP. gingivalisremain elusive. In this report, we found thatP. gingivalisencodes a neuraminidase, PG0352 (SiaPg). Transcriptional analysis showed thatPG0352is monocistronic and is regulated by a sigma70-like promoter. Biochemical analyses demonstrated that SiaPgis an exo-α-neuraminidase that cleaves glycosidic-linked sialic acids. Cryoelectron microscopy and tomography analyses revealed that thePG0352deletion mutant (ΔPG352) failed to produce an intact capsule layer. Compared to the wild type,in vitrostudies showed that ΔPG352 formed less biofilm and was less resistant to killing by the host complement.In vivostudies showed that while the wild type caused a spreading type of infection that affected multiple organs and all infected mice were killed, ΔPG352 only caused localized infection and all animals survived. Taken together, these results demonstrate that SiaPgis an important virulence factor that contributes to the biofilm formation, capsule biosynthesis, and pathogenicity ofP. gingivalis, and it can potentially serve as a new target for developing therapeutic agents againstP. gingivalisinfection.

scholarly journals Specific Control of Pseudomonas aeruginosa Surface-Associated Behaviors by Two c-di-GMP Diguanylate Cyclases

mBio ◽  
2010 ◽  
Vol 1 (4) ◽  
Author(s):  
Judith H. Merritt ◽  
Dae-Gon Ha ◽  
Kimberly N. Cowles ◽  
Wenyun Lu ◽  
Diana K. Morales ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Biofilm Formation ◽  
Extracellular Polysaccharide ◽  
Diguanylate Cyclase ◽  
Flagellar Motility ◽  
Critical Question ◽  
Cyclic Diguanylate ◽  
Critical Signal ◽  
Content Type ◽  
Wide Range

ABSTRACT The signaling nucleotide cyclic diguanylate (c-di-GMP) regulates the transition between motile and sessile growth in a wide range of bacteria. Understanding how microbes control c-di-GMP metabolism to activate specific pathways is complicated by the apparent multifold redundancy of enzymes that synthesize and degrade this dinucleotide, and several models have been proposed to explain how bacteria coordinate the actions of these many enzymes. Here we report the identification of a diguanylate cyclase (DGC), RoeA, of Pseudomonas aeruginosa that promotes the production of extracellular polysaccharide (EPS) and contributes to biofilm formation, that is, the transition from planktonic to surface-dwelling cells. Our studies reveal that RoeA and the previously described DGC SadC make distinct contributions to biofilm formation, controlling polysaccharide production and flagellar motility, respectively. Measurement of total cellular levels of c-di-GMP in ∆roeA and ∆sadC mutants in two different genetic backgrounds revealed no correlation between levels of c-di-GMP and the observed phenotypic output with regard to swarming motility and EPS production. Our data strongly argue against a model wherein changes in total levels of c-di-GMP can account for the specific surface-related phenotypes of P. aeruginosa. IMPORTANCE A critical question in the study of cyclic diguanylate (c-di-GMP) signaling is how the bacterial cell integrates contributions of multiple c-di-GMP-metabolizing enzymes to mediate its cognate functional outputs. One leading model suggests that the effects of c-di-GMP must, in part, be localized subcellularly. The data presented here show that the phenotypes controlled by two different diguanylate cyclase (DGC) enzymes have discrete outputs despite the same total level of c-di-GMP. These data support and extend the model in which localized c-di-GMP signaling likely contributes to coordination of the action of the multiple proteins involved in the synthesis, degradation, and/or binding of this critical signal.

scholarly journals Novel regulators of the csgD gene encoding the master regulator of biofilm formation in Escherichia coli K-12

Microbiology ◽  
2020 ◽  
Vol 166 (9) ◽  
pp. 880-890 ◽  
Author(s):  
Hiroshi Ogasawara ◽  
Toshiyuki Ishizuka ◽  
Shuhei Hotta ◽  
Michiko Aoki ◽  
Tomohiro Shimada ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Biofilm Formation ◽  
Cell Aggregation ◽  
Master Regulator ◽  
Gene Encoding ◽  
Content Type ◽  
Promoter Probe ◽  
K 12 ◽  
Specific Environmental Condition

Under stressful conditions, Escherichia coli forms biofilm for survival by sensing a variety of environmental conditions. CsgD, the master regulator of biofilm formation, controls cell aggregation by directly regulating the synthesis of Curli fimbriae. In agreement of its regulatory role, as many as 14 transcription factors (TFs) have so far been identified to participate in regulation of the csgD promoter, each monitoring a specific environmental condition or factor. In order to identify the whole set of TFs involved in this typical multi-factor promoter, we performed in this study ‘promoter-specific transcription-factor’ (PS-TF) screening in vitro using a set of 198 purified TFs (145 TFs with known functions and 53 hitherto uncharacterized TFs). A total of 48 TFs with strong binding to the csgD promoter probe were identified, including 35 known TFs and 13 uncharacterized TFs, referred to as Y-TFs. As an attempt to search for novel regulators, in this study we first analysed a total of seven Y-TFs, including YbiH, YdcI, YhjC, YiaJ, YiaU, YjgJ and YjiR. After analysis of curli fimbriae formation, LacZ-reporter assay, Northern-blot analysis and biofilm formation assay, we identified at least two novel regulators, repressor YiaJ (renamed PlaR) and activator YhjC (renamed RcdB), of the csgD promoter.

Environment in the lung of cystic fibrosis patients stimulates the expression of biofilm phenotype in Mycobacterium abscessus

2022 ◽  
Vol 71 (1) ◽  
Author(s):  
Bailey F. Keefe ◽  
Luiz E. Bermudez
Keyword(s):  
Cystic Fibrosis ◽  
Host Cell ◽  
Biofilm Formation ◽  
Type Species ◽  
Divalent Cations ◽  
Mycobacterium Abscessus ◽  
Content Type ◽  
Link Type ◽  
Populations At Risk

Introduction. Pulmonary infections caused by organisms of the Mycobacterium abscessus complex are increasingly prevalent in populations at risk, such as patients with cystic fibrosis, bronchiectasis and emphysema. Hypothesis. M. abscessus infection of the lung is not observed in immunocompetent individuals, which raises the possibility that the compromised lung environment is a suitable niche for the pathogen to thrive in due to the overproduction of mucus and high amounts of host cell lysis. Aim. Evaluate the ability of M. abscessus to form biofilm and grow utilizing in vitro conditions as seen in immunocompromised lungs of patients. Methodology. We compared biofilm formation and protein composition in the presence and absence of synthetic cystic fibrosis medium (SCFM) and evaluated the bacterial growth when exposed to human DNA. Results. M. abscessus is capable of forming biofilm in SCFM. By eliminating single components found in the medium, it became clear that magnesium works as a signal for the biofilm formation, and chelation of the divalent cations resulted in the suppression of biofilm formation. Investigation of the specific proteins expressed in the presence of SCFM and in the presence of SCFM lacking magnesium revealed many different proteins between the conditions. M. abscessus also exhibited growth in SCFM and in the presence of host cell DNA, although the mechanism of DNA utilization remains unclear. Conclusions. In vitro conditions mimicking the airways of patients with cystic fibrosis appear to facilitate M. abscessus establishment of infection, and elimination of magnesium from the environment may affect the ability of the pathogen to establish infection.

scholarly journals Quorum Sensing Influences Burkholderia thailandensis Biofilm Development and Matrix Production

2016 ◽  
Vol 198 (19) ◽  
pp. 2643-2650 ◽  
Author(s):  
Boo Shan Tseng ◽  
Charlotte D. Majerczyk ◽  
Daniel Passos da Silva ◽  
Josephine R. Chandler ◽  
E. Peter Greenberg ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Matrix Material ◽  
Close Relative ◽  
Wild Type ◽  
Flow Conditions ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
Biofilm Development

ABSTRACTMembers of the genusBurkholderiaare known to be adept at biofilm formation, which presumably assists in the survival of these organisms in the environment and the host. Biofilm formation has been linked to quorum sensing (QS) in several bacterial species. In this study, we characterizedBurkholderia thailandensisbiofilm development under flow conditions and sought to determine whether QS contributes to this process.B. thailandensisbiofilm formation exhibited an unusual pattern: the cells formed small aggregates and then proceeded to produce mature biofilms characterized by “dome” structures filled with biofilm matrix material. We showed that this process was dependent on QS.B. thailandensishas three acyl-homoserine lactone (AHL) QS systems (QS-1, QS-2, and QS-3). An AHL-negative strain produced biofilms consisting of cell aggregates but lacking the matrix-filled dome structures. This phenotype was rescued via exogenous addition of the three AHL signals. Of the threeB. thailandensisQS systems, we show that QS-1 is required for proper biofilm development, since abtaR1mutant, which is defective in QS-1 regulation, forms biofilms without these dome structures. Furthermore, our data show that the wild-type biofilm biomass, as well as the material inside the domes, stains with a fucose-binding lectin. ThebtaR1mutant biofilms, however, are negative for fucose staining. This suggests that the QS-1 system regulates the production of a fucose-containing exopolysaccharide in wild-type biofilms. Finally, we present data showing that QS ability during biofilm development produces a biofilm that is resistant to dispersion under stress conditions.IMPORTANCEThe saprophyteBurkholderia thailandensisis a close relative of the pathogenic bacteriumBurkholderia pseudomallei, the causative agent of melioidosis, which is contracted from its environmental reservoir. Since most bacteria in the environment reside in biofilms,B. thailandensisis an ideal model organism for investigating questions inBurkholderiaphysiology. In this study, we characterizedB. thailandensisbiofilm development and sought to determine if quorum sensing (QS) contributes to this process. Our work shows thatB. thailandensisproduces biofilms with unusual dome structures under flow conditions. Our findings suggest that these dome structures are filled with a QS-regulated, fucose-containing exopolysaccharide that may be involved in the resilience ofB. thailandensisbiofilms against changes in the nutritional environment.

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