Engineering a light-responsive, quorum quenching biofilm to mitigate biofouling on water purification membranes.
ABSTRACT: Quorum quenching (QQ) has been reported to be a promising approach for membrane biofouling control. Entrapment of QQ bacteria in porous matrices is required to retain them in continuously operated membrane processes and to prevent uncontrollable biofilm formation by the QQ bacteria on membrane surfaces. It would be more desirable if the formation and dispersal of biofilms by QQ bacteria could be controlled so that the QQ bacterial cells are self-immobilized, but the QQ biofilm itself still does not compromise membrane performance. In this study, we engineered a QQ bacterial biofilm whose growth and dispersal can be modulated by light through a dichromatic, optogenetic c-di-GMP gene circuit in which the bacterial cells sense near-infrared (NIR) light and blue light to adjust its biofilm formation by regulating the c-di-GMP level. We also demonstrated the potential application of the engineered light-responsive QQ biofilm in mitigating biofouling of water purification forward osmosis membranes. The c-di-GMP-targeted optogenetic approach for controllable biofilm development we have demonstrated here should prove widely applicable for designing other controllable biofilm-enabled applications such as biofilm-based biocatalysis.
Project description:Quorum quenching-membrane bioreactors (QQ-MBRs) have been studied widely in recent decades. However, limited information is known about the influence of QQ on the microbial community. In this study, the indigenous QQ bacterium Bacillus cereus HG10 was immobilized and used to control biofouling in a bioreactor. QQ beads caused extracellular polymeric substance reduction and significantly hindered biofilm formation on a submerged membrane. Community profiling of 16S rRNA gene amplicons revealed that QQ beads dramatically altered the bacterial community structure in activated sludge but not in biofilm. Bacterial structure in the presence of QQ beads showed a clear divergence from that of the control groups at phylum, class, order, family, and genus taxonomic ranks. A significant enrichment of several bacterial genera, including Acinetobacter, Aeromonas, Delftia, Bacillus, and Pseudomonas, and depletion of over 12 bacterial genera were observed. These findings would contribute to a better understanding of why and how immobilized QQ bacteria impair membrane biofouling in QQ-MBRs.
Project description:Quorum Quenching (QQ) has been developed over the last few years to overcome practical issues related to membrane biofouling, which is currently the major difficulty thwarting the extensive development of membrane bioreactors (MBRs). QQ is the disruption of Quorum Sensing (QS), cell-to-cell communication enabling the bacteria to harmonize their behavior. The production of biofilm, which is recognized as a major part of the biocake formed on a membrane surface, and which leads to biofouling, has been found to be one of the bacterial behaviors controlled by QS. Since the enzymatic disruption of QS was reported to be efficient as a membrane biofouling mitigation technique in MBRs, the application of QQ to lab-scale MBRs has been the subject of much research using different approaches under different operating conditions. This paper gives an overview of the effectiveness of QQ in mitigating membrane biofouling in MBRs. It is based on the results of previous studies, using two microbial strains, Rhodococcus sp. BH4 and Pseudomonas sp. 1A1. The effect of bacterial QQ on the physical phenomena of the MBR process is analyzed, adopting an original multi-scale approach. Finally, the potential influence of the MBR operating conditions on QQ effectiveness is discussed.
Project description:In Pseudomonas aeruginosa, the transition between planktonic and biofilm lifestyles is modulated by the intracellular secondary messenger cyclic dimeric-GMP (c-di-GMP) in response to environmental conditions. Here, we used gene deletions to investigate how the environmental stimulus nitric oxide (NO) is linked to biofilm dispersal, focusing on biofilm dispersal phenotype from proteins containing putative c-di-GMP turnover and Per-Arnt-Sim (PAS) sensory domains. We document opposed physiological roles for the genes ?rbdA and ?pa2072 that encode proteins with identical domain structure: while ?rbdA showed elevated c-di-GMP levels, restricted motility and promoted biofilm formation, c-di-GMP levels were decreased in ?pa2072, and biofilm formation was inhibited, compared to wild type. A second pair of genes, ?fimX and ?dipA, were selected on the basis of predicted impaired c-di-GMP turnover function: ?fimX showed increased, ?dipA decreased NO induced biofilm dispersal, and the genes effected different types of motility, with reduced twitching for ?fimX and reduced swimming for ?dipA. For all four deletion mutants we find that NO-induced biomass reduction correlates with increased NO-driven swarming, underlining a significant role for this motility in biofilm dispersal. Hence P. aeruginosa is able to differentiate c-di-GMP output using structurally highly related proteins that can contain degenerate c-di-GMP turnover domains.
Project description:Bacteria prefer to grow attached to themselves or an interface, and it is important for an array of applications to make biofilms disperse. Here we report simultaneously the discovery and protein engineering of BdcA (formerly YjgI) for biofilm dispersal using the universal signal 3,5-cyclic diguanylic acid (c-di-GMP). The bdcA deletion reduced biofilm dispersal, and production of BdcA increased biofilm dispersal to wild-type level. Since BdcA increases motility and extracellular DNA production while decreasing exopolysaccharide, cell length and aggregation, we reasoned that BdcA decreases the concentration of c-di-GMP, the intracellular messenger that controls cell motility through flagellar rotation and biofilm formation through synthesis of curli and cellulose. Consistently, c-di-GMP levels increase upon deleting bdcA, and purified BdcA binds c-di-GMP but does not act as a phosphodiesterase. Additionally, BdcR (formerly YjgJ) is a negative regulator of bdcA. To increase biofilm dispersal, we used protein engineering to evolve BdcA for greater c-di-GMP binding and found that the single amino acid change E50Q causes nearly complete removal of biofilms via dispersal without affecting initial biofilm formation.
Project description:Pseudomonas aeruginosa encodes many enzymes that are potentially associated with the synthesis or degradation of the widely conserved second messenger cyclic-di-GMP (c-di-GMP). In this study, we show that mutation of rbdA, which encodes a fusion protein consisting of PAS-PAC-GGDEF-EAL multidomains, results in decreased biofilm dispersal. RbdA contains a highly conserved GGDEF domain and EAL domain, which are involved in the synthesis and degradation of c-di-GMP, respectively. However, in vivo and in vitro analyses show that the full-length RbdA protein only displays phosphodiesterase activity, causing c-di-GMP degradation. Further analysis reveals that the GGDEF domain of RbdA plays a role in activating the phosphodiesterase activity of the EAL domain in the presence of GTP. Moreover, we show that deletion of the PAS domain or substitution of the key residues implicated in sensing low-oxygen stress abrogates the functionality of RbdA. Subsequent study showed that RbdA is involved in positive regulation of bacterial motility and production of rhamnolipids, which are associated with biofilm dispersal, and in negative regulation of production of exopolysaccharides, which are required for biofilm formation. These data indicate that the c-di-GMP-degrading regulatory protein RbdA promotes biofilm dispersal through its two-pronged effects on biofilm development, i.e., downregulating biofilm formation and upregulating production of the factors associated with biofilm dispersal.
Project description:When encountering surfaces, many bacteria produce adhesins to facilitate their initial attachment and to irreversibly glue themselves to the solid substrate. A central molecule regulating the processes of this motile-sessile transition is the second messenger c-di-GMP, which stimulates the production of a variety of exopolysaccharide adhesins in different bacterial model organisms. In Caulobacter crescentus, c-di-GMP regulates the synthesis of the polar holdfast adhesin during the cell cycle, yet the molecular and cellular details of this control are currently unknown. Here we identify HfsK, a member of a versatile N-acetyltransferase family, as a novel c-di-GMP effector involved in holdfast biogenesis. Cells lacking HfsK form highly malleable holdfast structures with reduced adhesive strength that cannot support surface colonization. We present indirect evidence that HfsK modifies the polysaccharide component of holdfast to buttress its cohesive properties. HfsK is a soluble protein but associates with the cell membrane during most of the cell cycle. Coincident with peak c-di-GMP levels during the C. crescentus cell cycle, HfsK relocalizes to the cytosol in a c-di-GMP-dependent manner. Our results indicate that this c-di-GMP-mediated dynamic positioning controls HfsK activity, leading to its inactivation at high c-di-GMP levels. A short C-terminal extension is essential for the membrane association, c-di-GMP binding, and activity of HfsK. We propose a model in which c-di-GMP binding leads to the dispersal and inactivation of HfsK as part of holdfast biogenesis progression.IMPORTANCE Exopolysaccharide (EPS) adhesins are important determinants of bacterial surface colonization and biofilm formation. Biofilms are a major cause of chronic infections and are responsible for biofouling on water-exposed surfaces. To tackle these problems, it is essential to dissect the processes leading to surface colonization at the molecular and cellular levels. Here we describe a novel c-di-GMP effector, HfsK, that contributes to the cohesive properties and stability of the holdfast adhesin in C. crescentus We demonstrate for the first time that c-di-GMP, in addition to its role in the regulation of the rate of EPS production, also modulates the physicochemical properties of bacterial adhesins. By demonstrating how c-di-GMP coordinates the activity and subcellular localization of HfsK, we provide a novel understanding of the cellular processes involved in adhesin biogenesis control. Homologs of HfsK are found in representatives of different bacterial phyla, suggesting that they play important roles in various EPS synthesis systems.
Project description:Bacteria in biofilms often undergo active dispersal events and revert to a free-swimming, planktonic state to complete the biofilm life cycle. The signaling molecule nitric oxide (NO) was previously found to trigger biofilm dispersal in the opportunistic pathogen Pseudomonas aeruginosa at low, nontoxic concentrations (N. Barraud, D. J. Hassett, S. H. Hwang, S. A. Rice, S. Kjelleberg, and J. S. Webb, J. Bacteriol. 188:7344-7353, 2006). NO was further shown to increase cell motility and susceptibility to antimicrobials. Recently, numerous studies revealed that increased degradation of the secondary messenger cyclic di-GMP (c-di-GMP) by specific phosphodiesterases (PDEs) triggers a planktonic mode of growth in eubacteria. In this study, the potential link between NO and c-di-GMP signaling was investigated by performing (i) PDE inhibitor studies, (ii) enzymatic assays to measure PDE activity, and (iii) direct quantification of intracellular c-di-GMP levels. The results suggest a role for c-di-GMP signaling in triggering the biofilm dispersal event induced by NO, as dispersal requires PDE activity and addition of NO stimulates PDE and induces the concomitant decrease in intracellular c-di-GMP levels in P. aeruginosa. Furthermore, gene expression studies indicated global responses to low, nontoxic levels of NO in P. aeruginosa biofilms, including upregulation of genes involved in motility and energy metabolism and downregulation of adhesins and virulence factors. Finally, site-directed mutagenesis of candidate genes and physiological characterization of the corresponding mutant strains uncovered that the chemotaxis transducer BdlA is involved in the biofilm dispersal response induced by NO.
Project description:Opportunistic pathogenic bacteria can engage in biofilm-based infections that evade immune responses and develop into chronic conditions. Because conventional antimicrobials cannot efficiently eradicate biofilms, there is an urgent need to develop alternative measures to combat biofilm infections. It has recently been established that the secondary messenger cyclic diguanosine monophosphate (c-di-GMP) functions as a positive regulator of biofilm formation in several different bacteria. In the present study we investigated whether manipulation of the c-di-GMP level in bacteria potentially can be used for biofilm control in vivo. We constructed a Pseudomonas aeruginosa strain in which a reduction in the c-di-GMP level can be achieved via induction of the Escherichia coli YhjH c-di-GMP phosphodiesterase. Initial experiments showed that induction of yhjH expression led to dispersal of the majority of the bacteria in in vitro-grown P. aeruginosa biofilms. Subsequently, we demonstrated that P. aeruginosa biofilms growing on silicone implants, located in the peritoneal cavity of mice, dispersed after induction of the YhjH protein. Bacteria accumulated temporarily in the spleen after induction of biofilm dispersal, but the mice tolerated the dispersed bacteria well. The present work provides proof of the concept that modulation of the c-di-GMP level in bacteria is a viable strategy for biofilm control.
Project description:Various important cellular processes in bacteria are controlled by c-di-GMP, such as motility, biofilm formation and virulence factors production. C-di-GMP is synthesized from two molecules of GTP by diguanylate cyclases (DGCs) and its actions are terminated by EAL or HD-GYP domain phosphodiesterases (PDEs), which hydrolyze c-di-GMP to linear pGpG or GMP. Thus far the majority of efforts have been dedicated to the development of inhibitors of DGCs but not PDEs. This is probably because the old view was that inhibiting any c-di-GMP PDE would lead to biofilm formation, an undesirable phenotype. Recent data however suggest that some PDEs only change the localized (not global) concentration of c-di-GMP to increase bacterial virulence and do not affect biofilm formation. A challenge therefore is to be able to develop selective PDE inhibitors that inhibit virulence-associated PDEs but not inhibit PDEs that regulate bacterial biofilm formation. Using high throughput docking experiments to screen a library of 250?000 commercially available compounds against E. coli YahA (also called PdeL), a benzoisothiazolinone derivative was found to bind to the c-di-GMP binding site of YahA with favorable energetics. Paradoxically the in silico identified inhibitor (a benzoisothiazolinone derivative) did not inhibit the hydrolysis of c-di-GMP by YahA, the model PDE that was used in the docking, but instead inhibited RocR, which is a PDE from the opportunistic pathogen P. aeruginosa (PA). RocR promotes bacterial virulence but not biofilm dispersal, making it an ideal PDE to target for anti-virulence purposes. This newly identified RocR ligand displayed some selectivity and did not inhibit other P. aeruginosa PDEs, such as DipA, PvrR and PA4108. DipA, PvrR and PA4108 are key enzymes that reduce global c-di-GMP concentration and promote biofilm dispersal; therefore the identification of an inhibitor of a PA PDE, such as RocR, that does not inhibit major PDEs that modulate global c-di-GMP is an important step towards the development of selective c-di-GMP PDEs that could have interesting biomedical applications. The identified RocR ligand could also inhibit P. aeruginosa (PAO1) swarming but not swimming or biofilm formation. Rhamnolipid production was decreased, explaining the inhibition of swarming.
Project description:Cyclic-di-GMP (c-di-GMP) is an intracellular secondary messenger which controls the biofilm life cycle in many bacterial species. High intracellular c-di-GMP content enhances biofilm formation via the reduction of motility and production of biofilm matrix, while low c-di-GMP content in biofilm cells leads to increased motility and biofilm dispersal. While the effect of high c-di-GMP levels on bacterial lifestyles is well studied, the physiology of cells at low c-di-GMP levels remains unclear. Here, we showed that Pseudomonas aeruginosa cells with high and low intracellular c-di-GMP contents possessed distinct transcriptome profiles. There were 535 genes being upregulated and 432 genes downregulated in cells with low c-di-GMP, as compared to cells with high c-di-GMP. Interestingly, both rhl and pqs quorum-sensing (QS) operons were expressed at higher levels in cells with low intracellular c-di-GMP content compared with cells with higher c-di-GMP content. The induced expression of pqs and rhl QS required a functional PqsR, the transcriptional regulator of pqs QS. Next, we observed increased production of pqs and rhl-regulated virulence factors, such as pyocyanin and rhamnolipids, in P. aeruginosa cells with low c-di-GMP levels, conferring them with increased intracellular survival rates and cytotoxicity against murine macrophages. Hence, our data suggested that low intracellular c-di-GMP levels in bacteria could induce QS-regulated virulence, in particular rhamnolipids that cripple the cellular components of the innate immune system.