Regulation Of Biofilm Formation Research Articles

Identification and characterization of a small molecule BFstatin inhibiting BrpR, the transcriptional regulator for biofilm formation of Vibrio vulnificus

Many pathogenic bacteria form biofilms that are resistant to not only host immune defenses but also antibiotics, posing a need for the development of strategies to control biofilms. In this study, to prevent biofilm formation of the fulminating foodborne pathogen Vibrio vulnificus, chemical libraries were extensively screened to identify a small molecule inhibiting the activity of BrpR, a transcriptional regulator for biofilm genes. Accordingly, the BrpR inhibitor BFstatin [N1-(2-chloro-5-fluorophenyl)-N3-propylmalonamide], with a half-maximal effective concentration of 8.01 μM, was identified. BFstatin did not interfere with bacterial growth or exhibit cytotoxicity to the human epithelial cell line. BFstatin directly bound to BrpR and interrupted its binding to the target promoter DNAs of the downstream genes. Molecular dynamics simulation of the interaction between BFstatin and BrpR proposed that BFstatin modifies the structure of BrpR, especially the DNA-binding domain. Transcriptomic analyses revealed that BFstatin reduces the expression of the BrpR regulon including the cabABC operon and brp locus which contribute to the production of biofilm matrix of V. vulnificus. Accordingly, BFstatin diminished the biofilm levels of V. vulnificus by inhibiting the matrix development in a concentration-dependent manner. Altogether, BFstatin could be an anti-biofilm agent targeting BrpR, thereby rendering V. vulnificus more susceptible to host immune defenses and antibiotics.

Double-Wing Motif Protein is a Novel Biofilm Regulatory Factor of the Plant Disease Biocontrol Agent, Bacillus subtilis.

Transposon mutagenesis screening of Bacillus subtilis YB-1471, a novel rhizosphere biocontrol agent of Fusarium crown rot (FCR) of wheat, resulted in the identification of orf04391, linked to reduced biofilm formation. The gene encodes a protein possessing a putative tertiary structure of a "double-wing" DNA-binding domain. Expression of orf04391 increased during biofilm development in stationary cultures and during rapid growth in shaking cultures. An orf04391 deletion strain showed reduced biofilm production related to lower levels of the extracellular matrix, and the mutant also had reduced sporulation, adhesion, root colonization, and FCR biocontrol efficiency. Transcriptome analysis of YB-1471 and Δorf04391 in stationary culture showed that the loss of orf04391 resulted in altered expression of numerous genes, including sinI, an initiator of biofilm formation. DNA binding was shown with his-tagged Orf04391 binding to the sinIR operon in vivo and in vitro. Orf04391 appears to be a transcriptional regulator of biofilm formation in B. subtilis through the Spo0A-SinI/SinR pathway.

Taxonomic and functional characterization of biofilms from a photovoltaic panel reveals high genetic and metabolic complexity of the communities.

Biofilms are complex microbial cell aggregates that attach to different surfaces in nature, industrial environments, or hospital settings. In photovoltaic panels (PVs), biofilms are related to significant energy conversion losses. In this study, our aim was to characterize the communities of microorganisms and the genes involved in biofilm formation. In this study, biofilm samples collected from a PV system installed in southeastern Brazil were analyzed through shotgun metagenomics, and the microbial communities and genes involved in biofilm formation were investigated. A total of 2030 different genera were identified in the samples, many of which were classified as extremophiles or producers of exopolysaccharides. Bacteria prevailed in the samples (89%), mainly the genera Mucilaginibacter, Microbacterium, Pedobacter, Massilia, and Hymenobacter. The functional annotation revealed >12 000 genes related to biofilm formation and stress response. Genes involved in the iron transport and synthesis of c-di-GMP and c-AMP second messengers were abundant in the samples. The pathways related to these components play a crucial role in biofilm formation and could be promising targets for preventing biofilm formation in the PV. In addition, Raman spectroscopy analysis indicated the presence of hematite, goethite, and ferrite, consistent with the mineralogical composition of the regional soil and metal-resistant bacteria. Taken together, our findings reveal that PV biofilms are a promising source of microorganisms of industrial interest and genes of central importance in regulating biofilm formation and persistence.

Heme pocket hydrogen bonding residue interactions within the Pectobacterium Diguanylate cyclase-containing globin coupled sensor: A resonance Raman study

Heme-based sensor proteins are used by organisms to control signaling and physiological effects in response to their gaseous environment. Globin-coupled sensors (GCS) are oxygen-sensing proteins that are widely distributed in bacteria. These proteins consist of a heme globin domain linked by a middle domain to various output domains, including diguanylate cyclase domains, which are responsible for synthesizing c-di-GMP, a bacterial second messenger crucial for regulating biofilm formation. To understand the roles of heme pocket residues in controlling activity of the diguanylate cyclase domain, variants of the Pectobacterium carotovorum GCS (PccGCS) were characterized by enzyme kinetics and resonance Raman (rR) spectroscopy. Results of these studies have identified roles for hydrogen bonding and heme edge residues in modulating heme pocket conformation and flexibility. Better understanding of the ligand-dependent GCS signaling mechanism and the residues involved may allow for future development of methods to control O2-dependent c-di-GMP production.

Deciphering the Intracellular Action of the Antimicrobial Peptide A11 via an In-Depth Analysis of Its Effect on the Global Proteome of Acinetobacter baumannii.

The potential antimicrobial activity and low propensity to induce the development of bacterial resistance have rendered antimicrobial peptides (AMPs) as novel and ideal candidate therapeutic agents for the treatment of infections caused by drug-resistant pathogenic bacteria. The targeting of bacterial membranes by AMPs has been typically considered their sole mode of action; however, increasing evidence supports the existence of multiple and complementary functions of AMPs that result in bacterial death. An in-depth characterization of their mechanism of action could facilitate further research and development of AMPs with higher potency. The current study employs biophysics and proteomics approaches to unveil the mechanisms underlying the antibacterial activity of A11, a potential candidate AMP, against Acinetobacter baumannii, a leading cause of hospital-acquired infections (HAIs) and consequently, a serious global threat. A11 peptide was found to induce membrane depolarization to a high extent, as revealed by flow cytometry and electron microscopy analyses. The prompt intracellular penetration of A11 peptide, observed using confocal microscopy, was found to occur concomitantly with a very low degree of membrane lysis, suggesting that its mode of action predominantly involves a nonlytic killing mechanism. Quantitative proteomics analysis employed for obtaining insights into the mechanisms underlying the antimicrobial activity of A11 peptide revealed that it disrupted energy metabolism, interfered with protein homeostasis, and inhibited fatty acid synthesis that is essential for cell membrane integrity; all these impacted the cellular functions of A. baumannii. A11 treatment also impacted signal transduction associated with the regulation of biofilm formation, hindered the stress response, and influenced DNA repair processes; these are all crucial survival mechanisms of A. baumannii. Additionally, robust antibacterial activity was exhibited by A11 peptide against multidrug-resistant (MDR) and extensively drug-resistant (XDR) clinical isolates of A. baumannii; moreover, A11 peptide exhibited synergy with levofloxacin and minocycline as well as low propensity for inducing resistance. Taken together, the findings emphasize the therapeutic potential of A11 peptide as an antibacterial agent against drug-resistant A. baumannii and underscore the need for further investigation.

A Brg1-Rme1 circuit in Candida albicans hyphal gene regulation.

Major Candida albicans virulence traits include its ability to make hyphae, to produce a biofilm, and to damage host cells. These traits depend upon expression of hypha-associated genes. A gene expression comparison among clinical isolates suggested that transcription factor Rme1, established by previous studies to be a positive regulator of chlamydospore formation, may also be a negative regulator of hypha-associated genes. Engineered RME1 overexpression supported this hypothesis, but no relevant rme1Δ/Δ mutant phenotype was detected. We reasoned that Rme1 may function within a specific regulatory pathway. This idea was supported by our finding that an rme1Δ/Δ mutation relieves the need for biofilm regulator Brg1 in biofilm formation. The impact of the rme1Δ/Δ mutation is most prominent under static or "biofilm-like" growth conditions. RNA sequencing (RNA-seq) of cells grown under biofilm-like conditions indicates that Brg1 activates hypha-associated genes indirectly via repression of RME1: hypha-associated gene expression levels are substantially reduced in a brg1Δ/Δ mutant and partially restored in a brg1Δ/Δ rme1Δ/Δ double mutant. An rme1Δ/Δ mutation does not simply bypass Brg1, because iron homeostasis genes depend upon Brg1 regardless of Rme1. Rme1 thus connects Brg1 to the targets relevant to hypha and biofilm formation under biofilm growth conditions.IMPORTANCECandida albicans is a major fungal pathogen of humans, and its ability to grow as a surface-associated biofilm on implanted devices is a common cause of infection. Here, we describe a new regulator of biofilm formation, RME1, whose activity is most prominent under biofilm-like growth conditions.

BmfR, a novel GntR family regulator, regulates biofilm formation in marine-derived, Bacillus methylotrophicus B-9987

Biofilms are common living states for microorganisms, allowing them to adapt to environmental changes. Numerous Bacillus strains can form complex biofilms that play crucial roles in biocontrol processes. However, our current understanding of the molecular mechanisms of biofilm formation in Bacillus is mainly based on studies of Bacillus subtilis. Knowledge regarding the biofilm formation of other Bacillus species remains limited. In this study, we identified a novel transcriptional regulator, BmfR, belonging to the GntR family, that regulates biofilm formation in marine-derived Bacillus methylotrophicus B-9987. We demonstrated that BmfR induces biofilm formation by activating the extracellular polysaccharide structural genes epsA-O and negatively regulating the matrix gene repressor, SinR; of note it positively affects the expression of the master regulator of sporulation, Spo0A. Furthermore, database mining for BmfR homologs has revealed their widespread distribution among many bacterial species, mainly Firmicutes and Proteobacteria. This study advances our understanding of the biofilm regulatory network of Bacillus strains, and provides a new target for exploiting and manipulating biofilm formation.

Effect of L-HSL on biofilm and motility of Pseudomonas aeruginosa and its mechanism

Pseudomonas aeruginosa (P. aeruginosa) biofilm formation is a crucial cause of enhanced antibiotic resistance. Quorum sensing (QS) is involved in regulating biofilm formation; QS inhibitors block the QS signaling pathway as a new strategy to address bacterial resistance. This study investigated the potential and mechanism of L-HSL (N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide) as a QS inhibitor for P. aeruginosa. The results showed that L-HSL effectively inhibited the biofilm formation and dispersed the pre-formed biofilm of P. aeruginosa. The production of extracellular polysaccharides and the motility ability of P. aeruginosa were suppressed by L-HSL. C. elegans infection experiment showed that L-HSL was non-toxic and provided protection to C. elegans against P. aeruginosa infection. Transcriptomic analysis revealed that L-HSL downregulated genes related to QS pathways and biofilm formation. L-HSL exhibits a promising potential as a therapeutic drug for P. aeruginosa infection.Key points• Chemical synthesis of N-(3-cyclic butyrolactone)-4-trifluorophenylacetamide, named L-HSL.• L-HSL does not generate survival pressure on the growth of P. aeruginosa and can inhibit the QS system.• KEGG enrichment analysis found that after L-HSL treatment, QS-related genes were downregulated.

EⅡB Mutation Reduces the Pathogenicity of Listeria monocytogenes by Negatively Regulating Biofilm Formation Ability, Infective Capacity, and Virulence Gene Expression.

To explore the role of the membrane permease ⅡB (EⅡB) gene of Listeria pathogenicity island 4 (LIPI-4) in the virulence of Listeria monocytogenes, both an EⅡB deletion strain (∆EⅡB) and a complemented strain were constructed. In vitro experiments demonstrated that EⅡB deletion affected the biofilm formation ability of the wild-type strain (Lm928). Moreover, this deletion decreased the intracellular proliferation abilities of L. monocytogenes. Mice infected with ∆EⅡB survived longer and experienced less weight loss on days 1, 2, and 3 post-infection. The bacterial load in the liver tissue of ∆EⅡB-infected mice was significantly reduced, and a considerable decrease in the blood levels of inflammatory cytokines IL-β, IL-6, IL-10, and TNF-α were observed. Following EⅡB deletion, 65% (13/20) of genes were downregulated, 25% (5/20) were upregulated, and 10% (2/20) showed no change. These findings suggest that EⅡB deletion may reduce both the in vivo and in vitro virulence levels as well as the biofilm formation ability of Lm928 by downregulating the transcription levels of genes associated with virulence and biofilm formation. These findings provide a foundation for further examining the pathogenic mechanisms of LIPI-4 and EⅡB in L. monocytogenes.

Enhancement of serrawettin W1-producing Serratia marcescens cell migration by resonant oscillation under alternating current electric field

Swarming migration is observed in flagellated bacteria on wet surfaces. As the secreted biosurfactant hydrates, friction between the cell and the wet surface is reduced, and weak flagellar movement appears to be a significant force for cell translocation. Developing an artificial swarming control method could greatly aid in establishing techniques for biofilm inhibition and detachment. In this study, the effect of forced cell vibration by an alternating current electric field (ACEF) on swarming motility was investigated using the serrawettin W1-producing Serratia marcescens strain. At frequencies close to the natural frequencies of microbial cells (12 MHz), swarming motion was effectively enhanced in biosurfactant-producing S. marcescens, but not in non-surfactant-producing Escherichia coli or non-flagellated Staphylococcus aureus. Electric field-assisted cell migration was significantly induced under swarming conditions with low friction resistance between the cell and gel surface. This finding suggests a direction for developing strategies to regulate biofilm formation and detachment using ACEF-assisted oscillation of cells attached to surfaces.

The arginine transporter Can1 negatively regulates biofilm formation in yeasts.

The arginine transporter Can1 is a multifunctional protein of the conventional yeast Saccharomyces cerevisiae. Apart from facilitating arginine uptake, Can1 plays a pivotal role in regulating proline metabolism and maintaining cellular redox balance. Here, we report a novel function of Can1 in the control of yeast biofilm formation. First, the S. cerevisiae CAN1 gene knockout strain displayed a significant growth delay compared to the wild-type strain. Our genetic screening revealed that the slow growth of the CAN1 knockout strain is rescued by a functional deficiency of the FLO8 gene, which encodes the master transcription factor associated with biofilm formation, indicating that Can1 is involved in biofilm formation. Intriguingly, the CAN1 knockout strain promoted the Flo11-dependent aggregation, leading to higher biofilm formation. Furthermore, the CAN1 knockout strain of the pathogenic yeast Candida glabrata exhibited slower growth and higher biofilm formation, similar to S. cerevisiae. More importantly, the C. glabrata CAN1 gene knockout strain showed severe toxicity to macrophage-like cells and nematodes. The present results could help to elucidate both the molecular mechanism underlying yeast biofilm formation and the role it plays. Future investigations may offer insights that contribute to development of antibiofilm agents.

A trans-acting sRNA SaaS targeting hilD, cheA and csgA to inhibit biofilm formation of S. Enteritidis

IntroductionSalmonella Enteritidis has brought great harm to public health, animal production and food safety worldwide. The biofilm formed by Salmonella Enteritidis plays a critical role in microbial cross-contamination. Small non-coding RNAs (sRNAs) have been demonstrated to be responsible for regulating the formation of biofilm. The sRNA SaaS has been identified previously, that promotes pathogenicity by regulating invasion and virulence factors. However, whether the SaaS is implicated in regulating biofilm formation in abiotic surfaces remains unclear. ObjectivesThis study aimed to clarify the effect of SaaS in Salmonella Enteritidis and explore the modulatory mechanism on the biofilm formation. MethodsMotility characteristics and total biomass of biofilm of test strains were investigated by the phenotypes in three soft agar plates and crystal violet staining in polystyrene microplates. Studies of microscopic structure and extracellular polymeric substances (EPS) of biofilm on solid surfaces were carried out using confocal laser scanning microscope (CLSM) and Raman spectra. Transcriptomics and proteomics were applied to analyze the changes of gene expression and EPS component. The RNA-protein pull-down and promoter–reporter β-galactosidase activity assays were employed to analyze RNA binding proteins and identify target mRNAs, respectively. ResultsSaaS inhibits biofilm formation by repressing the adhesion potential and the secretion of EPS components. Integration of transcriptomics and proteomics analysis revealed that SaaS strengthened the expression of the flagellar synthesis system and downregulated the expression of curli amyloid fibers. Furthermore, RNA-protein pull-down interactome datasets indicated that SaaS binds to Hfq (an RNA molecular chaperone protein, known as a host factor for phage Qbeta RNA replication) uniquely among 193 candidate proteins, and promoter–reporter β-galactosidase activity assay confirmed target mRNAs including hilD, cheA, and csgA. ConclusionSaaS inhibits the properties of bacterial mobility, perturbs the secretion of EPS, and contributes to the inhibition of biofilm formation by interacting with target mRNA (hilD, cheA, and csgA) through the Hfq-mediated pathway.

Role of LsrR in the regulation of biofilm formation in mammary pathogenic Escherichia coli

BackgroundMammary Pathogenic Escherichia coli (MPEC) is an important pathogen that can escape the attack of the host immune system through biofilm formation and proliferate in the mammary gland continuously, resulting in mastitis in cows and causing enormous economic losses. As an effector of AI-2 quorum sensing, LsrR extensively affects the expression levels of hundreds of genes related to multiple biological processes in model E. coli strain. However, the regulatory role of LsrR in MPEC and whether it is involved in pathogenesis has been seldom reported.ResultsIn this study, the function of LsrR in strain MPEC5, obtained from a milk sample in dairy cows with mastitis, was investigated by performing high-throughput sequencing (RNA-seq) assays. The results revealed that LsrR down-regulated the transcript levels of fimAICDFGH (encoding Type 1 pili), which have been reported to be associated with biofilm formation process. Biofilm assays confirmed that deletion of lsrR resulted in a significant increase in biofilm formation in vitro. In addition, electrophoretic mobility shift assay (EMSA) provided evidence that LsrR protein could directly bind to the promoter regions of fimAICDFGH in a dose-dependent manner.ConclusionsThese results indicate that LsrR protein inhibits the biofilm formation ability of MPEC5 by directly binding to the fimAICDFGH promoter region. This study presents a novel clue for further exploration of the prevention and treatment of MPEC.

CcdC Regulates Biofilm Dispersal in Bacillus velezensis FZB42.

Bacillus velezensis FZB42 is a plant growth-promoting rhizobacterium (PGPR) and a model microorganism for biofilm studies. Biofilms are required for the colonization and promotion of plant growth in the rhizosphere. However, little is known about how the final stage of the biofilm life cycle is regulated, when cells regain their motility and escape the mature biofilm to spread and colonize new niches. In this study, the non-annotated gene ccdC was found to be involved in the process of biofilm dispersion. We found that the ccdC-deficient strain maintained a wrinkled state at the late stage of biofilm formation in the liquid-gas interface culture, and the bottom solution showed a clear state, indicating that no bacterial cells actively escaped, which was further evidenced by the formation of a cellular ring (biofilm pellicle) located on top of the preformed biofilm. It can be concluded that dispersal, a biofilm property that relies on motility proficiency, is also positively affected by the unannotated gene ccdC. Furthermore, we found that the level of cyclic diguanylate (c-di-GMP) in the ccdC-deficient strain was significantly greater than that in the wild-type strain, suggesting that B. velezensis exhibits a similar mechanism by regulating the level of c-di-GMP, the master regulator of biofilm formation, dispersal, and cell motility, which controls the fitness of biofilms in Pseudomonas aeruginosain. In this study, we investigated the mechanism regulating biofilm dispersion in PGPR.

Counterclockwise rotation of the flagellum promotes biofilm initiation in Helicobacter pylori.

Chemotaxis signaling systems have been reported to contribute to biofilm formation in many bacteria; however, how they regulate biofilm formation remains largely unknown. Chemotaxis systems are composed of many distinct kinds of proteins, but most previous work analyzed the biofilm effect of loss of only a few. Here, we explored chemotaxis' role during biofilm formation in the human-associated pathogenic bacterium Helicobacter pylori. We found that chemotaxis proteins are involved in biofilm initiation in a manner that correlated with how they affected flagellar rotation. Biofilm initiation was high in mutants with counterclockwise (CCW) flagellar bias and low in those with clockwise bias. We supported the idea that a major driver of biofilm formation is flagellar rotational direction using a CCW-locked flagellar mutant, which stays CCW independent of chemotaxis input and showed elevated biofilm initiation. Our data suggest that CCW-rotating flagella, independent of chemotaxis inputs, are a biofilm-promoting signal.

Integrated Biofilm Modification and Transcriptional Analysis for Improving Fengycin Production in Bacillus amyloliquefaciens.

Although fengycin exhibits broad-spectrum antifungal properties, its application is hindered due to its low biosynthesis level and the co-existence of iturin A and surfactin in Bacillus amyloliquefaciens HM618, a probiotic strain. In this study, transcriptome analysis and gene editing were used to explore the potential mechanisms regulating fengycin production in B. amyloliquefaciens. The fengycin level of B. amyloliquefacien HM-3 (∆itu-ΔsrfAA) was 88.41mg/L after simultaneously inhibiting the biosyntheses of iturin A and surfactin. The knockout of gene eps associated with biofilm formation significantly increased the fengycin level of the strain HM618, whereas the fengycin level decreased 32.05% after knocking out sinI, a regulator of biofilm formation. Transcriptome analysis revealed that the differentially expressed genes, involved in pathways of amino acid and fatty acid syntheses, were significantly down-regulated in the recombinant strains, which is likely associated with a decrease of fengycin production. The knockout of gene comQXPA and subsequent transcriptome analysis revealed that the ComQXPA quorum sensing system played a positiveregulatory role in fengycin production. Through targeted genetic modifications and fermentation optimization, the fengycin production of the engineered strain HM-12 (∆itu-ΔsrfAA-ΔyvbJ) in a 5-L fermenter reached 1.172g/L, a 12.26-fold increase compared to the fengycin level in the strain HM-3 (∆itu-ΔsrfAA) in the Erlenmeyer flask. Taken together, these results reveal the underlying metabolic mechanisms associated with fengycin synthesis and provide a potential strategy for improving fengycin production in B. amyloliquefaciens.

The role of rcpA gene in regulating biofilm formation and virulence in Vibrio parahaemolyticus

Vibrio parahaemolyticus (V. parahaemolyticus) is a common seafood-borne pathogen that can colonize the intestine of host and cause gastroenteritis. Biofilm formation by V. parahaemolyticus enhances its persistence in various environments, which poses a series of threats to food safety. This work aims to investigate the function of rcpA gene in biofilm formation and virulence of V. parahaemolyticus. Deletion of rcpA significantly reduced motility, biofilm biomass, and extracellular polymeric substances, and inhibited biofilm formation on a variety of food and food contact surfaces. In mice infection model, mice infected with ∆rcpA strain exhibited a decreased rate of pathogen colonization, a lower level of inflammatory cytokines, and less tissue damage when compared to mice infected with wild type strain. RNA-seq analysis revealed that 374 genes were differentially expressed in the rcpA deletion mutant, which include genes related to quorum sensing, flagellar system, ribosome, type VI secretion system, biotin metabolism and transcriptional regulation. In conclusion, rcpA plays a role in determining biofilm formation and virulence of V. parahaemolyticus and further research is necessitated to fully understand its function in V. parahaemolyticus.

Co-regulation of Thermosensor Pathogenic Factors by C-di-GMP-Related Two-Component Systems and a cAMP Receptor-like Protein (Clp) in Stenotrophomonas maltophilia.

Stenotrophomonas maltophilia is a major threat to the food industry and human health owing to its strong protease production and biofilm formation abilities. However, information regarding regulatory factors or potential mechanisms is limited. Herein, we observed that temperature differentially regulates biofilm formation and protease production, and a cAMP receptor-like protein (Clp) negatively regulates thermosensor biofilm formation, in contrast to protease synthesis. Among four c-di-GMP-related two-component systems (TCSs), promoter fusion analysis revealed that clp transcription levels were predominantly controlled by LotS/LotR, partially controlled by both RpfC/RpfG and a novel TCS Sm0738/Sm0737, with no obvious effect caused by Sm1912/Sm1911. Biofilm formation in Δclp and ΔTCSs strains suggested that LotS/LotR controlled biofilm formation in a Clp-mediated manner, whereas both RpfC/RpfG and Sm0738/Sm0737 may occur in a distinct pathway. Furthermore, enzymatic activity analysis combined with c-di-GMP level indicated that the enzymatic activity of c-di-GMP-related metabolism proteins may not be a vital contributor to changes in c-di-GMP level, thus influencing physiological functions. Our findings elucidate that the regulatory pathway of c-di-GMP-related TCSs and Clp in controlling spoilage or the formation of potentially pathogenic factors in Stenotrophomonas expand the understanding of c-di-GMP metabolism and provide clues to control risk factors of S. maltophilia in food safety.

Bile-induced biofilm formation in Bacteroides thetaiotaomicron requires magnesium efflux by an RND pump.

Bacteroides thetaiotaomicron is a prominent member of the human gut microbiota contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm lifestyle, and it was recently shown that B. thetaiotaomicron biofilm formation is promoted by the presence of bile. This process also requires a B. thetaiotaomicron extracellular DNase, which is not, however, regulated by bile. Here, we showed that bile induces the expression of several Resistance-Nodulation-Division (RND) efflux pumps and that inhibiting their activity with a global competitive efflux inhibitor impaired bile-dependent biofilm formation. We then showed that, among the bile-induced RND-efflux pumps, only the tripartite BT3337-BT3338-BT3339 pump, re-named BipABC [for Bile Induced Pump A (BT3337), B (BT3338), and C (BT3339)], is required for biofilm formation. We demonstrated that BipABC is involved in the efflux of magnesium to the biofilm extracellular matrix, which leads to a decrease of extracellular DNA concentration. The release of magnesium in the biofilm matrix also impacts biofilm structure, potentially by modifying the electrostatic repulsion forces within the matrix, reducing interbacterial distance and allowing bacteria to interact more closely and form denser biofilms. Our study therefore, identified a new molecular determinant of B. thetaiotaomicron biofilm formation in response to bile salts and provides a better understanding on how an intestinal chemical cue regulates biofilm formation in a major gut symbiont.IMPORTANCEBacteroides thetaiotaomicron is a prominent member of the human gut microbiota able to degrade dietary and host polysaccharides, altogether contributing to nutrient exchange, gut function, and maturation of the host's immune system. This obligate anaerobe symbiont can adopt a biofilm community lifestyle, providing protection against environmental factors that might, in turn, protect the host from dysbiosis and dysbiosis-related diseases. It was recently shown that B. thetaiotaomicron exposure to intestinal bile promotes biofilm formation. Here, we reveal that a specific B. thetaiotaomicron membrane efflux pump is induced in response to bile, leading to the release of magnesium ions, potentially reducing electrostatic repulsion forces between components of the biofilm matrix. This leads to a reduction of interbacterial distance and strengthens the biofilm structure. Our study, therefore, provides a better understanding of how bile promotes biofilm formation in a major gut symbiont, potentially promoting microbiota resilience to stress and dysbiosis events.

Indole signaling enhances biofilm formation and quorum sensing in sequencing biofilm batch reactors

Indole is a heterocyclic organic compound that acts as a signaling molecule to regulate biofilm formation (BF) by microorganisms. However, the potential roles of indole in BF among microbial communities in wastewater treatment systems remain unknown. In this study, the effects of indole signaling on BF and quorum sensing (QS) of the complex microbial communities in sequencing batch biofilm reactors were systematically explored. The results showed that indole at 200 mg/L increased BF along with concentrations of homoserine lactones. High-throughput sequencing revealed the underlying mechanism of notable differences in the BF capacity of microbial communities between the indole-treated and control groups. The indole-related metabolic and QS functions of the genera in the biofilm were annotated based on the QS-related protein database and previous studies. Relative abundance analysis showed that the QS-related genera, such as Acinetobacter, that produce indole oxygenase were significantly enriched in the indole-treatment group. In addition, bioinformatics analyses revealed that indole at 200 mg/L enhanced BF, QS, and protein efflux. Based on a phylogenetic molecular ecological network, QS was positively correlated to indole treatment of Burkholderia, suggesting that indole promotes QS and BF. This study provides new insights into the effects of indole signaling on BF of microbes and sheds light on its potential regulatory mechanism in wastewater treatment plants.