Mutant Biofilms Research Articles

Exopolysaccharides matrix affects the process of extracellular electron transfer in electroactive biofilm

The applications of bioelectrochemical systems (BESs) in the field of environment and energy are achieved through the bioelectrocatalytic process of electroactive biofilms. As a primary component of biofilm, the role of exopolysaccharides in electroactive biofilm in BESs is poorly understood. This study constructed an exopolysaccharides-deficient Geobacter sulfurreducens-based BES to explore the role of exopolysaccharides in electroactive biofilm. Compared with the wild type, the mutant biofilm expressing less exopolysaccharides decreased the capacity of current generation. In the mutant biofilm, the content of exopolysaccharides decreased significantly, resulting in a thinner biofilm and lower cell viability compared with the wild-type biofilm. However, the mutant with overexpressed pili developed a mature biofilm with extended time, which indicating the importance of exopolysaccharides for early biofilm formation and the compensatory role of pili in biofilm formation. The mutant biofilm had less content of c-type cytochromes (c-Cyts) and lower electrochemical activity of extracellular polymeric substances than the wild-type biofilm, suggesting a function of exopolysaccharides anchoring extracellular c-Cyts that essential to extracellular electron transfer (EET) in electroactive biofilms. Our findings demonstrated the essential role of exopolysaccharides in the process of EET in electroactive biofilm, which contributed to a better understanding and optimization of the performance of BESs.

Salmonella Biofilms Tolerate Hydrogen Peroxide by a Combination of Extracellular Polymeric Substance Barrier Function and Catalase Enzymes

The ability of Salmonella enterica subspecies enterica serovar Typhi (S. Typhi) to cause chronic gallbladder infections is dependent on biofilm growth on cholesterol gallstones. Non-typhoidal Salmonella (e.g. S. Typhimurium) also utilize the biofilm state to persist in the host and the environment. How the pathogen maintains recalcitrance to the host response, and oxidative stress in particular, during chronic infection is poorly understood. Previous experiments demonstrated that S. Typhi and S. Typhimurium biofilms are tolerant to hydrogen peroxide (H2O2), but that mutations in the biofilm extracellular polymeric substances (EPSs) O antigen capsule, colanic acid, or Vi antigen reduce tolerance. Here, biofilm-mediated tolerance to oxidative stress was investigated using a combination of EPS and catalase mutants, as catalases are important detoxifiers of H2O2. Using co-cultured biofilms of wild-type (WT) bacteria with EPS mutants, it was demonstrated that colanic acid in S. Typhimurium and Vi antigen in S. Typhi have a community function and protect all biofilm-resident bacteria rather than to only protect the individual cells producing the EPSs. However, the H2O2 tolerance deficiency of a O antigen capsule mutant was unable to be compensated for by co-culture with WT bacteria. For curli fimbriae, both WT and mutant strains are tolerant to H2O2 though unexpectedly, co-cultured WT/mutant biofilms challenged with H2O2 resulted in sensitization of both strains, suggesting a more nuanced oxidative resistance alteration in these co-cultures. Three catalase mutant (katE, katG and a putative catalase) biofilms were also examined, demonstrating significant reductions in biofilm H2O2 tolerance for the katE and katG mutants. Biofilm co-culture experiments demonstrated that catalases exhibit a community function. We further hypothesized that biofilms are tolerant to H2O2 because the physical barrier formed by EPSs slows penetration of H2O2 into the biofilm to a rate that can be mitigated by intra-biofilm catalases. Compared to WT, EPS-deficient biofilms have a heighted response even to low-dose (2.5 mM) H2O2 challenge, confirming that resident bacteria of EPS-deficient biofilms are under greater stress and have limited protection from H2O2. Thus, these data provide an explanation for how Salmonella achieves tolerance to H2O2 by a combination of an EPS-mediated barrier and enzymatic detoxification.

AprD is important for extracellular proteolytic activity, physicochemical properties and spoilage potential in meat-borne Pseudomonas fragi

Pseudomonas fragi is a common spoilage agent of aerobically stored chilled meat. Its extracellular protease contributes to the spoilage of meat. AprD is considered an important component involved in the secretion of an alkaline extracellular protease in Pseudomonas. Thus, it is very possible that aprD plays an important role in spoilage regulation in P. fragi. The objective of the present study was to investigate the effects of aprD on bacterial proteolytic activity, biological properties, and in situ spoilage potential, as well as the mode of action of the protease AprA in spoiled meat. The results showed that aprD is important for surface-associated behaviors, as an ΔaprD mutant altered autoaggregation, swimming motility, and biofilm formation. aprD also contributes to the spoilage activities of P. fragi by affecting extracellular protease activity, total volatile basic nitrogen production, pH and the sensory traits of meat samples. These results highlight the presence of aprD-mediated regulation profiles in P. fragi and may provide new perspectives on chilled meat preservation.

Colonization and degradation of polyurethane coatings by Pseudomonas protegens biofilms is promoted by PueA and PueB hydrolases

The ability of bacteria and fungi to degrade polymeric substrates is often assessed in liquid-based assays conducted by exposing polymers to planktonic cells, or cell-free supernatants or enzymes generated from them. These assessments miss the opportunity to specifically examine the role of biofilm formation and physiology in degradative processes. In a previous study, we examined the ability of cell-free supernatants from wildtype and hydrolase-deficient Pseudomonas protegens Pf-5 strains to degrade a commercial polyester polyurethane (PU). In this study, we developed the methodology to conduct spatial-temporal analysis of both biofilm colonization and degradation of polyester PU coatings using the same strains. Wild-type Pf-5 grew as confluent biofilms that produced tendrils containing viable cells; in contrast, Pf-5ΔpueAB, a mutant lacking PueA and PueB hydrolases, colonized the PU only by growing as a confluent biofilm but lacked tendril expansion. Degradation of PU by the wild-type hydrolases may alter the substrata, facilitating colonization by the biofilm. Chemical analysis by in situ Fourier transform infrared (FTIR) and Raman spectroscopies revealed the polyester block of the PU was preferentially degraded to varying degrees by the wild-type Pf-5 and mutant biofilms, and showed degradation due to PueB that was undetectable in liquid-based assays. Raman spectroscopy analysis of biofilms of Pf-5ΔpueAB grown on PU revealed a detectable, but the lowest, level of PU degradation relative to other strains. Degradation of PU by this mutant had previously been undetectable and this study revealed the existence of biofilm-associated hydrolytic mechanisms other than those driven by PueA and PueB. Taken together, our data suggest that both PueA and PueB hydrolases play a role in the colonization and degradation of a model polyester PU by P. protegens biofilms. This study demonstrates the importance of using biofilm-based assays to identify mechanisms of microbiologically-influenced degradation.

High-resolution imaging of fungal biofilm-induced olivine weathering

Many microorganisms including free-living and symbiotic fungi weather minerals through the formation of biofilms on their surface. Weathering thus proceeds not only according to the mineral's chemistry and the environmental conditions but also according to the local biofilm chemistry. These processes can be dissected in experiments with defined environmental settings and by employing genetic tools to modify traits of the fungal biofilm. Biofilms of the rock-inhabiting fungus Knufia petricola strain A95 (wild-type, WT) and its melanin-deficient mutant (ΔKppks) were grown on polished olivine sections in subaerial (air-exposed) and subaquatic (submerged) conditions. After seven months of interaction at pH 6 and 25 °C, the fungus-mineral interface and abiotic olivine surface were compared using high resolution transmission electron microscopy (HRTEM). The abiotic, subaquatic olivine section showed a 25 nm thick, continuous amorphous layer, enriched in Fe and depleted in Si compared to the underlying crystalline olivine. This amorphous layer formed either through a coupled interfacial dissolution reprecipitation mechanism or through the adsorption of silicic acid on precipitated ferric hydroxides. Its thickness was likely enhanced by mechanical stresses of polishing. Directly underneath a fungal biofilm (WT and mutant alike), the surface remained mostly crystalline and was strongly etched and weathered, indicating enhanced olivine dissolution. The correlation between enhanced olivine dissolution and the absence of a continuous amorphous layer is a strong indication of the dissolution-inhibiting qualities of the latter. We propose that the fungal biofilm sequesters significant amounts of Fe, preventing formation of the amorphous layer and driving olivine dissolution onwards. The seemingly similar olivine surface underneath both WT and mutant biofilms illustrates the comparably insignificant role of specific biofilm traits in the weathering of olivine once biofilm attachment is imposed. Under subaerial conditions, the absence of water on the abiotic surface prohibited olivine dissolution. This was overcome by the water retention capacities of both the WT and mutant biofilm: the olivine surface underneath subaerial fungal biofilms was as weathered as the corresponding subaquatic olivine surface. Under the studied environmental settings, the effect of fungal biofilms on olivine weathering seems to be universal, independent of the production of melanin, the composition of extracellular polymeric substances (EPS) or air-exposure.

A Trimeric Autotransporter Enhances Biofilm Cohesiveness in Yersinia pseudotuberculosis but Not in Yersinia pestis.

Cohesion of biofilms made by Yersinia pestis and Yersinia pseudotuberculosis has been attributed solely to an extracellular polysaccharide matrix encoded by the hms genes (Hms-dependent extracellular matrix [Hms-ECM]). However, mutations in the Y. pseudotuberculosis BarA/UvrY/CsrB regulatory cascade enhance biofilm stability without dramatically increasing Hms-ECM production. We found that treatment with proteinase K enzyme effectively destabilized Y. pseudotuberculosiscsrB mutant biofilms, suggesting that cell-cell interactions might be mediated by protein adhesins or extracellular matrix proteins. We identified an uncharacterized trimeric autotransporter lipoprotein (YPTB2394), repressed by csrB, which has been referred to as YadE. Biofilms made by a ΔyadE mutant strain were extremely sensitive to mechanical disruption. Overexpression of yadE in wild-type Y. pseudotuberculosis increased biofilm cohesion, similar to biofilms made by csrB or uvrY mutants. We found that the Rcs signaling cascade, which represses Hms-ECM production, activated expression of yadE The yadE gene appears to be functional in Y. pseudotuberculosis but is a pseudogene in modern Y. pestis strains. Expression of functional yadE in Y. pestis KIM6+ weakened biofilms made by these bacteria. This suggests that although the YadE autotransporter protein increases Y. pseudotuberculosis biofilm stability, it may be incompatible with the Hms-ECM production that is essential for Y. pestis biofilm production in fleas. Inactivation of yadE in Y. pestis may be another instance of selective gene loss in the evolution of flea-borne transmission by this species.IMPORTANCE The evolution of Yersinia pestis from its Y. pseudotuberculosis ancestor involved gene acquisition and gene losses, leading to differences in biofilm production. Characterizing the unique biofilm features of both species may provide better understanding of how each adapts to its specific niches. This study identifies a trimeric autotransporter, YadE, that promotes biofilm stability of Y. pseudotuberculosis but which has been inactivated in Y. pestis, perhaps because it is not compatible with the Hms polysaccharide that is crucial for biofilms inside fleas. We also reveal that the Rcs signaling cascade, which represses Hms expression, activates YadE in Y. pseudotuberculosis The ability of Y. pseudotuberculosis to use polysaccharide or YadE protein for cell-cell adhesion may help it produce biofilms in different environments.

The ComX Quorum Sensing Peptide of Bacillus subtilis Affects Biofilm Formation Negatively and Sporulation Positively.

Quorum sensing (QS) is often required for the formation of bacterial biofilms and is a popular target of biofilm control strategies. Previous studies implicate the ComQXPA quorum sensing system of Bacillus subtilis as a promoter of biofilm formation. Here, we report that ComX signaling peptide deficient mutants form thicker and more robust pellicle biofilms that contain chains of cells. We confirm that ComX positively affects the transcriptional activity of the PepsA promoter, which controls the synthesis of the major matrix polysaccharide. In contrast, ComX negatively controls the PtapA promoter, which drives the production of TasA, a fibrous matrix protein. Overall, the biomass of the mutant biofilm lacking ComX accumulates more monosaccharide and protein content than the wild type. We conclude that this QS phenotype might be due to extended investment into growth rather than spore development. Consistent with this, the ComX deficient mutant shows a delayed activation of the pre-spore specific promoter, PspoIIQ, and a delayed, more synchronous commitment to sporulation. We conclude that ComX mediated early commitment to sporulation of the wild type slows down biofilm formation and modulates the coexistence of multiple biological states during the early stages of biofilm development.

Lactate production by Staphylococcus aureus biofilm inhibits HDAC11 to reprogramme the host immune response during persistent infection.

Staphylococcus aureus (S. aureus) is a leading cause of biofilm-associated prosthetic joint infection (PJI), resulting in significant disability and prolonged treatment. It is known that host leukocyte IL-10 production is required for S. aureus biofilm persistence in PJI. A S. aureus bursa aurealis Tn library consisting of 1,952 non-essential genes was screened for mutants that failed to induce IL-10 in myeloid-derived suppressor cells (MDSCs), which identified a critical role for bacterial lactic acid biosynthesis. We generated a S. aureus ddh/ldh1/ldh2 triple Tn mutant that cannot produce D- or L-lactate. Co-culture of MDSCs or macrophages with ddh/ldh1/ldh2 mutant biofilm produced substantially less IL-10 compared with wild type S. aureus, which was also observed in a mouse model of PJI and led to reduced biofilm burden. Using MDSCs recovered from the mouse PJI model and in vitro leukocyte-biofilm co-cultures we show that bacterial-derived lactate inhibits histone deacetylase 11 (HDAC11), causing unchecked HDAC6 activity and increased histone 3 acetylation at the Il-10 promoter, resulting in enhanced Il-10 transcription in MDSCs and macrophages. Finally, we show that synovial fluid of patients with PJI contains elevated amounts of D-lactate and IL-10 compared with control subjects, and bacterial lactate increases IL-10 production by human monocyte-derived macrophages.

Candida albicans Mrv8, is involved in epithelial damage and biofilm formation

ABSTRACT Candida albicans is the most common human fungal pathogen that can cause superficial and deep-seated infections in susceptible individuals. Despite its medical importance, the vast majority of C. albicans genes remain of unknown function. Here, we report a role for the lineage-specific gene, MRV8, in host pathogen interactions, mycelial microcolony maturation and biofilm formation. In silico analysis indicated that MRV8 encodes a four-pass transmembrane protein unique to the closely related pathogens C. albicans and Candida dubliniensis. Deletion of MRV8 did not affect C. albicans adherence to, or initial invasion into human oral epithelia, but inhibited mycelial development and strongly reduced epithelial damage. mrv8Δ/Δ cells exhibited a media-dependent defect in biofilm formation and mutant biofilm metabolic activity was enhanced by cyclosporin A. mrv8Δ/Δ biofilms were more tolerant to treatment with caspofungin, but not to fluconazole or amphotericin B. Co-stimulation with calcium chloride and calcofluor white rescued biofilm growth in the presence of caspofungin, and this rescue-effect was Mrv8-dependent. Together, our data demonstrate an important role for a lineage-specific gene (MRV8) in C. albicans biofilm formation, drug tolerance and host–pathogen interactions.

Investigation of LuxS-mediated quorum sensing in Klebsiella pneumoniae.

IntroductionAutoinducer-2 (AI-2) quorum sensing is a bacterial communication system that responds to cell density. The system requires luxS activity to produce AI-2, which can regulate gene expression and processes such as biofilm formation.AimTo investigate the role of luxS in biofilm formation and gene expression in the nosocomial pathogen Klebsiella pneumoniae .MethodologyA ΔluxS gene deletion was made in K. pneumoniae KP563, an extensively drug-resistant isolate. AI-2 production was assessed in wild-type and ΔluxS strains grown in media supplemented with different carbohydrates. Potential roles of luxS in biofilm formation were investigated using a microtiter plate biofilm assay and scanning electron microscopy. Quantitative RT-PCR evaluated the expression of lipopolysaccharide (wzm and wbbM), polysaccharide (pgaA), and type 3 fimbriae (mrkA) synthesis genes in wild-type and ΔluxS mutant biofilm extracts.ResultsAI-2 production was dependent on the presence of luxS. AI-2 accumulation was highest during early stationary phase in media supplemented with glucose, sucrose or glycerol. Changes in biofilm architecture were observed in the ΔluxS mutant, with less surface coverage and reduced macrocolony formation; however, no differences in biofilm formation between the wild-type and ΔluxS mutant using a microtiter plate assay were observed. In ΔluxS mutant biofilm extracts, the expression of wzm was down-regulated, and the expression of pgaA, which encodes a porin for poly-β−1,6-N-acetyl-d-glucosamine (PNAG) polysaccharide secretion, was upregulated.ConclusionRelationships among AI-2-mediated quorum sensing, biofilm formation and gene expression of outer-membrane components were identified in K. pneumoniae . These inter-connected processes could be important for bacterial group behaviour and persistence.

Biofilms Positively Contribute to Bacillus amyloliquefaciens 54-induced Drought Tolerance in Tomato Plants.

Drought stress is a major obstacle to agriculture. Although many studies have reported on plant drought tolerance achieved via genetic modification, application of plant growth-promoting rhizobacteria (PGPR) to achieve tolerance has rarely been studied. In this study, the ability of three isolates, including Bacillus amyloliquefaciens 54, from 30 potential PGPR to induce drought tolerance in tomato plants was examined via greenhouse screening. The results indicated that B. amyloliquefaciens 54 significantly enhanced drought tolerance by increasing survival rate, relative water content and root vigor. Coordinated changes were also observed in cellular defense responses, including decreased concentration of malondialdehyde and elevated concentration of antioxidant enzyme activities. Moreover, expression levels of stress-responsive genes, such as lea, tdi65, and ltpg2, increased in B. amyloliquefaciens 54-treated plants. In addition, B. amyloliquefaciens 54 induced stomatal closure through an abscisic acid-regulated pathway. Furthermore, we constructed biofilm formation mutants and determined the role of biofilm formation in B. amyloliquefaciens 54-induced drought tolerance. The results showed that biofilm-forming ability was positively correlated with plant root colonization. Moreover, plants inoculated with hyper-robust biofilm (ΔabrB and ΔywcC) mutants were better able to resist drought stress, while defective biofilm (ΔepsA-O and ΔtasA) mutants were more vulnerable to drought stress. Taken altogether, these results suggest that biofilm formation is crucial to B. amyloliquefaciens 54 root colonization and drought tolerance in tomato plants.

Statistics of correlated percolation in a bacterial community.

Signal propagation over long distances is a ubiquitous feature of multicellular communities, but cell-to-cell variability can cause propagation to be highly heterogeneous. Simple models of signal propagation in heterogenous media, such as percolation theory, can potentially provide a quantitative understanding of these processes, but it is unclear whether these simple models properly capture the complexities of multicellular systems. We recently discovered that in biofilms of the bacterium Bacillus subtilis, the propagation of an electrical signal is statistically consistent with percolation theory, and yet it is reasonable to suspect that key features of this system go beyond the simple assumptions of basic percolation theory. Indeed, we find here that the probability for a cell to signal is not independent from other cells as assumed in percolation theory, but instead is correlated with its nearby neighbors. We develop a mechanistic model, in which correlated signaling emerges from cell division, phenotypic inheritance, and cell displacement, that reproduces the experimentally observed correlations. We find that the correlations do not significantly affect the spatial statistics, which we rationalize using a renormalization argument. Moreover, the fraction of signaling cells is not constant in space, as assumed in percolation theory, but instead varies within and across biofilms. We find that this feature lowers the fraction of signaling cells at which one observes the characteristic power-law statistics of cluster sizes, consistent with our experimental results. We validate the model using a mutant biofilm whose signaling probability decays along the propagation direction. Our results reveal key statistical features of a correlated signaling process in a multicellular community. More broadly, our results identify extensions to percolation theory that do or do not alter its predictions and may be more appropriate for biological systems.

Clostridioides difficile LuxS mediates inter-bacterial interactions within biofilms

The anaerobic gut pathogen, Clostridioides difficile, forms adherent biofilms that may play an important role in recurrent C. difficile infections. The mechanisms underlying C. difficile community formation and inter-bacterial interactions are nevertheless poorly understood. C. difficile produces AI-2, a quorum sensing molecule that modulates biofilm formation across many bacterial species. We found that a strain defective in LuxS, the enzyme that mediates AI-2 production, is defective in biofilm development in vitro. Transcriptomic analyses of biofilms formed by wild type (WT) and luxS mutant (luxS) strains revealed a downregulation of prophage loci in the luxS mutant biofilms compared to the WT. Detection of phages and eDNA within biofilms may suggest that DNA release by phage-mediated cell lysis contributes to C. difficile biofilm formation. In order to understand if LuxS mediates C. difficile crosstalk with other gut species, C. difficile interactions with a common gut bacterium, Bacteroides fragilis, were studied. We demonstrate that C. difficile growth is significantly reduced when co-cultured with B. fragilis in mixed biofilms. Interestingly, the absence of C. difficile LuxS alleviates the B. fragilis-mediated growth inhibition. Dual species RNA-sequencing analyses from single and mixed biofilms revealed differential modulation of distinct metabolic pathways for C. difficile WT, luxS and B. fragilis upon co-culture, indicating that AI-2 may be involved in induction of selective metabolic responses in B. fragilis. Overall, our data suggest that C. difficile LuxS/AI-2 utilises different mechanisms to mediate formation of single and mixed species communities.

D-LL-31 in combination with ceftazidime synergistically enhances bactericidal activity and biofilm destruction in Burkholderia pseudomallei

Melioidosis is a severe disease caused by Burkholderia pseudomallei. The biofilm of B. pseudomallei acquires resistance to several antibiotics and may be related to relapse in melioidosis patients. Here, the killing activity of antimicrobial peptides (LL-37, LL-31) and the D-enantiomers (D-LL-37, D-LL-31) in combination with ceftazidime (CAZ) against B. pseudomallei 1026b, H777 and a biofilm mutant M10, derived from H777 grown under biofilm-stimulating conditions was observed. Using static conditions, D-LL-31 exhibited the strongest killing activity against the three isolates in a dose-dependent manner. IC50 values for D-LL-31 ranged from 1 to 6 µM, for isolates M10, H777, and 1026b, respectively. Moreover, D-LL-31 combined with CAZ synergistically decreased the IC50 values of the peptide and antibiotic and caused also disruption of biofilms of B. pseudomallei 1026b under flow conditions. Thus a combination of D-LL-31 and CAZ may enhance the efficacy of the currently used antibiotic treatments against B. pseudomallei.

Loss of the Two-Component System TctD-TctE in Pseudomonas aeruginosa Affects Biofilm Formation and Aminoglycoside Susceptibility in Response to Citric Acid.

The two-component system TctD-TctE is important for regulating the uptake of tricarboxylic acids in Pseudomonas aeruginosa TctD-TctE accomplishes this through derepression of the gene opdH, which encodes a tricarboxylic acid-specific porin. Previous work from our lab revealed that TctD-TctE in P. aeruginosa also has a role in resistance to aminoglycoside antibiotics. The aim of this study was to further characterize the role of TctD-TctE in P. aeruginosa in the presence of citric acid. Here it was found that deletion of P. aeruginosa PA14 TctD-TctE (ΔtctED) resulted in a 4-fold decrease in the biofilm bactericidal concentrations of the aminoglycosides tobramycin and gentamicin when citric acid was present in nutrient media. Tobramycin accumulation assays demonstrated that deletion of TctD-TctE resulted in an increase in the amount of tobramycin retained in biofilm cells. The PA14 wild type responded to increasing concentrations of citric acid by producing less biofilm. In contrast, the amount of ΔtctED mutant biofilm formation remained constant or enhanced. Furthermore, the ΔtctED strain was incapable of growing on citric acid as a sole carbon source and was highly reduced in its ability to grow in the presence of citric acid even when an additional carbon source was available. Use of phenotypic and genetic microarrays found that this growth deficiency of the ΔtctED mutant is unique to citric acid and that multiple metabolic genes are dysregulated. This work demonstrates that TctD-TctE in P. aeruginosa has a role in biofilm development that is dependent on citric acid and that is separate from the previously characterized involvement in resistance to antibiotics.IMPORTANCE Nutrient availability is an important contributor to the ability of bacteria to establish successful infections in a host. Pseudomonas aeruginosa is an opportunistic pathogen in humans causing infections that are difficult to treat. In part, its success is attributable to a high degree of metabolic versatility. P. aeruginosa is able to sense and respond to varied and limited nutrient stress in the host environment. Two-component systems are important sensors-regulators of cellular responses to environmental stresses, such as those encountered in the host. This work demonstrates that the response by the two-component system TctD-TctE to the presence of citric acid has a role in biofilm formation, aminoglycoside susceptibility, and growth in P. aeruginosa.

Deletion of D-Lactate Dehydrogenase A in Neisseria meningitidis Promotes Biofilm Formation Through Increased Autolysis and Extracellular DNA Release.

Neisseria meningitidis is a Gram-negative bacterium that asymptomatically colonizes the human nasopharyngeal mucosa. Pilus-mediated initial adherence of N. meningitidis to the epithelial mucosa is followed by the formation of three-dimensional aggregates, called microcolonies. Dispersal from microcolonies contributes to the transmission of N. meningitidis across the epithelial mucosa. We have recently discovered that environmental concentrations of host cell-derived lactate influences N. meningitidis microcolony dispersal. Here, we examined the ability of N. meningitidis mutants deficient in lactate metabolism to form biofilms. A lactate dehydrogenease A (ldhA) mutant had an increased level of biofilm formation. Deletion of ldhA increased the N. meningitidis cell surface hydrophobicity and aggregation. In this study, we used FAM20, which belongs to clonal complex ST-11 that forms biofilms independently of extracellular DNA (eDNA). However, treatment with DNase I abolished the increased biofilm formation and aggregation of the ldhA-deficient mutant, suggesting a critical role for eDNA. Compared to wild-type, the ldhA-deficient mutant exhibited an increased autolytic rate, with significant increases in the eDNA concentrations in the culture supernatants and in biofilms. Within the ldhA mutant biofilm, the transcription levels of the capsule, pilus, and bacterial lysis genes were downregulated, while norB, which is associated with anaerobic respiration, was upregulated. These findings suggest that the absence of ldhA in N. meningitidis promotes biofilm formation and aggregation through autolysis-mediated DNA release.

Influence of the major pilA transcriptional regulator in electrochemical responses of Geobacter sulfureducens PilR-deficient mutant biofilm formed on FTO electrodes

Geobacter sulfurreducens is a model organism for understanding the role of bacterial structures in extracellular electron transfer mechanism (EET). This kind of bacteria relies on different structures such as type IV pili and over 100 c-type cytochromes to perform EET towards soluble and insoluble electron acceptors, including electrodes. To our knowledge, this work is the first electrochemical study comparing a G. sulfurreducens PilR-deficient mutant and wild type biofilms developed on fluorine-doped tin oxide (FTO) electrodes. Open circuit potential (OCP), electrochemical impedance spectroscopy (EIS) and cyclic voltammetry (CV), were used to evaluate the electroactive properties of biofilms grown without externally imposed potential. Parallel studies of Confocal Laser Scanning Microscopy (CLSM) correlated with the electrochemical results. PilR is a transcriptional regulator involved in the expression of a wide variety of genes, including pilA (pilus structural protein) relevant c-type cytochromes and some other genes involved in biofilm formation and EET processes. Our findings suggest that PilR-deficient mutant forms a thinner (CLSM analysis) and less conductive biofilm (EIS analysis) than wild type, exhibiting different and irreversible redox processes at the interface (CV analysis). Additionally, this work reinforces some of the remarkable features described in previous reports about this G. sulfurreducens mutant.

Glycosyltransferase-Mediated Biofilm Matrix Dynamics and Virulence of Streptococcus mutans.

Streptococcus mutans is a key cariogenic bacterium responsible for the initiation of tooth decay. Biofilm formation is a crucial virulence property. We discovered a putative glycosyltransferase, SMU_833, in S. mutans capable of modulating dynamic interactions between two key biofilm matrix components, glucan and extracellular DNA (eDNA). The deletion of smu_833 decreases glucan and increases eDNA but maintains the overall biofilm biomass. The decrease in glucan is caused by a reduction in GtfB and GtfC, two key enzymes responsible for the synthesis of glucan. The increase in eDNA was accompanied by an elevated production of membrane vesicles, suggesting that SMU_833 modulates the release of eDNA via the membrane vesicles, thereby altering biofilm matrix constituents. Furthermore, glucan and eDNA were colocalized. The complete deletion of gtfBC from the smu_833 mutant significantly reduced the biofilm biomass despite the elevated eDNA, suggesting the requirement of minimal glucans as a binding substrate for eDNA within the biofilm. Despite no changes in overall biofilm biomass, the mutant biofilm was altered in biofilm architecture and was less acidic in vitro Concurrently, the mutant was less virulent in an in vivo rat model of dental caries, demonstrating that SMU_833 is a new virulence factor. Taken together, we conclude that SMU_833 is required for optimal biofilm development and virulence of S. mutans by modulating extracellular matrix components. Our study of SMU_833-modulated biofilm matrix dynamics uncovered a new target that can be used to develop potential therapeutics that prevent and treat dental caries.IMPORTANCE Tooth decay, a costly and painful disease affecting the vast majority of people worldwide, is caused by the bacterium Streptococcus mutans The bacteria utilize dietary sugars to build and strengthen biofilms, trapping acids onto the tooth's surface and causing demineralization and decay of teeth. As knowledge of our body's microbiomes increases, the need for developing therapeutics targeted to disease-causing bacteria has arisen. The significance of our research is in studying and identifying a novel therapeutic target, a dynamic biofilm matrix that is mediated by a new virulence factor and membrane vesicles. The study increases our understanding of S. mutans virulence and also offers a new opportunity to develop effective therapeutics targeting S. mutans In addition, the mechanisms of membrane vesicle-mediated biofilm matrix dynamics are also applicable to other biofilm-driven infectious diseases.

Analysis of Pseudomonas aeruginosa biofilm membrane vesicles supports multiple mechanisms of biogenesis.

Outer Membrane Vesicles (OMVs) are ubiquitous in bacterial environments and enable interactions within and between species. OMVs are observed in lab-grown and environmental biofilms, but our understanding of their function comes primarily from planktonic studies. Planktonic OMVs assist in toxin delivery, cell-cell communication, horizontal gene transfer, small RNA trafficking, and immune system evasion. Previous studies reported differences in size and proteomic cargo between planktonic and agar plate biofilm OMVs, suggesting possible differences in function between OMV types. In Pseudomonas aeruginosa interstitial biofilms, extracellular vesicles were reported to arise through cell lysis, in contrast to planktonic OMV biogenesis that involves the Pseudomonas Quinolone Signal (PQS) without appreciable autolysis. Differences in biogenesis mechanism could provide a rationale for observed differences in OMV characteristics between systems. Using nanoparticle tracking, we found that P. aeruginosa PAO1 planktonic and biofilm OMVs had similar characteristics. However, P. aeruginosa PA14 OMVs were smaller, with planktonic OMVs also being smaller than their biofilm counterparts. Large differences in Staphylococcus killing ability were measured between OMVs from different strains, and a smaller within-strain difference was recorded between PA14 planktonic and biofilm OMVs. Across all conditions, the predatory ability of OMVs negatively correlated with their size. To address biogenesis mechanism, we analyzed vesicles from wild type and pqsA mutant biofilms. This showed that PQS is required for physiological-scale production of biofilm OMVs, and time-course analysis confirmed that PQS production precedes OMV production as it does in planktonic cultures. However, a small sub-population of vesicles was detected in pqsA mutant biofilms whose size distribution more resembled sonicated cell debris than wild type OMVs. These results support the idea that, while a small and unique population of vesicles in P. aeruginosa biofilms may result from cell lysis, the PQS-induced mechanism is required to generate the majority of OMVs produced by wild type communities.

Oxidative stress response plays a role in antibiotic tolerance of Streptococcus mutans biofilms.

Knowledge about biofilm-associated antibiotic tolerance mechanisms is warranted in order to develop effective treatments against biofilm infections. We performed a screen of a Streptococcus mutans transposon mutant library for mutants with reduced biofilm-associated antimicrobial tolerance, and found that the spxA1 gene plays a role in tolerance towards gentamicin and other antibiotics such as vancomycin and linezolid. SpxA1 is a regulator of genes involved in the oxidative stress response in S. mutans. The oxidative stress response genes gor and ahpC were found to be up-regulated upon antibiotic treatment of S. mutans wild-type biofilms, but not spxA1 mutant biofilms. The gor gene product catalyses the formation of glutathione which functions as an important antioxidant during oxidative stress, and accordingly biofilm-associated antibiotic tolerance of the spxA1 mutant could be restored by exogenous addition of glutathione. Our results indicate that the oxidative stress response plays a role in biofilm-associated antibiotic tolerance of S. mutans, and add to the on-going debate on the role of reactive oxygen species in antibiotic mediated killing of bacteria.