The Innate Immune Protein Calprotectin Interacts With and Encases Biofilm Communities of Pseudomonas aeruginosa and Staphylococcus aureus.

Calprotectin is a transition metal chelating protein of the innate immune response known to exert nutritional immunity upon microbial infection. It is abundantly released during inflammation and is therefore found at sites occupied by pathogens such as Pseudomonas aeruginosa and Staphylococcus aureus. The metal limitation induced by this protein has previously been shown to mediate P. aeruginosa and S. aureus co-culture. In addition to the transition metal sequestration role of calprotectin, it has also been shown to have metal-independent antimicrobial activity via direct cell contact. Therefore, we sought to assess the impact of this protein on the biofilm architecture of P. aeruginosa and S. aureus in monomicrobial and polymicrobial culture. The experiments described in this report reveal novel aspects of calprotectin's interaction with biofilm communities of P. aeruginosa and S. aureus discovered using scanning electron microscopy and confocal laser scanning microscopy. Our results indicate that calprotectin can interact with microbial cells by stimulating encapsulation in mesh-like structures. This physical interaction leads to compositional changes in the biofilm extracellular polymeric substance (EPS) in both P. aeruginosa and S. aureus.

Comparative Transcriptome Analysis Reveals Regulatory Factors Involved in Vibrio Parahaemolyticus Biofilm Formation.

Vibrio parahaemolyticus biofilm poses a serious threat to food safety and human health. However, there is limited knowledge of transcriptional regulatory mechanism during the biofilm formation of this organism. Hence, the RNA sequencing technique was employed to compare the differences in transcriptome profiles between planktonic and biofilm state of V. parahaemolyticus ATCC33847 in this study. Collections of mRNA from planktonic and biofilm cells cultured at 25°C for 36 h were sequenced by studying their biological characteristics. The results showed that there were significant differences in the expression levels of 956 genes in biofilms compared with planktonic cells. These differences suggested that two-component regulatory system (TCS) and quorum sensing (QS) regulated V. parahaemolyticus biofilm formation by affecting important factors such as flagellar motility, Extracellular polymeric substance (EPS) secretion, tripartite ATP-independent (TRAP) transport system and ATP binding cassette (ABC) transport system. The present work in transcriptomics serves as a basis for future studies examining the complex network systems that regulate bacterial biofilm formation.

Propagation of antibiotic resistance genes during anaerobic digestion of thermally hydrolyzed sludge and their correlation with extracellular polymeric substances.

The positive impact of the thermal hydrolysis process (THP) of sewage sludge on antibiotic resistance genes (ARGs) removal during anaerobic digestion (AD) has been reported in the literature. However, little information is available on how changes in different extracellular polymeric substances (EPS) due to THP can influence ARG propagation during AD. This study focused on systematically correlating EPS components and ARG abundance in AD of sewage sludge pretreated with THP (80 °C, 110 °C, 140 °C, 170 °C). THP under different conditions improved sludge solubilization followed by improved methane yields in the biochemical methane potential (BMP) test. The highest methane yield of 275 ± 11.5 ml CH4/g COD was observed for THP-140 °C, which was 40.5 ± 2.5% higher than the control. Increasing THP operating temperatures showed a non-linear response of ARG propagation in AD due to the rebound effect. The highest ARGs removal in AD was achieved with THP at 140 °C. The multivariate analysis showed that EPS polysaccharides positively correlated with most ARGs and integrons, except for macrolides resistance genes. In contrast, EPS protein was only strongly correlated with ß-lactam resistance genes. These results suggest that manipulating THP operating conditions targeting specific EPS components will be critical to effectively mitigating the dissemination of particular ARG types in AD.

Screening and Characterization of Two Extracellular Polysaccharide-Producing Bacteria from the Biocrust of the Mu Us Desert.

The extracellular polysaccharide (EPS) matrix embedding microbial cells and soil particles plays an important role in the development of biological soil crusts (BSCs), which is widely recognized as beneficial to soil fertility in dryland worldwide. This study examined the EPS-producing bacterial strains YL24-1 and YL24-3 isolated from sandy soil in the Mu Us Desert in Yulin, Shaanxi province, China. The strains YL24-1 and YL24-3 were able to efficiently produce EPS; the levels of EPS were determined to be 257.22 µg/mL and 83.41 µg/mL in cultures grown for 72 h and were identified as Sinorhizobium meliloti and Pedobacter sp., respectively. When the strain YL24-3 was compared to Pedobacter yulinensis YL28-9T using 16S rRNA gene sequencing, the resemblance was 98.6% and the strain was classified as Pedobacter sp. using physiological and biochemical analysis. Furthermore, strain YL24-3 was also identified as a subspecies of Pedobacter yulinensis YL28-9T on the basis of DNA-DNA hybridization and polar lipid analysis compared with YL28-9T. On the basis of the EPS-related genes of relevant strains in the GenBank, several EPS-related genes were cloned and sequenced in the strain YL24-1, including those potentially involved in EPS synthesis, assembly, transport, and secretion. Given the differences of the strains in EPS production, it is possible that the differences in gene sequences result in variations in the enzyme/protein activities for EPS biosynthesis, assembly, transport, and secretion. The results provide preliminary evidence of various contributions of bacterial strains to the formation of EPS matrix in the Mu Us Desert.

Extracellular DNA (eDNA). A Major Ubiquitous Element of the Bacterial Biofilm Architecture.

After the first ancient studies on microbial slime (the name by which the biofilm matrix was initially indicated), multitudes of studies on the morphology, composition and physiology of biofilms have arisen. The emergence of the role that biofilms play in the pathogenesis of recalcitrant and persistent clinical infections, such as periprosthetic orthopedic infections, has reinforced scientific interest. Extracellular DNA (eDNA) is a recently uncovered component that is proving to be almost omnipresent in the extracellular polymeric substance (EPS) of biofilm. This macromolecule is eliciting unprecedented consideration for the critical impact on the pathogenesis of chronic clinical infections. After a systematic review of the literature, an updated description of eDNA in biofilms is presented, with a special focus on the latest findings regarding its fundamental structural role and the contribution it makes to the complex architecture of bacterial biofilms through interactions with a variety of other molecular components of the biofilm matrix.

Identification of Biomolecules Involved in the Adaptation to the Environment of Cold-Loving Microorganisms and Metabolic Pathways for Their Production.

Cold-loving microorganisms of all three domains of life have unique and special abilities that allow them to live in harsh environments. They have acquired structural and molecular mechanisms of adaptation to the cold that include the production of anti-freeze proteins, carbohydrate-based extracellular polymeric substances and lipids which serve as cryo- and osmoprotectants by maintaining the fluidity of their membranes. They also produce a wide diversity of pigmented molecules to obtain energy, carry out photosynthesis, increase their resistance to stress and provide them with ultraviolet light protection. Recently developed analytical techniques have been applied as high-throughoutput technologies for function discovery and for reconstructing functional networks in psychrophiles. Among them, omics deserve special mention, such as genomics, transcriptomics, proteomics, glycomics, lipidomics and metabolomics. These techniques have allowed the identification of microorganisms and the study of their biogeochemical activities. They have also made it possible to infer their metabolic capacities and identify the biomolecules that are parts of their structures or that they secrete into the environment, which can be useful in various fields of biotechnology. This Review summarizes current knowledge on psychrophiles as sources of biomolecules and the metabolic pathways for their production. New strategies and next-generation approaches are needed to increase the chances of discovering new biomolecules.

Contribution to determination of extracellular DNA origin in the biofilm matrix.

This study is focused on the analysis of extracellular DNA (eDNA) from a biofilm matrix formed by Staphylococcus aureus, Listeria monocytogenes, and Salmonella enterica. The presence of eDNA in the biofilm of all the studied strains was confirmed by confocal laser scanning microscopy using fluorescent dyes with high affinity to nucleic acid. The protocol for eDNA isolation from the biofilm matrix was established, and subsequent characterization of the eDNA was performed. The purified eDNA obtained from the biofilm matrix of all three microorganisms was compared to the genomic DNA (gDNA) isolated from relevant planktonic grown cells. The process of eDNA isolation consisted of biofilm cultivation, its collection, sonication, membrane filtration, dialysis, lyophilisation, and extraction of DNA separated from the biofilm matrix with cetyltrimethylammonium bromide. An amplified fragment length polymorphism (AFLP) was used for comparing eDNA and gDNA. AFLP profiles showed a significant similarity between eDNA and gDNA at the strain level. The highest similarity, with a profile concordance rate of 94.7% per strain, was observed for S. aureus, L. monocytogenes, and S. enterica exhibited lower profiles similarity (78% and 60%, respectively). The obtained results support the hypothesis that the eDNA of studied bacterial species has its origin in the gDNA.

Bacteria of eleven different species isolated from biofilms in a meat processing environment have diverse biofilm forming abilities.

Biofilms are formed by microorganisms protected by a self-produced matrix, most often attached to a surface. In the food processing environments biofilms endanger the product safety by the transmission of spoilage and pathogenic bacteria. In this study, we characterised the biofilm formation of the following eleven strains isolated from biofilms in a meat-processing environment: Acinetobacter harbinensis BF1, Arthrobacter sp. BF1, Brochothrix thermosphacta BF1, Carnobacterium maltaromaticum BF1, Kocuria salsicia BF1, Lactococcus piscium BF1, Microbacterium sp. BF1, Pseudomonas fragi BF1, Psychrobacter sp. BF1, Rhodococcus erythropolis BF1, Stenotrophomonas sp. BF1. We applied whole- genome sequencing and subsequent genome analysis to elucidate genetic features associated with the biofilm lifestyle. We furthermore determined the motility and studied biofilm formation on stainless steel using a static mono-species biofilm model mimicking the meat processing environment. The biomass and the EPS components carbohydrates, proteins and extracellular DNA (eDNA) of the biofilms were investigated after seven days at 10 °C. Whole-genome analysis of the isolates revealed that all strains except the Kocuria salsicia BF1 isolate, harboured biofilm associated genes, including genes for matrix production and motility. Genes involved in cellulose metabolism (present in 82% of the eleven strains) and twitching motility (present in 45%) were most frequently found. The capacity for twitching was confirmed using plate assays for all strains except Lactococcus piscium BF1, which showed the lowest motility behaviour. Differences in biofilm forming abilities could be demonstrated. The bacterial load ranged from 5.4 log CFU/cm2 (Psychrobacter sp. isolate) to 8.7 log CFU/cm2 (Microbacterium sp. isolate). The amount of the matrix components varied between isolates. In the biofilm of six strains we detected all three matrix components at different levels (carbohydrates, proteins and eDNA), in two only carbohydrates and eDNA, and in three only carbohydrates. Carbohydrates were detected in biofilms of all strains ranging from 0.5 to 4.3 µg glucose equivalents/cm2. Overall, the Microbacterium sp. strain showed the highest biofilm forming ability with high bacterial load (8.7 log CFU/cm2) and high amounts of carbohydrates (2.2 µg glucose equivalents/cm2), proteins (present in all experiments) and eDNA (549 ng/cm2). In contrast, Brochothrix thermosphacta was a weak biofilm former, showing low bacterial load and low levels of carbohydrates in the matrix (6.2 log CFU/cm2 and 0.5 µg glucose equivalents/cm2). This study contributes to our understanding of the biofilm forming ability of bacteria highly abundant in the meat processing environment, which is crucial to develop strategies to prevent and reduce biofilm formation in the food producing environment.

The biofilm matrix scaffold of Pseudomonas aeruginosa contains G-quadruplex extracellular DNA structures.

Extracellular DNA, or eDNA, is recognised as a critical biofilm component; however, it is not understood how it forms networked matrix structures. Here, we isolate eDNA from static-culture Pseudomonas aeruginosa biofilms using ionic liquids to preserve its biophysical signatures of fluid viscoelasticity and the temperature dependency of DNA transitions. We describe a loss of eDNA network structure as resulting from a change in nucleic acid conformation, and propose that its ability to form viscoelastic structures is key to its role in building biofilm matrices. Solid-state analysis of isolated eDNA, as a proxy for eDNA structure in biofilms, reveals non-canonical Hoogsteen base pairs, triads or tetrads involving thymine or uracil, and guanine, suggesting that the eDNA forms G-quadruplex structures. These are less abundant in chromosomal DNA and disappear when eDNA undergoes conformation transition. We verify the occurrence of G-quadruplex structures in the extracellular matrix of intact static and flow-cell biofilms of P. aeruginosa, as displayed by the matrix to G-quadruplex-specific antibody binding, and validate the loss of G-quadruplex structures in vivo to occur coincident with the disappearance of eDNA fibres. Given their stability, understanding how extracellular G-quadruplex structures form will elucidate how P. aeruginosa eDNA builds viscoelastic networks, which are a foundational biofilm property.

Weaponizing volatiles to inhibit competitor biofilms from a distance.

The soil bacterium Bacillus subtilis forms beneficial biofilms that induce plant defences and prevent the growth of pathogens. It is naturally found in the rhizosphere, where microorganisms coexist in an extremely competitive environment, and thus have evolved a diverse arsenal of defence mechanisms. In this work, we found that volatile compounds produced by B. subtilis biofilms inhibited the development of competing biofilm colonies, by reducing extracellular matrix gene expression, both within and across species. This effect was dose-dependent, with the structural defects becoming more pronounced as the number of volatile-producing colonies increased. This inhibition was mostly mediated by organic volatiles, and we identified the active molecules as 3-methyl-1-butanol and 1-butanol. Similar results were obtained with biofilms formed by phylogenetically distinct bacterium sharing the same niche, Escherichia coli, which produced the biofilm-inhibiting 3-methyl-1-butanol and 2-nonanon. The ability of established biofilms to inhibit the development and spreading of new biofilms from afar might be a general mechanism utilized by bacterial biofilms to protect an occupied niche from the invasion of competing bacteria.

Quantifying the occurrence and transformation potential of extracellular polymeric substances (EPS)-associated antibiotic resistance genes in activated sludge.

Antibiotic resistance has been regarded as a global concern and biological wastewater treatment plants (WWTPs) are ideal hotbeds for the emergence and propagation of antibiotic resistance genes (ARGs). Extracellular polymeric substances (EPS), one of the primary components of activated sludge, might affect the distribution of extracellular ARGs in supernatant and EPS matrix, and thus alter their uptake potential by microbial cells. Herein, the presence and significance of EPS-associated ARGs in activated sludge from four WWTPs were assessed. Seven typical ARGs (sulI, sulII, blaTEM-1, tetA, tetO, tetQ, tetW) and class I integron (intI1) in EPS-associated, cell-free, and intracellular DNA were quantified. Results show that the absolute abundances of EPS-associated, cell-free, and intracellular ARGs were 5.90 × 106-6.45 × 109, 5.53 × 104-4.58 × 106, and 2.68 × 108-1.79 × 1011 copies/g-volatile suspended solids, respectively. The absolute abundances of EPS-associated ARGs were 0.2-4.6 orders of magnitude higher than those of the corresponding cell-free ARGs. Considering the higher DNA contents in EPS, the transformation abilities of EPS-associated ARGs were 3.3-236.3 folds higher than those of cell-free ARGs. Therefore, EPS-associated ARGs are an important source of extracellular ARGs, and it may play a crucial role in horizontal gene transfer via transformation in WWTPs.

Temperature-regulated heterogeneous extracellular matrix gene expression defines biofilm morphology in Clostridium perfringens.

Cells in biofilms dynamically adapt to surrounding environmental conditions, which alters biofilm architecture. The obligate anaerobic pathogen Clostridium perfringens shows different biofilm structures in different temperatures. Here we find that the temperature-regulated production of extracellular polymeric substance (EPS) is necessary for morphological changes in biofilms. We identify BsaA proteins as an EPS matrix necessary for pellicle biofilm formation at lower temperature and find that extracellularly secreted BsaA protein forms filamentous polymers. We show that sipW-bsaA operon expression is bimodal, and the EPS-producing population size is increased at a lower temperature. This heterogeneous expression of the EPS gene requires a two-component system. We find that EPS-producing cells cover EPS-nonproducing cells attaching to the bottom surface. In the deletion mutant of pilA2, encoding a type IV pilin, the EPS gene expression is ON in the whole population. This heterogeneity is further regulated by the cleavage of the pilA2 mRNA by RNase Y, causing temperature-responsive EPS expression in biofilms. As temperature is an environmental cue, C. perfringens may modulate EPS expression to induce morphological changes in biofilm structure as a strategy for adapting to interhost and external environments.

Links between S-adenosylmethionine and Agr-based quorum sensing for biofilm development in Listeria monocytogenes EGD-e.

Listeria monocytogenes is the causative agent of human listeriosis which has high hospitalization and mortality rates for individuals with weakened immune systems. The survival and dissemination of L. monocytogenes in adverse environments can be reinforced by the formation of biofilms. Therefore, this study aimed to understand the mechanisms underlying listerial biofilm development. Given that both nutrient availability and quorum sensing (QS) have been known as the factors influencing biofilm development, we hypothesized that the signal from a sentinel metabolite S-adenosylmethionine (SAM) and Agr-based QS could be synchronous in L. monocytogenes to modulate nutrient availability, the synthesis of extracellular polymeric substances (EPSs), and biofilm formation. We performed biofilm assays and quantitative real-time PCR to investigate how biofilm volumes and the expression of genes for the synthesis of EPS were affected by SAM supplementation, agr deletion, or both. We found that exogenously applied SAM induced biofilm formation and that the expression of genes encoding the EPS synthesis machineries was regulated by SAM and/or Agr QS. Moreover, the gene transcription of components acting in the methyl cycle for SAM synthesis and Agr QS was affected by the signals from the other system. In summary, we reveal an interconnection at the transcriptional level between metabolism and QS in L. monocytogenes and highlight the critical role of metabolite-oriented QS in biofilm development.

Two genomic regions encoding exopolysaccharide production systems have complementary functions in B. cereus multicellularity and host interaction.

Bacterial physiology and adaptation are influenced by the exopolysaccharides (EPS) they produce. These polymers are indispensable for the assembly of the biofilm extracellular matrix in multiple bacterial species. In a previous study, we described the profound gene expression changes leading to biofilm assembly in B. cereus ATCC14579 (CECT148). We found that a genomic region putatively dedicated to the synthesis of a capsular polysaccharide (eps2) was overexpressed in a biofilm cell population compared to in a planktonic population, while we detected no change in the transcript abundance from another genomic region (eps1) also likely to be involved in polysaccharide production. Preliminary biofilm assays suggested a mild role for the products of the eps2 region in biofilm formation and no function for the products of the eps1 region. The aim of this work was to better define the roles of these two regions in B. cereus multicellularity. We demonstrate that the eps2 region is indeed involved in bacterial adhesion to surfaces, cell-to-cell interaction, cellular aggregation and biofilm formation, while the eps1 region appears to be involved in a kind of social bacterial motility. Consistent with these results, we further demonstrate using bacterial-host cell interaction experiments that the eps2 region is more relevant to the adhesion to human epithelial cells and the zebrafish intestine, suggesting that this region encodes a bacterial factor that may potentiate gut colonization and enhance pathogenicity against humans.

Various biofilm matrices of the emerging pathogen Staphylococcus lugdunensis: exopolysaccharides, proteins, eDNA and their correlation with biofilm mass.

Staphylococcus lugdunensis is an emerging high-virulent pathogen causative of hospital-acquired infections. Biofilm formation is a complex pathogenic process that leads to well-established bacterial communities. There is a paucity of data on the composition of the biofilm matrix among S. lugdunensis strains. Here, twenty-two S. lugdunensis clinical isolates, mainly from orthopaedic infections but also from other clinical sources, were sub-grouped by ribotyping and dendrogram analysis. Biofilms were analysed by fluorimetric methods based on FITC-Wheat Germ Agglutinin, SYPRO Ruby and TOTO-1 dyes to detect exopolysaccharides, proteins and extracellular DNA (eDNA), respectively. Biofilm morphology was investigated under confocal laser scanning microscopy (CLSM). Isolates displayed intriguing diversities in biofilm mass and matrix composition. The content of exopolysaccharides was found to be to be strongly associated with the biofilm mass (R2 = 0.882), while the content of proteins turned out to be weakly (R2 = 0.465) and that of eDNA very weakly associated (R2 = 0.202) to the biofilm mass.

Identification of Extracellular DNA-Binding Proteins in the Biofilm Matrix.

We developed a new approach that couples Southwestern blotting and mass spectrometry to discover proteins that bind extracellular DNA (eDNA) in bacterial biofilms. Using Staphylococcus aureus as a model pathogen, we identified proteins with known DNA-binding activity and uncovered a series of lipoproteins with previously unrecognized DNA-binding activity. We demonstrated that expression of these lipoproteins results in an eDNA-dependent biofilm enhancement. Additionally, we found that while deletion of lipoproteins had a minimal impact on biofilm accumulation, these lipoprotein mutations increased biofilm porosity, suggesting that lipoproteins and their associated interactions contribute to biofilm structure. For one of the lipoproteins, SaeP, we showed that the biofilm phenotype requires the lipoprotein to be anchored to the outside of the cellular membrane, and we further showed that increased SaeP expression correlates with more retention of high-molecular-weight DNA on the bacterial cell surface. SaeP is a known auxiliary protein of the SaeRS system, and we also demonstrated that the levels of SaeP correlate with nuclease production, which can further impact biofilm development. It has been reported that S. aureus biofilms are stabilized by positively charged cytoplasmic proteins that are released into the extracellular environment, where they make favorable electrostatic interactions with the negatively charged cell surface and eDNA. In this work we extend this electrostatic net model to include secreted eDNA-binding proteins and membrane-attached lipoproteins that can function as anchor points between eDNA in the biofilm matrix and the bacterial cell surface.IMPORTANCE Many bacteria are capable of forming biofilms encased in a matrix of self-produced extracellular polymeric substances (EPS) that protects them from chemotherapies and the host defenses. As a result of these inherent resistance mechanisms, bacterial biofilms are extremely difficult to eradicate and are associated with chronic wounds, orthopedic and surgical wound infections, and invasive infections, such as infective endocarditis and osteomyelitis. It is therefore important to understand the nature of the interactions between the bacterial cell surface and EPS that stabilize biofilms. Extracellular DNA (eDNA) has been recognized as an EPS constituent for many bacterial species and has been shown to be important in promoting biofilm formation. Using Staphylococcus aureus biofilms, we show that membrane-attached lipoproteins can interact with the eDNA in the biofilm matrix and promote biofilm formation, which suggests that lipoproteins are potential targets for novel therapies aimed at disrupting bacterial biofilms.

Exopolymeric substances (EPS) from Salmonella enterica: polymers, proteins and their interactions with plants and abiotic surfaces.

When Salmonella enterica is not in a planktonic state, it persists in organised communities encased in extracellular polymeric substances (EPS), defined as biofilms. Environmental conditions ultimately dictate the key properties of the biofilms such as porosity, density, water content, charge, sorption and ion exchange properties, hydrophobicity and mechanical stability. S. enterica has been extensively studied due to its ability to infect the gastrointestinal environment. However, only during the last decades studies on its persistence and replication in soil, plant and abiotic surfaces have been proposed. S. enterica is an environmental bacterium able to effectively persist outside the human host. It does so by using EPS as tools to cope with environmental fluctuations. We therefore address this mini-review to classify those EPS that are produced by Salmonella with focus on the environment (plant, soil, and abiotic surfaces) by using a classification of EPS proposed by Flemming and collaborators in 2007. The EPS are therefore classified as structural, sorptive, surface-active, active, and informative.

CalY is a major virulence factor and a biofilm matrix protein.

The extracellular biofilm matrix often contains a network of amyloid fibers which, in the human opportunistic pathogen Bacillus cereus, includes the two homologous proteins TasA and CalY. We show here, in the closely related entomopathogenic species Bacillus thuringiensis, that CalY also displays a second function. In the early stationary phase of planktonic cultures, CalY was located at the bacterial cell-surface, as shown by immunodetection. Deletion of calY revealed that this protein plays a major role in adhesion to HeLa epithelial cells, to the insect Galleria mellonella hemocytes and in the bacterial virulence against larvae of this insect, suggesting that CalY is a cell-surface adhesin. In mid-stationary phase and in biofilms, the location of CalY shifted from the cell surface to the extracellular medium, where it was found as fibers. The transcription study and the deletion of sipW suggested that CalY change of location is due to a delayed activity of the SipW signal peptidase. Using purified CalY, we found that the protein polymerization occurred only in the presence of cell-surface components. CalY is, therefore, a bifunctional protein, which switches from a cell-surface adhesin activity in early stationary phase, to the production of fibers in mid-stationary phase and in biofilms.

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.

Interaction between copper and extracellular nucleic acids in the EPS of unsaturated Pseudomonas putida CZ1 biofilm.

The role of extracellular DNA (eDNA) in biofilm in heavy metal complexation has been little reported. In this study, the interaction between the extracellular fraction of unsaturated biofilms and Cu2+ was studied using random amplified polymorphic DNA (RAPD) and synchrotron-based X-ray absorption spectroscopy (XAS) analyses. Under Cu2+ stress, the amount of eDNA was about 10-fold higher than the treatment without Cu2+ stress, which was substantially more than the amount of intracellular DNA (iDNA) present in the biofilm. The eDNA content increased significantly under Cu2+ stress and higher eDNA contents were found in colloidal extracellular polymeric substances (EPS) than in capsular EPS in Luria-Bertani medium. It was found that the composition of eDNA was distinctly changed under conditions of Cu2+ stress compared with the treatments without Cu2+ treatments, with specific eDNA bands appearing under Cu2+ treatments as revealed by RAPD analyses. X-ray absorption fine structure (XAFS) analysis assessing the molecular speciation of copper showed that copper in the secreted eDNA mainly existed as species resembling Cu3(PO4)2, followed by Cu-citrate species. This study investigated the interaction between copper and eDNA in unsaturated Pseudomonas putida CZ1 biofilms. Potential function of eDNA in biofilms under Cu2+ stress was found.