Exopolysaccharide Synthesis Research Articles

Tracking the homeostasis of second messenger cyclic-di-GMP in bacteria.

Cyclic-di-GMP (c-di-GMP) is an important second messenger in bacteria which regulates the bacterial transition from motile to sessile phase and also plays a major role in processes such as cell division, exopolysaccharide synthesis, and biofilm formation. Due to its crucial role in dictating the bacterial phenotype, the synthesis and hydrolysis of c-di-GMP is tightly regulated via multiple mechanisms. Perturbing the c-di-GMP homeostasis affects bacterial growth and survival, so it is necessary to understand the underlying mechanisms related to c-di-GMP metabolism. Most techniques used for estimating the c-di-GMP concentration lack single-cell resolution and do not provide information about any heterogeneous distribution of c-di-GMP inside cells. In this review, we briefly discuss how the activity of c-di-GMP metabolising enzymes, particularly bifunctional proteins, is modulated to maintain c-di-GMP homeostasis. We further highlight how fluorescence-based methods aid in understanding the spatiotemporal regulation of c-di-GMP signalling. Finally, we discuss the blind spots in our understanding of second messenger signalling and outline how they can be addressed in the future.

Enhanced rhamnolipids production in Pseudomonas aeruginosa SG by selectively blocking metabolic bypasses of glycosyl and fatty acid precursors.

To enhance rhamnolipids production in Pseudomonas aeruginosa, an optimization strategy based on selectively blocking the metabolic bypass that competed precursors with rhamnolipids biosynthesis pathway, containing exopolysaccharide (Psl and Pel) and polyhydroxyalkanoates (PHA) synthesis pathways. Blocking the synthesis of Psl and PHA by genes knockout, both mutants P. aeruginosa SG ∆pslAB and P. aeruginosa SG ∆phaC1DC2 can grow normally in fermentation medium and increase the production of rhamnolipids by 21% and 25.3%, respectively. While blocking the synthesis of Pel, the cell growth of the mutant strain P. aeruginosa SG ∆pelA was inhibited, thus its production yield of rhamnolipids was also decreased by 39.8%. In addition, simultaneously blocking the synthesis of Psl and PHA, a double mutant strain P. aeruginosa SG ∆pslAB ∆phaC1DC2 was constructed. Rhamnolipids production was significantly increased in strain SG ∆pslAB ∆phaC1DC2 by 69.7%. Through selectively blocking metabolic bypasses, increasing the amount of glycosyl and fatty acid precursors can significantly enhance rhamnolipids production in P. aeruginosa.

Initial Analysis on the Characteristics and Synthesis of Exopolysaccharides from Sclerotium rolfsii with Different Sugars as Carbon Sources.

Scleroglucan is widely used in the food and chemical industries because of its good rheological property, stability, and emulsification activity. To investigate the influence of different carbon sources on the properties and synthesis of exopolysaccharides (EPS), the three EPSs (GEPS, glucose was used as the carbon source; LEPS, lactose was used as the carbon source; and SEPS, sucrose was used as the carbon source) were determined, respectively. It was found that the yield and viscosity of exopolysaccharides were different. When sucrose and glucose were used as the carbon sources, the viscosity and yield of EPS were both higher than lactose. The scanning electron microscopy (SEM) images showed that the three EPSs had different morphologies, but the monosaccharide analysis showed that they were all composed of glucose units. Fourier transform infrared spectroscopy (FT-IR) proved that there were no additional substituents for the three EPSs. Furthermore, the high performance liquid chromatography (HPLC) results showed that SEPS and LEPS had two fractions. Through the analysis of proteomics data, there were few differences in the metabolic pathways between GEPS and SEPS, but a significant difference between LEPS and SEPS. Our study provides a theoretical basis and reference for understanding the biosynthesis of exopolysaccharides and the development of different types of EPS products.

LysR-Type Transcriptional Regulator MetR Controls Prodigiosin Production, Methionine Biosynthesis, Cell Motility, H2O2 Tolerance, Heat Tolerance, and Exopolysaccharide Synthesis in Serratia marcescens.

Prodigiosin, a secondary metabolite produced by Serratia marcescens, has attracted attention due to its immunosuppressive, antimicrobial, and anticancer properties. However, information on the regulatory mechanism behind prodigiosin biosynthesis in S. marcescens remains limited. In this work, a prodigiosin-hyperproducing strain with the BVG90_22495 gene disrupted (ZK66) was selected from a collection of Tn5G transposon insertion mutants. Using real-time quantitative PCR (RT-qPCR) analysis, β-galactosidase assays, transcriptomics analysis, and electrophoretic mobility shift assays (EMSAs), the LysR-type regulator MetR encoded by the BVG90_22495 gene was found to affect prodigiosin synthesis, and this correlated with MetR directly binding to the promoter region of the prodigiosin-synthesis positive regulator PigP and hence negatively regulated the expression of the prodigiosin-associated pig operon. More analyses revealed that MetR regulated some other important cellular processes, including methionine biosynthesis, cell motility, H2O2 tolerance, heat tolerance, exopolysaccharide synthesis, and biofilm formation in S. marcescens Although MetR protein is highly conserved in many bacteria, we report here on the LysR-type regulator MetR exhibiting novel roles in negatively regulating prodigiosin synthesis and positively regulating heat tolerance, exopolysaccharide synthesis, and biofilm formation.IMPORTANCESerratia marcescens, a Gram-negative bacterium, is found in a wide range of ecological niches and can produce several secondary metabolites, including prodigiosin, althiomycin, and serratamolide. Among them, prodigiosin shows diverse functions as an immunosuppressant, antimicrobial, and anticancer agent. However, the regulatory mechanisms behind prodigiosin synthesis in S. marcescens are not completely understood. Here, we adapted a transposon mutant library to identify the genes related to prodigiosin synthesis, and the BVG90_22495 gene encoding the LysR-type regulator MetR was found to negatively regulate prodigiosin synthesis. The molecular mechanism of the metR mutant hyperproducing prodigiosin was investigated. Additionally, we provided evidence supporting new roles for MetR in regulating methionine biosynthesis, cell motility, heat tolerance, H2O2 tolerance, and exopolysaccharide synthesis in S. marcescens Collectively, this work provides novel insight into regulatory mechanisms of prodigiosin synthesis and uncovers novel roles for the highly conserved MetR protein in regulating prodigiosin synthesis, heat tolerance, exopolysaccharide (EPS) synthesis, and biofilm formation.

Exopolysaccharides production by Lactobacillus rhamnosus strains – Optimization of synthesis and extraction conditions

Exopolysaccharides (EPS) synthesised by the tested strains of Lactobacillus rhamnosus bacteria (marked with symbols 0943, 0935 and OM-1) with probiotic properties are promising natural alternatives for commercial additives in the food, cosmetic and pharmaceutical industries. However, the low efficiency of synthesis and high acquisition cost make research and its industrial implementation much more difficult. In this paper, the environmental conditions and EPS extraction parameters were modelled. The culture medium used in the study was developed in the earlier stages of research on the basis of multi-parametric modelling. Thanks to a series of analyses, such cultivation parameters were developed which ensure high efficiency of EPS synthesis and a significantly reduced technological cost. The best results were obtained in stationary cultures cultivated for 24 h at 25 °C with the initial pH of 5.7 and the inoculum of 5% v/v. Unlike to other examined strains in case of Lb. rhamnosus ŁOCK 0943 prolongation of the incubation time and application of mixing (80 rpm) in the process of cultivation significantly increased the efficiency of EPS synthesis. The outcome sheds a new light on the possibility of the industrial application of the exopolysaccharides produced by Lb. rhamnosus bacteria in the “new generation” products.

Heavy Metals as a Factor Increasing the Functional Genetic Potential of Bacterial Community for Polycyclic Aromatic Hydrocarbon Biodegradation.

The bioremediation of areas contaminated with hydrocarbon compounds and heavy metals is challenging due to the synergistic toxic effects of these contaminants. On the other hand, the phenomenon of the induction of microbial secretion of exopolysaccharides (EPS) under the influence of heavy metals may contribute to affect the interaction between hydrophobic hydrocarbons and microbial cells, thus increasing the bioavailability of hydrophobic organic pollutants. The purpose of this study was to analyze the impact of heavy metals on the changes in the metapopulation structure of an environmental consortium, with particular emphasis on the number of copies of orthologous genes involved in exopolysaccharide synthesis pathways and the biodegradation of hydrocarbons. The results of the experiment confirmed that the presence of heavy metals at concentrations of 50 mg·L−1 and 150 mg·L−1 resulted in a decrease in the metabolic activity of the microbial consortium and its biodiversity. Despite this, an increase in the biological degradation rate of polycyclic aromatic hydrocarbons was noted of 17.9% and 16.9%, respectively. An assessment of the estimated number of genes crucial for EPS synthesis and biodegradation of polycyclic aromatic hydrocarbons confirmed the relationship between the activation of EPS synthesis pathways and polyaromatic hydrocarbon biodegradation pathways. It was established that microorganisms that belong to the Burkholderiales order are characterized by a high representation of the analyzed orthologs and high application potential in areas contaminated with heavy metals and hydrocarbons.

Exopolysaccharide production by lactic acid bacteria: the manipulation of environmental stresses for industrial applications

Exopolysaccharides (EPSs) are biological polymers secreted by microorganisms including Lactic acid bacteria (LAB) to cope with harsh environmental conditions. EPSs are one of the main components involved in the formation of extracellular biofilm matrix to protect microorganisms from adverse factors such as temperature, pH, antibiotics, host immune defenses, etc.. In this review, we discuss EPS biosynthesis; the role of EPSs in LAB stress tolerance; the impact of environmental stresses on EPS production and on the expression of genes involved in EPS synthesis. The evaluation results indicated that environmental stresses can alter EPS biosynthesis in LAB. For further studies, environmental stresses may be used to generate a new EPS type with high biological activity for industrial applications.

Carbohydrate Metabolism Regulated by Antisense vicR RNA in Cariogenicity

Streptococcus mutans is a major cariogenic pathogen that resides in multispecies oral microbial biofilms. The VicRK 2-component system is crucial for bacterial adaptation, virulence, and biofilm organization and contains a global and vital response regulator, VicR. Notably, we identified an antisense vicR RNA (ASvicR) associated with an adjacent RNase III–encoding (rnc) gene that was relevant to microRNA-size small RNAs (msRNAs). Here, we report that ASvicR overexpression significantly impeded bacterial growth, biofilm exopolysaccharide synthesis, and cariogenicity in vivo. Transcriptome analysis revealed that the ASvicR RNA mainly regulated carbohydrate metabolism. In particular, overproducing ASvicR demonstrated a reduction in galactose and glucose metabolism by monosaccharide composition analysis. The results of high-performance gel permeation chromatography revealed that the water-insoluble glucans isolated from ASvicR presented much lower molecular weights. Furthermore, direct evidence showed that total RNAs were disrupted by rnc-encoded RNase III. With the coexpression of T4 RNA ligase, putative msRNA1657, which is an rnc-related messenger RNA, was verified to bind to the 5′-UTR regions of the vicR gene. Furthermore, ASvicR regulation revealed a sponge regulatory-mediated network for msRNA associated with adjacent RNase III–encoding genes. There was an increase in ASvicR transcript levels in clinical S. mutans strains from caries-free children, while the expression of ASvicR was decreased in early childhood caries patients; this outcome may be explored as a potential strategy contributing to the management of dental caries. Taken together, our findings suggest an important role of ASvicR-mediated sponge regulation in S. mutans, indicating the characterization of lactose metabolism by a vital response regulator in cariogenicity. These findings have a number of implications and have reshaped our understanding of bacterial gene regulation from its transcriptional conception to the key roles of regulatory RNAs.

C-di-GMP Arms an Anti-σ to Control Progression of Multicellular Differentiation in Streptomyces.

SummaryStreptomyces are our primary source of antibiotics, produced concomitantly with the transition from vegetative growth to sporulation in a complex developmental life cycle. We previously showed that the signaling molecule c-di-GMP binds BldD, a master repressor, to control initiation of development. Here we demonstrate that c-di-GMP also intervenes later in development to control differentiation of the reproductive hyphae into spores by arming a novel anti-σ (RsiG) to bind and sequester a sporulation-specific σ factor (σWhiG). We present the structure of the RsiG-(c-di-GMP)2-σWhiG complex, revealing an unusual, partially intercalated c-di-GMP dimer bound at the RsiG-σWhiG interface. RsiG binds c-di-GMP in the absence of σWhiG, employing a novel E(X)3S(X)2R(X)3Q(X)3D motif repeated on each helix of a coiled coil. Further studies demonstrate that c-di-GMP is essential for RsiG to inhibit σWhiG. These findings reveal a newly described control mechanism for σ-anti-σ complex formation and establish c-di-GMP as the central integrator of Streptomyces development.

Virulence of methicillin-resistant Staphylococcus aureus modulated by the YycFG two-component pathway in a rat model of osteomyelitis

ObjectivesMethicillin-resistant Staphylococcus aureus (MRSA) strains present an urgent medical problem in osteomyelitis cases. Our previous study indicated that the YycFG two-component regulatory pathway is associated with the bacterial biofilm organization of MRSA strains. The aim of this study was to investigate the regulatory roles of ASyycG in the bacterial biofilm formation and the pathogenicity of MRSA strains using an antisense RNA strategy.MethodsAn ASyycG-overexpressing MRSA clinical isolate was constructed. The bacterial growth was monitored, and the biofilm biomass on bone specimens was examined using scanning electron microscopy and confocal laser scanning microscopy. Furthermore, quantitative RT-PCR (QRT-PCR) analysis was used to measure the expression of yycF/G/H and icaA/D in the MRSA and ASyycG strains. The expression of the YycG protein was quantified by Western blot assays. We validated the role of ASyycG in the invasive ability and pathogenicity of the strains in vivo using histology and peptide nucleic acid fluorescent in situ hybridization.ResultsThe results showed that overexpression of ASyycG lead to a reduction in biofilm formation and exopolysaccharide (EPS) synthesis compared to the control MRSA strains. The ASyycG strains exhibited decreased expression of the yycF/G/H and icaA/D genes. Furthermore, Western blot data showed that the production of the YycG protein was inhibited in the ASyycG strains. In addition, we demonstrated that ASyycG suppressed the invasive ability and pathogenicity of the strain in vivo using an SPF (specific pathogen free) rat model.ConclusionIn summary, the overexpression of ASyycG leads to a reduction in biofilm formation and bacterial pathogenicity in vivo, which provides a potential target for the management of MRSA-induced osteomyelitis.

The Expression of Virulence Factors in Vibrio anguillarum Is Dually Regulated by Iron Levels and Temperature.

Vibrio anguillarum causes a hemorrhagic septicemia that affects cold- and warm-water adapted fish species. The main goal of this work was to determine the temperature-dependent changes in the virulence factors that could explain the virulence properties of V. anguillarum for fish cultivated at different temperatures. We have found that although the optimal growth temperature is around 25°C, the degree of virulence of V. anguillarum RV22 is higher at 15°C. To explain this result, an RNA-Seq analysis was performed to compare the whole transcriptome profile of V. anguillarum RV22 cultured under low-iron availability at either 25 or 15°C, which would mimic the conditions that V. anguillarum finds during colonization of fish cultivated at warm- or cold-water temperatures. The comparative analysis of transcriptomes at high- and low-iron conditions showed profound metabolic adaptations to grow under low iron. These changes were characterized by a down-regulation of the energetic metabolism and the induction of virulence-related factors like biosynthesis of LPS, production of hemolysins and lysozyme, membrane transport, heme uptake, or production of siderophores. However, the expression pattern of virulence factors under iron limitation showed interesting differences at warm and cold temperatures. Chemotaxis, motility, as well as the T6SS1 genes are expressed at higher levels at 25°C than at 15°C. By contrast, hemolysin RTX pore-forming toxin, T6SS2, and the genes associated with exopolysaccharides synthesis were preferentially expressed at 15°C. Notably, at this temperature, the siderophore piscibactin system was strongly up-regulated. In contrast, at 25°C, piscibactin genes were down-regulated and the vanchrobactin siderophore system seems to supply all the necessary iron to the cell. The results showed that V. anguillarum adjusts the expression of virulence factors responding to two environmental signals, iron levels and temperature. Thus, the relative relevance of each virulence factor for each fish species could vary depending on the water temperature. The results give clues about the physiological adaptations that allow V. anguillarum to cause infections in different fishes and could be relevant for vaccine development against fish vibriosis.

Inhibition of biofilm formation and exopolysaccharide synthesis of Enterococcus faecalis by phenyllactic acid

This study aimed to evaluate the inhibitory activity of phenyllactic acid (PLA) against the biofilm formation of Enterococcus faecalis and to explore its potential molecular mechanism. The MIC value of PLA that inhibited the growth of E. faecalis R612-Z1 in BHI broth was 5 mg/mL. PLAs at subinhibitory concentrations of 1.25 and 2.50 mg/mL were found to inhibit biofilm formation by a crystal violet staining assay. The cell swimming and swarming motilities of E. faecalis were reduced in the presence of PLA. An apparent decrease in the thickness of PLA-treated biofilms was observed through confocal laser scanning microscopy analysis. The exopolysaccharide production in E. faecalis biofilms was inhibited by EPS quantification assay and scanning electron microscopy (SEM). qRT-PCR analyses showed that PLA down-regulated the transcription of Ebp pili genes (ebpABC) and Epa polysaccharide genes (epaABE). PLA inhibited the biofilm formation by interfering with cell mobility and EPS production of E. faecalis. In addition, PLA at concentrations of 10.0 mg/mL can effectively control the bacterial cells in a three-day-old mature biofilm of E. faecalis grown on 24-well flat-bottom polystyrene plates and stainless-steel surfaces. Thus, PLA is potentially an effective agent to control E. faecalis biofilms.

Inactivation of the Phosphatase CheZ Alters Cell-Surface Properties of Azorhizobium caulinodans ORS571 and Symbiotic Association with Sesbania rostrata.

Azorhizobium caulinodans can form root and stem nodules with the host plant Sesbania rostrata. The role of the CheZ phosphatase in the A. caulinodans chemotaxis pathway was previously explored using the nonchemotactic cheZ mutant strain (AC601). This mutant displayed stronger attachment to the root surface, enhancing early colonization; however, this did not result in increased nodulation efficiency. In this study, we further investigated the role of CheZ in the interaction between strain ORS571 and the roots of its host plant. By tracking long-term colonization dynamic of cheZ mutant marked with LacZ, we found a decrease of colonization of the cheZ mutant during this process. Furthermore, the cheZ mutant could not spread on the root surface freely and was gradually outcompeted by the wild type in original colonization sites. Quantitative reverse-transcription PCR analyses showed that exp genes encoding exopolysaccharides synthesis, including oac3, were highly expressed in the cheZ mutant. Construction of a strain carrying a deletion of both cheZ and oac3 resulted in a mutant strain defective in the colonization process to the same extent as found with the oac3 single-mutant strain. This result suggested that the enhanced colonization of the cheZ mutant may be achieved through regulating the formation of exopolysaccharides. This shows the importance of the chemotactic proteins in the interaction between rhizobia and host plants, and expands our understanding of the symbiosis interaction between rhizobium and host plant.

Optimization of Media Composition to Maximize the Yield of Exopolysaccharides Production by Lactobacillus rhamnosus Strains

Growth media composition is a critical factor influencing the yield of bacterial exopolysaccharides (EPSs), which have attracted the interest of researchers around the world due to their diverse physicochemical and biological properties. This work presents the optimization of media for EPS synthesis by three Lactobacillus rhamnosus strains, namely ŁOCK 0943, ŁOCK 0935, and OM-1. The optimized media led to a more than 13-fold increase in EPS yield for L. rhamnosus ŁOCK 0943 (from 85 to 1138.2 mg/L), an almost 9-fold increase for L. rhamnosus ŁOCK 0935 (from 103.67 to 900 mg/L), and a more than 7-fold increase for L. rhamnosus OM-1 (from 133.67 to 987.84 mg/L) as compared to cultures in standard MRS medium (de Man, Rogosa, and Sharpe). It has been found that the main medium-related determinant of EPS synthesis by the studied L. rhamnosus strains are the carbon source—in this case, it was fructose and sucrose.

Mitigation of salinity stress in plants using plant growth promoting bacteria

Salinity is one of the major constraints prevailing in environment that affects not only plant growth but also agriculture productivity and soil fertility. Salinity stress causes nutritional and hormonal imbalance, ion toxicity, oxidative and osmotic stress and increased susceptibility of plant to diseases. Increasing use of chemical based fertilizers creates demand for environmental friendly and ecological compatible alternative in agriculture. Plant growth promoting bacteria (PGPB) have tremendous ability to mitigate salinity stress and ameliorate plant growth, playing vital role in food security by increasing agriculture crop productivity. PGPB inoculation under salinity promotes plant growth by several traits including 1-aminocyclopropane 1 carboxylate (ACC) deaminase activity, synthesis of plant hormones (such as indole acetic acid (IAA), gibberellic acid (GA), abscisic acid (ABA), cytokinin) and exopolysaccharides. PGPB alleviates salinity stress in plants by providing nutrients, maintaining high potassium and sodium ratio, increasing accumulation of osmolytes, enhanced photosynthesis and activity of antioxidant enzymes. The present review highlights and discusses current knowledge on effect of salinity stress on plant growth, PGPB mechanisms resulting in mitigation of salinity stress and increasing plant growth. It assesses morphological, biochemical, physiological and molecular changes induced in plants suffering from salinity when supplemented with PGPB.

Antibacterial Silver Nanomaterial Synthesis From Mesoflavibacter zeaxanthinifaciens and Targeting Biofilm Formation.

Considering the significance of biological and eco-friendly nanomaterials, in the present study, we have synthesized silver nanoparticles from the exopolysaccharide of recently recovered bacterial strain CEES51 from the Red Sea coastal area of Jeddah, Saudi Arabia. 16S ribosomal RNA gene sequencing was used to characterize the isolated bacteria, and it was identified as Mesoflavibacter zeaxanthinifaciens and assigned an accession number MH707257.1 GenBank. The bacterial strain is an excellent exopolysaccharide producer and survived at hypersaline (30%) and high-temperature (50°C) conditions. The bacterial exopolysaccharides were employed for the fabrication of silver nanoparticles at room temperature. UV-visible spectrophotometer optimized the synthesized nanoparticles, and their size was determined by Nanophox particle size analyzer and dynamic light scattering. Additionally, the X-ray powder diffraction and Fourier-transform infrared spectroscopy studies also approved its crystalline nature and the involvement of organic functional groups in their formation. The synthesized nanomaterials were tested for their antibacterial and antibiofilm properties against pathogenic microorganisms Bacillus subtilis and methicillin-resistant Staphylococcus aureus. The antimicrobial property showed time, and dose-dependent response with a maximum of zone inhibition was observed at around 22 and 18 mm at a dose of 50 µg/well against B. subtilis and S. aureus and a minimum inhibitory concentration of 8 and 10 µg/ml, respectively. Furthermore, the synthesized silver nanoparticles possessed a substantial antibiofilm property and were also found to be biocompatible as depicted by red blood cell lysis assay and their interaction with peripheral blood mononuclear cells and human embryonic kidney 293 cells. Therefore, Mesoflavibacter zeaxanthinifaciens is found to be an excellent source for exopolysaccharide synthesis that assists in the silver nanoparticle production.

Extraction and optimization of exopolysaccharide from Lactobacillus sp. using response surface methodology and artificial neural networks

The microbial polysaccharides secreted and produced from various microbes into their extracellular environment is known as exopolysaccharide. These polysaccharides can be secreted from the microbes either in a soluble or insoluble form.Lactobacillus sp. is one of the organisms that have been found to produce exopolysaccharide. Exo-polysaccharides (EPS) have various applications such as drug delivery, antimicrobial activity, surgical implants and many more in different fields. Medium composition is one of the major aspects for the production of EPS from Lactobacillus sp., optimization of medium components can help to enhance the synthesis of EPS . In the present work, the production of exopolysaccharide with different medium composition was optimized by response surface methodology (RSM) followed by tested for fitting with artificial neural networks (ANN). Three algorithms of ANN were compared to investigate the highest yeild of EPS. The highest yeild of EPS production in RSM was achieved by the medium composition that consists of (g/L) dextrose 15, sodium dihydrogen phosphate 3, potassium dihydrogen phosphate 2.5, triammonium citrate 1.5, and, magnesium sulfate 0.25. The output of 32 sets of RSM experiments were tested for fitting with ANN with three algorithms viz. Levenberg–Marquardt Algorithm (LMA), Bayesian Regularization Algorithm (BRA) and Scaled Conjugate Gradient Algorithm (SCGA) among them LMA found to have best fit with the experiments as compared to the SCGA and BRA.

A Novel Dental Sealant Containing Dimethylaminohexadecyl Methacrylate Suppresses the Cariogenic Pathogenicity of Streptococcus mutans Biofilms.

Cariogenic oral biofilms are strongly linked to dental caries around dental sealants. Quaternary ammonium monomers copolymerized with dental resin systems have been increasingly explored for modulation of biofilm growth. Here, we investigated the effect of dimethylaminohexadecyl methacrylate (DMAHDM) on the cariogenic pathogenicity of Streptococcus mutans (S. mutans) biofilms. DMAHDM at 5 mass% was incorporated into a parental formulation containing 20 mass% nanoparticles of amorphous calcium phosphate (NACP). S. mutans biofilms were grown on the formulations, and biofilm inhibition and virulence properties were assessed. The tolerances to acid stress and hydrogen peroxide stress were also evaluated. Our findings suggest that incorporating 5% DMAHDM into 20% NACP-containing sealants (1) imparts a detrimental biological effect on S. mutans by reducing colony-forming unit counts, metabolic activity and exopolysaccharide synthesis; and (2) reduces overall acid production and tolerance to oxygen stress, two major virulence factors of this microorganism. These results provide a perspective on the value of integrating bioactive restorative materials with traditional caries management approaches in clinical practice. Contact-killing strategies via dental materials aiming to prevent or at least reduce high numbers of cariogenic bacteria may be a promising approach to decrease caries in patients at high risk.

Cadmium-affected synthesis of exopolysaccharides by rhizosphere bacteria.

Study is focused on the influence of cadmium addition to growth media on production yield, their size and molecular mass of exopolysaccharides (EPS) synthesized by three rhizosphere bacteria strains. Inhibition of bacterial growth by increasing concentrations of Cd2+ was also analysed. The highest impact of Cd2+ was noticed on the growth of Arthrobacter sp. and Rhizobium metallidurans. Chryseobacterium sp. and Arthrobacter sp. produced significantly lower when compared to R. metallidurans amounts of EPS under the influence of Cd2+ . In all bacterial strains both size and molecular mass decreased after addition of Cd2+ to growth media. It causes a change in EPS conformation to more planar, which minimizes the volume of liquid in the interglobular space next to the bacterial wall. Results confirmed strong effect of Cd2+ on the structure and synthesis of bacterial EPS what can be a key factor in the interactions between rhizosphere bacteria and host plants in heavy metal polluted soils. This work proves that due to the presence of cadmium ions, the size and conformation of EPS produced by selected bacterial strains is changed to minimize their impact on cell. We suggest that shifting in EPS conformation from bigger globular particles to the smaller planar ones could be one of the probable mechanisms of Cd resistance in metallotolerant bacteria, and finally explain increased efficiency of heavy metal phytoextraction by EPS-producing plant growth-promoting micro-organisms. One of the most promising remediation technique for Cd-contaminated areas is the phytoremediation in which rhizosphere bacteria play an important role by protecting plants' roots from toxic condition thus enhancing efficiency of intake. EPS secretion by bacteria is one of the most common mechanisms to protect the cell from impact of unpleasant environmental conditions, for example, toxicity of heavy metals like Cd.

Genetics and proteomics analyses reveal the roles of PhoB1 and PhoB2 regulators in bacterial responses to arsenite and phosphate

In bacteria, phosphate (Pi) stress response is governed by the two-component regulatory system, sensor kinase PhoR and its cognate response regulatory protein PhoB. The arsenite [As(III)]-oxidizing bacterium Agrobacterium tumefaciens GW4 contains two phoB genes, phoB1 and phoB2. phoB1 is adjacent to As(III)-oxidizing genes, however, the functions of PhoB1 and PhoB2 remain unclear. Here, phoB1 and phoB2 were each deleted in-frame, and proteomics, qRT-PCR and protein-DNA interaction were performed. We found that (1) phoB1 and phoB2 were both upregulated under low Pi conditions and phoB1 was induced by As(III), but phoB2 was not; (2) deletion of phoB1 reduced As(III)-oxidizing efficiency and protein-DNA interaction analysis showed PhoB1 could interact with aioXSR promoter to regulate As(III) oxidation; (3) deletions of phoB1 or phoB2 both reduced exopolysaccharides (EPS) synthesis; and (4) PhoB1 influenced Pi uptake, As(III) oxidation, EPS synthesis, TCA cycle, energy production and stress response with As(III), and PhoB2 was associated with Pi uptake and EPS synthesis in low Pi conditions. These results showed PhoB1 and PhoB2 were both involved in Pi acquisition, PhoB1 was more important with As(III) and PhoB2 played a major role without As(III). Strain GW4 uses these two regulators to survive under low Pi and arsenic-rich environments.