Exopolysaccharide Synthesis Research Articles

Enhancement of exopolysaccharides production and reactive oxygen species level of Nostoc flagelliforme in response to dehydration.

Nostoc flagelliforme is a remarkable drought-resistant terrestrial cyanobacterium whose exopolysaccharides (EPS) have been found to exert important physiological and ecological functions, and the EPS are known to improve soil physicochemical properties. In this study, we used physiological and molecular methods to investigate the influences of three moisture loss levels on EPS production and the antioxidant system in N. flagelliforme. The aim was to reveal the EPS production mechanism involved in the gene differential expression and antioxidant system of N. flagelliforme in response to drought. Our results showed that EPS contents increased by 13% and 22% after 6-h and 48-h dehydration (6HAD and 48HAD) compared with 4-h rehydration (4HAR), respectively. The same trends were also detected for most EPS synthesis genes, especially glycosyltransferases. Furthermore, the intracellular reactive oxygen species (ROS) levels in N. flagelliforme were generally higher at 6HAD and 48HAD than at 4HAR. Superoxide dismutase (SOD) and peroxidase (POD) activities were restricted in N. flagelliforme under 6HAD and 48HAD compared with 4HAR, but the opposite result was found in catalase (CAT) activity. These results provide a new foundation for understanding the mechanism of EPS accumulation in N. flagelliforme in response to drought.

Anti-Biofilm Activity of Cannabidiol against Candida albicans

Candida albicans is a common fungal pathogen in humans. Biofilm formation is an important virulence factor of C. albicans infections. We investigated the ability of the plant-derived cannabidiol (CBD) to inhibit the formation and removal of fungal biofilms. Further, we evaluated its mode of action. Our findings demonstrate that CBD exerts pronounced time-dependent inhibitory effects on biofilm formation as well as disruption of mature biofilm at a concentration range below minimal inhibitory and fungicidal concentrations. CBD acts at several levels. It modifies the architecture of fungal biofilm by reducing its thickness and exopolysaccharide (EPS) production accompanied by downregulation of genes involved in EPS synthesis. It alters the fungal morphology that correlated with upregulation of yeast-associated genes and downregulation of hyphae-specific genes. Importantly, it represses the expression of C. albicans virulence-associated genes. In addition, CBD increases ROS production, reduces the intracellular ATP levels, induces mitochondrial membrane hyperpolarization, modifies the cell wall, and increases the plasma membrane permeability. In conclusion, we propose that CBD exerts its activity towards C. albicans biofilm through a multi-target mode of action, which differs from common antimycotic agents, and thus can be explored for further development as an alternative treatment against fungal infections.

Design of a "Clean-Label" Gluten-Free Bread to Meet Consumers Demand.

The market of gluten-free (GF) products has been steadily increasing in last few years. Due to the technological importance of gluten, the GF food production is still a challenge for the industry. Indeed, large quantities of fat, sugars, structuring agents, and flavor enhancers are added to GF formulations to make textural and sensorial characteristics comparable to conventional products, leading to nutritional and caloric intake imbalances. The formulation of the novel “clean-label” GF bread included a commonly used mixture of maize and rice flour (ratio 1:1) fortified with selected protein-rich flours. Naturally hydrocolloids-containing flours (psyllium, flaxseed, chia) were included in the bread formulation as structuring agents. A type-II sourdough was obtained by using a selected Weissella cibaria P9 and a GF sucrose-containing flour as substrate for fermentation to promote the exo-polysaccharides synthesis by the starter lactic acid bacterium. A two-step protocol for bread-making was set-up: first, the GF sourdough was fermented (24 h at 30 °C); then, it was mixed with the other ingredients (30% of the final dough) and leavened with baker’s yeast before baking. Overall, the novel GF bread was characterized by good textural properties, high protein content (8.9% of dry matter) and in vitro protein digestibility (76.9%), low sugar (1.0% of dry matter) and fat (3.1% of dry matter) content, and an in vitro predicted glycemic index of 85.

Yeast exopolysaccharides and their physiological functions.

Mounting evidence indicated the capability of various microorganisms in biosynthesis of exopolysaccharides (EPSs). A wide range of evidence extensively investigated the ability of bacterial species for EPS synthesis and their favorable effects, so little is known regarding yeast species. Many factors like composition of growth media and fermentation conditions are related to the structural and physical properties of EPSs. The EPS protects the producer yeast strain against extreme environment. Researchers proposed that yeast EPSs have priority over bacterial EPSs because of high yields of EPS biosynthesis and easy separation methods from growth media. Besides, they have drawn increasing attention due to their interesting biological activities, food, pharmaceutical, and cosmetics applications. Although a limited number of studies exist, this review aims to highlight the EPS structure and various applications of known yeast species in detail.

Light Signaling Regulates Aspergillus niger Biofilm Formation by Affecting Melanin and Extracellular Polysaccharide Biosynthesis.

ABSTRACTLight is an important signal source in nature, which regulates the physiological cycle, morphogenetic pathways, and secondary metabolites of fungi. As an external pressure on Aspergillus niger, light signaling transmits stress signals into the cell via the mitogen-activated protein kinase (MAPK) signaling pathway. Studying the effect of light on the biofilm of A. niger will provide a theoretical basis for light in the cultivation of filamentous fungi and industrial applications. Here, the characterization of A. niger biofilm under different light intensities confirmed the effects of light signaling. Our results indicated that A. niger intensely accumulated protective mycelial melanin under light illumination. We also discovered that the RlmA transcription factor in the MAPK signaling pathway is activated by light signaling to promote the synthesis of melanin, chitin, and other exopolysaccharides. However, the importance of melanin to A. niger biofilm is rarely reported; therefore, we knocked out key genes of the melanin biosynthetic pathway—Abr1 and Ayg1. Changes in hydrophobicity and electrostatic forces resulted in the decrease of biofilm caused by the decrease of melanin in mutants.

Exopolysaccharide Characterization of Rhizobium favelukesii LPU83 and Its Role in the Symbiosis With Alfalfa.

One of the greatest inputs of available nitrogen into the biosphere occurs through the biological N2-fixation to ammonium as result of the symbiosis between rhizobia and leguminous plants. These interactions allow increased crop yields on nitrogen-poor soils. Exopolysaccharides (EPS) are key components for the establishment of an effective symbiosis between alfalfa and Ensifer meliloti, as bacteria that lack EPS are unable to infect the host plants. Rhizobium favelukesii LPU83 is an acid-tolerant rhizobia strain capable of nodulating alfalfa but inefficient to fix nitrogen. Aiming to identify the molecular determinants that allow R. favelukesii to infect plants, we studied its EPS biosynthesis. LPU83 produces an EPS I identical to the one present in E. meliloti, but the organization of the genes involved in its synthesis is different. The main gene cluster needed for the synthesis of EPS I in E. meliloti, is split into three different sections in R. favelukesii, which probably arose by a recent event of horizontal gene transfer. A R. favelukesii strain devoided of all the genes needed for the synthesis of EPS I is still able to infect and nodulate alfalfa, suggesting that attention should be directed to other molecules involved in the development of the symbiosis.

The effect of optimized carbon source on the synthesis and composition of exopolysaccharides produced by Lactobacillus paracasei

This study aimed to predict the optimal carbon source for higher production of exopolysaccharides (EPS) by Lactobacillus paracasei TD 062, and to evaluate the effect of this carbon source on the production and monosaccharide composition of EPS. We evaluated the EPS production capacity of 20 strains of L. paracasei under the same conditions. We further investigated L. paracasei TD 062, which showed the highest EPS-producing activity (0.609 g/L), by examining the associated biosynthesis pathways for EPS. Genomics revealed that fructose, mannose, trehalose, glucose, galactose, and lactose were carbon sources that L. paracasei TD 062 could use to produce EPS. We identified an EPS synthesis gene cluster that could participate in transport, export, and sugar chain synthesis, and generate 6 sugar nucleotides. Experimental results showed that the sugar content of the EPS produced using fermentation with the optimized carbon source (fructose, mannose, trehalose, glucose, galactose, and lactose) increased by 115%. Furthermore, use of the optimized carbon source changed the monosaccharide content of the associated EPS. The results of enzyme activity measurements showed significant increases in the activity of 2 key enzymes involved in the glycoside synthesis pathway. Our study revealed that optimizing the carbon source provided for fermentation not only increased the production of EPS, but also affected the composition of the monosaccharides by increasing enzyme activity in the underlying synthesis pathways, suggesting an important role for carbon source in the production of EPS by L. paracasei TD 062.

PBP4 and PBP5 are involved in regulating exopolysaccharide synthesis during Escherichia coli biofilm formation.

Escherichia coli low-molecular-mass (LMM) Penicillin-binding proteins (PBPs) help in hydrolysing the peptidoglycan fragments from their cell wall and recycling them back into the growing peptidoglycan matrix, in addition to their reported involvement in biofilm formation. Biofilms are external slime layers of extra-polymeric substances that sessile bacterial cells secrete to form a habitable niche for themselves. Here, we hypothesize the involvement of Escherichia coli LMM PBPs in regulating the nature of exopolysaccharides (EPS) prevailing in its extra-polymeric substances during biofilm formation. Therefore, this study includes the assessment of physiological characteristics of E. coli CS109 LMM PBP deletion mutants to address biofilm formation abilities, viability and surface adhesion. Finally, EPS from parent CS109 and its ΔPBP4 and ΔPBP5 mutants were purified and analysed for sugars present. Deletions of LMM PBP reduced biofilm formation, bacterial adhesion and their viability in biofilms. Deletions also diminished EPS production by ΔPBP4 and ΔPBP5 mutants, purification of which suggested an increased overall negative charge compared with their parent. Also, EPS analyses from both mutants revealed the appearance of an unusual sugar, xylose, that was absent in CS109. Accordingly, the reason for reduced biofilm formation in LMM PBP mutants may be speculated as the subsequent production of xylitol and a hindrance in the standard flow of the pentose phosphate pathway.

Bacterial tolerance strategies against lead toxicity and their relevance in bioremediation application.

Among heavy metals, lead (Pb) is a non-essential metal having a higher toxicity and without any crucial known biological functions. Being widespread, non-biodegradable and persistent in every sphere of soil, air and water, Pb is responsible for severe health and environmental issues, which need appropriate remediation measures. However, microbes inhabiting Pb-contaminated area are found to have evolved distinctive mechanisms to successfully thrive in the Pb-contaminated environment without exhibiting any negative effects on their growth and metabolism. The defensive strategies used by bacteria to ameliorate the toxic effects of lead comprise biosorption, efflux, production of metal chelators like siderophores and metallothioneins and synthesis of exopolysaccharides, extracellular sequestration and intracellular bioaccumulation. Lead remediation technologies by employing microbes may appear as potential advantageous alternatives to the conventional physical and chemical means due to specificity, suitability for applying in situ condition and feasibility to upgrade by genetic engineering. Developing strategies by designing transgenic bacterial strain having specific metal binding properties and metal chelating proteins or higher metal adsorption ability and using bacterial activity such as incorporating plant growth-promoting rhizobacteria for improved Pb resistance, exopolysaccharide and siderophores and metallothionein-mediated immobilization may prove highly effective for formulating bioremediation vis-a-vis phytoremediation strategies.

The Route of Sucrose Utilization by Streptococcus mutans Affects Intracellular Polysaccharide Metabolism.

Streptococcus mutans converts extracellular sucrose (Suc) into exopolysaccharides (EPS) by glucosyl-transferase and fructosyl-transferase enzymes and internalizes Suc for fermentation through the phosphotransferase system (PTS). Here, we examined how altering the routes for sucrose utilization impacts intracellular polysaccharide [IPS; glycogen, (glg)] metabolism during carbohydrate starvation. Strain UA159 (WT), a mutant lacking all exo-enzymes for sucrose utilization (MMZ952), and a CcpA-deficient mutant (∆ccpA) were cultured with sucrose or a combination of glucose and fructose, followed by carbohydrate starvation. At baseline (0h), and after 4 and 24h of starvation, cells were evaluated for mRNA levels of the glg operon, IPS storage, glucose-1-phosphate (G1P) concentrations, viability, and PTS activities. A pH drop assay was performed in the absence of carbohydrates at the baseline to measure acid production. We observed glg operon activation in response to starvation (p<0.05) in all strains, however, such activation was significantly delayed and reduced in magnitude when EPS synthesis was involved (p<0.05). Enhanced acidification and greater G1P concentrations were observed in the sucrose-treated group, but mostly in strains capable of producing EPS (p<0.05). Importantly, only the WT exposed to sucrose was able to synthesize IPS during starvation. Contrary to CcpA-proficient strains, IPS was progressively degraded during starvation in ∆ccpA, which also showed increased glg operon expression and greater PTS activities at baseline. Therefore, sucrose metabolism by secreted enzymes affects the capacity of S. mutans in synthesizing IPS and converting it into organic acids, without necessarily inducing greater expression of the glg operon.

Synthesis of microbial exopolysaccharides on non-traditional substrates

Synthesis of microbial exopolysaccharides on non-traditional substrates

Effects of an EPS Biosynthesis Gene Cluster of Paenibacillus polymyxa WLY78 on Biofilm Formation and Nitrogen Fixation under Aerobic Conditions

Exopolysaccharides (EPS) are of high significance in bacterial biofilm formation. However, the effects of EPS cluster(s) on biofilm formation in Paenibacillus species are little known. In this study, we have shown that Paenibacillus polymyxa WLY78, a N2-fixing bacterium, can form biofilm. EPS is the major component of the extracellular matrix. The genome of P. polymyxa WLY78 contains two putative gene clusters (designated pep-1 cluster and pep-2 cluster). The pep-1 cluster is composed of 12 putative genes (pepO-lytR) co-located in a 13 kb region. The pep-2 cluster contains 17 putative genes (pepA-pepN) organized as an operon in a 20 kb region. Mutation analysis reveals that the pep-2 cluster is involved in EPS biosynthesis and biofilm formation. Disruption of the pep-2 cluster also leads to the enhancement of motility and change of the colony morphology. In contrast, disruption of the pep-1 cluster does not affect EPS synthesis or biofilm formation. More importantly, the biofilm allowed P. polymyxa WLY78 to fix nitrogen in aerobic conditions, suggesting that biofilm may provide a microaerobic environment for nitrogenase synthesis and activity.

Removal of lithium from aqueous solutions using halotolerant bacteria from El Salar del Hombre Muerto

The interaction of lithium with halotolerant microorganisms was evaluated to determine its effect on bacterial isolates and its removal from the surrounding environment. The observed effects on bacterial cells included different biomass production, exopolysaccharide synthesis, variation in morphology, and spore formation under the presence of Li. They allowed microorganisms not only to tolerate 20–30 g/L LiCl but also to remove it from the surrounding environment. The microbial-metal interactions were verified through lithium removal from the growth medium and also from aqueous solutions using fresh cultures. Interestingly, in presence of LiCl, bacteria from the genus Bacillus sp. (strains FAMB1, SA313, HX11, and HA120a) produced similar amounts of biomass and higher amounts of exopolysaccharides in comparison to the condition without LiCl added. These isolates, except for FAMB1, were also efficient in Li removal in all the evaluated conditions, removing around 30% of initial Li during growth and 50% from aqueous solutions. Furthermore, fresh cultures of Micrococcus luteus SA211, Bacillus sp. FAMB1, Micrococcus sp. M12, Brevibacterium sp. SX139 and Kocuria sp. SA129b removed Li from the aqueous solutions, and only SA129b released the metal back to the solution after incubating for 6 h. Finally, Bacillus sp. HX11, Bacillus sp. HA120a, Kocuria sp. SA129b and Brevibacterium sp. SX139 developed an efficient and environmentally-friendly removal of lithium from aqueous matrices in short periods, showing specific lithium removal values between 1.0 and 1.4 mg Li/g cells.

Calcium (Ca2+)-regulated exopolysaccharide biosynthesis in probiotic Lactobacillus plantarum K25 as analyzed by an omics approach

Exopolysaccharide (EPS)-producing lactic acid bacteria have been widely used in dairy products, but how calcium, the main metal ion component in milk, regulates the EPS biosynthesis in lactic acid bacteria is not clear. In this study, the effect of Ca2+ on the biosynthesis of EPS in the probiotic Lactobacillus plantarum K25 was studied. The results showed that addition of CaCl2 at 20 mg/L in a semi-defined medium did not affect the growth of strain K25, but it increased the EPS yield and changed the microstructure of the polymer. The presence of Ca2+ also changed the monosaccharide composition of the EPS with decreased high molecular weight components and more content of rhamnose, though the functional groups of the polymer were not altered as revealed by Fourier transform infrared spectral analysis. These were further confirmed by analysis of the mRNA expression of cps genes, 9 of which were upregulated by Ca2+, including cps4F and rfbD associated with EPS biosynthesis with rhamnose. Proteomics analysis showed that Ca2+ upregulated most of the proteins related to carbon transport and metabolism, fatty acid synthesis, amino acid synthesis, ion transport, UMP synthesis. Specially, the increased expression of MelB, PtlIIBC, EIIABC, PtlIIC, PtlIID, Bgl, GH1, MalFGK, DhaK, and FBPase provided substrates for the EPS synthesis. Meanwhile, metabolomics analysis revealed significant change of the small molecular metabolites in tricarboxylic acid cycle, glucose metabolism and propionic acid metabolism. Among them the content of active small molecules such as polygalitol, lyxose, and 5-phosphate ribose increased, facilitating the EPS biosynthesis. Furthermore, Ca2+ activated HipB signaling pathway to inhibit the expression of manipulator repressor such as ArsR, LytR/AlgR, IscR, and RafR, and activated the expression of GntR to regulate the EPS synthesis genes. This study provides a basis for understanding the overall change of metabolic pathways related to the EPS biosynthesis in L. plantarum K25 in response to Ca2+, facilitating exploitation of its EPS-producing potential for application in probiotic dairy products.

Biosynthesis of exopolysaccharides by soil bacteria &lt;i&gt;Paenibacillus mucilaginosus&lt;/i&gt; on a nutrient molasses medium

Bacterial exopolysaccharides exhibiting water-retaining and cementing properties play an essential role in such processes as the formation and stabilization of soil aggregates, regulation of nutrients and water flow through plant roots, as well as reduction of salt stress in plants. When bacteria producing exopolysaccharides are introduced into a soil, plants become more resistant to water stress due to improved structure and significant accumulation of proline, sugars and free amino acids under the conditions of water deficiency. In this work, we set out to determine the efficiency of exopolysaccharide biosynthesis by the soil bacteria Paenibacillus mucilaginosus when cultivated on a nutrient molasses medium. The bacterial strains P. mucilaginosus 560 and 574 provided by the Departmental collection of non-pathogenic microorganisms for agricultural purposes (All-Russia Research Institute for Agricultural Microbiology, St. Petersburg) were used as exopolysaccharide producers. In order to determine the influence of cultivation conditions on the synthesis of exopolysaccharides by P. mucilaginosus, the one-factor-at-a-time (OFAT) method was used. On the basis of the obtained results, we determined the most effective producer of exopolysaccharides, the age and dose of the inoculum, the optimal values of the cultivation temperature and medium pH, the source of nitrogen and its concentration, as well as the aeration conditions affecting exopolysaccharide biosynthesis and the growth of the selected bacterial strain P. mucilaginosus. It was established that the P. mucilaginosus 574 strain is the most effective producer of exopolysaccharides. It was shown that the biosynthesis of exopolysaccharides is most effective and economical upon the cultivation of P. mucilaginosus 574 on a nutrient medium containing 2% molasses without addition of minerals and nitrogen. The maximum amount of exopolysaccharides can reach 9.55 g/L on a nutrient medium containing 2% molasses at a volume of 50 ml with the addition of 0.1% corn extract as an inducer of exopolysaccharide synthesis at a cultivation temperature of 30±1 °C, a medium pH of 6.0±0.2 with the introduction of 5% inoculum after 24 hours of inoculation. The obtained research results are recommended for use when creating new technologies for the production of microbiological fertilizers.

Physiological and genomic analysis of newly-isolated polysaccharide synthesizing cyanobacterium Chroococcus sp. FPU101 and chemical analysis of the exopolysaccharide.

A unicellular cyanobacterium that produces a large amount of exopolysaccharide (EPS) was isolated. The isolate, named Chroococcus sp. FPU101, grew between 20 and 30°C and at light intensities between 10 and 80 μmol m-2 s-1. Purified EPS from Chroococcus sp. FPU101 had a molecular size of 5.9 × 103 kDa and contained galactose, rhamnose, fucose, xylose, mannose, glucose, galacturonic acid, and glucuronic acid at a molar ratio of 17.2:15.9:14.1:11.0:9.6:9.5:13.0:9.7. The EPS content significantly increased when the NaCl concentration in the medium was increased from 1.7 to 100 mM. However, high NaCl concentrations did not significantly affect the molecular size or chemical composition of the EPS. The genes wza, wzb, wzc, wzx, wzy, and wzz that are involved in EPS synthesis were conserved in the genome of Chroococcus sp. FPU101, which was sequenced in this study. These results suggest that the Wzy-dependent pathway is potentially involved in EPS production in this organism.

Pantoea stewartii WceF is a glycan biofilm-modifying enzyme with a bacteriophage tailspike-like fold

Pathogenic microorganisms often reside in glycan-based biofilms. Concentration and chain length distribution of these mostly anionic exopolysaccharides (EPS) determine the overall biophysical properties of a biofilm and result in a highly viscous environment. Bacterial communities regulate this biofilm state via intracellular small-molecule signaling to initiate EPS synthesis. Reorganization or degradation of this glycan matrix, however, requires the action of extracellular glycosidases. So far, these were mainly described for bacteriophages that must degrade biofilms for gaining access to host bacteria. The plant pathogen Pantoea stewartii (P. stewartii) encodes the protein WceF within its EPS synthesis cluster. WceF has homologs in various biofilm forming plant pathogens of the Erwinia family. In this work, we show that WceF is a glycosidase active on stewartan, the main P. stewartii EPS biofilm component. WceF has remarkable structural similarity with bacteriophage tailspike proteins (TSPs). Crystal structure analysis showed a native trimer of right-handed parallel β-helices. Despite its similar fold, WceF lacks the high stability found in bacteriophage TSPs. WceF is a stewartan hydrolase and produces oligosaccharides, corresponding to single stewartan repeat units. However, compared with a stewartan-specific glycan hydrolase of bacteriophage origin, WceF showed lectin-like autoagglutination with stewartan, resulting in notably slower EPS cleavage velocities. This emphasizes that the bacterial enzyme WceF has a role in P. stewartii biofilm glycan matrix reorganization clearly different from that of a bacteriophage exopolysaccharide depolymerase.

Activity of Ligustrum robustum (Roxb.) Blume extract against the biofilm formation and exopolysaccharide synthesis of Streptococcus mutans.

Ligustrum robustum (Roxb.) Blume is utilized as a traditional Chinese herbal tea with various health benefits and protective effects. Streptococcus mutans is an important cariogenic oral bacteria species. The present study aimed to assess the influence of Ligustrum robustum extract (LRE) on the biofilm formation of S. mutans and the mechanism of its action, as well as to identify its chemical components. For chemical identification, HPLC-MS and nuclear magnetic resonance were applied and four identified phytochemicals were reported (Ligurobustoside B, Ligurobustoside N, Ligurobustoside J, and Ligurobustoside C). The dose-dependent (0.5 to 2.0μg/μL) antimicrobial toxicity of LRE against S. mutans biofilm formation and exopolysaccharide (EPS) synthesis was evaluated by confocal laser scanning microscopy (CLSM), Crystal violet stain, and CFU counting. The microstructure of S. mutans biofilm treated with LRE was investigated both on glass coverslips and ex vivo bovine dental enamel by scanning electron microscopy (SEM). Moreover, LRE downregulated the expression of S. mutans glucosyltransferase-encoding genes gtfB, gtfC, and gtfD, and the quorum sensing (QS) factors comD and comE, suggesting its toxic mechanism. In addition, the result of CCK-8 test on human oral cells revealed an acceptable biocompatibility of LRE. These findings indicated the possible application of this daily consumed herbal tea for caries prevention.

The pH-Responsive Property of Antimicrobial Peptide GH12 Enhances Its Anticaries Effects at Acidic pH

Dental caries is closely related to the acidification of the biofilms on the tooth surface, in which cariogenic bacteria bring about a dramatic pH decrease and disrupt remineralisation equilibrium upon the fermentation of dietary sugars. Thus, approaches targeting the acidified niches with enhanced anticaries activities at acidic pH are highly desirable. In our previous study, a cationic amphipathic α-helical antimicrobial peptide GH12 (Gly-Leu-Leu-Trp-His-Leu-Leu-His-His-Leu-Leu-His-NH₂) was designed with good stability, low cytotoxicity, and excellent antibacterial effects. Considering its potent antibacterial activity against the acidogenic bacteria and its histidine-rich sequence, it was speculated that GH12 might show enhanced antimicrobial effects at an acidic pH. In this study, the pH-responsive property of GH12 was determined to evaluate its potential as a smart acid-activated anticaries agent. GH12 possessed much lower minimal inhibitory concentrations and minimal bactericidal concentrations against various kinds of bacteria at pH 5.5 than at pH 7.2. Employing Streptococcus mutans, the principal caries pathogen, as the model system, it was found that GH12 showed much stronger bactericidal effects on both planktonic S. mutans and S. mutans embedded in the biofilm at pH 5.5. In addition, short-term treatment with GH12 showed much more effective inhibitory effects on water-insoluble exopolysaccharides synthesis and lactic acid production of the preformed S. mutans biofilm at pH 5.5. As for the mechanism exploration, it was found that the net positive charge of GH12 increased and the tryptophan fluorescence intensity heightened with the peak shifting towards the short wavelength at pH 5.5, which demonstrated that GH12 could be more easily attracted to the anionic microbial cell membranes and that GH12 showed stronger interactions with the lipid membranes. In conclusion, acidic pH enhanced the antibacterial and antibiofilm activities of GH12, and GH12 is a potential smart anticaries agent targeting the cariogenic acidic microenvironment.

Endogenous antisense walR RNA modulates biofilm organization and pathogenicity of Enterococcus faecalis.

Enterococcus faecalis (E. faecalis) is regarded as the major pathogen for persistent periapical periodontitis. The aim of the present study was to investigate the role of antisense walR RNA in the regulation of adjacent downstream genes. Reverse transcription-PCR assays were performed to validate walR. Adjacent downstream genes walK, EF1195, EF1196, and EF1197 were co-transcribed and detect antisense walR RNA. Northern blotting and 5'-rapid amplification of cDNA ends (5'-RACE) assays were conducted to detect and confirm a novel walR antisense (ASwalR) RNA. ASwalR overexpression mutants were constructed, and the biofilm biomass was determined using a crystal violet microtiter assay. The present study detected and confirmed a 550-bp noncoding antisense RNA with the potential to attenuate the activities of the essential response regulator WalR. The levels of antisense walR RNA transcripts were inversely associated with the production of WalR protein. It was showed that overexpression of ASwalR leads to reduced biofilm formation and exopolysaccharide synthesis. Furthermore, the pathogenicity of E. faecalis was markedly decreased by ASwalR overexpression in an in vivo periapical periodontitis model. In summary, the present study detected a novel antisense walR RNA that leads to a reduction in biofilm formation and the pathogenicity of E. faecalis. Collectively, the data suggest a role for ASwalR as a post-transcriptional modulator of the WalR regulator in E. faecalis.