RESUMO

Azospirillum baldaniorum is a plant growth-promoting rhizobacterium (PGPR) capable of fixing nitrogen, the synthesis of several phytohormones including indole-acetic acid, and induction of plant defenses against phytopathogens. To establish a successful and prolonged bacteria-plant interaction, A. baldaniorum can form biofilms, bacterial communities embedded in a self-made matrix formed by extracellular polymeric substances which provide favorable conditions for survival. A key modulator of biofilm formation is the second messenger bis-(3'-5')-cyclic-dimeric-GMP (c-di-GMP), which is synthesized by diguanylate cyclases (DGC) and degraded by specific phosphodiesterases. In this study, we analyzed the contribution of a previously uncharacterized diguanylate cyclase designated CdgC, to biofilm formation and bacterial-plant interaction dynamics. We showed that CdgC is capable of altering c-di-GMP levels in a heterologous host, strongly supporting its function as a DGC. The deletion of cdgC resulted in alterations in the three-dimensional structure of biofilms in a nitrogen-source dependent manner. CdgC was required for optimal colonization of wheat roots. Since we also observed that CdgC played an important role in exopolysaccharide production, we propose that this signaling protein activates a physiological response that results in the strong attachment of bacteria to the roots, ultimately contributing to an optimal bacterium-plant interaction. Our results demonstrate that the ubiquitous second messenger c-di-GMP is a key factor in promoting plant colonization by the PGPR A. baldaniorum by allowing proficient internalization in wheat roots. Understanding the molecular basis of PGPR-plant interactions will enable the design of better biotechnological strategies of agro-industrial interest.

RESUMO

Streptococcus mutans is considered to be a major bacterium involved in dental caries, and the control of virulence mechanisms is fundamental to prevent disease. Probiotics present a promising preventive method; however, the use of probiotics requires its incorporation into delivery materials to facilitate oral colonization. Thus, we performed a comprehensive study examining preventive effects of Lactobacillus paracasei 28.4-enriched gellan hydrogel materials to inhibit S. mutans in planktonic and biofilm states, addressing its influence in the production of extracellular polysaccharides (EPS) and altered gene expression of several cariogenic virulence factors. L. paracasei 28.4, a strain isolated from the oral cavity of a caries-free individual, was incorporated in three gellan hydrogels (0.5%, 0.75%, and 1% w/v). The pretreatment with probiotic-gellan formulations provided a release of L. paracasei cells over 24 h that was sufficient to inhibit the planktonic growth of S. mutans, independent of the gellan concentrations and pH variations. This pretreatment also had inhibitory activity against S. mutans biofilms, exhibiting a reduction of 0.57 to 1.54 log10 in CFU/mL (p < 0.0001) and a decrease of 68.8 to 71.3% in total biomass (p < 0.0001) compared with the control group. These inhibitory effects were associated with the decreased production of EPS by 80% (p < 0.0001) and the downregulation of luxS, brpA, gbpB, and gtfB genes. The gellan formulation containing L. paracasei 28.4 exhibited probiotic effects for preventing S. mutans growth, biofilm formation, and production of cariogenic factors to suggest possible use in tooth decay prevention.

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BACKGROUND: Rice sheath blight (caused by Rhizoctonia solani) and tobacco mosaic virus are very important plant diseases, causing a huge loss in global crop production. Paenibacillus kribbensis PS04 is a broad-spectrum biocontrol agent, used for controlling these diseases. Previously, extracellular polysaccharides (EPS) from P. kribbensis PS04 had been purified and their structure was inferred to be fructosan. This study aimed to evaluate the effects of exogenous EPS treatment on plant­pathogen interactions. RESULTS: Plant defense genes such as phenylalanine ammonia-lyase, catalase, chitinase, allene oxide synthase, and PR1a proteins were significantly induced by exogenous EPS treatment. Moreover, subsequent challenge of EPSpretreated plants with the pathogens (R. solani or tobacco mosaic virus) resulted in higher expression of defenseassociated genes. Increased activities of defense-associated enzymes, total phenols, and flavonoids were also observed in EPS pretreated plants. The contents of malondialdehyde in plants, which act as indicator of lipid peroxidation, were reduced by EPS treatment. CONCLUSIONS: This study comprehensively showed that EPS produced from P. kribbensis PS04 enhances disease resistance in plants by the activation of defense-associated genes as well as through the enhancement of activities of defense-related enzymes.

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AIM: Evidence of glucose supplementation effect on Candida biofilm metabolism has not been demonstrated. Therefore, the aim of the present study was to evaluate the effect of glucose concentration on Candida biofilms. METHODS: Single- and dual-species biofilms of Candida were grown on saliva-coated poly(methyl-methacrylate) disks for 72 hours. Biofilms (N = 8/group) were exposed to the following concentrations of glucose: 100 mmol/L (G100), 300 mmol/L (G300), and no glucose (G0: control). Biofilms were collected to determine the acidogenicity, viability, amount of soluble and insoluble extracellular polysaccharides (IEPS), and surface roughness. Data were analyzed using analysis of variance and Tukey's tests (α < 0.05). RESULTS: Single- and dual-species biofilms from G300 were more acidogenic at 48 and 72 hours compared to G100 and G0 (P < 0.05). The viability of the G100 and G300 groups did not differ (P > 0.05), but differed statistically from G0. The amount of IEPS in the G300 group was statistically higher than the G0 and G100 groups (P < 0.05). The G300 group also presented a higher IEPS proportion per number of viable cells compared to others. G300 presented greater surface roughness for both single- (mean roughness = 1460 µm) and dual-species (mean roughness = 1990 µm) biofilms. CONCLUSIONS: Higher glucose concentration (300 mmol/L) during biofilm development favors the growth of single- and dual-species biofilms of Candida.

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OBJECTIVE: The efficacy of a red propolis hydro-alcoholic extract (RP) in controlling Streptococcus mutans biofilm colonization was evaluated. The effect of RP on dental demineralization was also investigated. METHODS: Chemical composition was determined by High Performance Liquid Chromatography (HPLC). Minimum Inhibitory and Bactericidal Concentration (MIC and MBC, respectively) were investigated against Streptococcus mutans (ATCC 25175). The cytotoxic potential of 3% RP in oral fibroblasts was observed after 1 and 3 min. Bovine dental enamel blocks (N = 24) were used for S. mutans biofilm formation (48 h), simulating 'feast or famine' episodes. Blocks/biofilms were exposed 2×/day, for 3 days, to a cariogenic challenge with sucrose 10% (5 min) and treated (1 min) with: 0.85% saline solution (negative control), 0.12% Chlorhexidine (CHX, positive control for biofilm colonization), 0.05% Sodium Fluoride (NaF, positive control to avoid demineralization) and 3% RP. Biofilms were assessed for viability (CFU/mL), and to observe the concentration of soluble and insoluble extracellular polysaccharides (SEPS and IEPS). Dental demineralization was assessed by the percentage of surface hardness loss (%SHL) and through polarized light microscopy (PLM). RESULTS: The RP presented 4.0 pH and ºBrix = 4.8. The p-coumaric acid (17.2 µg/mL) and luteolin (15.23 µg/mL) were the largest contents of phenolic acids and flavonoids, respectively. MIC and MBC of RP were 293 µg/mL and 1172 µg/mL, respectively. The 3% RP showed 43% of viably cells after 1 min. Lower number (p < 0.05) of viable bacteria (CFU/mL) was observed after CHX (1.8 × 105) followed by RP (1.8 × 107) treatments. The lowest concentration (µg/CFU) of SEPS (12.6) and IEPS (25.9) was observed in CHX (p < 0.05) followed by RP (17.1 and 54.3), and both differed from the negative control (34.4 and 63.9) (p < 0.05). Considering the %SHL, all groups differed statistically (p < 0.05) from the negative control (46.6%); but NaF (13.9%), CHX (20.1%) and RP (20.7%) did not differ among them (p > 0.05). After all treatments, suggestive areas of caries lesions were observed by PLM, which were lower for CHX and NaF. CONCLUSION: The 3% RP reduced S. mutans colonization, decreased concentration of extracellular polysaccharides and reduced dental enamel demineralization.

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We assessed the occurrence of phenotypic variation in Azospirillum brasilense strains Sp7, Cd, Sp245, Az39 and phv2 during growth in rich media, screening for variants altered in colony pigmentation or extracellular polysaccharide (EPS) production. Previous studies showed that EPS-overproducing variants of Sp7 appear frequently following starvation or growth in minimal medium. In contrast, no such variants were detected during growth in rich media in the tested strains except for few variants of phv2. Regarding alteration in colony pigmentation (from pink to white in strain Cd and from white to pink in the others), strain Sp7 showed a relatively high frequency of variation (0.009-0.026%). Strain Cd showed a lower frequency of alteration in pigmentation (0-0.008%), and this type of variation was not detected in the other strains. In A. brasilense, carotenoid synthesis is controlled by two RpoE sigma factors and their cognate ChrR anti-sigma factors, the latter acting as negative regulators of carotenoid synthesis. Here, all tested (n = 28) pink variants of Sp7 carried mutations in one of the anti-sigma factor genes, chrR1. Our findings indicate that, in A. brasilense, phenotypic variation is strain- and environment-dependent and support the central role of ChrR1 in regulation of carotenoid production.

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Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L) and pH values (3.0-4.0) to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0) led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (Y P/S = 0.072) and maximum extracellular polysaccharide productivity (Q Pmax = 11.26 mg/L.h) were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (Y X/S = 0.24) and maximal biomass productivity (Q Xmax = 32.2 mg/L.h) were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm³ in untreated group and 1.6 cm³ in treated group (p = 0.05) with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells.

Assuntos

RESUMO

Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L) and pH values (3.0-4.0) to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0) led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (YP/S = 0.072) and maximum extracellular polysaccharide productivity (Q(Pmax) = 11.26 mg/L.h) were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (YX/S = 0.24) and maximal biomass productivity (Q(Xmax) = 32.2 mg/L.h) were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm(3) in untreated group and 1.6 cm(3) in treated group (p = 0.05) with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells.

Assuntos

RESUMO

Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L) and pH values (3.0-4.0) to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0) led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (Y P/S = 0.072) and maximum extracellular polysaccharide productivity (Q Pmax = 11.26 mg/L.h) were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (Y X/S = 0.24) and maximal biomass productivity (Q Xmax = 32.2 mg/L.h) were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm³ in untreated group and 1.6 cm³ in treated group (p = 0.05) with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells.

RESUMO

Polysaccharides with medicinal properties can be obtained from fruiting bodies, mycelium and culture broth of several fungus species. This work was carried out in batch culture using a stirred tank reactor with two different initial glucose concentrations (40-50 g/L) and pH values (3.0-4.0) to enhance extracellular polysaccharides production by Pleurotus djamor UNIVILLE 001 and evaluate antitumor effect of intraperitonial administration of Pleurotus djamor extract on sarcoma 180 animal model. According to factorial design, the low pH value (pH 3.0) led to a gain of 1.6 g/L on the extracellular polysaccharide concentration, while glucose concentration in the tested range had no significant effect on the concentration of polysaccharide. With 40 g/L initial glucose concentration and pH 3.0, it was observed that yield factor of extracellular polysaccharide on substrate (Y P/S = 0.072) and maximum extracellular polysaccharide productivity (Q Pmax = 11.26 mg/L.h) were about 188% and 321% respectively higher than those obtained in the experiment performed at pH 4.0. Under these conditions, the highest values of the yield factor of biomass on substrate (Y X/S = 0.24) and maximal biomass productivity (Q Xmax = 32.2 mg/L.h) were also reached. In tumor response study, mean tumor volume on the 21th day was 35.3 cm³ in untreated group and 1.6 cm³ in treated group (p = 0.05) with a tumor inhibition rate of 94%. These impressive results suggests an inhibitory effect of P.djamor extract on cancer cells.

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Glycolytic activities of eight enzymes in size-fractionated water samples from a eutrophic tropical reservoir are presented in this study, including enzymes assayed for the first time in a freshwater environment. Among these enzymes, rhamnosidase, arabinosidase and fucosidase presented high activity in the free-living fraction, while glucosidase, mannosidase and galactosidase exhibited high activity in the attached fraction. The low activity registered for rhamnosidase, arabinosidase and fucosidase in the attached fraction seemed contribute to the integrity of the aggregate and based on this fact, a protective role for these structures was proposed. The presented enzyme profiles and the differences in the relative activities probably reflected the organic matter composition as well as the metabolic requirements of the bacterial community, suggesting that bacteria attached to particulate matter had phenotypic traits distinct from those of free-living bacteria.

RESUMO

The activity of specific glycosidases during the degradation of the extracellular polysaccharide (EPS) produced by Anabaena spiroides was determined using MUF-substrates (MUF-monosaccharides). Polysaccharide degradation was found to occur in a two-phase process. The first consisted of high enzymatic activity that consumed 41% of the EPS at a relatively high rate, while the second consumed the remaining polysaccharide (59%) at a slower rate. A transition phase from the higher to the slower degradation rates was marked by a replacement of bacterial populations from coccoid to bacillus cells. During the degradation process, the bacterial biomass increased with the decrease of EPS, as revealed by bacterial cell counts. The enzymatic activity detected through the substrates MUF-alpha-D- and MUF-beta-D-glucoside was higher than that detected by other substrates tested. The remaining glycosides were MUF-alpha-L-rhamnopyranoside, MUF-beta-D-galactoside, MUF-alpha-D-mannopyranoside, MUF-beta-D-fucoside, MUF-beta-D-mannopyranoside, MUF-alpha-L-arabinopyranoside, and MUF-beta-L-fucoside. The fluorescence emitted by each MUF-substrate was proportional to the concentration of the corresponding monosaccharide in A. spiroides EPS. This demonstrates the susceptibility of EPS produced by A. spiroides to enzymatic attack by bacterial populations.

A atividade de glicosidases durante a degradação do polissacarídeo extracelular (EPS) produzido por Anabaena spiroides foi detectada e quantificada utilizando-se MUF-substratos (MUF-monossacarídeos). O consumo total do polissacarídeo efetuou-se em duas fases, uma primeira de alta atividade enzimática que rapidamente consumiu 41% do polissacarídeo e uma segunda, mais lenta, que consumiu o polissacarídeo restante (59%). A mudança de fase coincidiu com a sucessão de uma população de bactérias cocóides por outra de bacilos. A biomassa bacteriana, quantificada por contagens de células, aumentou com a degradação do EPS. As atividades registradas através dos substratos 4-MUF-alfa-D- e 4-MUF-beta-D- glicosídeo foram mais altas quando comparadas aos demais substratos testados que foram: MUF-alfa-L-ramnopiranosídeo, MUF-beta-D-galactosídeo, MUF-alfa-D-manopiranosídeo, MUF-beta-D-fucosídeo, MUF-beta-D-manopiranosídeo, MUF-alfa-L-arabinopiranosídeo, e MUF-beta-L-fucosídeo. A fluorescência emitida a partir de cada um dos diferentes MUF-substratos foi, de modo geral, proporcional à concentração dos monossacarídeos correspondentes constituintes do polissacarídeo, um indício da susceptibilidade ao ataque enzimático microbiano do EPS produzido por A. spiroides.

RESUMO

The activity of specific glycosidases during the degradation of the extracellular polysaccharide (EPS) produced by Anabaena spiroides was determined using MUF-substrates (MUF-monosaccharides). Polysaccharide degradation was found to occur in a two-phase process. The first consisted of high enzymatic activity that consumed 41% of the EPS at a relatively high rate, while the second consumed the remaining polysaccharide (59%) at a slower rate. A transition phase from the higher to the slower degradation rates was marked by a replacement of bacterial populations from coccoid to bacillus cells. During the degradation process, the bacterial biomass increased with the decrease of EPS, as revealed by bacterial cell counts. The enzymatic activity detected through the substrates MUF-alpha-D- and MUF-beta-D-glucoside was higher than that detected by other substrates tested. The remaining glycosides were MUF-alpha-L-rhamnopyranoside, MUF-beta-D-galactoside, MUF-alpha-D-mannopyranoside, MUF-beta-D-fucoside, MUF-beta-D-mannopyranoside, MUF-alpha-L-arabinopyranoside, and MUF-beta-L-fucoside. The fluorescence emitted by each MUF-substrate was proportional to the concentration of the corresponding monosaccharide in A. spiroides EPS. This demonstrates the susceptibility of EPS produced by A. spiroides to enzymatic attack by bacterial populations.

A atividade de glicosidases durante a degradação do polissacarídeo extracelular (EPS) produzido por Anabaena spiroides foi detectada e quantificada utilizando-se MUF-substratos (MUF-monossacarídeos). O consumo total do polissacarídeo efetuou-se em duas fases, uma primeira de alta atividade enzimática que rapidamente consumiu 41% do polissacarídeo e uma segunda, mais lenta, que consumiu o polissacarídeo restante (59%). A mudança de fase coincidiu com a sucessão de uma população de bactérias cocóides por outra de bacilos. A biomassa bacteriana, quantificada por contagens de células, aumentou com a degradação do EPS. As atividades registradas através dos substratos 4-MUF-alfa-D- e 4-MUF-beta-D- glicosídeo foram mais altas quando comparadas aos demais substratos testados que foram: MUF-alfa-L-ramnopiranosídeo, MUF-beta-D-galactosídeo, MUF-alfa-D-manopiranosídeo, MUF-beta-D-fucosídeo, MUF-beta-D-manopiranosídeo, MUF-alfa-L-arabinopiranosídeo, e MUF-beta-L-fucosídeo. A fluorescência emitida a partir de cada um dos diferentes MUF-substratos foi, de modo geral, proporcional à concentração dos monossacarídeos correspondentes constituintes do polissacarídeo, um indício da susceptibilidade ao ataque enzimático microbiano do EPS produzido por A. spiroides.