Extracellular Polysaccharide Synthesis Research Articles

Inhibition of Streptococcus mutans biofilm formation, extracellular polysaccharide production, and virulence by an oxazole derivative.

Dental caries, a biofilm-related oral disease, is a result of disruption of the microbial ecological balance in the oral environment. Streptococcus mutans, which is one of the primary cariogenic bacteria, produces glucosyltransferases (Gtfs) that synthesize extracellular polysaccharides (EPSs). The EPSs, especially water-insoluble glucans, contribute to the formation of dental plaque, biofilm stability, and structural integrity, by allowing bacteria to adhere to tooth surfaces and supplying the bacteria with protection against noxious stimuli and other environmental attacks. The identification of novel alternatives that selectively inhibit cariogenic organisms without suppressing oral microbial residents is required. The goal of the current study is to investigate the influence of an oxazole derivative on S. mutans biofilm formation and the development of dental caries in rats, given that oxazole and its derivatives often exhibit extensive and pharmacologically important biological activities. Our data shows that one particular oxazole derivative, named 5H6, inhibited the formation of S. mutans biofilms and prevented synthesis of extracellular polysaccharides by antagonizing Gtfs in vitro, without affecting the growth of the bacteria. In addition, topical applications with the inhibitor resulted in diminished incidence and severity of both smooth and sulcal surface caries in vivo with a lower percentage of S. mutans in the animals' dental plaque compared to the control group (P < 0.05). Our results showed that this oxazole derivative has the capacity to inhibit biofilm formation and cariogenicity of S. mutans.

Molecule Targeting Glucosyltransferase Inhibits Streptococcus mutans Biofilm Formation and Virulence.

Dental plaque biofilms are responsible for numerous chronic oral infections and cause a severe health burden. Many of these infections cannot be eliminated, as the bacteria in the biofilms are resistant to the host's immune defenses and antibiotics. There is a critical need to develop new strategies to control biofilm-based infections. Biofilm formation in Streptococcus mutans is promoted by major virulence factors known as glucosyltransferases (Gtfs), which synthesize adhesive extracellular polysaccharides (EPS). The current study was designed to identify novel molecules that target Gtfs, thereby inhibiting S. mutans biofilm formation and having the potential to prevent dental caries. Structure-based virtual screening of approximately 150,000 commercially available compounds against the crystal structure of the glucosyltransferase domain of the GtfC protein from S. mutans resulted in the identification of a quinoxaline derivative, 2-(4-methoxyphenyl)-N-(3-{[2-(4-methoxyphenyl)ethyl]imino}-1,4-dihydro-2-quinoxalinylidene)ethanamine, as a potential Gtf inhibitor. In vitro assays showed that the compound was capable of inhibiting EPS synthesis and biofilm formation in S. mutans by selectively antagonizing Gtfs instead of by killing the bacteria directly. Moreover, the in vivo anti-caries efficacy of the compound was evaluated in a rat model. We found that the compound significantly reduced the incidence and severity of smooth and sulcal-surface caries in vivo with a concomitant reduction in the percentage of S. mutans in the animals' dental plaque (P < 0.05). Taken together, these results represent the first description of a compound that targets Gtfs and that has the capacity to inhibit biofilm formation and the cariogenicity of S. mutans.

Role of DetR in defence is critical for virulence of Xanthomonas oryzae pv. oryzae

Like other bacteria, Xanthomonas oryzae pv. oryzae (Xoo), the causal agent of bacterial leaf blight disease in rice, possesses intracellular signalling systems, known as two-component regulatory systems (TCSs), which regulate pathogenesis and biological processes. Completion of the genome sequences of three Xoo strains has facilitated the functional study of genes, including those of TCSs, but the biological functions of most Xoo TCSs have not yet been uncovered. To identify TCSs involved in Xoo pathogenesis, we generated knockout strains lacking response regulators (RRs, a cytoplasmic signalling component of the TCS) and examined the virulence of the RR knockout strains. This study presents a knockout strain (detR(-) ) lacking the PXO_04659 gene which shows dramatically reduced virulence relative to the wild-type. Our studies to elucidate detR function in Xoo pathogenesis revealed a reduction in extracellular polysaccharide (EPS), intolerance to reactive oxygen species (ROS) and deregulation of iron homeostasis in the detR(-) strain. Moreover, gene expression of regulatory factors, including other RRs and transcription factors (TFs), was altered in the absence of DetR protein, as determined by reverse transcription-polymerase chain reaction (RT-PCR) and/or real-time quantitative RT-PCR analyses. All evidence leads to the conclusion that DetR is essential for Xoo virulence through the regulation of the Xoo defence system including EPS synthesis, ROS detoxification and iron homeostasis, solely or cooperatively with other regulatory factors.

Improved poly-γ-glutamic acid production in Bacillus amyloliquefaciens by modular pathway engineering

A Bacillus amyloliquefaciens strain with enhanced γ-PGA production was constructed by metabolically engineering its γ-PGA synthesis-related metabolic networks: by-products synthesis, γ-PGA degradation, glutamate precursor synthesis, γ-PGA synthesis and autoinducer synthesis. The genes involved in by-products synthesis were firstly deleted from the starting NK-1 strain. The obtained NK-E7 strain with deletions of the epsA-O (responsible for extracellular polysaccharide synthesis), sac (responsible for levan synthesis), lps (responsible for lipopolysaccharide synthesis) and pta (encoding phosphotransacetylase) genes, showed increased γ-PGA purity and slight increase of γ-PGA titer from 3.8 to 4.15g/L. The γ-PGA degrading genes pgdS (encoding poly-gamma-glutamate depolymerase) and cwlO (encoding cell wall hydrolase) were further deleted. The obtained NK-E10 strain showed further increased γ-PGA production from 4.15 to 9.18g/L. The autoinducer AI-2 synthetase gene luxS was deleted in NK-E10 strain and the resulting NK-E11 strain showed comparable γ-PGA titer to NK-E10 (from 9.18 to 9.54g/L). In addition, we overexpressed the pgsBCA genes (encoding γ-PGA synthetase) in NK-E11 strain; however, the overexpression of these genes led to a decrease in γ-PGA production. Finally, the rocG gene (encoding glutamate dehydrogenase) and the glnA gene (glutamine synthetase) were repressed by the expression of synthetic small regulatory RNAs in NK-E11 strain. The rocG-repressed NK-anti-rocG strain exhibited the highest γ-PGA titer (11.04g/L), which was 2.91-fold higher than that of the NK-1 strain. Fed-batch cultivation of the NK-anti-rocG strain resulted in a final γ-PGA titer of 20.3g/L, which was 5.34-fold higher than that of the NK-1 strain in shaking flasks. This work is the first report of a systematically metabolic engineering approach that significantly enhanced γ-PGA production in a B. amyloliquefaciens strain. The engineering strategies explored here are also useful for engineering cell factories for the production of γ-PGA or of other valuable metabolites.

EXOPOLYSACCHARIDE SYNTHESIS PRODUCTIVITY BY BACILLUS MUCILAGINOSUS DEPENDING ON THE NITROGEN SOURCE AND ORIGIN OF INOCULUM

It was established that Bacillus mucilaginosus C-3 requires introduction of nitrogen sourceinto the culture medium in form of nitrate as themost optimal for maximal accumulation of bacterial biomass and sporulation process. Introduction of the ammonium form was less efficient. It was shown that the intensity of nitrogensource use by B. mucilaginosus is highly depended on the physiological state of the inoculum cells. At sowing of spore inoculum theoptimal concentration of potassium nitrate was2.5 g/l resulting in 1010 cells/ml, while under theuse of vegetative cells the optimal concentrationof potassium nitrate was 1.0 g/l leading to theaccumulation of 109 cells/ml.Use of spore inoculum had ensured themaximum productivity of extracellular polysaccharide synthesis. With the higher number ofB. mucilaginosus subcultures in a vegetativestate the efficiency of the exopolysaccharidesynthesis has decreased: after the second passage — by 20.0 %, after the fourth — by 56.5 %,after the sixth — at 127.8 %. After eighth passage the culture loses its ability to synthesizepolysaccharide, especially on the medium withthe N : C ratio 1 : 3 and resume this ability onlyafter sporulation and the significant increase ofthe N : C ratio.

Nicotine promotes Streptococcus mutans extracellular polysaccharide synthesis, cell aggregation and overall lactate dehydrogenase activity

Several epidemiology studies have reported a positive relationship between smoking and dental caries. Nicotine, an alkaloid component of tobacco, has been demonstrated to stimulate biofilm formation and metabolic activity of Streptococcus mutans, one of the most important pathogens of dental caries. The first aim of the present study was to explore the possible mechanisms leading to increased biofilm by nicotine treatment from three aspects, extracellular polysaccharides (EPS) synthesis, glucosyltransferase (Gtf) synthesis and glucan-binding protein (Gbp) synthesis at the mRNA and protein levels. The second aim was to investigate how nicotine affects S. mutans virulence, particular in lactate dehydrogenase (LDH) activity. Confocal laser scanning microscopy results demonstrated that both biofilm bacterial cell numbers and EPS were increased by nicotine. Gtf and GbpA protein expression of S. mutans planktonic cells were upregulated while GbpB protein expression of biofilm cells were downregulated by nicotine. The mRNA expression trends of those genes were mostly consistent with results on protein level but not statistically significant, and gtfD and gbpD of biofilm cells were inhibited. Nicotine was not directly involved in S. mutans LDH activity. However, since it increases the total number of bacterial cells in biofilm, the overall LDH activity of S. mutans biofilm is increased. In conclusion, nicotine stimulates S. mutans planktonic cell Gtf and Gbp expression. This leads to more planktonic cells attaching to the dental biofilm. Increased cell numbers within biofilm results in higher overall LDH activity. This contributes to caries development in smokers.

Unexpected Properties of Micromonosporae from Marine Origin

Members of the genus Micromonospora show a complex life cycle which normally involves the presence of substrate or vegetative mycelia and sporulation with single spores born on the veget- ative hyphae followed by the synthesis of a dark extracellular polysaccharide. Bergey's Manual states that micromonosporae rarely produces aerial mycelia (AM) and if so, is considered sterile. During the characterisation of novel micromonosporae from the Sea of Cortes, it was observed that AM is produced reproducibly in the presence of certain carbon and/or nitrogen sources. Mi- cromanipulation of the AM subcultured onto fresh media produced colonies; hence, this structure should not be called sterile. TEM of the AM producing isolates suggests that the spores also show activity as reported for bacilli of marine origin. This would be the first report of the presence of inducible AM in micromonosporae of marine sources and that the spores of this genus have a role other than just dispersal.

Effect of copper treatment on the composition and function of the bacterial community in the sponge Haliclona cymaeformis.

ABSTRACTMarine sponges are the most primitive metazoan and host symbiotic microorganisms. They are crucial components of the marine ecological system and play an essential role in pelagic processes. Copper pollution is currently a widespread problem and poses a threat to marine organisms. Here, we examined the effects of copper treatment on the composition of the sponge-associated bacterial community and the genetic features that facilitate the survival of enriched bacteria under copper stress. The 16S rRNA gene sequencing results showed that the sponge Haliclona cymaeformis harbored symbiotic sulfur-oxidizing Ectothiorhodospiraceae and photosynthetic Cyanobacteria as dominant species. However, these autotrophic bacteria decreased substantially after treatment with a high copper concentration, which enriched for a heterotrophic-bacterium-dominated community. Metagenomic comparison revealed a varied profile of functional genes and enriched functions, including bacterial motility and chemotaxis, extracellular polysaccharide and capsule synthesis, virulence-associated genes, and genes involved in cell signaling and regulation, suggesting short-period mechanisms of the enriched bacterial community for surviving copper stress in the microenvironment of the sponge. Microscopic observation and comparison revealed dynamic bacterial aggregation within the matrix and lysis of sponge cells. The bacteriophage community was also enriched, and the complete genome of a dominant phage was determined, implying that a lytic phage cycle was stimulated by the high copper concentration. This study demonstrated a copper-induced shift in the composition of functional genes of the sponge-associated bacterial community, revealing the selective effect of copper treatment on the functions of the bacterial community in the microenvironment of the sponge.

Effect of sucrose concentration on sucrose-dependent adhesion and glucosyltransferase expression of S. mutans in children with severe early-childhood caries (S-ECC).

Sucrose, extracellular polysaccharide, and glucosyltransferases (GTFs) are key factors in sucrose-dependent adhesion and play important roles in the process of severe early-childhood caries (S-ECC). However, whether sucrose concentration regulates gtf expression, extracellular polysaccharide synthesis, and sucrose-dependent adhesion is related to the different genotypes of S. mutans isolated from ECC in children and still needs to be investigated. In this study, 52 strains of S. mutans were isolated from children with S-ECC and caries-free (CF) children. Water-insoluble glucan (WIG) synthesis was detected by the anthrone method, adhesion capacity by the turbidimetric method, and expression of gtf by RT-PCR in an in vitro model containing 1%–20% sucrose. The genotypes of S. mutans were analyzed by AP-PCR. The results showed that WIG synthesis, adhesion capacity, and gtf expression increased significantly when the sucrose concentration was from 1% to 10%. WIG synthesis and gtfB as well as gtfC expression of the 1% and 5% groups were significantly lower than those of the 10% and 20% groups (p < 0.05). There were no significant differences between the 10% and 20% groups. The fingerprints of S. mutans detected from individuals in the S-ECC group exhibited a significant difference in diversity compared with those from CF individuals (p < 0.05). Further, the expression of gtfB and gtfC in the S-ECC group was significantly different among the 1- to 5-genotype groups (p < 0.05). It can be concluded that sucrose-dependent adhesion might be related to the diversity of genotypes of S. mutans, and the 10% sucrose level can be seen as a “turning point” and essential factor for the prevention of S-ECC.

Cryptococcus laurentii extracellular biopolymer production for application in wound management.

Cryptococcus laurentii growth and extracellular polysaccharide (EPS) production in bioreactor were studied. Biomass yield 14.3g/L and EPS synthesis 4.3g/L in 144h of submerged cultivation were achieved. EPS synthesis and cell growth had different optima. For EPS formation, pH3, 25°C and low aeration (1% < pO2 < 10%) were advantageous, while cell growth optimum was at pH6, 20°C, and high aeration (pO2 > 30%). As medium pH changed from pH3 to pH6, glucuronic acid (GluAc) content in EPS increased, while galactose, xylose, and glucose decreased. Twenty-five degrees Celsius was optimal for GluAc containing polysaccharide synthesis, while lower temperature (15°C) increased glucose content in EPS. Aeration intensity and time of cultivation had little effect on EPS composition. Molecular mass distribution of raw C. laurentii EPS was determined by SEC-MALS as 1.352. The row EPS was composed of acidic glucuronoxylomannan for more than 85%. In the in vivo experiments, EPS significantly improved excisional wound healing in healthy rats. The results suggest that C. laurentii EPS is a promising biotechnological product and an advanced material for application in wound management.

The protein tyrosine kinases EpsB and PtkA differentially affect biofilm formation in Bacillus subtilis

The Gram-positive soil bacterium Bacillus subtilis is able to choose between motile and sessile lifestyles. The sessile way of life, also referred to as biofilm, depends on the formation of an extracellular polysaccharide matrix and some extracellular proteins. Moreover, a significant proportion of cells in a biofilm form spores. The first two genes of the 15-gene operon for extracellular polysaccharide synthesis, epsA and epsB, encode a putative transmembrane modulator protein and a putative protein tyrosine kinase, respectively, with similarity to the TkmA/PtkA modulator/kinase couple. Here we show that the putative kinase EpsB is required for the formation of structured biofilms. However, an epsB mutant is still able to form biofilms. As shown previously, a ptkA mutant is also partially defective in biofilm formation, but this defect is related to spore formation in the biofilm. The absence of both kinases resulted in a complete loss of biofilm formation. Thus, EpsB and PtkA fulfil complementary functions in biofilm formation. The activity of bacterial protein tyrosine kinases depends on their interaction with modulator proteins. Our results demonstrate the specific interaction between the putative kinase EpsB and its modulator protein EpsA and suggest that EpsB activity is stimulated by its modulator EpsA.

Cyclic di‐GMP inhibits Vibrio cholerae motility by repressing induction of transcription and inducing extracellular polysaccharide production

Cyclic di-GMP (c-di-GMP) controls the transition between sessility and motility in many bacterial species. This regulation is achieved by a variety of mechanisms including alteration of transcription initiation and inhibition of flagellar function. How c-di-GMP inhibits the motility of Vibrio cholerae has not been determined. FlrA, a homologue of the c-di-GMP binding Pseudomonas aeruginosa motility regulator FleQ, is the master regulator of the V. cholerae flagellar biosynthesis regulon. Here we show that binding of c-di-GMP to FlrA abrogates binding of FlrA to the promoter of the flrBC operon, deactivating expression of the flagellar biosynthesis regulon. FlrA does not regulate expression of extracellular Vibrio polysaccharide (VPS) synthesis genes. Mutation of the FlrA amino acids R135 and R176 to histidine abrogates binding of c-di-GMP to FlrA, rendering FlrA active in the presence of high levels of c-di-GMP. Surprisingly, c-di-GMP still inhibited the motility of V. cholerae only expressing the c-di-GMP blind FlrA(R176H) mutant. We determined that this flagellar transcription-independent inhibition is due to activation of VPS production by c-di-GMP. Therefore, c-di-GMP prevents motility of V. cholerae by two distinct but functionally redundant mechanisms.

Cellulose synthases: new insights from crystallography and modeling

Detailed information about the structure and biochemical mechanisms of cellulose synthase (CelS) proteins remained elusive until a complex containing the catalytic subunit (BcsA) of CelS from Rhodobacter sphaeroides was crystalized. Additionally, a 3D structure of most of the cytosolic domain of a plant CelS (GhCESA1 from cotton, Gossypium hirsutum) was produced by computational modeling. This predicted structure contributes to our understanding of how plant CelS proteins may be similar and different as compared with BcsA. In this review, we highlight how these structures impact our understanding of the synthesis of cellulose and other extracellular polysaccharides. We show how the structures can be used to generate hypotheses for experiments testing mechanisms of glucan synthesis and translocation in plant CelS.

English

Introduction Photodynamic antimicrobial chemotherapy studies regarding dental caries have been present more frequently in the literature. However, photodynamic antimicrobial chemotherapy depends on the adjustment of variables such as the type of light source and, photosensitisers target microorganism; this makes it difficult to draw meaningful comparisons. The purpose of this paper was to provide a critical review related to this coadjuvant approach in the prevention and treatment of dental caries. Materials and Methods A database search was made via Medline/PubMed (keywords: photodynamic therapy and dental caries) and 33 articles were found. Results Twelve articles were included after using the filter tool, being excluded reviews and manuscripts reporting works not related to the studied area. Conclusion The manuscripts showed that photodynamic therapy presents optimal results against dental caries, even though better understanding of photodynamic antimicrobial chemotherapy and its components are necessary before the clinical application of this alternative modality in the dental practice. Photodynamic antimicrobial chemotherapy for prevention and treatment of dental caries: a critical review MA Paschoal1, S Duarte2*, L Santos-Pinto1* Introduction The human oral cavity is heavily colonised by a complex, relatively specific and highly interrelated range of microorganisms (as many as 1000 different species have been detected) collectively known as normal oral microflora1. A peculiarity of this environment is that most of the bacteria found here are present in complex aggregates (known as biofilms)2. Microbial biofilms are composed of microorganisms adhered both to each other and to dental surfaces (or interfaces) and embedded in an extracellular polymeric matrix, which includes water and nutrient channels3. A change in a key environmental factor will trigger a shift in the balance of the resident microflora, which will promote the emergence of acidogenic/aciduric bacteria. The constant accumulation of these kind of bacteria change the equilibrium towards dental demineralisation (dental caries lesions)4. The constituents of diet present an important role in the development of dental caries. Sucrose is considered the most cariogenic dietary carbohydrate, because it is fermentable and serves as a substrate for the synthesis of extracellular polysaccharides (EPS) and intracellular polysaccharides in cariogenic dental plaque4. In addition, the presence of EPS (mainly insoluble glucan) promote bacterial adherence to the tooth surface and contribute to the structural integrity of dental biofilms. Yet, there is a clear evidence showing that sucrose exposure and insoluble EPS lead to a more cariogenic biofilm5. Dental caries is among the most significant human chronicle infectious diseases and results in the progressive dissolution of enamel. With the disease progression, it can lead the underlying dentine compromising the vitality of the element and its fixation in the maxillomandibular complex6. Prevention of dental caries can be achieved by controlling the accumulation of dental plaque by mechanical removal7. In cases of insufficient biofilm disorganisation, the association with antimicrobial chemical agents, such as chlorhexidine may help in the decreasing of pathogenic bacteria levels7. Unfortunately, this preventive approach does not reach the population as a whole, allowing dental cavity formation. Treatment of the carious lesion involves the removal of infected dentine with posterior restoration of the affected tooth with any of the variety of materials, for example mercury amalgam, resin composite and glass ionomer cements8. Due to emergence of antibiotic resistant strains, alteration in taste, burning sensation, increase of calculus formation and staining of the teeth and restorative materials stimulated a search for alternative treatments9. Recently, approaches that might offer the possibility of efficient intraoral bacterial count reduction with minimum damage to systemic health (preventive approach) and avoid secondary caries development reducing the chance of material substitution and pulp inflammation as well (curative approach) are necessary. For these circumstances, photodynamic antimicrobial chemotherapy (PACT) offers the possibility of a novel modality to reduce pathogenic bacteria, and consequently, prevent against (new) dental caries lesions10. * Corresponding authors Emails: marcobpaschoal@hotmail.com; lspinto@foar.unesp.br; sd84@nyu.edu 1 Department of Pediatric Dentistry, Araraquara School of Dentistry, Univ Estadual Paulista (UNESP), Araraquara, SP, Brazil 2 Department of Basic Science and Craniofacial Biology, College of Dentistry, New York University (NYU), New York, NY, USA

Methylovorus sp. MP688 exopolysaccharides contribute to oxidative defense and bacterial survival under adverse condition

Methylovorus sp. MP688 is an aerobic bacterium that can grow on reduced C1 compounds such as methanol, being regarded as an attractive producer for many commercial materials including polysaccharides. The aim of the study was to learn more information about the biochemical and physiological functions of extracellular polysaccharides (EPS) produced by Methylovorus sp. MP688. Firstly, gene clusters involved in EPS synthesis were identified by whole genome sequence analysis. Then EPS produced by Methylovorus sp. MP688 were isolated and purified by centrifugation, precipitation and deproteinization. Purified EPS displayed antioxidant activity towards DPPH free radical, hydroxyl radical and superoxide anion radical. Glucose, galactose and mannose were identified to be main component monosaccharides in EPS. One mutant with defect in EPS production was obtained by knocking out epsA gene within EPS synthesis cluster. Strain with deletion of epsA exhibited compromised growth ability in the presence of oxidative stress due to the sharp reduction in EPS synthesis. Meanwhile, the intracellular antioxidant scavengers were activated to a higher level in order to counteract with the excess harmful radicals. In addition, EPS were assimilated by Methylovorus sp. MP688 to survive under disadvantage condition when the preferred carbon source was exhausted. It was reasonable to conclude that EPS produced by Methylovorus sp. MP688 contributed to oxidative defense and bacterial survival under adverse condition.

Optimization of cultivation conditions to maximize extracellular investments of two Nostoc strains

An investigation was undertaken on two Nostoc strains isolated from the paddy fi elds of Iran and having different morphologies of polysaccharide sheaths. Ex tracellular polysaccharides (EPS) presumably play a major (protective) role in cyanobacteria but there are no clear reports that link EPS production with (selected) abiotic stresses. The aim here is to investigate key factors enhancing EPS biosynthesis. The relationship between cell growth and the amount of EPS were compared under photoautotrophic, mixotrophic and heterotrophic growth conditions as well as under different salt concentrations, and under nitrogen-deplete and nitrogen-replete conditions Cell growth and EPS production are both highly dependent on culture conditions, but there is no correlation between cell growth and EPS production in cultures grown in different sources of nitrogen. In contrast, light inten si ty and cel l growth in cultures experiencing mixotrophic conditions at either 150 or 50 μmol photon m–2 s–1 have strong positive effect on EPS production. In salt-rich cult ures, in the ASN_S strain the outer cell layers p rotect the inner cells from NaCl s tress and hence its growth is maintained without inhibition under the NaCl stress. Each strain of Nostoc exhibits a different pattern of EPS synthesis, making it essential that the optimal culture conditions are determined for each species reading to the development of an effi cient technology for the maximum production of cyanobacterial EPS.

Biosorption of Cu (II) ions by indigenous copper-resistant bacteria isolated from polluted coastal environment

The concentrations of copper in water and sediment samples of Cuddalore coastal area, Tamil Nadu, India were examined. The isolation, characterization, screening, and metal sorption capabilities of copper-resistant bacteria (CURB) also were investigated. Biosorption capabilities and growth time varied among the resistant isolates. All isolates reduced more than 80% of copper from copper-treated broth. However, Bacillus cereus CURB-4 strain absorbed more copper ions (99.9%) than other resistant isolates. Changes in structural group position and synthesis of extracellular polysaccharides (EPS) of CURB-4 strain under copper stress and controlled conditions also were examined. Under copper stress condition, CURB-4 produced more EPS (71 ± 0.6%) compared to control conditions (66 ± 0.4%). Fourier transform infrared spectroscopy revealed that the changes in functional group position of EPS were observed in stressed condition. Overall, synthesis of EPS in bacteria is a protective barrier against environmental stress.

The HD-GYP Domain Protein RpfG of Xanthomonas oryzae pv. oryzicola Regulates Synthesis of Extracellular Polysaccharides that Contribute to Biofilm Formation and Virulence on Rice

Bacterial leaf streak caused by Xanthomonas oryzae pv. oryzicola (Xoc) is one of the most important diseases in rice. However, little is known about the pathogenicity mechanisms of Xoc. Here we have investigated the function of three HD-GYP domain regulatory proteins in biofilm formation, the synthesis of virulence factors and virulence of Xoc. Deletion of rpfG resulted in altered production of extracellular polysaccharides (EPS), abolished virulence on rice and enhanced biofilm formation, but had little effect on the secretion of proteases and motility. In contrast, mutational analysis showed that the other two HD-GYP domain proteins had no effect on virulence factor synthesis and tested phenotypes. Mutation of rpfG led to up-regulation of the type III secretion system and altered expression of three putative glycosyltransferase genes gumD, pgaC and xagB, which are part of operons directing the synthesis of different extracellular polysaccharides. The pgaABCD and xagABCD operons were greatly up-regulated in the Xoc ΔrpfG mutant, whereas the expression of the gum genes was unaltered or slightly enhanced. The elevated biofilm formation of the Xoc ΔrpfG mutant was dramatically reduced upon deletion of gumD, xagA and xagB, but not when pgaA and pgaC were deleted. Interestingly, only the ΔgumD mutant, among these single gene mutants, exhibits multiple phenotype alterations including reduced biofilm and EPS production and attenuated virulence on rice. These data indicate that RpfG is a global regulator that controls biofilm formation, EPS production and bacterial virulence in Xoc and that both gumD- and xagB-dependent EPS contribute to biofilm formation under different conditions.

Novel route of tannic acid biotransformation and their effect on major biopolymer synthesis inAzotobactersp. SSB81

To examine tannic acid (TA) utilization capacity by nitrogen-fixing bacteria, Azotobacter sp. SSB81, and identify the intermediate products during biotransformation. Another aim of this work is to investigate the effects of TA on major biopolymers like extracellular polysaccharide (EPS) and polyhydroxybutyrate (PHB) synthesis. Tannic acid utilization and tolerance capacity of the strain was determined according to CLSI method. Intermediate products were identified using high-performance liquid chromatography, LC-MS/MS and (1) H NMR analysis. Intermediates were quantified by multiple reactions monitoring using LC-MS/MS. The strain was able to tolerate a high level of TA and utilized through enzymatic system. Growth of Azotobacter in TA-supplemented medium was characterized by an extended lag phase and decreased growth rate. Presence of TA catalytic enzymes as tannase, polyphenol oxidase (PPO) and phenol decarboxylase was confirmed in cell lysate using their specific substrates. PPO activity was more prominent in TA-supplemented mineral medium after 48h of growth when gallic to ellagic acid (EA) reversible reaction was remarkable. Phase contrast and scanning electron microscopic analysis revealed elongated and irregular size of Azotobacter cells in response to TA. (1) H NMR analysis indicated that TA was transformed into gallic acid (GA), EA and pyrogallol. Biopolymer (EPS and PHB) production was decreased several folds in the presence of TA compared with cells grown in only glucose medium. This is the first evidence on the biotransformation of TA by Azotobacter and also elevated level of EA production from gallotannins. Azotobacter has developed the mechanism to utilize TA for their carbon and energy source. The widespread occurrence and exploitation of Azotobacter sp. strain SSB81 in agricultural and forest soil have an additional advantage to utilize the soil-accumulated TA and detoxifies the allelopathic effect of constant accumulated TA in soil.

Effects of UV-B Radiation and Periodic Desiccation on the Morphogenesis of the Edible Terrestrial Cyanobacterium Nostoc flagelliforme

The terrestrial cyanobacterium Nostoc flagelliforme Berk. et M. A. Curtis has been a popular food and herbal ingredient for hundreds of years. To meet great market demand and protect the local ecosystem, for decades researchers have tried to cultivate N. flagelliforme but have failed to get macroscopic filamentous thalli. In this study, single trichomes with 50 to 200 vegetative cells were induced from free-living cells by low light and used to investigate the morphogenesis of N. flagelliforme under low UV-B radiation and periodic desiccation. Low-fluence-rate UV-B (0.1 W m(-2)) did not inhibit trichome growth; however, it significantly increased the synthesis of extracellular polysaccharides and mycosporine-like amino acids and promoted sheath formation outside the trichomes. Under low UV-B radiation, single trichomes developed into filamentous thalli more than 1 cm long after 28 days of cultivation, most of which grew separately in liquid BG11 medium. With periodic desiccation treatment, the single trichomes formed flat or banded thalli that grew up to 2 cm long after 3 months on solid BG11 medium. When trichomes were cultivated on solid BG11 medium with alternate treatments of low UV-B and periodic desiccation, dark and scraggly filamentous thalli that grew up to about 3 cm in length after 40 days were obtained. In addition, the cultivation of trichomes on nitrogen-deficient solid BG11 medium (BG11(0)) suggested that nitrogen availability could affect the color and lubricity of newly developed thalli. This study provides promising techniques for artificial cultivation of N. flagelliforme in the future.