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We describe sample preparation and visualization of fluorescently tagged cellulose synthases in cellulose synthase complexes at the plasma membrane of Arabidopsis hypocotyl epidermal cells using live-cell imaging via spinning disk microscopy. We present a technique for sample mounting that may be suitable for imaging other samples. Additionally, we offer free, open-source solutions for image analysis and provide extensive troubleshooting suggestions. For complete information on the use and execution of this protocol, please refer to McFarlane et al., 2021., Competing Interests: The authors declare no competing interests., (© 2021 The Author(s).)

Trehalose synthase converts maltose into trehalose in a single conversion step via intramolecular transformation and is thus useful for industrial production. In this study, we synthesized a thermophilic trehalose synthase from Thermus antranikianii (TaTS), which was recombinantly expressed in Escherichia coli BL21(DE3). The recombinant TaTS showed the highest activity at pH 7.0 and 60°C, with the maximum trehalose yield (76.8%) obtained at pH 7.0 and 30°C. TaTS activity was stable over a wide pH and temperature range of 6-10 and 4-70°C, respectively, over 6 h of incubation. The enzyme activity was strongly inhibited by Co 2+ , Cu 2+ , Zn 2+ , sodium dodecyl sulfate, and Tris. TaTS showed a 1.48-fold higher catalytic efficiency (k cat /K m ) for maltose than for trehalose. Overall, these results demonstrate the good application potential of the recombinant enzyme TaTS in the efficient conversion of trehalose from maltose, with superior environmental tolerance to other trehalose synthases reported., (Copyright © 2019 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

The cyclodextrin glycosyltransferase (CGTase) is an important enzyme for cyclodextrin (CD) production, and is also widely used in the biotechnology, food, and pharmaceuticals industries. Secretory CGTase production by recombinant Komagataella phaffii using defined medium is a promising approach because of low cost, less impurity protein. It was found that no CGTase was expressed using traditional defined medium (basal salt medium [BSM]) because of pH value decreasing significantly. CGTase was expressed by recombinant K. phaffii through pH maintenance in range of 5.5-7.0. β-CGTase activity increased to 122.0 U/mL after optimization of glycerol, phosphate buffer, pH value, ammonium sulfate, temperature, methanol, and additives based on BSM, establishing a modified defined medium. These results showed that it was necessary to establish recombinant K. phaffii-based special defined medium although the same host cell used for different heterologous protein expression., (© 2019 American Institute of Chemical Engineers.)

Cyclocarya paliurus (Batalin) Iljinsk is a medicinal plant belonging to the Juglandaceae family, and its leaves are used for a traditional sweet herbal tea with bioactivity against obesity and hyperglycaemia in China. It contains various bioactive specialised metabolites, such as flavonoids, triterpenes and their glucosides, while no glycosyltransferases (GTs) have been reported in C. paliurus to date. Herein, we identified and cloned the first glucosyltransferase C. paliurus GT1. The expression profiles of C. paliurus GT1 showed very high expression in young leaves, callus and branches, but relatively low expression in old leaves and bark and no expression in root. The recombinant C. paliurus GT1 protein was heterologously expressed in Escherichia coli and exhibited catalytic activity towards multiple flavonoids favouring substrate- and regio-specific biosynthesis. Further enzyme assays indicated a preference for certain hydroxyl group glucosylation by C. paliurus GT1. C. paliurus GT1 actively catalysed the glucosylation of flavones and flavonols, but it was less active towards isoflavones, flavanones or triterpenes. C. paliurus GT1 was also able to catalyse the attachment of sugars to the thiol (S-) or amine (N-) sites on aromatic compounds but not on aliphatic compounds. Molecular docking and site-directed mutagenesis analyses indicated that A43F, V84P, and M201Y dramatically altered the regio-selectivity and activity, and the W283M mutation and deletion of the V309-D320 region enhanced the activity and the formation of disaccharides. Herein, we present the identification and characterization of the first multi-functional glucosyltransferase in C. paliurus and provide a basis for understanding the biosynthesis of flavonoid glucosides. C. paliurus GT1 could be utilized as a synthetic biology tool for the synthesis of O-, N-, or S-glucosylated natural/unnatural products., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

We report on a naphthalimide ratiometric fluorescent probe for the real-time sensing and imaging of pathogenic bacterial glucosyltransferases, which are associated with the development of dental caries. Using a high-throughput screening method, we identified that several natural polyphenols from green tea were GTFs inhibitors that could eventually lead to suitable oral treatments to prevent the development of dental caries.

Bisphenol A glycidyl methacrylate (bis-GMA), which is released into the oral environment by dental composites through incomplete polymerization, hydrolysis, and mechanical degradation, can significantly influence oral ecology around resin-based materials. The purpose of this study was to investigate how bis-GMA changes the virulence properties of Streptococcus mutans, a major cariogenic bacterium in humans. The results show that bis-GMA not only inhibited the planktonic growth of cells in medium containing glucose, fructose, or mannose, but also reduced the viability of S. mutans. However, the presence of bis-GMA increased sugar transport and intracellular polysaccharide accumulation in S. mutans, thereby increasing the potential of cell persistence. In addition, bis-GMA could enhance S. mutans's adhesion to hard surfaces and glucan synthesis, which could contribute to biofilm formation. Although free bis-GMA made cells vulnerable to acidic stress, it also provided increased resistance to hydrogen peroxide, which might confer an advantage in competition with other oral microorganisms during the early stage of biofilm development. Interestingly, the presence of bis-GMA did not change the ability of S. mutans to interact with saliva. The results suggest that leachable bis-GMA could contribute to biofilm-related secondary dental caries at the marginal interface between resin-based materials and teeth by altering the virulent properties of S. mutans, although bis-GMA reduced the planktonic growth and viability of S. mutans., (© 2018 S. Karger AG, Basel.)

Assembly of polysaccharide-based walls by plant cells involves the rapid synthesis, trafficking, and deposition of complex biopolymers, but how these events are controlled and coordinated to achieve a strong, resilient extracellular matrix has remained obscure for decades. Recent quantitative analyses of fluorescence microscopy data have revealed details of the trafficking and synthetic activity of cellulose synthases, and new methods for labeling matrix polymers have unveiled aspects of their regulated deposition in the wall. Detailed studies of the identity, architecture, activity, and trafficking of the proteins and protein complexes that synthesize wall polymers, combined with advances in image acquisition and analysis, will aid future efforts to dissect wall assembly., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

As is well-known, fungi are an important biocatalysis model of glucosylation and have been widely applied for bioactive compounds glucosylation mediated by the intracellular glucosytransferases (GTs). However, there is no efficient method for the real-time detection of GTs and the rapid isolation of the target fungi strains with the high expression of GTs. In the present work, we first developed a two-photon ratiometric fluorescent probe N-( n-butyl)-4-hydroxy-1,8-naphthalimide (NHN) for detecting the glucosyltransferases activity and intracellular imaging of GTs. Under UV light (365 nm), the transformed product of NHN mediated by intracellular glucosyltransferase displayed blue emission to guide the rapid isolation of fungal strains possessing overexpression of GTs from complex soil samples. Finally, by using the fluorescent probe, two target fungi were isolated and identified to be Rhizopus oryzae and Mucor circinelloides by molecular analysis, and they exhibited a robust capability for regio- and stereospecific O-glycosylation. Our results fully demonstrated that NHN may be a promising tool for guiding real-time GTs activity in fungal strains and even for developing natural fungal strains with GTs overexpression.

In the highly aluminum-resistant tree Eucalyptus camaldulensis, hydrolyzable tannins are proposed to play a role in internal detoxification of aluminum, which is a major factor inhibiting plant growth on acid soils. To understand and modulate the molecular mechanisms of aluminum detoxification by hydrolyzable tannins, the biosynthetic genes need to be identified. In this study, we identified and characterized genes encoding UDP-glucose:gallate glucosyltransferase, which catalyzes the formation of 1-O-galloyl-β-d-glucose (β-glucogallin), the precursor of hydrolyzable tannins. By homology-based cloning, seven full-length candidate cDNAs were isolated from E. camaldulensis and expressed in Escherichia coli as recombinant N-terminal His-tagged proteins. Phylogenetic analysis classified four of these as UDP glycosyltransferase (UGT) 84A subfamily proteins (UGT84A25a, -b, UGT84A26a, -b) and the other three as UGT84J subfamily proteins (UGT84J3, -4, -5). In vitro enzyme assays showed that the UGT84A proteins catalyzed esterification of UDP-glucose and gallic acid to form 1-O-galloyl-β-d-glucose, whereas the UGT84J proteins were inactive. Further analyses with UGT84A25a and -26a indicated that they also formed 1-O-glucose esters of other structurally related hydroxybenzoic and hydroxycinnamic acids with a preference for hydroxybenzoic acids. The UGT84A genes were expressed in leaves, stems, and roots of E. camaldulensis, regardless of aluminum stress. Taken together, our results suggest that the UGT84A subfamily enzymes of E. camaldulensis are responsible for constitutive production of 1-O-galloyl-β-d-glucose, which is the first step of hydrolyzable tannin biosynthesis., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Helicobacter suis (H. suis), formerly called Helicobacter heilmannii type 1 (H. heilmannii), is a gram-negative bacterium of the Helicobacter species. This pathogen infects the stomach of humans and animals such as dogs, cats, pigs, and rodents, the latter giving rise to zoonotic infection. Here, we generated a H. suis-specific antibody useful for immunohistochemistry with formalin-fixed, paraffin-embedded tissue sections. To do so, we began by cloning the gene encoding H. suis cholesterol α-glucosyltransferase (αCgT). αCgT is the key enzyme responsible for biosynthesis of cholesteryl α-D-glucopyranoside (CGL), a major cell wall component of Helicobacter species including H. suis. The deduced amino acid sequence of H. suis αCgT had 56% identity with the corresponding Helicobacter pylori (H. pylori). We then developed a polyclonal antibody (anti-Hh-I205R) by immunizing rabbits with a 205 amino acid H. suis αCgT fragment. Immunohistochemistry with the anti-Hh-I205R antibody could differentiate H. suis from H. pylori in gastric mucosa sections derived from mice infected with either pathogen. We then probed formalin-fixed, paraffin-embedded sections of human gastric mucosa positive for H. suis infection with the anti-Hh-I205R antibody and detected positive staining. These results indicate that anti-Hh-I205R antibody is specific for H. suis αCgT and useful to detect H. suis in gastric specimens routinely analyzed in pathological examinations.

The present study compared IgA specificity against oral streptococci in colostrum and saliva samples. Sixty-two mother-and-child pairs were included; samples of colostrum (C) and saliva (MS) were collected from the mothers and saliva samples were collected from babies (BS). The specificity of IgA against Streptococcus mutans and S. mitis were analyzed by western blot. Only 30% of babies' samples presented IgA reactivity to S. mutans, while 74 and 80% of MS and C, respectively, presented this response. IgA reactivity to S. mutans virulence antigens (Ag I/II, Gtf and GbpB) in positive samples showed differences between samples for Gtf and especially for GbpB (p < 0.05), but responses to Ag I/II were similar (p > 0.05). The positive response of Gtf-reactive IgA was different between C (90%) and MS (58%) samples (p < 0.05), but did not differ from BS (p > 0.05). GbpB was the least detected, with 48 and 26% of C and MS, and only 5% of BS samples presenting reactivity (p > 0.05). Eight percent of MS and C samples presented identical bands to SM in the same time-point. In conclusion, the differences of IgA response found between C and MS can be due to the different ways of stimulation, proliferation and transportation of IgA in those secretions. The colostrum has high levels of IgA against S. mutans virulence antigens, which could affect the installation and accumulation process of S. mutans, mainly by supplying anti-GbpB IgA to the neonate.

Background: Glucosylceramide synthase (GCS) and P-glycoprotein (P-gp) overexpression are associated with multidrug resistance in several human cancers. This study investigated the prognostic value of GCS and P-gp in oral cavity squamous cell carcinoma (OSCC)., Methods: The association between GCS and P-gp overexpression and clinical outcomes was assessed in 186 human clinical specimens of primary tumors obtained from curative surgery. Immunohistochemistry staining results were scored as high or low for GCS, and positive or negative for P-gp. Univariate and multivariate analyses using the Cox proportional hazards model were conducted to assess the significance of differences in recurrence or survival outcomes between variables., Results: GCS overexpression was observed in 128 (68.8 %) patients and P-gp overexpression in 43 (23.1 %) patients. High GCS expression was significantly correlated with P-gp immunopositivity (P = 0.005). GCS and P-gp overexpression was significantly correlated with cervical nodal metastasis (P < 0.05). Univariate analyses showed that tumor lymphovascular invasion, positive neck lymph nodes, advanced overall TNM stage, high GCS expression, and P-gp immunopositivity were associated with poor locoregional control (LRC), disease-free survival (DFS), and overall survival (OS) (P < 0.05). Multivariate analyses showed that lymphovascular invasion, nodal positivity, and P-gp overexpression remained independent prognostic variables for LRC, DFS, and OS, and that GCS expression was an independent predictor of LRC and DFS (P < 0.05)., Conclusion: GCS and P-gp expression is associated with poor prognosis, suggesting suitability as novel biomarkers in OSCC.

Flavonoid and limonoid glycosides influence taste properties as well as marketability of Citrus fruit and products, particularly grapefruit. In this work, nine grapefruit putative natural product glucosyltransferases (PGTs) were resolved by either using degenerate primers against the semiconserved PSPG box motif, SMART-RACE RT-PCR, and primer walking to full-length coding regions; screening a directionally cloned young grapefruit leaf EST library; designing primers against sequences from other Citrus species; or identifying PGTs from Citrus contigs in the harvEST database. The PGT proteins associated with the identified full-length coding regions were recombinantly expressed in Escherichia coli and/or Pichia pastoris and then tested for activity with a suite of substrates including flavonoid, simple phenolic, coumarin, and/or limonoid compounds. A number of these compounds were eliminated from the predicted and/or potential substrate pool for the identified PGTs. Enzyme activity was detected in some instances with quercetin and catechol glucosyltransferase activities having been identified.

In this study, we report the distribution and abundance of cold-adaptation proteins in microbial mat communities in the perennially ice-covered Lake Joyce, located in the McMurdo Dry Valleys, Antarctica. We have used MG-RAST and R code bioinformatics tools on Illumina HiSeq2000 shotgun metagenomic data and compared the filtering efficacy of these two methods on cold-adaptation proteins. Overall, the abundance of cold-shock DEAD-box protein A (CSDA), antifreeze proteins (AFPs), fatty acid desaturase (FAD), trehalose synthase (TS), and cold-shock family of proteins (CSPs) were present in all mat samples at high, moderate, or low levels, whereas the ice nucleation protein (INP) was present only in the ice and bulbous mat samples at insignificant levels. Considering the near homogeneous temperature profile of Lake Joyce (0.08-0.29 °C), the distribution and abundance of these proteins across various mat samples predictively correlated with known functional attributes necessary for microbial communities to thrive in this ecosystem. The comparison of the MG-RAST and the R code methods showed dissimilar occurrences of the cold-adaptation protein sequences, though with insignificant ANOSIM (R = 0.357; p-value = 0.012), ADONIS (R(2) = 0.274; p-value = 0.03) and STAMP (p-values = 0.521-0.984) statistical analyses. Furthermore, filtering targeted sequences using the R code accounted for taxonomic groups by avoiding sequence redundancies, whereas the MG-RAST provided total counts resulting in a higher sequence output. The results from this study revealed for the first time the distribution of cold-adaptation proteins in six different types of microbial mats in Lake Joyce, while suggesting a simpler and more manageable user-defined method of R code, as compared to a web-based MG-RAST pipeline.

This study examined polymorphism of the glucosyltransferase gene (ycjM) in fecal Escherichia coli isolates and evaluated the use of the sequence polymorphism for measuring human fecal pollution in water. Significant nucleotide variations were observed through comparative analysis of the ycjM sequences of 70 E. coli strains isolated from the feces of humans, domestic livestock, and wild animals. Three distinct types of ycjM sequences were found: universal-ycjM, human/chicken-ycjM, and human-ycjM. Using the human-ycjM sequences, both a polymerase chain reaction (PCR), Hycj-PCR and a quantitative PCR, Hycj-qPCR, were developed. As shown by the Hycj-PCR amplification, the human-ycjM marker appeared to be highly associated with the E. coli strains isolated from human feces, based on the analysis of 370 E. coli strains isolated from humans and seven other animal species. Similarly, the human-ycjM marker was highly linked with human feces, as demonstrated by the Hycj-PCR assay, when using 337 fecal DNA samples from 16 host animal sources, including both domestic and wild animals. Overall, the specificity and sensitivity of the human-ycjM marker for differentiating between the feces of humans and those of nonhuman groups were 99.7% and 100%, respectively; the prevalence of the marker appeared to be greater than 50% in the human-feces-associated E. coli population. In addition, our study showed that the quantification of human E. coli by the Hycj-qPCR was linearly correlated with the anthropogenic activity within a watershed. Our study suggests that this novel human-ycjM marker and the resulting PCR-based methods developed should be useful for measuring human-associated E. coli and human fecal pollution in water., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Cellulose biosynthesis in plant secondary cell walls forms the basis of vascular development in land plants, with xylem tissues constituting the vast majority of terrestrial biomass. We used plant lines that contained an inducible master transcription factor controlling xylem cell fate to quantitatively image fluorescently tagged cellulose synthase enzymes during cellulose deposition in living protoxylem cells. The formation of secondary cell wall thickenings was associated with a redistribution and enrichment of CESA7-containing cellulose synthase complexes (CSCs) into narrow membrane domains. The velocities of secondary cell wall-specific CSCs were faster than those of primary cell wall CSCs during abundant cellulose production. Dynamic intracellular of endomembranes, in combination with increased velocity and high density of CSCs, enables cellulose to be synthesized rapidly in secondary cell walls., (Copyright © 2015, American Association for the Advancement of Science.)

We selected the immunogenic cell wall ß-(1,3)-glucosyltransferase Bgl2p from Candida albicans as a target protein for the production of antibodies. We identified a unique peptide sequence in the protein and generated monoclonal anti- C. albicans Bgl2p antibodies, which bound in particular to whole C. albicans cells., (Copyright © 2014 Elsevier B.V. All rights reserved.)

Background and Objectives: Breast cancer is one of the most common causes of cancer-related deaths in women worldwide. Studies on glucosylceramide synthase (GCS) activity suggest that this enzyme has a role in the development of multidrug resistance in many cancer cells. However, few studies have shown the expression of GCS in invasive ductal breast cancer and breast intraductal proliferative lesions., Methods: In total, 196 samples from patients with invasive ductal breast cancer and 61 samples of breast intraductal proliferative lesions were collected. Immunohistochemical analyses were conducted to determine the expression of GCS and other related proteins., Results: Expression of GCS was high in estrogen receptor (ER)-positive and HER-2 negative samples. In contrast, the expression of GCS in invasive ductal cancer was significantly lower than that in intraductal proliferative lesions., Conclusion: Our data demonstrates a correlation between the expression of the GCS protein and ER-positive/HER-2 negative breast cancer. Furthermore, in contrast to previous reports, the expression of GCS protein was shown to be much higher in ductal carcinoma in-situ than that in invasive ductal cancer., Virtual Slides: The virtual slide(s) for this article can be found here: http://www.diagnosticpathology.diagnomx.eu/vs/1559854430111589.

The biological function of mannosylerythritol lipids (MELs) towards their producer, Pseudozyma antarctica, on plant surfaces was investigated. MEL-producing wild-type strain and its MEL production-defective mutant strain (ΔPaEMT1) were compared in terms of their phenotypic traits on the surface of plastic plates, onion peels, and fresh leaves of rice and wheat. While wild-type cells adhering on plastic surfaces and onion peels changed morphologically from single cells to elongated ones for a short period of about 4 h and 1 day, respectively, ΔPaEMT1 cells did not. Microscopic observation of both strains grown on plant leaf surfaces verified that the wild type colonized a significantly bigger area than that of ΔPaEMT1. However, when MELs were exogenously added to the mutant cells on plant surfaces, their colonized area became enlarged. High-performance liquid chromatography analysis revealed a secretion of higher amount of MELs in the cell suspension incubated with wheat leaf cuttings compared to that in the suspension without cuttings. Transcriptional analysis by real-time reverse transcriptase PCR verified that the expression of erythritol/mannose transferase gene and MELs transporter gene of P. antarctica increased in the cells inoculated onto wheat leaves at 4, 6, and 8 days of incubation, indicating a potential of P. antarctica to produce MELs on the leaves. These findings demonstrate that MELs produced by P. antarctica on plant surfaces could be expected to play a significant role in fungal morphological development and propagation on plant surfaces.

Streptococcus mutans is considered the primary etiologic agent of dental caries and contributes significantly to the virulence of dental plaque, especially in the presence of sucrose. To avoid the role of sucrose on the virulence factors of S. mutans, sugar substitutes are commonly consumed because they lead to lower or no production of acids and interfere with biofilm formation. This study aimed to investigate the contribution of sugar substitutes in the cariogenic potential of S. mutans biofilms. Thus, in the presence of sucrose, glucose, sucralose and sorbitol, the biofilm mass was quantified up to 96 h, the pH of the spent culture media was measured, the expression of biofilm-related genes was determined, and demineralization challenge experiments were conduct in enamel fragments. The presence of sugars or sugar substitutes profoundly affected the expression of spaP, gtfB, gtfC, gbpB, ftf, vicR and vicX in either biofilm or planktonic cells. The substitution of sucrose induced a down-regulation of most genes involved in sucrose-dependent colonization in biofilm cells. When the ratio between the expression of biofilm and planktonic cells was considered, most of those genes were down-regulated in biofilm cells in the presence of sugars and up-regulated in the presence of sugar substitutes. However, sucralose but not sorbitol fulfilled the purpose of reducing the cariogenic potential of the diet since it induced the biofilm formation with the lowest biomass, did not change the pH of the medium and led to the lowest lesion depth in the cariogenic challenge.

Antibiotic prophylaxis for maxillofacial surgical wounds remains common practice. Surgeons must weigh the risks (e.g., Clostridium difficile colitis) against the benefits before administering antibiotics for any reason and the relative risk and morbidity of C difficile colitis against those of a potential postoperative wound infection. In addition, the possibility of C difficile infection as a complication of perioperative antibiotic prophylaxis should be discussed with patients before surgery, especially those with concomitant baseline risk factors. This report describes the case of a young healthy patient with few risk factors for C difficile infection who received a standard perioperative course of antibiotic therapy. Subsequently, the patient developed severe fulminant C difficile infection that required a protracted hospital admission, subtotal colectomy, and ileostomy. This case underscores that antibiotic prophylaxis continues in widespread use and is not benign therapy., (Copyright © 2013 American Association of Oral and Maxillofacial Surgeons. Published by Elsevier Inc. All rights reserved.)

Clathrin-mediated endocytosis (CME) is the best-characterized type of endocytosis in eukaryotic cells. Plants appear to possess all of the molecular components necessary to carry out CME; however, functional characterization of the components is still in its infancy. A yeast two-hybrid screen identified μ2 as a putative interaction partner of CELLULOSE SYNTHASE6 (CESA6). Arabidopsis (Arabidopsis thaliana) μ2 is homologous to the medium subunit 2 of the mammalian ADAPTOR PROTEIN COMPLEX2 (AP2). In mammals, the AP2 complex acts as the central hub of CME by docking to the plasma membrane while concomitantly recruiting cargo proteins, clathrin triskelia, and accessory proteins to the sites of endocytosis. We confirmed that μ2 interacts with multiple CESA proteins through the μ-homology domain of μ2, which is involved in specific interactions with endocytic cargo proteins in mammals. Consistent with its role in mediating the endocytosis of cargos at the plasma membrane, μ2-YELLOW FLUORESCENT PROTEIN localized to transient foci at the plasma membrane, and loss of μ2 resulted in defects in bulk endocytosis. Furthermore, loss of μ2 led to increased accumulation of YELLOW FLUORESCENT PROTEIN-CESA6 particles at the plasma membrane. Our results suggest that CESA represents a new class of CME cargo proteins and that plant cells might regulate cellulose synthesis by controlling the abundance of active CESA complexes at the plasma membrane through CME.

The cellulose complementing factor (Ccp) is known to be involved in cellulose production in the Acetobacter species. However, its precise functions remain unclear. In the current study, we identified the coding region of the ccpAx gene (ccp gene from Acetobacter xylinum) and the localization of the CcpAx in cells by generating fusion proteins tagged to an enhanced green fluorescent protein (EGFP). From the results of N-terminal sequencing of CcpAx-EGFP-fusion protein, which recovered 65% of cellulose-producing abilities of the wild-type to the ccpAx gene-knockout mutant, the ccpAx gene was determined to encode a protein with the molecular weight of 8 kDa. The amino acid sequence deduced had high similarities with the C-terminal regions of Ccp proteins from other Acetobacter species. Fluorescence microscopy analysis showed that CcpAx was longitudinally localized along with one side of the cell membrane. Additionally, the localization of AxCeSD, which is thought to be a member of the cellulose synthase complex [terminal complex (TC)] in A. xylinum, was determined in the same manner as CcpAx. Fluorescence microscopy analysis showed that AxCeSD had a localization pattern similar to that of CcpAx. Pulldown assays and isothermal titration calorimetry analysis clearly showed a significant interaction between CcpAx and AxCeSD. Taken together, these data strongly suggest that CcpAx functions as a member of the TC in A. xylinum., (Copyright © 2013 The Society for Biotechnology, Japan. Published by Elsevier B.V. All rights reserved.)

Objective: Antibacterial primer and adhesive are promising to help combat biofilms and recurrent caries. The objectives of this study were to compare novel bonding agent containing quaternary ammonium dimethacrylate (QADM) with bonding agent containing nanoparticles of silver (NAg) in antibacterial activity, contact-inhibition vs. long-distance inhibition, glucosyltransferases (gtf) gene expressions, and cytotoxicity for the first time., Methods: QADM and NAg were incorporated into Scotchbond Multi-Purpose adhesive and primer. Microtensile dentin bond strength was measured. Streptococcus mutans (S. mutans) biofilm on resin surface (contact-inhibition) as well as S. mutans in culture medium away from the resin surface (long-distance inhibition) were tested for metabolic activity, colony-forming units (CFUs), lactic acid production, and gtf gene expressions. Eluents from cured primer/adhesive samples were used to examine cytotoxicity against human gingival fibroblasts., Results: Bonding agent with QADM greatly reduced CFU and lactic acid of biofilms on the resin surface (p<0.05), while having no effect on S. mutans in culture medium away from the resin surface. In contrast, bonding agent with NAg inhibited not only S. mutans on the resin surface, but also S. mutans in culture medium away from the resin surface. Bonding agent with QADM suppressed gtfB, gtfC and gtfD gene expressions of S. mutans on its surface, but not away from its surface. Bonding agent with NAg suppressed S. mutans gene expressions both on its surface and away from its surface. Bonding agents with QADM and NAg did not adversely affect microtensile bond strength or fibroblast cytotoxicity, compared to control (p>0.1)., Significance: QADM-containing adhesive had contact-inhibition and inhibited bacteria on its surface, but not away from its surface. NAg-containing adhesive had long-distance killing capability and inhibited bacteria on its surface and away from its surface. The novel antibacterial adhesives are promising for caries-inhibition restorations, and QADM and NAg could be complimentary agents in inhibiting bacteria on resin surface as well as away from resin surface., (Published by Elsevier Ltd.)

Amylose, a linear polymer of α(1,4)-linked glucosyl units and a major constituent of starch granules, can also be enzymatically synthesized in vitro from sucrose by bacterial amylosucrases. Depending on the initial sucrose concentration and the enzyme used, amylose oligomers (or polymers) are formed and self-associate during synthesis into various semicrystalline morphologies. This work describes for the first time a synchrotron SAXS study of the structure in solution of two amylosucrases, namely, NpAS and the thermostable DgAS, under conditions of polymer synthesis and, simultaneously, the amylose conformation. The structure in solution of both amylosucrases during the reaction was shown to be similar to the known crystallographic structures. The conformation of amylose produced at an early stage consists of a mixture of wormlike chains and double helical cylindrical structures. In the case of NpAS, in a second stage, individual double helices pack into clusters before crystallizing and precipitating. Amylose produced by DgAS never self-associates in such clusters due to the higher temperature used for amylose synthesis. All the dimensions determined for wormlike chains and cylindrical conformations at different times of NpAS synthesis are in very good agreement with structural features usually observed on gels of amylose extracted from starch. This provides new insights in understanding the mechanisms of amylose gelation.

Purpose: Glucosyltransferase B enzyme (GtfB) plays an essential role in the pathogenesis of dental caries. It catalyzes the synthesis of water-insoluble glucan from sucrose, which is essential for accumulation of Streptococcus mutans in the dental biofilm. On the other hand, salivary immunoglobulin A (sIgA) plays a protective role in the same process. Therefore, the purpose of this study was to attempt to correlate glucosyltransferase B enzyme and immunoglobulin A levels in whole saliva with caries experience in preschool children., Methods: Forty-five 3- to 6-year-old were examined clinically, and their caries experiences were recorded as decayed, missing, and filled teeth (dmft) scores. Unstimulated whole saliva was collected and analyzed for GtfB and IgA using enzyme-linked immunosorbent assay., Results: Mixed-model analysis revealed that both GtfB and IgA are covariants regarding the effect on dmft scores (P =.008). GtfB levels displayed a simultaneous increase in caries experience (dmft) and number of carious lesions (d), while IgA levels were high in caries-free children and significantly lower values were obtained in the high caries group., Conclusion: Salivary IgA was negatively correlated with caries experience and inversely proportional with GtfB levels in the saliva of preschool children.

Objective: This study aimed to evaluate the influence of NaF (2, 10, 50 and 125 ppm F(-)) on the virulence factors and composition of Streptococcus mutans biofilms., Methods: S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite discs. To assess the influence of NaF on the virulence factors of S. mutans biofilm cells, glycolytic pH drop, proton-permeability and F-ATPase activity assay were performed using 74 h old S. mutans biofilms. Glucosyltransferase (GTF) activity assay in suspension was also performed. To examine the influence of NaF on S. mutans biofilm composition, the biofilms were treated twice daily (5 min exposure/treatment) a total of five times during biofilm formation. After a total of 5 treatments, the biomass, colony forming unit (CFU) and polysaccharide composition of the treated 74h old S. mutans biofilms were analysed by microbiological and biochemical methods, and scanning electron microscopy., Results: NaF showed inhibitory effects on the acid production and acid tolerance of S. mutans biofilm cells at 10, 50 and 125 ppm F(-), compared to the vehicle control (P<0.05) and the treatments at these concentrations also affected the biomass, water-insoluble extracellular polysaccharides and intracellular iodophilic polysaccharides of the biofilms, compared to the vehicle control (P<0.05)., Conclusions: These results indicate that NaF (10, 50 and 125 ppm F(-)) has inhibitory effects on the virulence factors and composition of S. mutans biofilms, suggesting the potential use of these concentrations as an effective measure for controlling dental biofilms., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Purpose: Plants can absorb a diversity of natural and man-made toxic compounds for which they have developed diverse detoxification mechanisms. Plants are able to metabolize and detoxify a wide array of xenobiotics by oxidation, sugar conjugation, glutathione conjugation, and more complex reactions. In this study, detoxification mechanisms of dimethomorph, a fungicide currently found in aquatic media were investigated in Elodea canadensis., Methods: Cytochrome P450 (P450) activity was measured by an oxygen biosensor system, glucosyltransferases (GTs) by HPLC, glutathione S-transferases (GSTs), and ascorbate peroxidase (APOX) were assayed spectrophotometrically., Results: Incubation of Elodea with dimethomorph induced an increase of the P450 activity. GST activity was not stimulated by dimethomorph suggesting that GST does not participate in dimethomorph detoxification. In plants exposed to dimethomorph, comparable responses were observed for GST and APOX activities showing that the GST was more likely to play a role in response to oxidative stress. Preincubation with dimethomorph induced a high activity of O- and N-GT, it is therefore likely that both enzymes participate in the phase II (conjugation) of dimethomorph detoxification process., Conclusions: For the first time in aquatic plants, P450 activity was shown to be induced by a fungicide suggesting a role in the metabolization of dimethomorph. Moreover, our finding is the first evidence of dimethomorph and isoproturon activation of cytochrome P450 multienzyme family in an aquatic plant, i.e., Elodea (isoproturon was taken here as a reference molecule). The detoxification of dimetomorph seems to proceed via hydroxylation, and subsequent glucosylation, and might yield soluble as well as cell wall bound residues.

Helicobacter pylori (H. pylori) is the causative pathogen underlying gastric diseases such as chronic gastritis and gastric cancer. Previously, the authors revealed that α1,4-linked N-acetylglucosamine-capped O-glycan (αGlcNAc) found in gland mucin suppresses H. pylori growth and motility by inhibiting catalytic activity of cholesterol α-glucosyltransferase (CHLαGcT), the enzyme responsible for biosynthesis of the major cell wall component cholesteryl-α-D-glucopyranoside (CGL). Here, the authors developed a polyclonal antibody specific for CHLαGcT and then undertook quantitative ultrastructural analysis of the enzyme's localization in H. pylori. They show that 66.3% of CHLαGcT is detected in the cytoplasm beneath the H. pylori inner membrane, whereas 24.7% is present on the inner membrane. In addition, 2.6%, 5.0%, and 1.4% of the protein were detected in the periplasm, on the outer membrane, and outside microbes, respectively. By using an in vitro CHLαGcT assay with fractionated H. pylori proteins, which were used as an enzyme source for CHLαGcT, the authors demonstrated that the membrane fraction formed CGL, whereas other fractions did not. These data combined together indicate that CHLαGcT is originally synthesized in the cytoplasm of H. pylori as an inactive form and then activated when it is associated with the cell membrane. This article contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Lipids have an established role as structural components of membranes or as signalling molecules, but their role as molecular actors in protein secretion is less clear. The complex sphingolipid glucosylceramide (GlcCer) is enriched in the plasma membrane and lipid microdomains of plant cells, but compared to animal and yeast cells, little is known about the role of GlcCer in plant physiology. We have investigated the influence of GlcCer biosynthesis by glucosylceramide synthase (GCS) on the efficiency of protein transport through the plant secretory pathway and on the maintenance of normal Golgi structure. We determined that GlcCer is synthesized at the beginning of the plant secretory pathway [mainly endoplasmic reticulum (ER)] and that D,L-threo-1-phenyl-2-decanoyl amino-3-morpholino-propanol (PDMP) is a potent inhibitor of plant GCS activity in vitro and in vivo. By an in vivo confocal microscopy approach in tobacco leaves infiltrated with PDMP, we showed that the decrease in GlcCer biosynthesis disturbed the transport of soluble and membrane secretory proteins to the cell surface, as these proteins were partly retained intracellularly in the ER and/or Golgi. Electron microscopic observations of Arabidopsis thaliana root cells after high-pressure freezing and freeze substitution evidenced strong morphological changes in the Golgi bodies, pointing to a link between decreased protein secretion and perturbations of Golgi structure following inhibition of GlcCer biosynthesis in plant cells.

Natural resistance of wheat against Fusarium head blight (FHB) is inadequate and new strategies for controlling the disease are required. Chitin synthases that catalyze chitin biosynthesis would be an ideal target for antifungal agents. In this study, a class I chitin synthase gene (CHS1) from Fusarium asiaticum, the predominant species of FHB pathogens on wheat in China, was functionally disrupted via Agrobacterium tumefaciens-mediated transformation. Specific disruption of the CHS1 gene resulted in a 58% reduction of chitin synthase activity, accompanied by decreases of 35% in chitin content, 22% in conidiation, and 16% in macroconidium length. The Deltachs1 mutant strain had a growth rate comparable to that of the wild-type on PDA medium but had a 35% increase in the number of nuclear cellulae and exhibited a remarkably increased sensitivity to osmosis stresses. Electron microscopy revealed substantial changes occurring in cell wall structures of the macroconidium, ascospore, and mycelium, with the most profound changes in the mycelium. Furthermore, the Deltachs1 mutant displayed significantly reduced pathogenicity on wheat spikes and seedlings. Re-introduction of a functional CHS1 gene into the Deltachs1 mutant strain restored the wild-type phenotype. These results reveal an important in vivo role played by a CHS1 gene in a FHB pathogen whose mycelial chitin could serve as a target for controlling the disease., (Copyright (c) 2009 Elsevier Inc. All rights reserved.)

Introduction: Streptococcus mutans, known to be a pathogen of dental caries as well as bacteremia and infective endocarditis, is classified into four serotypes, c, e, f and k, based on the structures of serotype-specific polysaccharides. Serotype k was recently designated using blood isolates from Japanese subjects and such strains are considered to be virulent in the bloodstream. The purpose of the present study was to analyse the serotype distribution of strains isolated from Thai subjects and determine whether serotype k strains were present., Methods: A total of 250 S. mutans strains were isolated from 50 Thai subjects, and serotypes of all strains were determined. Then, molecular and biological analyses were carried out for serotype k strains., Results: Immunodiffusion and polymerase chain reaction analyses showed that serotype c was the most prevalent (70%), followed by serotypes e (22.8%), f (4.4%) and k (2.8%), which indicated that serotype k S. mutans strains occurred in Thai individuals at a similar rate to that previously reported for Japanese and Finnish populations. Molecular analyses of the seven serotype k strains showed extremely low expression of rgpE, which is related to glucose side-chain formation in serotype-specific rhamnose-glucose polymers, similar to previous reports for those other populations. In addition, analysis of the biological properties of the seven serotype k strains demonstrated low levels of sucrose-dependent adhesion, cellular hydrophobicity, dextran-binding activity and phagocytosis susceptibility by human polymorphonuclear leukocytes, which are characteristics similar to those of serotype k strains previously isolated in Japan., Conclusion: Our results indicate the possibility of a worldwide prevalence of serotype k strains with properties in common with those of previously reported strains.

The entry of carbon from sucrose into cellular metabolism in plants can potentially be catalyzed by either sucrose synthase (SUS) or invertase (INV). These 2 routes have different implications for cellular metabolism in general and for the production of key metabolites, including the cell-wall precursor UDPglucose. To examine the importance of these 2 routes of sucrose catabolism in Arabidopsis thaliana (L.), we generated mutant plants that lack 4 of the 6 isoforms of SUS. These mutants (sus1/sus2/sus3/sus4 mutants) lack SUS activity in all cell types except the phloem. Surprisingly, the mutant plants are normal with respect to starch and sugar content, seed weight and lipid content, cellulose content, and cell-wall structure. Plants lacking the remaining 2 isoforms of SUS (sus5/sus6 mutants), which are expressed specifically in the phloem, have reduced amounts of callose in the sieve plates of the sieve elements. To discover whether sucrose catabolism in Arabidopsis requires INVs rather than SUSs, we further generated plants deficient in 2 closely related isoforms of neutral INV predicted to be the main cytosolic forms in the root (cinv1/cinv2 mutants). The mutant plants have severely reduced growth rates. We discuss the implications of these findings for our understanding of carbon supply to the nonphotosynthetic cells of plants.

Infective endocarditis is characterized by inflammatory infiltrates of mononuclear cells in infected cardiac valve leaflets. To delineate the role of valve interstitial cells (VICs) in leukocyte recruitment, we stimulated human VICs with glucosyltransferase, a modulin from viridians streptococci. Interstitial cells were activated directly by glucosyltransferase in a dose-dependent manner through concerted mitogen-activated protein kinase and nuclear factor-kappaB signaling pathways; activation resulted in up-regulation of synthesis and release of interleukin-6, interleukin-8, or monocyte chemoattractant protein-1 and enhanced transwell migration of U937 monocytic cells or primary mononuclear cells. The expression of glucosyltransferases and activation of VICs (nuclear localization of RelA) were detected in a rat model of experimental endocarditis. Proinflammatory cytokines also were detected in VICs from diseased human autopsy specimens but not in VICs from normal specimens. These results indicate that interstitial cells in the cardiac valve can be activated directly by bacterial modulins to recruit and retain mononuclear cells, likely contributing to the persistent inflammation characteristic of infective endocarditis.

The pathways responsible for cell wall polysaccharide biosynthesis are vital in eukaryotic microorganisms. The corresponding synthases are potential targets of inhibitors such as fungicides. Despite their fundamental and economical importance, most polysaccharide synthases are not well characterized, and their molecular mechanisms are poorly understood. With the example of Saprolegnia monoica as a model organism, we show that chitin and (1-->3)-beta-d-glucan synthases are located in detergent-resistant membrane microdomains (DRMs) in oomycetes, a phylum that comprises some of the most devastating microorganisms in the agriculture and aquaculture industries. Interestingly, no cellulose synthase activity was detected in the DRMs. The purified DRMs exhibited similar biochemical features as lipid rafts from animal, plant, and yeast cells, although they contained some species-specific lipids. This report sheds light on the lipid environment of the (1-->3)-beta-d-glucan and chitin synthases, as well as on the sterol biosynthetic pathways in oomycetes. The results presented here are consistent with a function of lipid rafts in cell polarization and as platforms for sorting specific sets of proteins targeted to the plasma membrane, such as carbohydrate synthases. The involvement of DRMs in the biosynthesis of major cell wall polysaccharides in eukaryotic microorganisms suggests a function of lipid rafts in hyphal morphogenesis and tip growth.

Sucrose synthase (Sus; EC 2.4.1.13) is a key enzyme of sucrose metabolism in plant cells, providing carbon for respiration and for the synthesis of cell wall polymers and starch. Since Sus is important for plant cell growth, insights into its structure, localization, and features are useful for defining the relationships between nutrients, growth, and cell morphogenesis. We used the pollen tube of tobacco (Nicotiana tabacum) as a cell model to characterize the main features of Sus with regard to cell growth and cell wall synthesis. Apart from its role during sexual reproduction, the pollen tube is a typical tip-growing cell, and the proper construction of its cell wall is essential for correct shaping and direction of growth. The outer cell wall layer of pollen tubes consists of pectins, but the inner layer is composed of cellulose and callose; both polymers require metabolic precursors in the form of UDP-glucose, which is synthesized by Sus. We identified an 88-kD polypeptide in the soluble, plasma membrane and Golgi fraction of pollen tubes. The protein was also found in association with the cell wall. After purification, the protein showed an enzyme activity similar to that of maize (Zea mays) Sus. Distribution of Sus was affected by brefeldin A and depended on the nutrition status of the pollen tube, because an absence of metabolic sugars in the growth medium caused Sus to distribute differently during tube elongation. Analysis by bidimensional electrophoresis indicated that Sus exists as two isoforms, one of which is phosphorylated and more abundant in the cytoplasm and cell wall and the other of which is not phosphorylated and is specific to the plasma membrane. Results indicate that the protein has a role in the construction of the extracellular matrix and thus in the morphogenesis of pollen tubes.

Introduction: The combination of starch and sucrose has been shown to be potentially more cariogenic than either alone. The aim of this study was to examine the influence of starch and sucrose, alone or in combinations, on formation, polysaccharide composition, gene expression, and acidogenicity of Streptococcus mutans biofilms., Methods: S. mutans UA159 biofilms were formed on saliva-coated hydroxyapatite (sHA) discs in batch culture for 5 days in the presence of 1% (weight/volume) starch, 1% sucrose, 1% starch plus 1% sucrose, 1% starch plus 0.5% fructose plus 0.5% glucose, or 1% sucrose plus 1% glucose., Results: Amylase activity from sHA disks was detected up to 48 h, thereby increasing the availability of reducing sugars and acidogenicity in the early stages of biofilm development. S. mutans grown in the presence of sucrose alone or in combinations formed well-defined and tightly adherent biofilms comprised of mostly water-insoluble polysaccharides (INS); in contrast, the presence of starch or starch + glucose + fructose resulted in little biofilm formation with minimal amounts of INS. However, the combination of starch + sucrose produced biofilms with more biomass and acidogenicity, and a higher content of INS than those grown in sucrose or sucrose + glucose (P < 0.05). The INS extracted from biofilms formed in the presence of starch + sucrose displayed a higher percentage of 3-linked branching (3,4-, 3,6-, and 3,4,6-linked glucose) compared to those from biofilms grown in sucrose or sucrose + glucose. Furthermore, biofilms grown in starch + sucrose expressed significantly higher levels of gtfB messenger RNA than sucrose-grown or sucrose + glucose-grown biofilms (P < 0.05)., Conclusion: The combination of starch and sucrose has profound effects not only on the composition and structure of the polysaccharide matrix but also on gene expression of S. mutans within biofilms, which may enhance the cariogenic potential of dental biofilms.

An enzyme reactor installed with ultrafiltration membrane was developed to produce alpha-, beta-, and gamma-cyclodextrins (CDs) from soluble starch by Bacillus macerans cyclodextrin glycosyltransferase (CGTase) tagged with 10 lysines at its C-terminus (CGTK10ase). Ultrafiltration membrane YM10 with 10,000 of molecular cutoff was chosen for membrane modification and CD production. A repeated-batch type of the enzyme reaction with free CGTK10ase resulted in a alpha-CD yield of 24.0 (+/-1.5)% and a productivity of 4.68 (+/-0.88) g/l-h, which were 7 times higher that those for CGTK10ase immobilized on modified YM10 membrane. Addition of 1- nonanol increased CD yields by 30% relative to the control, which might be due to prevention of the reversible hydrolysis of CDs.

There is no systematic examination of affinity tag utility in Gram-positive bacteria, which limits the investigation of protein function in this important group of bacteria as specific antibodies for many of native proteins are generally not available. In this study, we utilized an E. coli-streptococcal shuttle vector pVT1666 and constructed two sets of expression plasmids pVPT-CTag and pVPT-NTag, with each set containing five affinity tags (GST, GFP, HSV, T7 and Nano) that can be fused to either the C- or N-terminus of a target protein. A putative glycosyltransferase (Gtf2) essential for Fap1 glycosylation was used to demonstrate the utility of the cassettes in detection of Gtf2 fusion proteins, and the biological relevance of the proteins in our working strain Streptococcus parasanguinis. GFP and T7 tags were readily expressed in S. parasanguinis as either an N- or C-terminal fusion to Gtf2. Only the C- terminal fusion of GST and HSV were able to be identified in S. parasanguinis. The Nano tag was not detected in either E. coli or S. parasanguinis. Genetic complementation experiments indicated that all the tagged Gtf2 fusion proteins could restore the Gtf2 function in the null mutant except for the Nano-tagged Gtf2 at its N-terminal fusion. Using a T7-tagged Gtf2 fusion construct, we demonstrated that the fusion cassette is also useful in detection of the fusion tag expression in other streptococci including S. mutans, S. pneumoniae and S. sanguinis. Therefore, the expression cassettes we constructed will be a useful tool not only to investigate protein-protein interactions in Fap1 biogenesis in S. parasanguinis, but also to study protein functions in other gram-positive bacteria in which pVT1666 replicates.

This study investigated the roles of sucrose synthase (SUS) in developing seeds and siliques of Arabidopsis thaliana. Enzyme activity assays showed that SUS activity was highest in developing whole siliques and young rosette leaves compared with other tissues including mature leaves, stems, and flowers. Surprisingly, quantitative PCR analyses revealed little correlation between SUS activity and transcript expression, which indicated the importance of examining the role of SUS at the protein level. Therefore, immunolocalization was performed over a developmental time course to determine the previously unreported cellular localization of SUS in Arabidopsis seed and silique tissues. At 3 d and 10 d after flowering (daf), SUS protein localized to the silique wall, seed coat, funiculus, and endosperm. By 13 daf, SUS protein was detected in the embryo and aleurone layer, but was absent from the seed coat and funiculus. Starch grains were also present in the seed coat at 3 and 10 daf, but were absent at 13 daf. Co-localization of SUS protein and starch grains in the seed coat at 3 and 10 daf indicates that SUS may be involved in temporary starch deposition during the early stages of seed development, whilst in the later stages SUS metabolizes sucrose in the embryo and cotyledon. Within the silique wall, SUS localized specifically to the companion cells, indicating that SUS activity may be required to provide energy for phloem transport activities in the silique wall. The results highlight the diverse roles that SUS may play during the development of silique and seed in Arabidopsis.

The cariogenicity of starch alone or in combination with sucrose is controversial and the effect on dentine demineralization and on the dental biofilm formed has not been explored under controlled conditions. A crossover, single-blind study was conducted in four steps of 14 days each, during which 11 volunteers wore palatal appliance containing 10 slabs of root dentine to which the following treatments were applied extraorally: 2% starch gel-like solution (starch group); 10% sucrose solution (sucrose group); a solution containing 2% starch and 10% sucrose (starch + sucrose group), or 2% starch solution followed by 10% sucrose solution (starch --> sucrose group). On the 14th day of each phase the biofilms were collected for biochemical and microbiological analyses, and dentine demineralization was assessed by hardness. A higher demineralization was found in dentine exposed to sucrose and starch sucrose combinations than to starch alone (p < 0.01), but the sucrose-containing groups did not differ significantly from each other (p > 0.05). The concentrations of soluble and insoluble extracellular polysaccharides (EPS), and the proportion of insoluble EPS, were lower in the biofilm formed in presence of starch (p < 0.01) than in those formed in the presence of sucrose or sucrose/starch combinations; however, no significant difference was observed among the groups containing sucrose (p > 0.05). RNA was successfully isolated and purified from in situ biofilms and only biofilms formed in response to sucrose and starch/sucrose combinations showed detectable levels of gtfB and gtfC mRNA. The findings suggest that the combination of starch with sucrose may not be more cariogenic to dentine than sucrose alone., (Copyright 2008 S. Karger AG, Basel.)

* The cellulose synthase (CesA) gene family encodes the catalytic subunits of a large protein complex responsible for the deposition of cellulose into plant cell walls. Early in vascular plant evolution, the gene family diverged into distinct members with conserved structures and functions (e.g. primary or secondary cell wall biosynthesis). Although the functions and expression domains of CesA genes have been extensively studied in plants, little is known about transcriptional regulation and promoter evolution in this gene family. * Here, comparative sequence analysis of orthologous CesA promoters from three angiosperm genera, Arabidopsis, Populus and Eucalyptus, was performed to identify putative cis-regulatory sequences. The promoter sequences of groups of Arabidopsis genes that are co-expressed with the primary or secondary cell wall-related CesA genes were also analyzed. * Reporter gene analysis of newly isolated promoter regions of six E. grandis CesA genes in Arabidopsis revealed the conserved functionality of the promoter sequences. Comparative sequence analysis identified 71 conserved sequence motifs, of which 66 were significantly over-represented in either primary or secondary wall-associated promoters. * The presence of conserved cis-regulatory elements in the evolutionary distant CesA promoters of Arabidopsis, Populus and Eucalyptus suggests an ancient transcriptional network regulating cellulose biosynthesis in vascular plants.

Members of a large family of cellulose synthase-like genes (CSLs) are predicted to encode glycosyl transferases (GTs) involved in the biosynthesis of plant cell walls. The CSLA and CSLF families are known to contain mannan and glucan synthases, respectively, but the products of other CSLs are unknown. Here we report the effects of disrupting ATCSLD5 expression in Arabidopsis. Both stem and root growth were significantly reduced in ATCSLD5 knock-out plants, and these plants also had increased susceptibility to the cellulose synthase inhibitor isoxaben. Antibody and carbohydrate-binding module labelling indicated a reduction in the level of xylan in stems, and in vitro GT assays using microsomes from stems revealed that ATCSLD5 knock-out plants also had reduced xylan and homogalacturonan synthase activity. Expression in Nicotiana benthamiana of ATCSLD5 and ATCSLD3, fluorescently tagged at either the C- or the N-terminal, indicated that these GTs are likely to be localized in the Golgi apparatus. However, the position of the fluorescent tag affected the subcellular localization of both proteins. The work presented provides a comprehensive analysis of the effects of disrupting ATCSLD5 in planta, and the possible role(s) of this gene and other ATCSLDs in cell wall biosynthesis are discussed.

This article describes the design and validation of a general procedure for the high-throughput isolation of amylosucrase variants displaying higher thermostability or increased resistance to organic solvents. This procedure consists of 2 successive steps: an in vivo selection that eliminates inactive variants followed by automated screening of active variants to isolate mutants displaying enhanced features. The authors chose an Escherichia coli expression vector, allowing a high production rate of the recombinant enzyme in miniaturized culture conditions. The screening assay was validated by minimizing variability for various parameters of the protocol, especially bacterial growth and protein production in cultures in 96-well microplates. Recombinant amylosucrase production was normalized by decreasing the coefficient of variance from 27% to 12.5%. Selective screening conditions were defined to select variants displaying higher thermostability or increased resistance to organic solvents. A first-generation amylosucrase variant library, constructed by random mutagenesis, was subjected to this procedure, yielding a mutant displaying a 25-fold increased stability at 50 degrees C compared to the parental wild-type enzyme.

A novel method using an HPAE-PAD system, which is routinely applied to detect carbohydrates at low levels (ng per sample injection), has been applied to the measurement of key sucrose metabolising enzyme activities in partially purified extracts of sugarcane tissues. Extraction and assay procedures tailored for the HPAE-PAD system enabled the accurate measurement of enzyme activities in more mature internodes than had previously been possible using enzyme coupled assay methodology. A major advantage of the HPAE-PAD method is the capability to monitor a broad range of sugars in each assay and provides an overarching perspective of the mix of competing enzymes that may be operating simultaneously in crude extracts. The technique has been successfully applied to measuring the activity of key sucrose metabolising enzymes in sugarcane stem tissue that is generally low in protein and high in endogenous sugars, primarily sucrose.

Bacteria-derived glucosyltransferases (Gtf) (EC 2.4.1.5), through synthesizing glucan polymers from sucrose and starch hydrolysates, play an essential role in the etiology and pathogenesis of caries. We attempted to correlate the levels of Gtf in whole saliva with the prevalence of carious lesions in young children. We examined saliva from children who were either free of overt carious lesions, or had severe early childhood caries (mean dmfs = 18.72 +/- 9.0 SD), for Gtf by direct enzyme assay. The levels of GtfB, GtfC and GtfD from Streptococcus mutans in the saliva using monoclonal/specific antibodies in an enzyme-linked immunosorbent assay were determined. Multiple logistic regression analyses with model selection showed that GtfB levels correlated with dmfs values of the subjects (p = 0.006). There was no correlation between total Gtf activity as measured by direct enzyme assay and dmfs values. There was a strong correlation between mutans streptococci populations in saliva and caries activity. Collectively, these data show that GtfB levels in saliva correlate strongly with presence of clinical caries and with number of carious lesions in young children. It is also possible to measure different Gtfs, separately, in whole saliva. These observations may have important clinical implications, may lead to development of a chair side caries activity test and support the importance of GtfB in the pathogenesis of dental caries., ((c) 2007 S. Karger AG, Basel)

New aqueous-aqueous two-phase systems composed of relatively low molecular weight polymers such as polyethylene glycol (PEG) (Mr: 1000-4000) and dextran (Mr: 10,000 and 40,000) were evaluated for purification of proteins by counter-current chromatography (CCC). The compositions of aqueous two-phase systems were optimized by measuring parameters such as viscosity and volume ratio between the two phases. CCC purification of a glucosyltransferase (GTF) from Streptococcus mutans (SM) cell-lysate was successfully demonstrated with a 7.5% PEG 3350-10% dextran T40 system containing 10mM potassium phosphate buffer at pH 9.0. After CCC purification, both PEG and dextran contained in the CCC fractions were easily removed by ultrafiltration in a short period of time. The fractionated column contents containing GTF were analyzed by enzymatic activity as well as sodium dodecyl sulfate (SDS) polyacrylamide gel electrophoresis. The recovery of the enzyme from CCC fraction was over 95% as estimated by enzymatic activities.