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Frozen aquatic products undergo unavoidable quality changes owing to temperature fluctuations during frozen storage and distribution. This study investigated the effects of 1% cellobiose (CB), and 0.5 and 1% carboxylated cellulose nanofibers (CNF) on ice crystal growth and recrystallization of frozen large yellow croaker fillets exposed to temperature fluctuations. Denser and more uniformly distributed ice crystals were observed in the CB- and CNF-treated samples than in the water-treated samples. Furthermore, the addition of CB and CNF suppressed the conversion of bound water to frozen water in the samples during temperature fluctuation cycles, played a positive role in fixing the ionic and hydrogen bonds that stabilize the protein structure, limited the conformational transition from α-helix to β-sheet, and improved protein thermal stability. Based on turbidity, zeta potential, and confocal laser scanning microscopy (CLSM) analyses, the presence of CB and CNF restricted the protein aggregation. Compared with CB, CNF molecules with abundant carboxyl functional groups and longer morphology exhibited better cryoprotective effects. Moreover, the fillets were more improved protected from mechanical damage induced by large ice crystals at a higher CNF concentration. This study reveals the potential of CB and CNF as novel cryoprotectants., (Copyright © 2021 Elsevier B.V. All rights reserved.)

The development of spots or lesions symptomatic of common scab on root and tuber crops is caused by few pathogenic Streptomyces with Streptomyces scabiei 87-22 as the model species. Thaxtomin phytotoxins are the primary virulence determinants, mainly acting by impairing cellulose synthesis, and their production in S. scabiei is in turn boosted by cello-oligosaccharides released from host plants. In this work we aimed to determine which molecules and which biosynthetic gene clusters (BGCs) of the specialized metabolism of S. scabiei 87-22 show a production and/or a transcriptional response to cello-oligosaccharides. Comparative metabolomic analyses revealed that molecules of the virulome of S. scabiei induced by cellobiose and cellotriose include (i) thaxtomin and concanamycin phytotoxins, (ii) desferrioxamines, scabichelin and turgichelin siderophores in order to acquire iron essential for housekeeping functions, (iii) ectoine for protection against osmotic shock once inside the host, and (iv) bottromycin and concanamycin antimicrobials possibly to prevent other microorganisms from colonizing the same niche. Importantly, both cello-oligosaccharides reduced the production of the spore germination inhibitors germicidins thereby giving the 'green light' to escape dormancy and trigger the onset of the pathogenic lifestyle. For most metabolites - either with induced or reduced production - cellotriose was revealed to be a slightly stronger elicitor compared to cellobiose, supporting an earlier hypothesis which suggested the trisaccharide was the real trigger for virulence released from the plant cell wall through the action of thaxtomins. Interestingly, except for thaxtomins, none of these BGCs' expression seems to be under direct control of the cellulose utilization repressor CebR suggesting the existence of a yet unknown mechanism for switching on the virulome. Finally, a transcriptomic analysis revealed nine additional cryptic BGCs that have their expression awakened by cello-oligosaccharides, suggesting that other and yet to be discovered metabolites could be part of the virulome of S. scabiei .

Three new spherical sulfated cellobiose-polylysine dendrimers of increasing generations bearing negatively charged sulfate groups were prepared by sulfating the corresponding cellobiose-polylysine dendrimers. The first, second, and third-generation derivatives exhibited potent anti-HIV activity with EC 50 values of 3.7, 0.6, and 1.5 μg/mL, respectively, in constant to sulfated oligosaccharides with low anti-HIV activity, while the second-generation sulfated dendrimer was the most active. Surface plasmon resonance measurements with poly-l-lysine bearing positively charged amino acids as a model of the HIV surface glycoprotein gp120, indicated that the second-generation dendrimer had the lowest dissociation constant (K D  = 1.86 × 10 -12  M). Both the particle size and ζ potential increased in the presence of poly-l-lysine. It was proven that the moderate distance between the terminal sulfated cellobiose units in the second-generation dendrimer favored the high anti-HIV activity, owing to the electrostatic interactions developed due to the cluster effect., (Copyright © 2020. Published by Elsevier Ltd.)

Dark fermentative biohydrogen (H 2 ) production could become a key technology for providing renewable energy. Until now, the H 2 yield is restricted to 4 moles of H 2 per mole of glucose, referred to as the "Thauer limit". Here we show, that precision design of artificial microbial consortia increased the H 2 yield to 5.6 mol mol -1 glucose, 40% higher than the Thauer limit. In addition, the volumetric H 2 production rates of our defined artificial consortia are superior compared to any mono-, co- or multi-culture system reported to date. We hope this study to be a major leap forward in the engineering of artificial microbial consortia through precision design and provide a breakthrough in energy science, biotechnology and ecology. Constructing artificial consortia with this drawing-board approach could in future increase volumetric production rates and yields of other bioprocesses. Our artificial consortia engineering blueprint might pave the way for the development of a H 2 production bioindustry.

Streptomyces scabies is responsible for common scab disease on root and tuber vegetables. Production of its main phytotoxin thaxtomin A is triggered upon transport of cellulose byproducts cellotriose and cellobiose, which disable the repression of the thaxtomin biosynthesis activator gene txtR by the cellulose utilization regulator CebR. To assess the intracellular response under conditions where S. scabies develops a virulent behavior, we performed a comparative proteomic analysis of wild-type S. scabies 87-22 and its cebR null mutant (hyper-virulent phenotype) grown in the absence or presence of cellobiose. Our study revealed significant changes in abundance of proteins belonging to metabolic pathways known or predicted to be involved in pathogenicity of S. scabies. Among these, we identified proteins of the cello-oligosaccharide-mediated induction of thaxtomin production, the starch utilization system required for utilization of the carbohydrate stored in S. scabies's hosts, and siderophore synthesis utilization systems, which are key features of pathogens to acquire iron once they colonized the host. Thus, proteomic analysis supported by targeted mass spectrometry-based metabolite quantitative analysis revealed the central role of CebR as a regulator of virulence of S. scabies.

The aim of this study was to examine whether the combination of dietary soluble fiber and cellobiose exerts a synergistic effect on growth performance, health status, fermentation traits, and immune response in rabbits. Six treatments in a 3 × 2 factorial arrangement were used: 3 cellobiose concentrations in drinking water (0.0, 7.5, and 15.0 g/L) × 2 dietary levels of soluble fiber (84.0 and 130 g/kg DM, for the low soluble fiber [LSF] and high soluble fiber [HSF] diets, respectively). A total of 318 young rabbits (53/treatment) were weaned at 34 d of age and had ad libitum access to feed and water. At 46 d of age, 9 rabbits/treatment were slaughtered and ileal and cecal digesta were collected to analyze VFA profile and the immune response in the cecal appendix mucosa. At 48 d of age, the cellobiose supplementation was withdrawn and the experimental diets were replaced by a standard commercial diet until 61 d of age. From 34 to 48 d of age, there was a linear increase of mortality with the level of cellobiose in the HSF group (0% vs. 17.1%; P = 0.017). In contrast, a quadratic effect of cellobiose level on mortality was observed in the LSF group, the rabbits offered 7.5-cellobiose showing the lowest mortality (5.7% vs. 21.4%; P = 0.030). Cellobiose level had a quadratic effect on ADFI, ADG, and G:F in this period (P ≤ 0.047), with the 7.5-cellobiose groups having the best growth performance. In contrast, only minor changes on these traits were observed from 48 d of age onwards. Cellobiose level influenced quadratically the ileal VFA concentrations (P = 0.014), showing the maximal value in the 7.5-cellobiose groups. In rabbits fed 7.5-cellobiose-LSF, a change of acetate to propionate, butyrate, and valerate was observed in the ileum. Increasing cellobiose levels reduced linearly cecal VFA concentrations in HSF fed rabbits, but no effect was detected in LSF groups (P = 0.046). The level of soluble fiber increased VFA concentrations in both the ileum (by 22%; P < 0.001), and the cecum (by 11%; P = 0.005). The relative gene expression of IL-6, IL-10, TNF-α, iNOS, MUC-1, and toll-like receptors (TLR-2 and TLR-4) in the cecal appendix increased linear and quadratically with increasing levels of cellobiose (P ≤ 0.063). In conclusion, in rabbits fed LSF diets, a dose of 7.5 g cellobiose/L drinking water would be recommended, whereas these levels of cellobiose supplementation should be avoided in rabbits fed HSF diets.

Neurotoxic methylmercury (MeHg) is produced by anaerobic Bacteria and Archaea possessing the genes hgcAB , but it is unknown how organic substrate and electron acceptor availability impacts the distribution and abundance of these organisms. We evaluated the impact of organic substrate amendments on mercury (Hg) methylation rates, microbial community structure, and the distribution of hgcAB + microbes with sediments. Sediment slurries were amended with short-chain fatty acids, alcohols, or a polysaccharide. Minimal increases in MeHg were observed following lactate, ethanol, and methanol amendments, while a significant decrease (∼70%) was observed with cellobiose incubations. Postincubation, microbial diversity was assessed via 16S rRNA amplicon sequencing. The presence of hgcAB + organisms was assessed with a broad-range degenerate PCR primer set for both genes, while the presence of microbes in each of the three dominant clades of methylators ( Deltaproteobacteria , Firmicutes , and methanogenic Archaea ) was measured with clade-specific degenerate hgcA quantitative PCR (qPCR) primer sets. The predominant microorganisms in unamended sediments consisted of Proteobacteria , Firmicutes , Bacteroidetes , and Actinobacteria Clade-specific qPCR identified hgcA + Deltaproteobacteria and Archaea in all sites but failed to detect hgcA + Firmicutes Cellobiose shifted the communities in all samples to ∼90% non- hgcAB -containing Firmicutes (mainly Bacillus spp. and Clostridium spp.). These results suggest that either expression of hgcAB is downregulated or, more likely given the lack of 16S rRNA gene presence after cellobiose incubation, Hg-methylating organisms are largely outcompeted by cellobiose degraders or degradation products of cellobiose. These results represent a step toward understanding and exploring simple methodologies for controlling MeHg production in the environment. IMPORTANCE Methylmercury (MeHg) is a neurotoxin produced by microorganisms that bioacummulates in the food web and poses a serious health risk to humans. Currently, the impact that organic substrate or electron acceptor availability has on the mercury (Hg)-methylating microorganisms is unclear. To study this, we set up microcosm experiments exposed to different organic substrates and electron acceptors and assayed for Hg methylation rates, for microbial community structure, and for distribution of Hg methylators. The sediment and groundwater was collected from East Fork Poplar Creek in Oak Ridge, TN. Amendment with cellobiose (a lignocellulosic degradation by-product) led to a drastic decrease in the Hg methylation rate compared to that in an unamended control, with an associated shift in the microbial community to mostly nonmethylating Firmicutes This, along with previous Hg-methylating microorganism identification methods, will be important for identifying strategies to control MeHg production and inform future remediation strategies., (Copyright © 2018 Christensen et al.)

Tritrophic interactions involving a biocontrol agent, a pathogen and a plant have been analyzed predominantly from the perspective of the biocontrol agent. We have conducted the first comprehensive transcriptomic analysis of all three organisms in an effort to understand the elusive properties of Pseudozyma flocculosa in the context of its biocontrol activity against Blumeria graminis f.sp. hordei as it parasitizes Hordeum vulgare. After inoculation of P. flocculosa, the tripartite interaction was monitored over time and samples collected for scanning electron microscopy and RNA sequencing. Based on our observations, P. flocculosa indirectly parasitizes barley, albeit transiently, by diverting nutrients extracted by B. graminis from barley leaves through a process involving unique effectors. This brings novel evidence that such molecules can also influence fungal-fungal interactions. Their release is synchronized with a higher expression of powdery mildew haustorial effectors, a sharp decline in the photosynthetic machinery of barley and a developmental peak in P. flocculosa. The interaction culminates with a collapse of B. graminis haustoria, thereby stopping P. flocculosa growth, as barley plants show higher metabolic activity. To conclude, our study has uncovered a complex and intricate phenomenon, described here as hyperbiotrophy, only achievable through the conjugated action of the three protagonists., (© 2017 The Authors. New Phytologist © 2017 New Phytologist Trust.)

Fermentation with acetogens can be affected by cultivation gas phase, but to date, there is not enough evidence on that matter for Clostridium thermocellum and Moorella thermoacetica. In this work, the effects of sparged CO 2 as well as sparged and non-sparged N 2 on these microorganisms were studied using glucose and cellobiose as substrates. It was revealed that sparged CO 2 and non-sparged N 2 supported growth and acetic acid production by C. thermocellum and M. thermoacetica, while sparged N 2 inhibited both of the microorganisms. Notably, part of the sparged CO 2 was fermented by the co-culture system and contributed to an overestimation of the products from the actual substrate as well as an erring material balance. The best condition for the co-culture was concluded to be N 2 without sparging. These results demonstrate the importance of cultivation conditions for efficient fermentation by anaerobic clostridia species.

Synbiotics are always considered to be beneficial in healthy manipulation of gut environment; however, the purpose of this research was to investigate the dominance of synbiotic over the individual potential of probiotics and prebiotics. Four different types of prebiotics, fructo-oligosaccharides, raffinose, inulin, and cellobiose, were evaluated based on their varying degree of polymerization, combined each with 2 different Lactobacilli strains, including Lactobacillus paracasei 276 and Lactobacillus plantarum WCFS1. The effects of synbiotics combination on the surface structure were evaluated by analyzing auto-aggregation, membrane hydrophobicity, and adhesion to Caco-2 cells. Our results showed that both Lactobacilli exhibited significantly greater degree of attachment to Caco-2 cells (23.31% and 16.85%, respectively) when using cellobiose as a substrate than with other prebiotics (P < 0.05). Intestinal adhesion ability was in correlation with the percent of auto-aggregation, both Lactobacillus exhibited higher percent of auto-aggregation in cellobiose compared to other prebiotics. These behavioral changes in terms of attachment and auto-aggregation were further supported with the changes noticed from infrared spectra (FT-IR)., (© 2017 Institute of Food Technologists®.)

Shewanella sp. G5, a psychrotolerant marine bacterium, has a cold-shock protein (CspA) and three β-glucosidases, two of which were classified in the glycosyl hydrolase families 1 and 3 and are encoded by bgl-A and bgl genes, respectively. Shewanella sp. G5 was cultured on Luria-Bertani (LB) and Mineral Medium Brunner (MMB) media with glucose and cellobiose at various temperatures and pH 6 and 8. Relative quantification of the expression levels of all three genes was studied by real-time PCR with the comparative Ct method (2(-ΔΔCt)) using the gyrB housekeeping gene as a normalizer. Results showed that the genes had remarkably different genetic expression levels under the conditions evaluated, with increased expression of all genes obtained on MMB with cellobiose at 30 °C. Specific growth rate and specific β-glucosidase activity were also determined for all the culture conditions. Shewanella sp. G5 was able to grow on both media at 4 °C, showing the maximum specific growth rate on LB with cellobiose at 37 °C. The specific β-glucosidase activity obtained on MMB with cellobiose at 30 °C was 25 to 50 % higher than for all other conditions. At pH 8, relative activity was 34, 60, and 63 % higher at 30 °C than at 10 °C, with three peaks at 10, 25, and 37 °C on both media. Enzyme activity increased by 61 and 47 % in the presence of Ca(2+) and by 24 and 31 % in the presence of Mg(2+) on LB and MMB at 30 °C, respectively, but it was totally inhibited by Hg(2+), Cu(2+), and EDTA. Moreover, this activity was slightly decreased by SDS, Zn(2+), and DTT, all at 5 mM. Ethanol (14 % v/v) and glucose (100 mM) also reduced the activity by 63 and 60 %, respectively.

Responses in the amiloride-insensitive (AI) pathway, one of the two pathways mediating salty taste in mammals, are modulated by the size of the anion of a salt. This "anion effect" has been hypothesized to result from inhibitory transepithelial potentials (TPs) generated across the lingual epithelium as cations permeate through tight junctions and leave their larger and less permeable anions behind (Ye et al., 1991). We tested directly the necessity of TPs for the anion effect by measuring responses to NaCl and Na-gluconate (small and large anion sodium salts, respectively) in isolated taste cells from mouse circumvallate papillae. Using calcium imaging, we identified AI salt-responsive type III taste cells and demonstrated that they compose a subpopulation of acid-responsive taste cells. Even in the absence of TPs, many (66%) AI salt-responsive type III taste cells still exhibited the anion effect, demonstrating that some component of the transduction machinery for salty taste in type III cells is sensitive to anion size. We hypothesized that osmotic responses could explain why a minority of type III cells (34%) had AI salt responses but lacked anion sensitivity. All AI type III cells had osmotic responses to cellobiose, which were significantly modulated by extracellular sodium concentration, suggesting the presence of a sodium-conducting osmotically sensitive ion channel. However, these responses were significantly larger in AI type III cells that did not exhibit the anion effect. These findings indicate that multiple mechanisms could underlie AI salt responses in type III taste cells, one of which may contribute to the anion effect., Significance Statement: Understanding the mechanisms underlying salty taste will help inform strategies to combat the health problems associated with NaCl overconsumption by humans. Of the two pathways underlying salty taste in mammals, the amiloride-insensitive (AI) pathway is the least understood. Using calcium imaging of isolated mouse taste cells, we identify two separate populations of AI salt-responsive type III taste cells distinguished by their sensitivity to anion size and show that these cells compose subpopulations of acid-responsive taste cells. We also find evidence that a sodium-conducting osmotically sensitive mechanism contributes to salt responses in type III taste cells. Our data not only provide new insights into the transduction mechanisms of AI salt taste but also have important implications for general theories of taste encoding., (Copyright © 2016 the authors 0270-6474/16/361942-12$15.00/0.)

Thermal inactivation kinetics of Hypocrea orientalis β-glucosidase and effect of glucose on thermostability of the enzyme have been determined in this paper. Kinetic studies showed that the thermal inactivation was irreversible and first-order reaction. The microscopic rate constants for inactivation of free enzyme and substrate-enzyme complex were both determined, which suggested that substrates can protect β-glucosidase against thermal deactivation effectively. On the other hand, glucose was found to protect β-glucosidase from heat inactivation to remain almost whole activity below 70°C at 20mM concentration, whereas the apparent inactivation rate of BG decreased to be 0.3×10(-3)s(-1) in the presence of 5mM glucose, smaller than that of sugar-free enzyme (1.91×10(-3)s(-1)). The intrinsic fluorescence spectra results showed that glucose also had stabilizing effect on the conformation of BG against thermal denaturation. Docking simulation depicted the interaction mode between glucose and active residues of the enzyme to produce stabilizing effect., (Copyright © 2015 Elsevier B.V. All rights reserved.)

Clostridium termitidis CT1112 is an anaerobic, mesophilic, cellulolytic bacterium with potential applications in consolidated bioprocessing of lignocellulosic biomass. To understand how C. termitidis degrades lignocellulose, iTRAQ-based 2D HPLC-MS/MS proteomics was used to measure protein expression in cell lysates and extracellular (secretome) fractions of C. termitidis grown on α-cellulose and cellobiose at both exponential and stationary growth phases. Exoglucanases (GH48, GH9), endoglucanases (GH5, GH8, GH9), hemicellulases including xylanases (GH8, GH10, GH11, GH30) and mannanase (GH26) as well as extracellular adhesion proteins and cellulosome associated proteins, exhibited higher expression on cellulose-grown cells. The expression of these proteins increased with a decrease in growth rate. Non-cellulosomal proteins however did not change significantly between substrate conditions, although there were a few exceptions. Collectively, these would contribute to hydrolysis of lignocellulosic material for uptake through ABC sugar transport proteins. On cellobiose, chitinases (GH18) were expressed abundantly. Although a large number of proteins were shared between the fractions analyzed, some proteins were detected exclusively in the cellular fraction, while others were detected in the secretome. This study reports for the first time on the cellulolytic machinery employed by C. termitidis to hydrolyze cellulosic substrate and provides an understanding of how this microbe deconstructs biomass., Biological Significance: The genome of C. termitidis CT1112 contains genes for a wide variety of carbohydrate active enzymes. Based on bioinformatics analyses, many of these genes appear to encode cellulosome-associated proteins, while others may be secreted extracellularly. To understand how C. termitidis degrades and depolymerizes cellulosic substrates, cells were grown on simple and complex carbohydrates, and quantitative 4-plex iTRAQ-based 2D HPLC-MS/MS proteomics was applied to measure protein expression levels in biological replicates of both cell lysates and extracellular protein (secretome) fractions, at exponential and stationary phases of growth. The resulting data have provided insight into the range of substrates that may be hydrolyzed by C. termitidis, and may be useful in determining potential industrial applications of C. termitidis in biomass to bioenergy production via consolidated bioprocessing., (Copyright © 2015 Elsevier B.V. All rights reserved.)

In this work, a newly isolated marine thraustochytrid strain, Schizochytrium sp. DT3, was used for omega-3 fatty acid production by growing on lignocellulose biomass obtained from local hemp hurd (Cannabis sativa) biomass. Prior to enzymatic hydrolysis, hemp was pretreated with sodium hydroxide to open the biomass structure for the production of sugar hydrolysate. The thraustochytrid strain was able to grow on the sugar hydrolysate and accumulated polyunsaturated fatty acids (PUFAs). At the lowest carbon concentration of 2%, the PUFAs productivity was 71% in glucose and 59% in the sugars hydrolysate, as a percentage of total fatty acids. Saturated fatty acids (SFAs) levels were highest at about 49% of TFA using 6% glucose as the carbon source. SFAs of 41% were produced using 2% of SH. This study demonstrates that SH produced from lignocellulose biomass is a potentially useful carbon source for the production of omega-3 fatty acids in thraustochytrids, as demonstrated using the new strain, Schizochytrium sp. DT3., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Probiotics, prebiotics, and combinations thereof, that is synbiotics, have been reported to modulate gut microbiota of humans. In this study, effects of a novel synbiotic on the composition and metabolic activity of human gut microbiota were investigated. Healthy volunteers (n = 18) were enrolled in a double-blinded, randomized, and placebo-controlled cross-over study and received synbiotic [Lactobacillus acidophilus NCFM (10(9) CFU) and cellobiose (5 g)] or placebo daily for 3 weeks. Fecal samples were collected and lactobacilli numbers were quantified by qPCR. Furthermore, 454 tag-encoded amplicon pyrosequencing was used to monitor the effect of synbiotic on the composition of the microbiota. The synbiotic increased levels of Lactobacillus spp. and relative abundances of the genera Bifidobacterium, Collinsella, and Eubacterium while the genus Dialister was decreased (P < 0.05). No other effects were found on microbiota composition. Remarkably, however, the synbiotic increased concentrations of branched-chain fatty acids, measured by gas chromatography, while short-chain fatty acids were not affected., (© 2014 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved.)

4'-O-β-d-Glucopyranosyl-quercetin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyra-noside (3) was isolated from Helminthostachys zeylanica root extract as a melanogenesis acceleration compound and was synthesized using rutin as the starting material. Related compounds were also synthesized to understand the structure-activity relationships in melanin biosynthesis. Melanogenesis activities of the glycosides were determined by measuring intracellular melanin content in B16 melanoma cells. Among the synthesized quercetin glycosides, quercetin-3-O-β-d-glucopyranoside (1), quercetin-3-O-β-d-glucopyranosyl-(1→4)-β-d-glucopyranoside (2), and 3 showed more potent intracellular melanogenesis acceleration activities than theophyline used as positive control in a dose-dependent manner with no cytotoxic effect., (Copyright © 2014. Published by Elsevier Ltd.)

Antibiotic activity was compared for Cryptococcus humicola cellobiose lipids, the mixture of 2,3,4-О-triacetyl-β-D-glucopyranosyl-(1→4)-(6-О-acetyl-β-D-glucopyranosyl-(1→16)-2,16-dihydroxyhexodecanoic acid and 2,3,4-О-triacetyl-β-D-glucopyranosyl-(1→4)-(6-О-acetyl-β-D-glucopyranosyl-(1→16)-2,17,18-trihydroxyoctotodecanoic acid, and the commercial sophorose lipid mixture of a mono-acetylated acidic sophorose lipid and a di-acetylated acidic sophorose lipid, both containing the C18:1 fatty acid residue. The MIC values of cellobiose lipids were 0.005 and 0.04 mg/mL for Filobasidiella neoformans and Candida tropicalis, respectively. The MIC values of sophorose lipids were 1 and 15 mg/mL for F. neoformans and C. tropicalis, respectively. MIC values for some bacteria were in the range of 10-30 mg/mL for both glycolipid preparations. Both sophorose lipids and cellobiose lipids displayed a membrane-damaging activity against F. neoformans. The treatment with these glycolipids reduces the content of ATP in the cells of test cultures and results in their staining with ethidium bromide.

In this study, cellobiose, a reducing disaccharide was used to produce succinic acid by Actinobacillus succinogenes NJ113. A final succinic acid concentration of 30.3g/l with a yield of 67.8% was achieved from an initial cellobiose concentration of 50 g/l via batch fermentation in anaerobic bottles. The cellobiose uptake mechanism was investigated and the results of enzyme assays revealed that the phosphoenolpyruvate phosphotransferase system (PEP-PTS) played an important role in the cellobiose uptake process. In batch fermentation with 18 g/l of cellobiose and 17 g/l of other sugars from sugarcane bagasse cellulose hydrolysates, a succinic acid concentration of 20.0 g/l was obtained, with a corresponding yield of 64.7%. This study found that cellobiose from incomplete hydrolysis of cellulose could be a potential carbon source for economical and efficient succinic acid production by A. succinogenes., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Product inhibition of cellulolytic enzymes has been deemed a critical factor in the industrial saccharification of cellulosic biomass. Several investigations have addressed this problem using crude enzyme preparations or commercial (mixed) cellulase products, but quantitative information on individual cellulases hydrolyzing insoluble cellulose remains insufficient. Such knowledge is necessary to pinpoint and quantify inhibitory weak-links in cellulose hydrolysis, but has proven challenging to come by. Here we show that product inhibition of mono-component cellulases hydrolyzing unmodified cellulose may be monitored by calorimetry. The key advantage of this approach is that it directly measures the rate of hydrolysis while being essentially blind to the background of added product. We investigated the five major cellulases from Hypocrea jecorina (anamorph: Tricoderma reesei), Cel7A (formerly CBH1), Cel6A (CBH2), Cel7B (EG1), Cel5A (EG2) and Cel12A (EG3), for their sensitivity to the products glucose and cellobiose. The strongest inhibition was found for Cel7A, which showed a 50% activity-loss in 19 mM cellobiose (IC(50)=19 mM). The other exoglucanase, Cel6A, was much less inhibited by cellobiose, but showed the highest sensitivity to glucose among all investigated enzymes. The endoglucanases Cel12A and Cel7B were moderately inhibited by cellobiose (IC(50)=60-80 mM), and weakly inhibited by glucose (IC(50)=350-380 mM). The highest resistance to both products was found for Cel5A, which retained about 75% of its activity at the highest investigated concentrations (respectively 65 mM cellobiose and 1000 mM glucose)., (Copyright © 2013 Elsevier Inc. All rights reserved.)

Hydrolysates of lignocellulosic biomass contain glucose, xylose, arabinose, cellobiose, among other sugars. Effective utilization of these sugars remains challenging for microbial conversion, because most microorganisms consume such sugars sequentially with a strong preference for glucose. In the present study, the oleaginous yeast, Lipomyces starkeyi, was shown to consume cellobiose and xylose simultaneously and to produce intracellular lipids from cellobiose, xylose and glucose. In flask cultures with glucose, cellobiose or a mixture of cellobiose/xylose as carbon sources, overall substrate consumption rates were close to 0.6 g/L/h, and lipid coefficients were 0.19 g lipid/g sugar, respectively. This cellobiose/xylose co-fermentation strategy provides an opportunity to efficiently utilize lignocellulosic biomass for microbial lipid production, which is important for biorefinery and biofuel production., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

Neurospora crassa colonizes burnt grasslands in the wild and metabolizes both cellulose and hemicellulose from plant cell walls. When switched from a favored carbon source such as sucrose to cellulose, N. crassa dramatically upregulates expression and secretion of a wide variety of genes encoding lignocellulolytic enzymes. However, the means by which N. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. Here, we show that an N. crassa mutant carrying deletions of two genes encoding extracellular β-glucosidase enzymes and one intracellular β-glucosidase lacks β-glucosidase activity, but efficiently induces cellulase gene expression in the presence of cellobiose, cellotriose, or cellotetraose as a sole carbon source. These data indicate that cellobiose, or a modified version of cellobiose, functions as an inducer of lignocellulolytic gene expression in N. crassa. Furthermore, the inclusion of a deletion of the catabolite repressor gene, cre-1, in the triple β-glucosidase mutant resulted in a strain that produces higher concentrations of secreted active cellulases on cellobiose. Thus, the ability to induce cellulase gene expression using a common and soluble carbon source simplifies enzyme production and characterization, which could be applied to other cellulolytic filamentous fungi.

Aims: The effects of different freeze-drying protective agents on the viabilities of biocontrol strains Bacillus cereus AR156, Burkholderia vietnamiensis B418 and Pantoea agglomerans 2Re40 were investigated., Method and Results: Several concentrations of protective and rehydration media were tested to improve the survival of biocontrol agents after freeze-drying. The subsequent survival rates during storage and rehydration media of freeze-dried biocontrol strains were also examined., Conclusions: The results indicated that cellobiose (5%) and d-galactose (5%) gave maximum viability of strains Bu. vietnamiensis B418 and P. agglomerans 2Re40 (98 and 54·3% respectively) while the perfect one (100%) of strain B. cereus AR156 was obtained with sucrose (5%) during freeze-drying, and the highest survival of the three strains was reached when they were rehydrated with 10% nonfat skim milk. In the following storage, the survival rates showed that B. cereus AR156 could still reach 50% after 12 months., Significance and Impact of the Study: This study showed that freeze-drying could be used to stabilize cells of these three biocontrol strains. Further studies should focus on the scale-up possibilities and formulation development., (© Nanjing Agricultural University. Letters in Applied Microbiology © 2011 The Society for Applied Microbiology.)

Our lab and most others have not been able to close a carbon balance for fermentation by the thermophilic, cellulolytic anaerobe, Clostridium thermocellum. We undertook a detailed accounting of product formation in C. thermocellum ATCC 27405. Elemental analysis revealed that for both cellulose (Avicel) and cellobiose, ≥92% of the substrate carbon utilized could be accounted for in the pellet, supernatant and off-gas when including sampling. However, 11.1% of the original substrate carbon was found in the liquid phase and not in the form of commonly-measured fermentation products--ethanol, acetate, lactate, and formate. Further detailed analysis revealed all the products to be <720 da and have not usually been associated with C. thermocellum fermentation, including malate, pyruvate, uracil, soluble glucans, and extracellular free amino acids. By accounting for these products, 92.9% and 93.2% of the final product carbon was identified during growth on cellobiose and Avicel, respectively., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The human gastrointestinal tract can be positively modulated by dietary supplementation of probiotic bacteria in combination with prebiotic carbohydrates. Here differential transcriptomics and functional genomics were used to identify genes in Lactobacillus acidophilus NCFM involved in the uptake and catabolism of 11 potential prebiotic compounds consisting of α- and β-linked galactosides and glucosides. These oligosaccharides induced genes encoding phosphoenolpyruvate-dependent sugar phosphotransferase systems (PTS), galactoside pentose hexuronide (GPH) permease, and ATP-binding cassette (ABC) transporters. PTS systems were upregulated primarily by di- and tri-saccharides such as cellobiose, isomaltose, isomaltulose, panose and gentiobiose, while ABC transporters were upregulated by raffinose, Polydextrose, and stachyose. A single GPH transporter was induced by lactitol and galactooligosaccharides (GOS). The various transporters were associated with a number of glycoside hydrolases from families 1, 2, 4, 13, 32, 36, 42, and 65, involved in the catabolism of various α- and β-linked glucosides and galactosides. Further subfamily specialization was also observed for different PTS-associated GH1 6-phospho-β-glucosidases implicated in the catabolism of gentiobiose and cellobiose. These findings highlight the broad oligosaccharide metabolic repertoire of L. acidophilus NCFM and establish a platform for selection and screening of both probiotic bacteria and prebiotic compounds that may positively influence the gastrointestinal microbiota.

Biological utilization of cellulose is a complex process involving the coordinated expression of different cellulases, often in a synergistic manner. One possible means of inducing an organism-level change in cellulase activity is to use laboratory adaptive evolution. In this study, evolved strains of the cellulolytic actinobacterium, Thermobifida fusca, were generated for two different scenarios: continuous exposure to cellobiose (strain muC) or alternating exposure to cellobiose and glucose (strain muS). These environmental conditions produced a phenotype specialized for growth on cellobiose (muC) and an adaptable, generalist phenotype (muS). Characterization of cellular phenotypes and whole genome re-sequencing were conducted for both the muC and muS strains. Phenotypically, the muC strain showed decreased cell yield over the course of evolution concurrent with decreased cellulase activity, increased intracellular ATP concentrations, and higher end-product secretions. The muS strain increased its cell yield for growth on glucose and exhibited a more generalist phenotype with higher cellulase activity and growth capabilities on different substrates. Whole genome re-sequencing identified 48 errors in the reference genome and 18 and 14 point mutations in the muC and muS strains, respectively. Among these mutations, the site mutation of Tfu&#95;1867 was found to contribute the specialist phenotype and the site mutation of Tfu&#95;0423 was found to contribute the generalist phenotype. By conducting and characterizing evolution experiments on Thermobifida fusca, we were able to show that evolutionary changes balance ATP energetic considerations with cellulase activity. Increased cellulase activity is achieved in stress environments (switching carbon sources), otherwise cellulase activity is minimized to conserve ATP.

Stachybotrys microspora is a filamentous fungus that produces various β-glucosidases, of which two have already been characterized. The present study reports on the production of a third one, named bglG, in the presence of d-glucose used as a sole carbon source, and on its subsequent purification and characterization. Although efficiently produced in the presence of d-glucose, bglG continues to be highly inhibited by this sugar. In fact, the addition of d-glucose significantly decreases the glucose formation rates during the hydrolysis of pNPG. This work reports on the effect of various carbohydrates on bglG activity in order to understand the mechanisms adopted by d-glucose to inhibit this enzyme. The findings indicate that bglG is strongly inhibited by d-glucose (44% of the relative activity at 5mM), d-glucitol (96% of the relative activity), d-mannose (56% of the relative activity), cellobiose and maltose (72% and 71% of the relative activity, respectively). On the other hand, d-galactose, d-fructose, lactose, and sucrose have no effect on bglG activity. Similarly, several isomers, such as 2-acetamido-2-deoxy-d-glucose and 2-deoxy-d-arabino-hexose (2-deoxy-d-glucose) were noted to bring no change on the relative activity of bglG. d-xylose and xylitol, on the other hand, enhanced bglG activity up to 123% and 120% of relative activity, respectively. Accordingly, the configuration, epimerisation, isomerisation, and substitutions played key roles in bglG inhibition. The effect of the combination of iron (the best activator of bglG, 161%) with some of those additives was also investigated. The findings revealed that, while a combination of iron at a concentration of 10mM with d-glucose resulted in a two-fold decrease in bglG inhibition (84% at 5mM), iron maintained the same effect with the remainder of the additives being tested., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

The hydrolysis of p-nitrophenyl-β-1,4-cellobioside (pNP-G2) by the catalytic domain of the retaining-family 5-2 endocellulase Cel5A from Thermobifida fusca (Cel5Acd) was studied. The dominant reaction pathway involves hydrolysis of the aglyconic bond, producing cellobiose (G2) and a 'reporter' species p-nitrophenol (pNP), which was monitored spectrophotometrically to track the reaction. We also detected the production of cellotriose (G3) and p-nitrophenyl-glucoside (pNP-G1), confirming the presence of a competing transglycosylation pathway. We use a mechanistic model of hydrolysis and transglycosylation to derive an expression for the rate of pNP-formation as a function of enzyme concentration, substrate concentration, and several lumped kinetics parameters. The derivation assumes that the quasi-steady-state assumption (QSSA) applies for three intermediate species in the mechanism; we determine conditions under which this assumption is rigorously justified. We integrate the rate expression and compare its integral form to pNP-versus-time data collected for a range of enzyme and substrate concentrations. The integral comparison gives a stringent test of the mechanistic model, and it serves to quantify the lumped kinetics parameters with good statistical precision, particularly a previously unidentified parameter that determines the selectivity of hydrolysis versus transglycosylation. The integrated rate expression accounts well for pNP-versus-time data under all circumstances we have investigated., (Copyright © 2010 Elsevier Ltd. All rights reserved.)

Trichoderma harzianum ETS 323 secretes two glucanases, a 23.5 kDa endoglucanase (EG Th1) and a 61 kDa exoglucanase (ExG Th1). They were identified by their hydrolysis products and were purified to homogeneity. The optimal temperature and pH for both EG Th1 (7.3-fold purification, 5.0% yield) and ExG Th1 (33.7-fold purification, 0.15% yield) were 50 °C and pH 4.5, respectively. The kinetic parameters of EG Th1 (K(m) = 23 mg mL(-1), V(max) = 294 μM min(-1), specific activity = 7.4 U mg(-1)) and ExG Th1 (K(m) = 85 mg mL(-1), V(max) = 385 μM min(-1), specific activity = 24.6 U mg(-1)) toward carboxymethyl cellulose were determined. Both enzymes favored CMC and maintained 100% activity for 10 days at 38 °C. KCl, MgCl(2), HgCl(2), and FeCl(3) showed approximately 30% inhibition against EG Th1 but not ExG Th1. They catalyzed transglycosylation of glucose in the presence of cellobiose, but ExG Th1 exhibited better activity and higher product diversity.

The objective of this study was to investigate the effect of dietary supplementation with the disaccharides trehalose and cellobiose on antioxidant activity in rumen fluid, blood, and milk of dairy cows. Nine Holstein dairy cows housed in a free-stall barn were divided into 3 groups, with each group receiving a different dietary treatment (a control diet, a 1% trehalose-supplemented diet, or a 1% cellobiose-supplemented diet) following a 3x3 Latin square design. Feed intake and milk production increased in cows receiving the trehalose-supplemented diet compared with those receiving the control and cellobiose-supplemented diets. The total protozoa numbers in the rumen fluid of cows fed trehalose- or cellobiose-supplemented diets were greater than those of the control group. The C18:0 and C18:1 fatty acid content was increased in the milk of cows fed the trehalose-supplemented diet compared with that of the control group, and the C18:3n-3 fatty acid content in the milk of cows fed the cellobiose-supplemented diet was less than that of the control group. Plasma biochemical parameters were unchanged among the different treatments. In rumen fluid, 1,1-diphenyl-2-picrylhydrazyl radical scavenging activity and superoxide dismutase activity were increased 2h after feeding in cows receiving the cellobiose-supplemented diet compared with the control group, and the concentration of thiobarbituric acid reactive substances in the rumen fluid of cows fed the cellobiose-supplemented diet was decreased. In contrast, the values of these parameters measured in the milk of cows fed the cellobiose-supplemented diet were no different from those of control cows. Dietary supplementation with trehalose did, however, bring about an improvement of the oxidative status of milk and blood in these animals compared with controls. These results provide the first evidence supporting the use of dietary disaccharides to decrease lipid peroxide levels and increase the antioxidant content of dairy cow milk. The findings suggest that disaccharides, particularly trehalose, might be useful as supplements for reducing oxidative stress and improving the quality of milk for human consumption, as well as possibly impairing the processes that give rise to lipid oxidation odor in dairy cow milk., (Copyright (c) 2010 American Dairy Science Association. Published by Elsevier Inc. All rights reserved.)

The yeast Trichosporon porosum suppresses growth of ascomycetes and basidiomycetes belonging to 52 genera. It is due to secretion of a thermostable fungicidal agent. The suppression was maximal at pH 3.5-4.0. Fungicidal preparation obtained from the culture broth was shown to be a mixture of cellobiosides of dihydrodecane acid with different degree of acetylation of cellobiose residue. The preparation caused the death of Candida albicans and Filobasidiella neoformans cells in the concentrations of 0.2 and 0.03 mM, respectively.

Streptomyces scabies is a gram-positive soil bacterium recognized as the main causal agent of common scab. Pathogenicity in Streptomyces spp. depends on their capacity to synthesize phytotoxins called thaxtomins. Genes involved in biosynthesis of these secondary metabolites are known to be induced by cellobiose, a plant disaccharide. However, growth of S. scabies in a minimal medium containing cellobiose as a carbon source is very poor and only generates traces of thaxtomins. The effect of suberin, a lipid plant polymer, on thaxtomin A biosynthesis and the expression of genes involved in its biosynthetic pathway was analyzed. S. scabies was grown in a starch-containing minimal medium supplemented with cellobiose (0.5%), suberin (0.1%), or both. The presence of both cellobiose and suberin doubled bacterial growth and triggered thaxtomin A production, which correlated with the upregulation (up to 342-fold) of genes involved in thaxtomins synthesis. The addition of either suberin or cellobiose alone did not affect these parameters. Suberin appeared to stimulate the onset of secondary metabolism, which is a prerequisite to the production of molecules such as thaxtomin A, while cellobiose induced the biosynthesis of this secondary metabolite.

The reaction mechanism of cellobiose dehydrogenase (CDH) from Phanerochaete chrysosporium, adsorbed on graphite electrodes, was investigated by following its catalytic reaction with cellobiose registered in both direct and mediated electron transfer modes between the enzyme and the electrode. A wall-jet flow through amperometric cell housing the CDH-modified graphite electrode was connected to a single line flow injection system. In the present study, it is proven that cellobiose, at concentrations higher than 200 microM, competes for the reduced state of the FAD cofactor and it slows down the transfer of electrons to any 2e(-)/H(+) acceptors or further to the heme cofactor, via the internal electron transfer pathway. Based on and proven by electrochemical results, a kinetic model of substrate inhibition is proposed and supported by the agreement between simulation of plots and experimental data. The implications of this kinetic model, called pseudo-ping-pong mechanism, on the possible functions CDH are also discussed. The enzyme exhibits catalytic activity also for lactose, but in contrast to cellobiose, this sugar does not inhibit the enzyme. This suggests that even if some other substrates are coincidentally oxidized by CDH, however, they do not trigger all the possible natural functions of the enzyme. In this respect, cellobiose is regarded as the natural substrate of CDH.

Aims: To investigate the in vitro antibacterial activity and antifungal mode of action of flocculosin, a cellobiose lipid produced by Pseudozyma flocculosa., Methods and Results: When tested against clinical bacterial isolates, the compound was particularly active against Gram-positive bacteria and its effect was not mitigated against isolates known as resistant to other antibiotics. The antifungal activity of flocculosin was found to be rapid and concentration-dependent. At lethal concentrations against Candida albicans, flocculosin caused a rapid leakage of intracellular potassium and inhibited acidification of the medium by plasma membrane ATPases suggesting a physical rather than a biochemical effect. TEM observations of cells exposed 6 h to flocculosin revealed disrupted membranes and disorganized mitochondria., Conclusions: Data obtained in this study confirm that flocculosin acts by disrupting the membrane surface of sensitive micro-organisms., Significance and Impact of the Study: The elucidation of an antifungal mode of action of flocculosin can be exploited in furthering its antimicrobial potential against fungi and bacteria whose cell membranes are particularly sensitive to the action of the molecule.

We searched the genome database of the basidiomycete Coprinopsis cinerea (Coprinus cinereus) and found five genes encoding the glycoside hydrolase family 6 (GH6) enzyme, CcCel6A, CcCel6B, CcCel6C, CcCel6D, and CcCel6E, designated in order of increasing locus number (CC1G&#95;01107.1, CC1G&#95;04166.1, CC1G&#95;08276.1, CC1G&#95;08277.1, and CC1G&#95;10605.1). The amino acid sequence of CcCel6A suggests a two-domain structure consisting of an N-terminal family 1 carbohydrate-binding module (CBM1) and a GH6 catalytic domain, while the other genes lack CBM1. The transcripts of CcCel6A were observed at the active growth stage in cellulose culture, whereas they were absent from glucose culture. Cellobiose strongly induced transcription of CcCel6A. On the other hand, transcripts of CcCel6B, -D, and -E were detected in both glucose and cellulose cultures, and transcription of them was induced weakly by cellobiose. The transcript level of CcCel6C was not influenced by glucose or cellobiose.

As part of the effort to find better cellulases for bioethanol production processes, we were looking for novel GH-7 family cellobiohydrolases, which would be particularly active on insoluble polymeric substrates and participate in the rate-limiting step in the hydrolysis of cellulose. The enzymatic properties were studied and are reported here for family 7 cellobiohydrolases from the thermophilic fungi Acremonium thermophilum, Thermoascus aurantiacus, and Chaetomium thermophilum. The Trichoderma reesei Cel7A enzyme was used as a reference in the experiments. As the native T. aurantiacus Cel7A has no carbohydrate-binding module (CBM), recombinant proteins having the CBM from either the C. thermophilum Cel7A or the T. reesei Cel7A were also constructed. All these novel acidic cellobiohydrolases were more thermostable (by 4-10 degrees C) and more active (two- to fourfold) in hydrolysis of microcrystalline cellulose (Avicel) at 45 degrees C than T. reesei Cel7A. The C. thermophilum Cel7A showed the highest specific activity and temperature optimum when measured on soluble substrates. The most effective enzyme for Avicel hydrolysis at 70 degrees C, however, was the 2-module version of the T. aurantiacus Cel7A, which was also relatively weakly inhibited by cellobiose. These results are discussed from the structural point of view based on the three-dimensional homology models of these enzymes., ((c) 2008 Wiley Periodicals, Inc.)

Several outbreaks of foodborne yersiniosis have been documented and this disease continues to be source of infections transmitted through foods. The selective agars most commonly used to isolate Yersinia enterocolitica in clinical, food and environmental samples, cefsulodin-irgasan-novobiocin (CIN) and MacConkey (MAC) agars, lack the ability to differentiate potentially virulent Y. enterocolitica from other Yersinia that may be present as well as some other bacterial spp. This study proposes the use of an agar medium, Y. enterocolitica chromogenic medium (YeCM), for isolation of potentially virulent Y. enterocolitica. This agar contains cellobiose as the fermentable sugar, a chromogenic substrate and selective inhibitors for suppression of colony formation by many competing bacteria. All strains of potentially virulent Yersinia of biotypes 1B, and biotypes 2-5 formed convex, red bulls-eye colonies on YeCM that were very similar to those described for CIN agar. However, Y. enterocolitica biotype 1A and other related Yersinia formed colonies that were purple/blue on YeCM while they formed typical red bulls-eye colonies on CIN agar. When a mixture of potentially virulent Y. enterocolitica biotype 1B, Y. enterocolitica biotype 1A and 5 other bacterial species was used to artificially contaminate tofu and then spread-plated on three selective agars, Y. enterocolitica biotype 1B colonies were easily distinguished from other strains on YeCM. However, Y. enterocolitica biotype 1B colonies were indistinguishable from many other colonies on CIN and only distinguishable from those of C. freundii on MAC. When colonies were picked and identified from these agars, typical colonies from YeCM were confirmed only as Y. enterocolitica biotype 1B. Typical colonies on CIN and MAC were found to belong to several competing species and biotypes.

The phytopathogenic basidiomycetous fungus Ustilago maydis secretes large amounts of the glycolipid biosurfactant ustilagic acid (UA). UA consists of 15,16-dihydroxypalmitic or 2,15,16-trihydroxypalmitic acid, which is O-glycosidically linked to cellobiose at its terminal hydroxyl group. In addition, the cellobiose moiety is acetylated and acylated with a short-chain hydroxy fatty acid. We have identified a 58 kb spanning gene cluster that contains 12 open reading frames coding for most, if not all, enzymes needed for UA biosynthesis. Using a combination of genetic and mass spectrometric analysis we were able to assign functional roles to three of the proteins encoded by the gene cluster. This allowed us to propose a biosynthesis route for UA. The Ahd1 protein belongs to the family of non-haem diiron reductases and is required for alpha-hydroxylation of palmitic acid. Two P450 monooxygenases, Cyp1 and Cyp2, catalyse terminal and subterminal hydroxylation of palmitic acid. We could demonstrate that infection of tomato leaves by the plant pathogenic fungus Botrytis cinerea is prevented by co-inoculation with wild-type U. maydis sporidia. U. maydis mutants defective in UA biosynthesis were unable to inhibit B. cinerea infection indicating that UA secretion is critical for antagonistic activity.

A metabolic isotope-labeling strategy was used in conjunction with nano-liquid chromatography-electrospray ionization mass spectrometry peptide sequencing to assess quantitative alterations in the expression patterns of subunits within cellulosomes of the cellulolytic bacterium Clostridium thermocellum, grown on either cellulose or cellobiose. In total, 41 cellulosomal proteins were detected, including 36 type I dockerin-containing proteins, which count among them all but three of the known docking components and 16 new subunits. All differential expression data were normalized to the scaffoldin CipA such that protein per cellulosome was compared for growth between the two substrates. Proteins that exhibited higher expression in cellulosomes from cellulose-grown cells than in cellobiose-grown cells were the cell surface anchor protein OlpB, exoglucanases CelS and CelK, and the glycoside hydrolase family 9 (GH9) endoglucanase CelJ. Conversely, lower expression in cellulosomes from cells grown on cellulose than on cellobiose was observed for the GH8 endoglucanase CelA; GH5 endoglucanases CelB, CelE, CelG; and hemicellulases XynA, XynC, XynZ, and XghA. GH9 cellulases were the most abundant group of enzymes per CipA when cells were grown on cellulose, while hemicellulases were the most abundant group on cellobiose. The results support the existing theory that expression of scaffoldin-related proteins is coordinately regulated by a catabolite repression type of mechanism, as well as the prior observation that xylanase expression is subject to a growth rate-independent type of regulation. However, concerning transcriptional control of cellulases, which had also been previously shown to be subject to catabolite repression, a novel distinction was observed with respect to endoglucanases.

A new yeast species, Candida gelsemii, is described to accommodate three isolates recovered in Georgia, USA, from the toxic nectar of the Carolina jessamine (Gelsemium sempervirens). The species resembles other members of the Metschnikowiaceae clade that have been recovered from nectar, but differs in a number of morphological and physiological characteristics. Analysis of rDNA sequences places the new species well into the clade, but in a basal position with respect to a group of Metschnikowia and Candida species known to occur in association with nectars and bees, as well as marine invertebrates. The type is strain UWOPS 06-24.1(T) (CBS 10509(T), NRRL Y-48212(T).

Cellobiose lipids of yeast fungi Cryptococcus humicola and Pseudozyma fusiformata have similar fungicidal activities against different yeast, including pathogenic Cryptococcus and Candida species. Basidiomycetic yeast reveals maximum sensitivity to these preparations; e.g., cells of cryptococcus Filobasidiella neoformans almost completely die after 30-min incubation in a glycolipid solution at a concentration of 0.02 mg/ml. The same effect toward ascomycetous yeast, including pathogenic Candida species, is achieved only at five to eight times higher concentrations of glycolipids. The cellobiose lipid from P. fusiformata, which, unlike glycolipid from Cr. humicola, has hydroxycaproic acid residue as O-subtituent of cellobiose and additional 15-hydroxy group in aglycone, inhibits the growth of the studied mycelial fungi more efficiently than the cellobiose lipid from Cr. humicola.

Artificial diet was developed for rearing of lower termites (workers) Coptotermes formosanus. C. formosanus was fed with either wood powder of Japanese red pine, cellulose, cellobiose, or glucose for 30 days. The effect of carbon sources in the diet on the structure and function of the symbiotic intestinal microbial community and on the physiological activity of C. formosanus was studied. Three symbiont protozoa, Pseudotrichonympha grassi, Holomastigotoides hartmanni, and Spirotrichonympha leidyi, were found in the hindgut of C. formosanus that fed on the diets containing carbon sources with high molecular weight (MW). However, when artificial diets containing carbohydrate with low MW were used, both P. grassi and H. hartmanni disappeared, and only few S. leidyi were alive. This suggested that both P. grassi and H. hartmanni play important roles in the digestion and utilization of carbohydrate with high MW. The denaturing gradient gel electrophoresis analysis of bacterial community in the hindgut of termites showed that the similarity between intestinal bacteria community in termites fed with diets containing high-MW carbon sources and those with low MW was only about 40%. It was apparent that changes in diets resulted to changes in intestinal microbial community, and this in turn affected cellulase activity in C. formosanus.

The integrated forms of the Michaelis-Menten equation assuming variable substrate (depletion) or constant substrate concentration were used to study the effect of the simultaneous presence of two exoglucanase Cel7A inhibitors (cellobiose and ethanol) on the kinetics of cellulose hydrolysis. The kinetic parameters obtained, assuming constant substrate (K(m) = 21 mM, Kic = 0.035 mM; K(icl) = 1.5 x 1,015 mM; k(cat) = 12 h-1) or assuming variable substrate (K(m) = 16 mM, Kic = 0.037 mM; K(icl) = 5.8 x 1,014 mM; k(cat) = 9 h-1), showed a good similarity between these two alternative methodologies and pointed out that both ethanol and cellobiose are competitive inhibitors. Nevertheless, ethanol is a very weak inhibitor, as shown by the large value estimated for the kinetic constant K(icl). In addition, assuming different concentrations of initial accessible substrate present in the reaction, both inhibition and velocity constants are at the same order of magnitude, which is consistent with the obtained values. The possibility of using this kind of methodology to determine kinetic constants in general kinetic studies is discussed, and several integrated equations of different Michaelis-Menten kinetic models are presented. Also examined is the possibility of determining inhibition constants without knowledge of the true accessible substrate concentration.

Among carbon sources studied, cellobiose and mannitol provided the highest laccase (Lac) activity (648 and 742 U1(-1), respectively) of Trametes versicolor 775 while glucose gave maximum manganese peroxidase (MnP) and peroxidase activities (44 and 114 U1(-1), respectively). Citrus fruit peel as growth substrate enhanced Lac activity 7-fold when compared to the medium with cellobiose, whereas grape vine sawdust increased MnP and peroxidase activity up to 148 and 677 U1(-1), respectively.

Flocculosin, a glycolipid isolated from the yeast-like fungus Pseudozyma flocculosa, was investigated for in vitro antifungal activity. The compound displayed antifungal properties against several pathogenic yeasts. Synergistic activity was observed between flocculosin and amphotericin B, and no significant cytotoxicity was demonstrated when tested against human cell lines.

Regulation of cell-specific cellulase synthesis (expressed in milligrams of cellulase per gram [dry weight] of cells) by Clostridium thermocellum was investigated using an enzyme-linked immunosorbent assay protocol based on antibody raised against a peptide sequence from the scaffoldin protein of the cellulosome (Zhang and Lynd, Anal. Chem. 75:219-227, 2003). The cellulase synthesis in Avicel-grown batch cultures was ninefold greater than that in cellobiose-grown batch cultures. In substrate-limited continuous cultures, however, the cellulase synthesis with Avicel-grown cultures was 1.3- to 2.4-fold greater than that in cellobiose-grown cultures, depending on the dilution rate. The differences between the cellulase yields observed during carbon-limited growth on cellulose and the cellulase yields observed during carbon-limited growth on cellobiose at the same dilution rate suggest that hydrolysis products other than cellobiose affect cellulase synthesis during growth on cellulose and/or that the presence of insoluble cellulose triggers an increase in cellulase synthesis. Continuous cellobiose-grown cultures maintained either at high dilution rates or with a high feed substrate concentration exhibited decreased cellulase synthesis; there was a large (sevenfold) decrease between 0 and 0.2 g of cellobiose per liter, and there was a much more gradual further decrease for cellobiose concentrations >0.2 g/liter. Several factors suggest that cellulase synthesis in C. thermocellum is regulated by catabolite repression. These factors include: (i) substantially higher cellulase yields observed during batch growth on Avicel than during batch growth on cellobiose, (ii) a strong negative correlation between the cellobiose concentration and the cellulase yield in continuous cultures with varied dilution rates at a constant feed substrate concentration and also with varied feed substrate concentrations at a constant dilution rate, and (iii) the presence of sequences corresponding to key elements of catabolite repression systems in the C. thermocellum genome.

An experimental study of cellobiose inhibition in cellulose hydrolysis by synergism of cellobiohydrolyse I and endoglucanase I is presented. Cellobiose is the structural unit of cellulose molecules and also the main product in enzymatic hydrolysis of cellulose. It has been identified that cellobiose can strongly inhibit hydrolysis reaction of cellulase, whereas it has no effect on the adsorption of cellulase on cellulose surface. The experimental data of FT-IR spectra, fluorescence spectrum and circular dichroism suggested that cellobiose can be combined with tryptophan residue located near the active site of cellobiohydrolase and then form steric hindrance, which prevents cellulose molecule chains from diffusing into active site of cellulase. In addition, the molecular conformation of cellobiohydrolase changes after cellobiose binding, which also causes most of the non-productive adsorption. Under these conditions, microfibrils cannot be separated from cellulose chains, thus further hydrolysis of cellulose can hardly proceed.

The sigma(54) subunit of the RNA polymerase directs the expression of specific operons in association with cognate activators. Three different activators have been detected in the Listeria monocytogenes genome on the basis of the high conservation of a specific domain. Among them, the LacR activator, of the LevR family, was found just upstream from a newly described sigma(54)-dependent operon, lpo, which presents a classical -24/-12 consensus promoter. The lpo operon encodes proteins similar to subunits of a PTS permease (EII) of the lactose family, namely LpoA (IIA) and LpoB (IIB). It also encodes a third putative protein, LpoO, with an unknown function but sharing high similarity with proteins also encoded within PTS operons from other bacteria and bearing a RGD motif. The expression of lpo was clearly dependent on LacR and sigma(54), and was induced by cellobiose, chitobiose and lactose. It underlies that the lpo operon likely encodes proteins involved in the utilization of these sugars by L. monocytogenes.

Insertional mutagenesis was applied for the first time to a fungal biocontrol agent, Pseudozyma flocculosa, in an attempt to obtain mutants with altered antagonistic properties. Transformants were obtained via DNA-mediated transformation. Molecular analyses of the transformants revealed that multiple copies of the plasmid were integrated in tandem at one to many chromosomal loci. The transformants were screened for their biocontrol properties using standard bioassays, and the 160 tested transformants were classified into four groups: group I mutants (22 transformants) showed a stronger antagonistic effect than the wild type (WT) while those of group II (107 transformants) had a comparable antagonistic effect; group III mutants (17 transformants) had a decreased antagonistic effect relative to WT and group IV mutants (14 transformants) had lost their biocontrol properties. Culture extracts of the mutants (group IV) and WT were analyzed and compared for the presence of active metabolites which were then separated by solid-phase extraction and purified using conventional methods. Nuclear magnetic resonance experiments and analytical studies on a metabolite specifically produced by the WT revealed the presence of 2-(2',4'-diacetoxy-5'-carboxy-pentanoyl) octadecyl cellobioside (flocculosin), a novel glycolipid with strong antifungal properties; the production of this compound would account for the biocontrol activity of P. flocculosa.

Three forms of cellobiohydrolase (EC 3.2.1.91), CBH IA, CBH IB and CBH II, were isolated to apparent homogeneity from culture filtrates of the aerobic fungus Talaromyces emersonii. The three enzymes are single sub-unit glycoproteins, and unlike most other fungal cellobiohydrolases are characterised by noteworthy thermostability. The kinetic properties and mode of action of each enzyme against polymeric and small soluble oligomeric substrates were investigated in detail. CBH IA, CBH IB and CBH II catalyse the hydrolysis of microcrystalline cellulose, albeit to varying extents. Hydrolysis of a soluble cellulose derivative (CMC) and barley 1,3;1,4-beta-D-glucan was not observed. Cellobiose (G2) is the main reaction product released by CBH IA, CBH IB, and CBH II from microcrystalline cellulose. All three CBHs are competitively inhibited by G2; inhibition constant values (K(i)) of 2.5 and 0.18 mM were obtained for CBH IA and CBH IB, respectively (4-nitrophenyl-beta-cellobioside as substrate), while a K(i) of 0.16 mM was determined for CBH II (2-chloro-4-nitrophenyl-beta-cellotrioside as substrate). Bond cleavage patterns were determined for each CBH on 4-methylumbelliferyl derivatives of beta-cellobioside and beta-cellotrioside (MeUmbG(n)). While the Tal. emersonii CBHs share certain properties with their counterparts from Trichoderma reesei, Humicola insolens and other fungal sources, distinct differences were noted.