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Cellvibrio japonicus is a saprophytic bacterium proficient at environmental polysaccharide degradation for carbon and energy acquisition. Genetic, enzymatic, and structural characterization of C. japonicus carbohydrate active enzymes, specifically those that degrade plant and animal-derived polysaccharides, demonstrated that this bacterium is a carbohydrate-bioconversion specialist. Structural analyses of these enzymes identified highly specialized carbohydrate binding modules that facilitate activity. Steady progress has been made in developing genetic tools for C. japonicus to better understand the function and regulation of the polysaccharide-degrading enzymes it possesses, as well as to develop it as a biotechnology platform to produce renewable fuels and chemicals.

Importance: Understanding the bacterial metabolism of starch is important as this polysaccharide is a ubiquitous ingredient in foods, supplements, and medicines, all of which influence gut microbiome composition and health. Our RNAseq and growth data set provides a valuable resource to those who want to better understand the regulation of starch utilization in Gram-negative bacteria. These data are also useful as they provide an example of how to approach studying a starch-utilizing bacterium that has many putative amylases by coupling transcriptomic data with growth assays to overcome the potential challenges of functional redundancy. The RNAseq data can also be used as a part of larger meta-analyses to compare how C. japonicus regulates carbohydrate active enzymes, or how this bacterium compares to gut microbiome constituents in terms of starch utilization potential., Competing Interests: The authors declare no conflict of interest.

Chitin utilization by microbes plays a significant role in biosphere carbon and nitrogen cycling, and studying the microbial approaches used to degrade chitin will facilitate our understanding of bacterial strategies to degrade a broad range of recalcitrant polysaccharides. The early stages of chitin depolymerization by the bacterium Cellvibrio japonicus have been characterized and are dependent on one chitin-specific lytic polysaccharide monooxygenase and nonredundant glycoside hydrolases from the family GH18 to generate chito-oligosaccharides for entry into metabolism. Here, we describe the mechanisms for the latter stages of chitin utilization by C. japonicus with an emphasis on the fate of chito-oligosaccharides. Our systems biology approach combined transcriptomics and bacterial genetics using ecologically relevant substrates to determine the essential mechanisms for chito-oligosaccharide transport and catabolism in C. japonicus. Using RNAseq analysis we found a coordinated expression of genes that encode polysaccharide-degrading enzymes. Mutational analysis determined that the hex20B gene product, predicted to encode a hexosaminidase, was required for efficient utilization of chito-oligosaccharides. Furthermore, two gene loci (CJA_0353 and CJA_1157), which encode putative TonB-dependent transporters, were also essential for chito-oligosaccharides utilization. This study further develops our model of C. japonicus chitin metabolism and may be predictive for other environmentally or industrially important bacteria., (© 2021 John Wiley & Sons Ltd.)

The release of glucose from lignocellulosic waste for subsequent fermentation into biofuels holds promise for securing humankind's future energy needs. The discovery of a set of copper-dependent enzymes known as lytic polysaccharide monooxygenases (LPMOs) has galvanised new research in this area. LPMOs act by oxidatively introducing chain breaks into cellulose and other polysaccharides, boosting the ability of cellulases to act on the substrate. Although several proteins have been implicated as electron sources in fungal LPMO biochemistry, no equivalent bacterial LPMO electron donors have been previously identified, although the proteins Cbp2D and E from Cellvibrio japonicus have been implicated as potential candidates. Here we analyse a small c-type cytochrome (CjX183) present in Cellvibrio japonicus Cbp2D, and show that it can initiate bacterial CuII/I LPMO reduction and also activate LPMO-catalyzed cellulose-degradation. In the absence of cellulose, CjX183-driven reduction of the LPMO results in less H2O2 production from O2, and correspondingly less oxidative damage to the enzyme than when ascorbate is used as the reducing agent. Significantly, using CjX183 as the activator maintained similar cellulase boosting levels relative to the use of an equivalent amount of ascorbate. Our results therefore add further evidence to the impact that the choice of electron source can have on LPMO action. Furthermore, the study of Cbp2D and other similar proteins may yet reveal new insight into the redox processes governing polysaccharide degradation in bacteria., (© 2021 The Author(s).)

Understanding the complex growth and metabolic dynamics in microorganisms requires advanced kinetic models containing both metabolic reactions and enzymatic regulation to predict phenotypic behaviors under different conditions and perturbations. Most current kinetic models lack gene expression dynamics and are separately calibrated to distinct media, which consequently makes them unable to account for genetic perturbations or multiple substrates. This challenge limits our ability to gain a comprehensive understanding of microbial processes towards advanced metabolic optimizations that are desired for many biotechnology applications. Here, we present an integrated computational and experimental approach for the development and optimization of mechanistic kinetic models for microbial growth and metabolic and enzymatic dynamics. Our approach integrates growth dynamics, gene expression, protein secretion, and gene-deletion phenotypes. We applied this methodology to build a dynamic model of the growth kinetics in batch culture of the bacterium Cellvibrio japonicus grown using either cellobiose or glucose media. The model parameters were inferred from an experimental data set using an evolutionary computation method. The resulting model was able to explain the growth dynamics of C. japonicus using either cellobiose or glucose media and was also able to accurately predict the metabolite concentrations in the wild-type strain as well as in β-glucosidase gene deletion mutant strains. We validated the model by correctly predicting the non-diauxic growth and metabolite consumptions of the wild-type strain in a mixed medium containing both cellobiose and glucose, made further predictions of mutant strains growth phenotypes when using cellobiose and glucose media, and demonstrated the utility of the model for designing industrially-useful strains. Importantly, the model is able to explain the role of the different β-glucosidases and their behavior under genetic perturbations. This integrated approach can be extended to other metabolic pathways to produce mechanistic models for the comprehensive understanding of enzymatic functions in multiple substrates., (© 2020 Wiley Periodicals LLC.)

Lignocellulose degradation by microbes plays a central role in global carbon cycling, human gut metabolism and renewable energy technologies. While considerable effort has been put into understanding the biochemical aspects of lignocellulose degradation, much less work has been done to understand how these enzymes work in an in vivo context. Here, we report a systems level study of xylan degradation in the saprophytic bacterium Cellvibrio japonicus. Transcriptome analysis indicated seven genes that encode carbohydrate active enzymes were up-regulated during growth with xylan containing media. In-frame deletion analysis of these genes found that only gly43F is critical for utilization of xylo-oligosaccharides, xylan, and arabinoxylan. Heterologous expression of gly43F was sufficient for the utilization of xylo-oligosaccharides in Escherichia coli. Additional analysis found that the xyn11A, xyn11B, abf43L, abf43K, and abf51A gene products were critical for utilization of arabinoxylan. Furthermore, a predicted transporter (CJA_1315) was required for effective utilization of xylan substrates, and we propose this unannotated gene be called xntA (xylan transporter A). Our major findings are (i) C. japonicus employs both secreted and surface associated enzymes for xylan degradation, which differs from the strategy used for cellulose degradation, and (ii) a single cytoplasmic β-xylosidase is essential for the utilization of xylo-oligosaccharides., (© 2017 John Wiley & Sons Ltd.)

A bacterial strain, designated TPY-10T, was isolated from calla lily roots in Taiwan and characterized by using a polyphasic taxonomy approach. Cells of strain TPY-10T were Gram-stain-negative, strictly aerobic, motile and creamy white rods. Growth occurred at 15-35 °C (optimum, 25-30 °C), at pH 6-7 (optimum, pH 6) and with 0-1 % NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain TPY-10T belonged to the genus Cellvibrio and was most closely related to Cellvibriomixtus ACM 2601T with sequence similarity of 97.8 %. Strain TPY-10T contained C16 : 0, summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c) and C18 : 1ω7c as the predominant fatty acids. The only isoprenoid quinone was Q-9. The major polar lipids were phosphatidylethanolamine and phosphatidylglycerol. The DNA G+C content of the genomic DNA was 49.8 mol%. The DNA-DNA hybridization value for strain TPY-10T with Cellvibriomixtus ACM 2601T was less than 21 %. On the basis of the phylogenetic inference and phenotypic data, strain TPY-10T should be classified as a novel species, for which the name Cellvibrio zantedeschiae sp. nov. is proposed. The type strain is TPY-10T (=BCRC 80525T=LMG 27291T=KCTC 32239T).

A bacterial strain, designated MVW-40T, was isolated from Maolin Spring in Taiwan and characterized using a polyphasic taxonomy approach. Cells of strain MVW-40T were Gram-negative, strictly aerobic, motile by a single polar flagellum and bright yellow-pigmented rods with pointed ends. Growth occurred at 15-40 °C (optimum, 20-30 °C), at pH 6-9 (optimum, pH 6) and with 0-2 % NaCl (optimum, 0 %). Phylogenetic analyses based on 16S rRNA gene sequences showed that strain MVW-40T belonged to the genus Cellvibrio and showed the highest levels of sequence similarity with respect to Cellvibrio mixtussubsp. mixtus ACM 2601T (98.1 %) and Cellvibrio fibrivorans R-4079T (97.2 %). Strain MVW-40T contained summed feature 3 (C16 : 1ω7c and/or C16 : 1ω6c), C16 : 0 and C18 : 1ω7c as the predominant fatty acids. The polar lipid profile consisted of phosphatidylethanolamine, phosphatidylglycerol, two uncharacterized aminophospholipids, two uncharacterized phospholipids and an uncharacterized lipid. The DNA G+C content of the genomic DNA was 52.8 mol%. The DNA-DNA hybridization value for strain MVW-40T with C. mixtussubsp. mixtus ACM 2601T and C. fibrivorans R-4079T was less than 45 %. On the basis of the phylogenetic inference and phenotypic data, strain MVW-40T should be classified as a novel species, for which the name Cellvibrio fontiphilus sp. nov. is proposed. The type strain is MVW-40T (=BCRC 80977T=LMG 29557T=KCTC 52237T).

Cellvibrio sp. PR1 is a xylanolytic and agarolytic bacterium isolated from the Pearl River. Strain PR1 is closely related to Cellvibrio fibrivorans and C. ostraviensis (identity > 98%). The xylanase and agarase contents of strain PR1 reach up to 15.4 and 25.9 U/mL, respectively. The major cellular fatty acids consisted of C16:0 (36.7%), C18:0 (8.8%), C20:0 (6.8%), C 15:0 iso 2-OH or/and C 16:1 ω 7c (17.4%), and C 18:1 ω 7c or/and C 18:1 ω 6c (6.7%). A total of 251 CAZyme modules (63 CBMs, 20 CEs, 128 GHs, 38 GTs, and 2 PLs) were identified from 3,730 predicted proteins. Genomic analysis suggested that strain PR1 has a complete xylan-hydrolyzing (5 β -xylanases, 16 β -xylosidases, 17 α -arabinofuranosidases, 9 acetyl xylan esterases, 4 α -glucuronidases, and 2 ferulic acid esterases) and agar-hydrolyzing enzyme system (2 β -agarases and 2 α -neoagarooligosaccharide hydrolases). In addition, the main metabolic pathways of xylose, arabinose, and galactose are established in the genome-wide analysis. This study shows that strain PR1 contains a large number of glycoside hydrolases.

Physiological studies of recalcitrant polysaccharide degradation are challenging for several reasons, one of which is the difficulty in obtaining a reproducibly accurate real-time measurement of bacterial growth using insoluble substrates. Current methods suffer from several problems including (i) high background noise due to the insoluble material interspersed with cells, (ii) high consumable and reagent cost and (iii) significant time delay between sampling and data acquisition. A customizable substrate and cell separation device would provide an option to study bacterial growth using optical density measurements. To test this hypothesis we used 3-D printing to create biomass containment devices that allow interaction between insoluble substrates and microbial cells but do not interfere with spectrophotometer measurements. Evaluation of materials available for 3-D printing indicated that UV-cured acrylic plastic was the best material, being superior to nylon or stainless steel when examined for heat tolerance, reactivity, and ability to be sterilized. Cost analysis of the 3-D printed devices indicated they are a competitive way to quantitate bacterial growth compared to viable cell counting or protein measurements, and experimental conditions were scalable over a 100-fold range. The presence of the devices did not alter growth phenotypes when using either soluble substrates or insoluble substrates. We applied biomass containment to characterize growth of Cellvibrio japonicus on authentic lignocellulose (non-pretreated corn stover), and found physiological evidence that xylan is a significant nutritional source despite an abundance of cellulose present., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Study of recalcitrant polysaccharide degradation by bacterial systems is critical for understanding biological processes such as global carbon cycling, nutritional contributions of the human gut microbiome, and the production of renewable fuels and chemicals. One bacterium that has a robust ability to degrade polysaccharides is the Gram-negative saprophyte Cellvibrio japonicus. A bacterium with a circuitous history, C. japonicus underwent several taxonomy changes from an initially described Pseudomonas sp. Most of the enzymes described in the pre-genomics era have also been renamed. This review aims to consolidate the biochemical, structural, and genetic data published on C. japonicus and its remarkable ability to degrade cellulose, xylan, and pectin substrates. Initially, C. japonicus carbohydrate-active enzymes were studied biochemically and structurally for their novel polysaccharide binding and degradation characteristics, while more recent systems biology approaches have begun to unravel the complex regulation required for lignocellulose degradation in an environmental context. Also included is a discussion for the future of C. japonicus as a model system, with emphasis on current areas unexplored in terms of polysaccharide degradation and emerging directions for C. japonicus in both environmental and biotechnological applications.

Carbohydrate isomerases/epimerases are essential in carbohydrate metabolism, and have great potential in industrial carbohydrate conversion. Cellobiose 2-epimerase (CE) reversibly epimerizes the reducing end d-glucose residue of β-(1→4)-linked disaccharides to d-mannose residue. CE shares catalytic machinery with monosaccharide isomerases and epimerases having an (α/α)6-barrel catalytic domain. Two histidine residues act as general acid and base catalysts in the proton abstraction and addition mechanism. β-Mannoside hydrolase and 4-O-β-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in β-mannan metabolism, where it epimerizes β-d-mannopyranosyl-(1→4)-d-mannose to β-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other β-mannoside phosphorylases and hydrolases. Structural analysis of GH130 enzymes revealed an unusual catalytic mechanism involving a proton relay and the molecular basis for substrate and reaction specificities. Epilactose, efficiently produced from lactose using CE, has superior physiological functions as a prebiotic oligosaccharide.

Lignocellulosic biomass is a sustainable industrial substrate. Copper-dependent lytic polysaccharide monooxygenases (LPMOs) contribute to the degradation of lignocellulose and increase the efficiency of biofuel production. LPMOs can contain non-catalytic carbohydrate binding modules (CBMs), but their role in the activity of these enzymes is poorly understood. Here we explored the importance of CBMs in LPMO function. The family 2a CBMs of two monooxygenases,CfLPMO10 andTbLPMO10 fromCellulomonas fimiandThermobispora bispora, respectively, were deleted and/or replaced with CBMs from other proteins. The data showed that the CBMs could potentiate and, surprisingly, inhibit LPMO activity, and that these effects were both enzyme-specific and substrate-specific. Removing the natural CBM or introducingCtCBM3a, from theClostridium thermocellumcellulosome scaffoldin CipA, almost abolished the catalytic activity of the LPMOs against the cellulosic substrates. The deleterious effect of CBM removal likely reflects the importance of prolonged presentation of the enzyme on the surface of the substrate for efficient catalytic activity, as only LPMOs appended to CBMs bound tightly to cellulose. The negative impact ofCtCBM3a is in sharp contrast with the capacity of this binding module to potentiate the activity of a range of glycoside hydrolases including cellulases. The deletion of the endogenous CBM fromCfLPMO10 or the introduction of a family 10 CBM fromCellvibrio japonicusLPMO10B intoTbLPMO10 influenced the quantity of non-oxidized products generated, demonstrating that CBMs can modulate the mode of action of LPMOs. This study demonstrates that engineered LPMO-CBM hybrids can display enhanced industrially relevant oxygenations., (© 2016 by The American Society for Biochemistry and Molecular Biology, Inc.)

An α-neoagarooligosaccharide hydrolase, AgaNash, was purified from Cellvibrio sp. OA-2007, which utilizes agarose as a substrate. The agaNash gene, which encodes AgaNash, was obtained by comparing the N-terminal amino acid sequence of AgaNash with that deduced from the nucleotide sequence of the full-length OA-2007 genome. The agaNash gene combined with the Saccharomyces cerevisiae signal peptide α-mating factor was transformed into the YPH499 strain of S. cerevisiae to generate YPH499/pTEF-MF-agaNash, and the recombinant yeast was confirmed to produce AgaNash, though it was mainly retained within the recombinant cell. To enhance AgaNash secretion from the cell, the signal peptide was replaced with a combination of the signal peptide and a threonine- and serine-rich tract (T-S region) of the S. diastaticus STA1 gene. The new recombinant yeast, YPH499/pTEF-STA1SP-agaNash, was demonstrated to secrete AgaNash and hydrolyze neoagarobiose with an efficiency of as high as 84%, thereby producing galactose, which is a fermentable sugar for the yeast, and ethanol, at concentrations of up to 1.8 g/L, directly from neoagarobiose., (Copyright © 2015 Elsevier Inc. All rights reserved.)

In this work, a Cellvibrio pealriver-microalga co-cultivation mode was used to promote the growths of four microalgae by using xylan as feedstock. After 12days of cultivation, the biomass concentrations of Chlorella sacchrarophila, Chlorella pyrenoidosa and Chlamydomonas reinhardtii in co-cultivation were equal to those in mixotrophic growth on glucose, and the Dunaliella was about 1.6-fold higher than that on glucose. The comparative transcriptomes analysis demonstrated that the xylose and xylan hydrolysates were catalyzed to some active substrates by C. pealriver via some functional enzymes; these active substrates are possibly responsible for the promotion of microalgal growth. This C. pealriver-microalga co-cultivation mode is a potential method to produce low-cost microalgal biodiesel by using hemicellulose as feedstock., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

Cellvibrio pealriver PR1 (CGMCC 1.14955=NBRC 110968) was isolated from a freshwater sample from the Pearl River in China. It is able to degrade various carbohydrates such as starch, xylan, agar, cellulose or chitin. The genomic feature and polysaccharide hydrolases of this strain were described in this paper. The total genome size of C. pealriver PR1 is 4,427,922 bp with 3986 coding sequences (CDS), 53 tRNAs, 16 rRNAs and 1 sRNA. The annotated full genome sequence of this strain provides the genetic basis for revealing its role as a xylanolytic and agarolytic bacterium., (Copyright © 2015. Published by Elsevier B.V.)

The depolymerization of the recalcitrant polysaccharides found in lignocellulose has become an area of intense interest due to the role of this process in global carbon cycling, human gut microbiome nutritional contributions, and bioenergy production. However, underdeveloped genetic tools have hampered study of bacterial lignocellulose degradation, especially outside model organisms. In this report, we describe an in-frame deletion strategy for the Gram-negative lignocellulose-degrading bacterium Cellvibrio japonicus. This method leverages optimized growth conditions for conjugation and sacB counterselection for the generation of markerless in-frame deletions. This method produces mutants in as few as 8 days and allows for the ability to make multiple gene deletions per strain. It is also possible to remove large sections of the genome, as shown in this report with the deletion of the nine-gene (9.4-kb) gsp operon in C. japonicus. We applied this system to study the complex phenotypes of cellulose degradation in C. japonicus. Our data indicated that a Δcel5B Δcel6A double mutant is crippled for cellulose utilization, more so than by either single mutation alone. Additionally, we deleted individual genes in the two-gene cbp2ED operon and showed that both genes contribute to cellulose degradation in C. japonicus. Overall, these described techniques substantially enhance the utility of C. japonicus as a model system to study lignocellulose degradation., (Copyright © 2015, American Society for Microbiology. All Rights Reserved.)

Cellvibrio mixtus strain J3-8 is a gram-negative, xylanase-producing aerobic soil bacterium isolated from giant snails in Singapore. It is able to produce up to 10.1 U ml(-1) of xylanase, which is comparable to xylanase production from known bacterial and fungal strains. Genome sequence analysis of strain J3-8 reveals that the assembled draft genome contains 5,171,890 bp with a G + C content of 46.66%, while open reading frame (ORF) annotations indicate a high density of genes encoding glycoside hydrolase (GH) families involved in (hemi)cellulose hydrolysis. On the basis of 15 identified putative xylanolytic genes, one metabolic pathway in strain J3-8 is constructed for utilization of xylan. In addition, a 1,083 bp xylanase gene from strain J3-8 represents a new member of GH11 family. This gene is verified to be novel via phylogenetic analysis. To utilize this novel gene for hydrolysis of xylan to xylose, it is expressed in recombinant E. coli and characterized for its hydrolytic activity. This study shows that strain J3-8 is a potential candidate for hydrolysis of lignocellulosic materials.

The degradation of plant biomass by saprophytes is an ecologically important part of the global carbon cycle, which has also inspired a vast diversity of industrial enzyme applications. The xyloglucans (XyGs) constitute a family of ubiquitous and abundant plant cell wall polysaccharides, yet the enzymology of XyG saccharification is poorly studied. Here, we present the identification and molecular characterization of a complex genetic locus that is required for xyloglucan utilization by the model saprophyte Cellvibrio japonicus. In harness, transcriptomics, reverse genetics, enzyme kinetics, and structural biology indicate that the encoded cohort of an α-xylosidase, a β-galactosidase, and an α-l-fucosidase is specifically adapted for efficient, concerted saccharification of dicot (fucogalacto)xyloglucan oligosaccharides following import into the periplasm via an associated TonB-dependent receptor. The data support a biological model of xyloglucan degradation by C. japonicus with striking similarities - and notable differences - to the complex polysaccharide utilization loci of the Bacteroidetes., (© 2014 The Authors. Molecular Microbiology published by John Wiley & Sons Ltd.)

Two Gram-reaction-negative, aerobic, nitrogen-fixing, rod-shaped bacteria, designated strains E20 and E50(T), were isolated from the rhizosphere of salt meadow plants Plantago winteri and Hordeum secalinum, respectively, near Münzenberg, Germany. Based on the 16S rRNA gene sequence analysis both strains E20 and E50(T) are affiliated with the genus Cellvibrio, sharing the highest similarity with Cellvibrio gandavensis LMG 18551(T) (96.4%) and (97.1%), respectively. Strains E20 and E50(T) were oxidase and catalase-positive, grew at a temperature range between 16 and 37 °C and in the presence of 0-5% NaCl (w/v). The DNA G+C contents were 52.1 mol% (E20) and 51.6 mol% (E50(T)). Major fatty acids of strains E20 and E50(T) were summed feature 3 (C16 : 1ω7c and/or iso-C(15 : 0) 2-OH), C(16 : 0), C(18 : 1)ω7c, C(12 : 0), C(18 : 0) and C(12 : 0) 3-OH. The DNA-DNA relatedness of the strains to Cellvibrio gandavensis LMG 18551(T) was 39% for strain E20 and 58% for strain E50(T). The nitrogen fixation capability of strains E20 and E50(T) was confirmed by the acetylene reduction assay. On the basis of our polyphasic taxonomic study, strains E20 and E50(T) represent a novel species of the genus Cellvibrio, for which the name Cellvibrio diazotrophicus is proposed. The type strain of Cellvibrio diazotrophicus is E50(T) ( = LMG 27267(T) = KACC 17069(T)). An emended description of the genus Cellvibrio is proposed based on the capability of fixing nitrogen and growth in presence of up to 5% NaCl (w/v).

Actinobacteria in the genus Cellulomonas are the only known and reported cellulolytic facultative anaerobes. To better understand the cellulolytic strategy employed by these bacteria, we sequenced the genome of the Cellulomonas fimi ATCC 484(T). For comparative purposes, we also sequenced the genome of the aerobic cellulolytic "Cellvibrio gilvus" ATCC 13127(T). An initial analysis of these genomes using phylogenetic and whole-genome comparison revealed that "Cellvibrio gilvus" belongs to the genus Cellulomonas. We thus propose to assign "Cellvibrio gilvus" to the genus Cellulomonas. A comparative genomics analysis between these two Cellulomonas genome sequences and the recently completed genome for Cellulomonas flavigena ATCC 482(T) showed that these cellulomonads do not encode cellulosomes but appear to degrade cellulose by secreting multi-domain glycoside hydrolases. Despite the minimal number of carbohydrate-active enzymes encoded by these genomes, as compared to other known cellulolytic organisms, these bacteria were found to be proficient at degrading and utilizing a diverse set of carbohydrates, including crystalline cellulose. Moreover, they also encode for proteins required for the fermentation of hexose and xylose sugars into products such as ethanol. Finally, we found relatively few significant differences between the predicted carbohydrate-active enzymes encoded by these Cellulomonas genomes, in contrast to previous studies reporting differences in physiological approaches for carbohydrate degradation. Our sequencing and analysis of these genomes sheds light onto the mechanism through which these facultative anaerobes degrade cellulose, suggesting that the sequenced cellulomonads use secreted, multidomain enzymes to degrade cellulose in a way that is distinct from known anaerobic cellulolytic strategies.

Agarase genes of non-marine agarolytic bacterium Cellvibrio sp. were cloned into Escherichia coli and one of the genes obtained using HindIII was sequenced. From nucleotide and putative amino acid sequences (713 aa, molecular mass; 78,771 Da) of the gene, designated as agarase AgaA, the gene was found to have closest homology to the Saccharophagus degradans (formerly, Microbulbifer degradans) 2-40 aga86 gene, belonging to glycoside hydrolase family 86 (GH86). The putative protein appears to be a non-secreted protein because of the absence of a signal sequence. The recombinant protein was purified with anion exchange and gel filtration columns after ammonium sulfate precipitation and the molecular mass (79 kDa) determined by SDS-PAGE and subsequent enzymography agreed with the estimated value, suggesting that the enzyme is monomeric. The optimal pH and temperature for enzymatic hydrolysis of agarose were 6.5 and 42.5 degrees C, and the enzyme was stable under 40 degrees C. LC-MS and NMR analyses revealed production of a neoagarobiose and a neoagarotetraose with a small amount of a neoagarohexaose during hydrolysis of agarose, indicating that the enzyme is a beta-agarase.

We have developed a simple, rapid, quantitative colorimetric assay to measure cellulose degradation based on the absorbance shift of Congo red dye bound to soluble cellulose. We term this assay "Congo Red Analysis of Cellulose Concentration," or "CRACC." CRACC can be performed directly in culture media, including rich and defined media containing monosaccharides or disaccharides (such as glucose and cellobiose). We show example experiments from our laboratory that demonstrate the utility of CRACC in probing enzyme kinetics, quantifying cellulase secretion, and assessing the physiology of cellulolytic organisms. CRACC complements existing methods to assay cellulose degradation, and we discuss its utility for a variety of applications.

The genus Pseudomonas has gone through many taxonomic revisions over the past 100 years, going from a very large and diverse group of bacteria to a smaller, more refined and ordered list having specific properties. The relationship of the Pseudomonas genus to Azotobacter vinelandii is examined using three genomic sequence-based methods. First, using 16S rRNA trees, it is shown that A. vinelandii groups within the Pseudomonas close to Pseudomonas aeruginosa. Genomes from other related organisms (Acinetobacter, Psychrobacter, and Cellvibrio) are outside the Pseudomonas cluster. Second, pan genome family trees based on conserved gene families also show A. vinelandii to be more closely related to Pseudomonas than other related organisms. Third, exhaustive BLAST comparisons demonstrate that the fraction of shared genes between A. vinelandii and Pseudomonas genomes is similar to that of Pseudomonas species with each other. The results of these different methods point to a high similarity between A. vinelandii and the Pseudomonas genus, suggesting that Azotobacter might actually be a Pseudomonas.

Microbial degradation of plant cell walls is a critical contributor to the global carbon cycle, and enzymes derived from microbes play a key role in the sustainable biofuels industry. Despite its biological and biotechnological importance, relatively little is known about how microbes degrade plant cell walls. Much of this gap in knowledge has resulted from difficulties in extending modern molecular tools to the study of plant cell wall-degrading microbes. The bacterium Cellvibrio japonicus has recently emerged as a powerful model system for the study of microbial plant cell wall degradation. C. japonicus is unique among microbial model systems in that it possesses the ability to carry out the complete degradation of plant cell wall polysaccharides. Furthermore, an extensive array of genetic and molecular tools exists for functional genomic analysis. In this review, we describe progress in the development of methodology for the functional genomic study of plant cell wall degradation by this microbe, and discuss future directions for research., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Clostridium thermocellum cellodextrin phosphorylase (CtCDP), a single-module protein without an apparent carbohydrate-binding module, has reported activities on soluble cellodextrin with a degree of polymerization (DP) from two to five. In this study, CtCDP was first discovered to have weak activities on weakly water-soluble celloheptaose and insoluble regenerated amorphous cellulose (RAC). To enhance its activity on solid cellulosic materials, four cellulose binding modules, e.g., CBM3 (type A) from C. thermocellum CbhA, CBM4-2 (type B) from Rhodothermus marinus Xyn10A, CBM6 (type B) from Cellvibrio mixtus Cel5B, and CBM9-2 (type C) from Thermotoga maritima Xyn10A, were fused to the C terminus of CtCDP. Fusion of any selected CBM with CtCDP did not influence its kinetic parameters on cellobiose but affected the binding and catalytic properties on celloheptaose and RAC differently. Among them, addition of CBM9 to CtCDP resulted in a 2.7-fold increase of catalytic efficiency for degrading celloheptaose. CtCDP-CBM9 exhibited enhanced specific activities over 20% on the short-chain RAC (DP = 14) and more than 50% on the long-chain RAC (DP = 164). The chimeric protein CtCDP-CBM9 would be the first step to construct a cellulose phosphorylase for in vitro hydrogen production from cellulose by synthetic pathway biotransformation (SyPaB).

The microbial deconstruction of the plant cell wall is a critical biological process, which also provides important substrates for environmentally sustainable industries. Enzymes that hydrolyze the plant cell wall generally contain non-catalytic carbohydrate binding modules (CBMs) that contribute to plant cell wall degradation. Here we report the biochemical properties and crystal structure of a family of CBMs (CBM60) that are located in xylanases. Uniquely, the proteins display broad ligand specificity, targeting xylans, galactans, and cellulose. Some of the CBM60s display enhanced affinity for their ligands through avidity effects mediated by protein dimerization. The crystal structure of vCBM60, displays a β-sandwich with the ligand binding site comprising a broad cleft formed by the loops connecting the two β-sheets. Ligand recognition at site 1 is, exclusively, through hydrophobic interactions, whereas binding at site 2 is conferred by polar interactions between a protein-bound calcium and the O2 and O3 of the sugar. The observation, that ligand recognition at site 2 requires only a β-linked sugar that contains equatorial hydroxyls at C2 and C3, explains the broad ligand specificity displayed by vCBM60. The ligand-binding apparatus of vCBM60 displays remarkable structural conservation with a family 36 CBM (CBM36); however, the residues that contribute to carbohydrate recognition are derived from different regions of the two proteins. Three-dimensional structure-based sequence alignments reveal that CBM36 and CBM60 are related by circular permutation. The biological and evolutionary significance of the mechanism of ligand recognition displayed by family 60 CBMs is discussed.

A novel agarolytic bacterium KY-YJ-3, producing extracellular agarase, was isolated from the freshwater sediment of the Sincheon River in Daegu, Korea. On the basis of gram-staining data, morphology, and phylogenetic analysis of the 16S rDNA sequence, the isolate was identified as Cellvibrio sp. By ammonium sulfate precipitation followed by Toyopearl QAE-550C, Toyopearl HW-55F, and Mono-Q column chromatography, the extracellular agarase in the culture fluid could be purified 120.2-fold with yield of 8.1%. The specific activity of the purified agarase was 84.2 U/mg. The molecular mass of the purified agarase was 70 kDa as determined by dodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The optimal temperature and pH of the purified agarase were 35 degrees C and pH 7.0, respectively. The purified agarase failed to hydrolyze the other polysaccharide substrates, including carboxymethyl (CM)-cellulose, dextran, soluble starch, pectin, and polygalacturonic acid. Kinetic analysis of the agarose-hydrolysis catalyzed by the purified agarase using thin layer chromatography (TLC) exhibited that the main products were neoagarobiose, neoagarotetraose, and neoagarohexaose. These results demonstrated that the newly isolated freshwater agarolytic bacterium KY-YJ-3 was a Cellvibrio sp., and could produce an extracellular beta-agarase, which hydrolyzed agarose to yield neoagarobiose, neoagarotetraose, and neoagarohexaose as the main products.

Cellulosic biofuels represent a powerful alternative to petroleum but are currently limited by the inefficiencies of the conversion process. While gram-positive and fungal organisms have been widely explored as sources of cellulases and hemicellulases for biomass degradation, gram-negative organisms have received less experimental attention. We investigated the ability of Cellvibrio japonicus, a recently sequenced gram-negative cellulolytic bacterium, to degrade bioenergy-related feedstocks. Using a newly developed biomass medium, we showed that C. japonicus is able to utilize corn stover and switchgrass as sole sources of carbon and energy for growth. We also developed tools for directed gene disruptions in C. japonicus and used this system to construct a mutant in the gspD gene, which is predicted to encode a component of the type II secretion system. The gspD::pJGG1 mutant displayed a greater-than-2-fold decrease in endoglucanase secretion compared to wild-type C. japonicus. In addition, the mutant strain showed a pronounced growth defect in medium with biomass as a carbon source, yielding 100-fold fewer viable cells than the wild type. To test the potential of C. japonicus to undergo metabolic engineering, we constructed a strain able to produce small amounts of ethanol from biomass. Collectively, these data suggest that C. japonicus is a useful platform for biomass conversion and biofuel production.

Noncatalytic carbohydrate-binding modules (CBMs), which are found in a variety of carbohydrate-degrading enzymes, have been grouped into sequence-based families. CBMs, by recruiting their appended enzymes onto the surface of the target substrate, potentiate catalysis particularly against insoluble substrates. Family 6 CBMs (CBM6s) display unusual properties in that they present two potential ligand-binding sites termed clefts A and B, respectively. Cleft B is located on the concave surface of the beta-sandwich fold while cleft A, the more common binding site, is formed by the loops that connect the inner and the outer beta-sheets. Here, we report the biochemical properties of CBM6-1 from Cellvibrio mixtus CmCel5A. The data reveal that CBM6-1 specifically recognizes beta1,3-glucans through residues located both in cleft A and in cleft B. In contrast, a previous report showed that a CBM6 derived from a Bacillus halodurans laminarinase binds to beta1,3-glucans only in cleft A. These studies reveal a different mechanism by which a highly conserved protein platform can recognize beta1,3-glucans.

The microbial degradation of lignocellulose biomass is not only an important biological process but is of increasing industrial significance in the bioenergy sector. The mechanism by which the plant cell wall, an insoluble composite structure, activates the extensive repertoire of microbial hydrolytic enzymes required to catalyze its degradation is poorly understood. Here we have used a transposon mutagenesis strategy to identify a genetic locus, consisting of two genes that modulate the expression of xylan side chain-degrading enzymes in the saprophytic bacterium Cellvibrio japonicus. Significantly, the locus encodes a two-component signaling system, designated AbfS (sensor histidine kinase) and AbfR (response regulator). The AbfR/S two-component system is required to activate the expression of the suite of enzymes that remove the numerous side chains from xylan, but not the xylanases that hydrolyze the beta1,4-linked xylose polymeric backbone of this polysaccharide. Studies on the recombinant sensor domain of AbfS (AbfS(SD)) showed that it bound to decorated xylans and arabinoxylo-oligosaccharides, but not to undecorated xylo-oligosaccharides or other plant structural polysaccharides/oligosaccharides. The crystal structure of AbfS(SD) was determined to a resolution of 2.6A(.) The overall fold of AbfS(SD) is that of a classical Per Arndt Sim domain with a central antiparallel four-stranded beta-sheet flanked by alpha-helices. Our data expand the number of molecules known to bind to the sensor domain of two-component histidine kinases to include complex carbohydrates. The biological rationale for a regulatory system that induces enzymes that remove the side chains of xylan, but not the hydrolases that cleave the backbone of the polysaccharide, is discussed.

The plant cell wall, which consists of a highly complex array of interconnecting polysaccharides, is the most abundant source of organic carbon in the biosphere. Microorganisms that degrade the plant cell wall synthesize an extensive portfolio of hydrolytic enzymes that display highly complex molecular architectures. To unravel the intricate repertoire of plant cell wall-degrading enzymes synthesized by the saprophytic soil bacterium Cellvibrio japonicus, we sequenced and analyzed its genome, which predicts that the bacterium contains the complete repertoire of enzymes required to degrade plant cell wall and storage polysaccharides. Approximately one-third of these putative proteins (57) are predicted to contain carbohydrate binding modules derived from 13 of the 49 known families. Sequence analysis reveals approximately 130 predicted glycoside hydrolases that target the major structural and storage plant polysaccharides. In common with that of the colonic prokaryote Bacteroides thetaiotaomicron, the genome of C. japonicus is predicted to encode a large number of GH43 enzymes, suggesting that the extensive arabinose decorations appended to pectins and xylans may represent a major nutrient source, not just for intestinal bacteria but also for microorganisms that occupy terrestrial ecosystems. The results presented here predict that C. japonicus possesses an extensive range of glycoside hydrolases, lyases, and esterases. Most importantly, the genome of C. japonicus is remarkably similar to that of the gram-negative marine bacterium, Saccharophagus degradans 2-40(T). Approximately 50% of the predicted C. japonicus plant-degradative apparatus appears to be shared with S. degradans, consistent with the utilization of plant-derived complex carbohydrates as a major substrate by both organisms.

Hydrolysis of plant cell wall polysaccharides, a process which is of intrinsic biological and biotechnological importance, requires the concerted action of an extensive repertoire of microbial cellulases and hemicellulases. Here, we report the identification of the gene cluster unk16A, regA and cel5B in the aerobic soil bacterium Cellvibrio mixtus, encoding a family 16 (CmUnk16A) glycoside hydrolase (GH), an AraC/XylS transcription activator (CmRegA) and a family 5 (CmCel5B) endo-glucanase, respectively. CmUnk16A is a modular enzyme comprising, in addition to the catalytic domain, two family 32 carbohydrate-binding modules (CBMs), termed CBM32-1 and CBM32-2, a CBM4 and a domain of unknown function. We show that CBM32-2 binds weakly to laminarin and pustulan. CmRegA is also a modular protein containing a highly hydrophobic N-terminal domain and a C-terminal DNA-binding domain of the AraC/XylS family. The role of the identified enzymes in the hydrolysis of cell wall polysaccharides by aerobic bacteria is discussed.

The enzymatic hydrolysis of the glycosidic bond is central to numerous biological processes. Glycoside hydrolases, which catalyze these reactions, are grouped into families based on primary sequence similarities. One of the largest glycoside hydrolase families is glycoside hydrolase family 5 (GH5), which contains primarily endo-acting enzymes that hydrolyze beta-mannans and beta-glucans. Here we report the cloning, characterization, and three-dimensional structure of the Cellvibrio mixtus GH5 beta-mannosidase (CmMan5A). This enzyme releases mannose from the nonreducing end of mannooligosaccharides and polysaccharides, an activity not previously observed in this enzyme family. CmMan5A contains a single glycone (-1) and two aglycone (+1 and +2) sugar-binding subsites. The -1 subsite displays absolute specificity for mannose, whereas the +1 subsite does not accommodate galactosyl side chains but will bind weakly to glucose. The +2 subsite is able to bind to decorated mannose residues. CmMan5A displays similar activity against crystalline and amorphous mannans, a property rarely attributed to glycoside hydrolases. The 1.5 A crystal structure reveals that CmMan5A adopts a (beta/alpha)(8) barrel fold, and superimposition with GH5 endo-mannanases shows that dramatic differences in the length of three loops modify the active center accessibility and thus modulate the specificity from endo to exo. The most striking and significant difference is the extended loop between strand beta8 and helix alpha8 comprising residues 378-412. This insertion forms a "double" steric barrier, formed by two short beta-strands that function to "block" the substrate binding cleft at the edge of the -1 subsite forming the "exo" active center topology of CmMan5A.

Thirty-one cellulolytic bacterial isolates from soils that were phenotypically very similar and phylogenetically highly related to Cellvibrio strains were further characterized using a polyphasic taxonomic approach. By using repetitive extragenic palindromic DNA-PCR fingerprinting, six different fingerprints could be recognized among the isolates. Representative strains and four reference strains of the genus Cellvibrio were used for DNA-DNA hybridization, which yielded eight DNA hybridization groups at a cut-off level of 70% DNA binding. One group was formed by three isolates and Cellvibrio vulgaris LMG 2848T and a second group consisted of Cellvibrio mixtus strains ACM 2601T and ACM 2603. Two isolates and Cellvibrio fulvus LMG 2847T constituted single-member groups. For the remaining groups, three novel species are proposed: Cellvibrio fibrivorans sp. nov. (six strains, type strain LMG 18561T =ACM 5172T), Cellvibrio ostraviensis sp. nov. (eight strains, type strain LMG 19434T =ACM 5173T) and Cellvibrio gandavensis sp. nov. (12 strains, type strain LMG 18551T =ACM 5174T). The novel Cellvibrio species could be differentiated from each other and from C. mixtus, C. vulgaris and C. fulvus on the basis of phenotypic features, their fatty acid compositions and the G + C content of their DNA.

'Pseudomonas fluorescens subsp. cellulosa' NCIMB 10462 has been demonstrated by a polyphasic taxonomic approach to be a member of the genus Cellvibrio. 16S rDNA sequence analysis suggests that this is the only genus that could accept this specimen. The sequence is 95.5% similar to that of Cellvibrio mixtus subsp. mixtus ACM 2601T (the type strain of the type species of the genus), which is its closest relation. The genomic DNA G + C content was determined to be 53.3 mol%, which is similar to the values obtained for the validly described Cellvibrio species. DNA-DNA hybridization experiments have shown that strain NCIMB 10462T (= NCDO 2697T) represents a novel species; therefore, it is proposed that it be designated as the type strain of the novel species Cellvibrio japonicus sp. nov. This study also used 16S rDNA analysis, DNA-DNA hybridization experiments and phenotypic testing to revive the species Cellvibrio vulgaris sp. nov., nom. rev. and Cellvibrio fulvus sp. nov., nom. rev. C. vulgaris NCIMB 8633T (=LMG 2848T) and C. fulvus NCIMB 8634T (=LMG 2847T) are the proposed type strains.

Enzyme-catalyzed beta-elimination of sugar uronic acids, exemplified by the degradation of plant cell wall pectins, plays an important role in a wide spectrum of biological processes ranging from the recycling of plant biomass through to pathogen virulence. The three-dimensional crystal structure of the catalytic module of a "family PL-10" polysaccharide lyase, Pel10Acm from Cellvibrio japonicus, solved at a resolution of 1.3 A, reveals a new polysaccharide lyase fold and is the first example of a polygalacturonic acid lyase that does not exhibit the "parallel beta-helix" topology. The "Michaelis" complex of an inactive mutant in association with the substrate trigalacturonate/Ca2+ reveals the catalytic machinery harnessed by this polygalacturonate lyase, which displays a stunning resemblance, presumably through convergent evolution, to the tetragalacturonic acid complex observed for a structurally unrelated polygalacturonate lyase from family PL-1. Common coordination of the -1 and +1 subsite saccharide carboxylate groups by a protein-liganded Ca2+ ion, the positioning of an arginine catalytic base in close proximity to the alpha-carbon hydrogen and numerous other conserved enzyme-substrate interactions, considered in light of mutagenesis data for both families, suggest a generic polysaccharide anti-beta-elimination mechanism.

Hydrolysis of the plant cell wall polysaccharides cellulose and xylan requires the synergistic interaction of a repertoire of extracellular enzymes. Recently, evidence has emerged that anaerobic bacteria can synthesize high levels of periplasmic xylanases which may be involved in the hydrolysis of small xylo-oligosaccharides absorbed by the micro-organism. Cellvibrio mixtus, a saprophytic aerobic soil bacterium that is highly active against plant cell wall polysaccharides, was shown to express internal xylanase activity when cultured on media containing xylan or glucose as sole carbon source. A genomic library of C. mixtus DNA, constructed in lambdaZAPII, was screened for xylanase activity. The nucleotide sequence of the genomic insert from a xylanase-positive clone that expressed intracellular xylanase activity in Escherichia coli revealed an ORF of 1137 bp (xynC), encoding a polypeptide with a deduced M(r) of 43413, defined as xylanase C (XylC). Probing a gene library of Pseudomonas fluorescens subsp. cellulosa with C. mixtus xynC identified a xynC homologue (designated xynG) encoding XylG; XylG and xynG were 67% and 63% identical to the corresponding C. mixtus sequences, respectively. Both XylC and XylG exhibit extensive sequence identity with family 10 xylanases, particularly with non-modular enzymes, and gene deletion studies on xynC supported the suggestion that they are single-domain xylanases. Purified recombinant XylC had an M(r) of 41000, and displayed biochemical properties typical of family 10 polysaccharidases. However, unlike previously characterized xylanases, XylC was particularly sensitive to proteolytic inactivation by pancreatic proteinases and was thermolabile. C. mixtus was grown to late-exponential phase in the presence of glucose or xylan and the cytoplasmic, periplasmic and cell envelope fractions were probed with anti-XylC antibodies. The results showed that XylC was absent from the culture media but was predominantly present in the periplasm of C. mixtus cells grown on glucose, xylan, CM-cellulose or Avicel. These data suggest that C. mixtus can express non-modular internal xylanases whose potential roles in the hydrolysis of plant cell wall components are discussed.