Microbe Profile: Cellvibrio japonicus: living the sweet life via biomass break-down.

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.

RNAseq analysis of Cellvibrio japonicus during starch utilization differentiates between genes encoding carbohydrate active enzymes controlled by substrate detection or growth rate.

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.

C-type cytochrome-initiated reduction of bacterial lytic polysaccharide monooxygenases.

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.

Efficient chito-oligosaccharide utilization requires two TonB-dependent transporters and one hexosaminidase in Cellvibrio japonicus.

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.

Kinetic modeling of microbial growth, enzyme activity, and gene deletions: An integrated model of ß-glucosidase function in Cellvibrio japonicus.

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 ß-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 ß-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.

The complex physiology of Cellvibrio japonicus xylan degradation relies on a single cytoplasmic ß-xylosidase for xylo-oligosaccharide utilization.

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 ß-xylosidase is essential for the utilization of xylo-oligosaccharides.

Cellvibrio zantedeschiae sp. nov., isolated from the roots of Zantedeschia aethiopica.

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).

Cellvibrio fontiphilus sp. nov., isolated from a spring.

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).

Comparative Phenotype and Genome Analysis of Cellvibrio sp. PR1, a Xylanolytic and Agarolytic Bacterium from the Pearl River.

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%), C15:0 iso 2-OH or/and C16:1ω7c (17.4%), and C18:1ω7c or/and C18:1ω6c (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 ß-xylanases, 16 ß-xylosidases, 17 α-arabinofuranosidases, 9 acetyl xylan esterases, 4 α-glucuronidases, and 2 ferulic acid esterases) and agar-hydrolyzing enzyme system (2 ß-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.

Custom fabrication of biomass containment devices using 3-D printing enables bacterial growth analyses with complex insoluble substrates.

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.

Polysaccharide degradation systems of the saprophytic bacterium Cellvibrio japonicus.

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.

Functions, structures, and applications of cellobiose 2-epimerase and glycoside hydrolase family 130 mannoside phosphorylases.

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 ß-(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. ß-Mannoside hydrolase and 4-O-ß-d-mannosyl-d-glucose phosphorylase (MGP) were found as neighboring genes of CE, meaning that CE is involved in ß-mannan metabolism, where it epimerizes ß-d-mannopyranosyl-(1→4)-d-mannose to ß-d-mannopyranosyl-(1→4)-d-glucose for further phosphorolysis. MGPs form glycoside hydrolase family 130 (GH130) together with other ß-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.

Direct production of ethanol from neoagarobiose using recombinant yeast that secretes α-neoagarooligosaccharide hydrolase.

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.

The Contribution of Non-catalytic Carbohydrate Binding Modules to the Activity of Lytic Polysaccharide Monooxygenases.

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.

Promotion of microalgal growth by co-culturing with Cellvibrio pealriver using xylan as feedstock.

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.

Genome sequence of Cellvibrio pealriver PR1, a xylanolytic and agarolytic bacterium isolated from freshwater.

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.

In-Frame Deletions Allow Functional Characterization of Complex Cellulose Degradation Phenotypes in Cellvibrio japonicus.

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.

Characterization of a xylanase-producing Cellvibrio mixtus strain J3-8 and its genome analysis.

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.

A complex gene locus enables xyloglucan utilization in the model saprophyte Cellvibrio japonicus.

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 ß-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.

Cellvibrio diazotrophicus sp. nov., a nitrogen-fixing bacteria isolated from the rhizosphere of salt meadow plants and emended description of the genus Cellvibrio.

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).