Cellvibrio polysaccharolyticus sp. nov., a cellulolytic bacterium isolated from agricultural soil.

A novel Gram-reaction-negative bacterial strain, designated Ka43T, was isolated from agricultural soil and characterised using a polyphasic approach to determine its taxonomic position. On the basis of 16S rRNA gene sequence analysis, the strain shows highest similarity (97.1 %) to Cellvibrio diazotrophicus E50T. Cells of strain Ka43T are aerobic, motile, short rods. The major fatty acids are summed feature 3 (C16 : 1 ω7c and/or iso-C15 : 0 2-OH), C18 : 1 ω7c and C16 : 0. The only isoprenoid quinone is Q-8. The polar lipid profile includes phosphatidylethanolamine, phosphatidylglycerol, four phospholipids, two lipids and an aminolipid. The assembled genome of strain Ka43T has a total length of 4.2 Mb and the DNA G+C content is 51.6 mol%. Based on phenotypic data, including chemotaxonomic characteristics and analysis of the 16S rRNA gene sequences, it was concluded that strain Ka43T represents a novel species in the genus Cellvibrio, for which the name Cellvibrio polysaccharolyticus sp. nov. is proposed. The type strain of the species is strain Ka43T (=LMG 31577T=NCAIM B.02637T).

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).

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.

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).

The genome sequences of Cellulomonas fimi and "Cellvibrio gilvus" reveal the cellulolytic strategies of two facultative anaerobes, transfer of "Cellvibrio gilvus" to the genus Cellulomonas, and proposal of Cellulomonas gilvus sp. nov.

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.

Defining the Pseudomonas genus: where do we draw the line with Azotobacter?

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.

Isolation of a novel freshwater agarolytic Cellvibrio sp. KY-YJ-3 and characterization of its extracellular beta-agarase.

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.

Identification and specific detection of a novel pseudomonadaceae cluster associated with soils from winter wheat plots of a long-term agricultural field experiment.

The genus Pseudomonas (sensu stricto) represents a group of microorganisms directly involved in functions conferring plant health. We performed a study in the DOK long-term agricultural field experiment on the basis of previously published Pseudomonas-selective PCR primers in order to investigate the community structure of the microbial groups defined by the target range of these primers. Three different agricultural management systems, i.e., conventional, biodynamic, and bio-organic, along with mineral and unfertilized controls were investigated, with each system planted with either winter wheat or a grass-clover ley. Amplified small-subunit rRNA gene fragments were analyzed using the genetic profiling techniques restriction fragment length polymorphism (RFLP) and denaturing gradient gel electrophoresis (DGGE), revealing distinct differences between soils planted with winter wheat and grass clover but only minor differences between the management systems. Phylogenetic analyses of 59 clone sequences retrieved from bio-organic and unfertilized systems identified sequences related to Pseudomonas fluorescens and a novel cluster termed Cellvibrio-related Pseudomonadaceae (CRP). The CRP clones were exclusively isolated from winter wheat soil samples and were responsible for the crop-specific differences observed in RFLP and DGGE profiles. New primers were designed for the amplification of CRP targets directly from soil DNA, yielding strong signals exclusively for winter wheat soils. We concluded that crop-associated CRP exist in agricultural soils and that genetic profiling followed by specific probe design represents a valuable approach for identification as well as sensitive and rapid monitoring of novel microbial groups in the environment.

Reclassification of 'Pseudomonas fluorescens subsp. cellulosa' NCIMB 10462 (Ueda et al. 1952) as Cellvibrio japonicus sp. nov. and revival of Cellvibrio vulgaris sp. nov., nom. rev. and Cellvibrio fulvus sp. nov., nom. rev.

'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.

Taxonomic study of Cellvibrio strains and description of Cellvibrio ostraviensis sp. nov., Cellvibrio fibrivorans sp. nov. and Cellvibrio gandavensis sp. nov.

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.