Mesorhizobial strains nodulating Anagyris latifolia and Lotus berthelotii in Tamadaya ravine (Tenerife, Canary Islands) are two symbiovars of the same species, Mesorhizobium tamadayense sp. nov

Barranco de Tamadaya is a deep ravine located in southern Tenerife, which is included within a protected area where several endemic plants grow. Among them, two legumes are catalogued as critically endangered, Anagyris latifolia and Lotus berthelotii. Rhizobial strains isolated from their root nodules grown in soil samples from this ravine harboured symbiotic genes belonging to two distant symbiovars, but they shared identical 16S rRNA gene sequences (rrs). The phylogeny based on the rrs sequences placed these isolates in a separate subbranch that did not include any of the currently recognised Mesorhizobium species, but the resolution of the ribosomal tree did not permit further taxonomic conclusions. Nevertheless, multilocus sequence analysis (MLSA) of four housekeeping genes (atpD, recA, glnII and dnaK) and the rrs gene generated a highly supported Bayesian phylogeny, identifying these isolates as a new Mesorhizobium lineage. DNA-DNA hybridisation homology percentages were lower than 30% compared to type strains of the closest related species, and supported the phylogenetic data. Phenotypic characterisation also distinguished this lineage from the other closest Mesorhizobium species. The polyphasic approach thus confirmed that the isolates represented a novel species for which we propose the name Mesorhizobium tamadayense sp. nov. The type strain is Ala-3(T) (CECT 8040(T), LMG 26736(T)).     

Shivajiella indica gen. nov., sp. nov., a marine bacterium of the family "Cyclobacteriaceae" with nitrate reducing activity

Novel orange pigmented, Gram-negative-staining, rod-shaped, non-motile, strictly aerobic strains designated NIO-S1(T) and NIO-S2 were isolated from the water sample of a pond adjacent to the coast and an algal mat from a fish pond, respectively, at Kakinada, India. Both strains were positive for oxidase, catalase and β-galactosidase activities. The predominant fatty acids in NIO-S1(T) were iso-C(15:0) (39.6%), anteiso-C(15:0) (9.9%), iso-C(17:0) 3OH (10.9%) and C(16:1)ω7c/C(16:1)ω6c (summed feature 3) (5.7%). The strains contained MK-7 as the major respiratory quinine, and diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and three unidentified lipids as the polar lipids. Phylogenetic analysis indicated that strain NIO-S1(T) was a member of the family "Cyclobacteriaceae" of the class "Sphingobacteriia" and it clustered with the genera Fontibacter, Cecembia and Aquiflexum with phylogenetic distances of 6.8, 9.0 and 12.2% (93.2, 91.0 and 87.8% similarity), respectively. DNA-DNA hybridization between strains NIO-S1(T) and NIO-S2 showed a relatedness of 93% and rep-PCR banding patterns were similar. Based on data from the current polyphasic study, it is proposed that the new isolates be placed in a new genus and species with the name Shivajiella indica gen. nov., sp. nov. The type strain of Shivajiella indica is NIO-S1(T) (= KCTC 19812(T)=MTCC 11065(T)).     

Bradyrhizobium rifense sp. nov. isolated from effective nodules of Cytisus villosus grown in the Moroccan Rif

Two bradyrhizobial strains, CTAW71(T) and CTAW69, previously isolated from root nodules of Cytisus villosus, have been analysed using a polyphasic approach. These strains have identical 16S rRNA genes and their closest relative species is Bradyrhizobium cytisi, whose type strain CTAW11(T) presented 99.8% identity with respect to strain CTAW71(T). Despite the closeness of the 16S rRNA genes, the housekeeping genes recA, atpD and glnII harboured by strain CTAW71(T) were divergent to those from B. cytisi CTAW11(T), with identity values of 93%, 95% and 97%, respectively. These differences were congruent with DNA-DNA hybridization analysis that revealed an average of 37% relatedness between strain CTAW71(T) and B. cytisi CTAW11(T). Phenotypic characteristics were identical for strains CTAW71(T) and CTAW69, but differed from those of the described species from genus Bradyrhizobium. Based on the genotypic and phenotypic data obtained in this study, we propose that strains CTAW71(T) and CTAW69 should be classified into a new species for which the name Bradyrhizobium rifense sp. nov. is proposed (type strain CTAW71(T)=LMG 26781(T)=CECT 8066(T)).     

Chryseobacterium oncorhynchi sp. nov., isolated from rainbow trout (Oncorhynchus mykiss)

Genotypic and phenotypic analyses were performed on five Gram-negative, catalase and oxidase-positive, rod-shaped bacteria isolated from the gill and liver of four rainbow trout. Studies based on comparative 16S rRNA gene sequence analysis showed that the five new isolates shared 99.8-100% sequence similarity and that they belong to the genus Chryseobacterium. The nearest phylogenetic neighbours of the strain 701B-08(T) were Chryseobacterium ureilyticum F-Fue-04IIIaaaa(T) (99.1% 16S rRNA gene sequence similarity) and Chryseobacterium joosteii LMG 18212(T) (98.6%). DNA-DNA hybridization values between the five isolates were 91-99% and ranged from 2 to 53% between strain 701B-08(T) and the type strains of phylogenetically closely related species of Chryseobacterium. Strain 701B-08(T) had a DNA G+C content of 36.3 mol%, the major fatty acids were iso-C(15:0), iso-C(17:1)ω9c, C(16:1)ω6c and iso-C(17:0) 3-OH and the predominant respiratory quinone was MK-6. The novel isolates were distinguished from related Chryseobacterium species by physiological and biochemical tests. The genotypic and phenotypic properties of the isolates from rainbow trout suggest their classification as representatives of a novel species of the genus Chryseobacterium, for which the name Chryseobacterium oncorhynchi sp. nov. is proposed. The type strain is 701B-08(T) (=CECT 7794(T)=CCUG 60105(T)).     

Legionella impletisoli sp. nov. and Legionella yabuuchiae sp. nov., isolated from soils contaminated with industrial wastes in Japan

In this study, we tried to isolate legionellae from nine Legionella DNA-positive soil samples collected from four different sites contaminated with industrial wastes in Japan. Using culture methods with or without Acanthamoeba culbertsoni, a total of 22 isolates of legionellae were obtained from five of the nine samples. Identification of species and/or serogroups (SGs), performed by DNA-DNA hybridization and agglutination tests, revealed that the 22 isolates consisted of ten isolates of Legionella pneumophila including five SGs, five Legionella feeleii, and one each of Legionella dumoffii, Legionella longbeachae, and Legionella jamestownensis. The species of the remaining four isolates (strains OA1-1, -2, -3, and -4) could not be determined, suggesting that these isolates may belong to new species. The 16S rDNA sequences (1476-1488bp) of the isolates had similarities of less than 95.0% compared to other Legionella species. A phylogenetic tree created by analysis of the 16S rRNA (1270bp) genes demonstrated that the isolates formed distinct clusters within the genus Legionella. Quantitative DNA-DNA hybridization tests on the OA1 strains indicated that OA1-1 should be categorized as a new taxon, whereas OA1-2, -3, and -4 were also genetically independent in another taxon. Based on the evaluated phenotypic and phylogenetic characteristics, it is proposed that one of these isolates from the soils, OA1-1, be classified as a novel species, Legionella impletisoli sp. nov.; the type strain is strain OA1-1(T) (=JCM 13919(T)=DSMZ 18493(T)). The remaining three isolates belong to another novel Legionella species, Legionella yabuuchiae sp. nov.; the type strain is strain OA1-2(T) (=JCM 14148(T)=DSMZ 18492(T)). This is the first report on the isolation of legionellae from soils contaminated with industrial wastes.     

Fusobacterium canifelinum sp. nov., from the oral cavity of cats and dogs

Fourteen strains of Gram-negative, anaerobic, fluoroquinolone-resistant, non-sporulating rods were isolated from various infections in cats and dogs, as well as from wounds in humans after cat- or dog-bites. These strains were characterized by sequencing of the 16S-23S rDNA internal transcribed spacer (ITS) regions, 16S rDNA, DNA-DNA hybridization, phylogenetic analysis, and phenotypic tests. The results indicate that the novel strains belong to a distinct species, closely related to Fusobacterium nucleatum. The species Fusobacterium canifelinum sp. nov. is proposed, with strain ATCC BAA 689T as the type strain.     

Description of Gibbsiella quercinecans gen. nov., sp. nov., associated with Acute Oak Decline

Gram-negative, facultatively anaerobic bacterial strains were consistently isolated from oak trees displaying symptoms of extensive stem bleeding. In Britain, this disorder is called Acute Oak Decline (AOD). A similar condition has been noted on species of Mediterranean oak in Spain. The identity of bacterial isolates from symptomatic trees in both countries was investigated using molecular techniques and phenotypic assays. 16S rRNA gene sequencing indicated that the strains were most closely related to the genera Serratia, Kluyvera, Klebsiella and Raoultella (all>97%). Phylogenetic analysis revealed that the strains formed a distinct lineage within the family Enterobacteriaceae, which was confirmed by both gyrB- and rpoB-gene sequencing. DNA-DNA hybridization confirmed that the strains belonged to a single taxon which could also be differentiated phenotypically from its closest phylogenetic neighbours. The phylogenetic and phenotypic data both demonstrated that the strains isolated from oak represented a novel genus and species within the family Enterobacteriaceae for which the name Gibbsiella quercinecans gen. nov., sp. nov. (type strain=FRB 97(T)=LMG 25500(T)=NCPPB 4470(T)) is proposed.     

Microbacterium marinum sp. nov., isolated from deep-sea water

Two Gram-positive, rod-shaped bacterial strains, H101(T) and H207, were isolated from deep sea water collected from South-West Indian Ocean. Phylogenetic analysis of 16S rRNA gene sequences showed that the two strains were closely related to one another (100% similarity), and had the closest relationship with Microbacterium hominis NBRC 15708(T) and Microbacterium insulae KCTC 19247(T) (98.2-98.3% similarities). DNA-DNA hybridization value between strains H101(T) and H207 was 87.2 ± 3.7%, and the values between the two strains and the closely related type strains were well below 70%. The two strains also shared a number of physiological and biochemical characteristics that were distinct from the closely related species, and grew at 2-37 ° C, pH 5-11 and 0-8% (w/v) NaCl. Both strains contained MK-12, MK-13 and MK-11 as the detected menaquinones. The peptidoglycan was of type B1γ with an interpeptide bridge D-Glu(Hyg)→ Gly(2)→ l-Lys. The major cellular fatty acids were anteiso-C(15:0), anteiso-C(17:0), and iso-C(16:0). Based on the genetic and phenotypic properties, it is proposed that strains H101(T) and H207 be classified as representatives of a novel species of the genus Microbacterium, with the name Microbacterium marinum sp. nov. The type strain is H101(T) (= CGMCC 4.6941(T) = DSM 24947(T)).     

Molecular and phenotypic characterization of strains nodulating Anthyllis vulneraria in mine tailings, and proposal of Aminobacter anthyllidis sp. nov., the first definition of Aminobacter as legume-nodulating bacteria

Bacterial strains from Zn-Pb mine tailings were isolated by trapping with Anthyllis vulneraria, a legume-host suitable for mine substratum phytostabilisation. Sequence analysis of the 16S rRNA gene and three housekeeping genes (atpD, dnaK and recA) showed that they were related to those of the genus Aminobacter. DNA-DNA relatedness of representative isolates supported the placement of novel strains in Aminobacter as a new species. Phenotypic data emphasize their differentiation from the other related species of Aminobacter and Mesorhizobium. Aminobacter isolates exhibited nodA sequences tightly related with M. loti as the closest nodA relative. By contrast, their nodA sequences were highly divergent from those of M. metallidurans, another species associated with A. vulneraria that carries two complete copies of nodA. Therefore, the novel bacterial strains efficient on A. vulneraria represented the first occurrence of legume symbionts in the genus Aminobacter. They represent a new species for which the name Aminobacter anthyllidis sp. nov. is proposed (type strain STM4645(T)=LMG26462(T)=CFBP7437(T)).     

Natrinema salaciae sp. nov., a halophilic archaeon isolated from the deep, hypersaline anoxic Lake Medee in the Eastern Mediterranean Sea

Two halophilic archaea, strains MDB25(T) and MDB20, were isolated from a sample of the brine from Lake Medee, at a depth of 3050m, in the Mediterranean Sea. Cells of the organisms were Gram-negative, non-motile and pleomorphic, and colonies were red pigmented. Strains MDB25(T) and MDB20 showed optimum growth at 45°C, in 2.6-3.4M NaCl and at pH 7.0-8.0. The major polar lipids of the two strains were phosphatidylglycerol (PG1 and PG2), phosphatidylglycerol phosphate methyl ester (PGP-Me) and mannose-2,6-dissulfate (1→2)-glucose glycerol diether (S(2)-DGD). Menaquinone MK-8 and MK-8(H(2)) were the major respiratory quinones. The DNA G+C content of strain MDB25(T) was 63.0%. The strains were facultatively anaerobic but grew better under aerobic conditions, nitrate served as electron acceptor. Analysis of the almost complete 16S rRNA gene sequence indicated that the strains MDB25(T) and MDB20 represented a member of the genus Natrinema in the family Halobacteriaceae. Both strains formed a distinct cluster and were most closely related to Natrinema ejinorense JCM 13890(T) and Haloterrigena longa JCM 13562(T) (98.0% and 97.9% sequence similarity, respectively). Based on 16S rRNA gene sequence analysis, DNA-DNA hybridization results, physiological and biochemical characteristics we describe a new species represented by strain MDB25(T) (=DSM 25055(T) =JCM 17869(T)) for which we propose the name Natrinema salaciae sp. nov.     

Klebsiella variicola, a novel species with clinical and plant-associated isolates

A new Klebsiella species, K. variicola, is proposed on the basis of total DNA-DNA hybridization, on the monophyly observed in the phylogenetic analysis derived from the sequences of rpoB, gyrA, mdh, infB, phoE and nifH genes and on distinct phenotypic traits. The bacteria from this new species seem to be genetically isolated from K. pneumoniae strains, do not ferment adonitol and were obtained from plants (such as banana, rice, sugar cane and maize) and hospitals. The type strain is F2R9T (= ATCC BAA-830T = CFNE 2004T).     

Hymenobacter perfusus sp. nov., Hymenobacter flocculans sp. nov. and Hymenobacter metalli sp. nov. three new species isolated from an uranium mine waste water treatment system

Three red-pink pigmented strains, designated A1-12(T), A2-50A(T) and A2-91(T), were recovered from two different sites in a uranium mine. For all strains, the optimum growth temperature was 25°C, the optimum pH was 6.0-6.5 and the DNA G+C contents were between 60 and 63.4 mol%. The major respiratory quinone was menaquinone 7 (MK-7) and the fatty acid profiles contained iso- and anteiso-branched C15 fatty acids, summed feature 3 (16:1 ω6c and/or ω7c and/or 15:0 iso 2-OH), summed feature 4 (17:1 anteiso B and/or iso I) and the unsaturated fatty acid 16:1 ω5c as the major components. Phylogenetic analysis of the 16S rRNA gene sequences showed that these organisms represented three distinct branches within the family Flexibacteraceae most closely related to the members of the genus Hymenobacter. Strain A1-12(T) formed a distinct phylogenetic line along with H. rigui KCTC 12533(T) and they shared approximately 98.9% 16S rRNA gene sequence similarity. However, these two strains shared only 14.7% pairwise similarity in their genomic DNA. Strains A2-50A(T) and A2-91(T) formed two distinct lineages, related to the species H. soli KCTC 12607(T), sharing about 95.5% 16S rRNA gene sequence similarity between themselves, and 88.3 and 92.0% with other members of the genus Hymenobacter. Based on the phylogenetic analysis and physiological and biochemical characteristics, these isolates were considered to represent three novel species for which we propose the names Hymenobacter perfusus for strain A1-12(T) (=CIP 110166=LMG 26000), Hymenobacter flocculans for strain A2-50A(T) (=CIP 110139=LMG 25699) and Hymenobacter metalli for strain A2-91(T) (=CIP 110140=LMG 25700).     

Neoscardovia arbecensis gen. nov., sp. nov., isolated from porcine slurries

Three Gram-positive, anaerobic, pleomorphic strains (PG10(T), PG18 and PG22), were selected among five strains isolated from pig slurries while searching for host specific bifidobacteria to track the source of fecal pollution in water. Analysis of the 16S rRNA gene sequence showed a maximum identity of 94% to various species of the family Bifidobacteriaceae. However, phylogenetic analyses of 16S rRNA and HSP60 gene sequences revealed a closer relationship of these strains to members of the recently described Aeriscardovia, Parascardovia and Scardovia genera, than to other Bifidobacterium species. The names Neoscardovia gen. nov. and Neoscardovia arbecensis sp. nov. are proposed for a new genus and for the first species belonging to this genus, respectively, and for which PG10(T) (CECT 8111(T), DSM 25737(T)) was designated as the type strain. This new species should be placed in the Bifidobacteriaceae family within the class Actinobacteria, with Aeriscardovia aeriphila being the closest relative. The prevailing cellular fatty acids were C(16:0) and C(18:1)ω9c, and the major polar lipids consisted of a variety of glycolipids, diphosphatidyl glycerol, two unidentified phospholipids, and phosphatidyl glycerol. The peptidoglycan structure was A1γmeso-Dpm-direct. The GenBank accession numbers for the 16S rRNA gene and HSP60 gene sequences of strains PG10(T), PG18 and PG22 are JF519691, JF519693, JQ767128 and JQ767130, JQ767131, JQ767133, respectively.     

Taxonomic characterisation of Pseudomonas strain L48 and formal proposal of Pseudomonas entomophila sp. nov

An entomopathogenic, Gram-negative bacterium isolated from a female specimen of the fruit fly Drosophila melanogaster was taxonomically characterised. Strain L48(T) was strictly aerobic, non-fermentative, oxidase and catalase positive, rod-shaped, and motile due to a polar inserted flagellum. Phylogenetic analysis of the 16S rRNA gene and three other housekeeping genes placed strain L48 (T) in the Pseudomonas putida phylogenetic group. DNA-DNA hybridisation studies together with phenotypic metabolic tests and MALDI-TOF MS analysis justified the proposal of strain L48(T) as a representative of a novel species, for which the name Pseudomonas entomophila sp. nov. is proposed. The type strain is deposited in culture collections under accession numbers CCUG 61470(T) and CECT 7985(T).     

Malate dehydrogenase: a useful phylogenetic marker for the genus Aeromonas

The reconstruction of correct genealogies among biological entities, the estimation of the divergence time between organisms or the study of the different events that occur along evolutionary lineages are not always based on suitable genes. For reliable results, it is necessary to look at full-length sequences of genes under stabilizing selection (neutral or purifying) and behaving as good molecular clocks. In bacteria it has been proved that the malate dehydrogenase gene (mdh) can be used to determine the inter- and intraspecies divergence, and hence this gene constitutes a potential marker for phylogeny and bacterial population genetics. We have sequenced the full-length mdh gene in 36 type and reference strains of Aeromonas. The species grouping obtained in the phylogenetic tree derived from mdh sequences was in agreement with that currently accepted for the genus Aeromonas. The maximum likelihood models applied to our sequences indicated that the mdh gene is highly conserved among the Aeromonas species and the main evolutionary force acting on it is purifying selection. Only two sites under potential diversifying selection were identified (T 108 and S 193). In order to determine if these two residues could have an influence on the MDH structure, we mapped them in a three-dimensional model constructed from the sequence of A. hydrophila using the human mitochondrial MDH as a template. The presence of purifying selection together with the linear relationship between substitutions and gene divergence makes the mdh an excellent candidate gene for a phylogeny of Aeromonas and probably for other bacterial groups.