- Volume 54, Issue 6, 2004

In an ecological survey of nitrogen-fixing bacteria isolated from the rhizosphere and as endophytes of sugarcane, maize and teosinte plants in Brazil, Mexico and South Africa, a new phylogenetically homogeneous group of N2-fixing bacteria was identified within the genus Burkholderia. This polyphasic taxonomic study included microscopic and colony morphology, API 20NE tests and growth on different culture media at different pH and temperatures, as well as carbon source assimilation tests and whole-cell protein pattern analysis. Analysis of 16S rRNA gene sequences showed 99·2–99·9 % similarity within the novel species and 97·2 % similarity to the closest related species, Burkholderia sacchari. The novel species was composed of four distinct amplified 16S rDNA restriction analysis groups. The DNA–DNA reassociation values within the novel species were greater than 70 % and less than 42 % for the closest related species, B. sacchari. Based on these results and on many phenotypic characteristics, a novel N2-fixing species is proposed for the genus Burkholderia, Burkholderia tropica sp. nov., with the type strain Ppe8T (=ATCC BAA-831T=LMG 22274T=DSM 15359T). B. tropica was isolated from plants grown in geographical regions with climates ranging from temperate subhumid to hot humid.

The taxonomic position of a halo- and organo-sensitive, oligotrophic soil bacterium, strain S213T, was investigated. Cells were Gram-negative, non-motile, strictly aerobic, yellow-pigmented rods of short to medium length on diluted nutrient broth. When 0·1–0·4 % (w/v) NaCl was added to diluted media composed of peptone and meat extract, growth was inhibited with increasing NaCl concentration and the cells became long aberrant forms. When 6 mM CaCl2 was added, the cells grew quite normally and aberrant cells were no longer found at 0·1–0·5 % (w/v) NaCl. Chemotaxonomically, strain S213T contains chemical markers that indicate its assignment to the Sphingomonadaceae: the presence of ubiquinone Q-10 as the predominant respiratory quinone, C18 : 1 and C16 : 0 as major fatty acids, C14 : 0 2-OH as the major 2-hydroxy fatty acid and sphingoglycolipids. 16S rRNA gene sequence analysis indicated that strain S213T belongs to the genus Sphingomonas, exhibiting high sequence similarity to the 16S rRNA gene sequences of Sphingomonas mali IFO 15500T (98·3 %), Sphingomonas pruni IFO 15498T (98·0 %), Sphingomonas asaccharolytica IFO 15499T (97·9 %) and Sphingomonas echinoides DSM 1805T (97·8 %). The results of DNA–DNA hybridization experiments and its phenotypic characteristics clearly distinguished the strain from its nearest neighbours and demonstrate that strain S213T represents a novel Sphingomonas species, for which the name Sphingomonas oligophenolica sp. nov. is proposed. The type strain is S213T (=JCM 12082T=CIP 107926T).

While Helicobacter pylori is accepted as the major bacterial agent of gastric disease in humans, some patients and many animals are infected with a larger, tightly helical-shaped bacterium previously referred to as ‘Helicobacter heilmannii’ or ‘Gastrospirillum hominis’. Taxonomic classification of these bacteria has been hampered by the inability to cultivate them in vitro and by the inadequate discriminatory power of 16S rRNA gene sequence analysis. This study describes the detection and phylogenetic analysis of 26 different gastrospirillum isolates from humans and animals, which incorporates sequence data based on the 16S rRNA and urease genes. Fifteen gastrospirilla detected in humans, primates and pigs clustered with ‘Candidatus Helicobacter suis’, thus expanding the host range for this organism. By comparison, based on 16S rRNA data, the remaining 11 gastrospirilla could not be differentiated from Helicobacter felis, Helicobacter bizzozeronii and Helicobacter salomonis. However, urease gene sequence analysis allowed for the discrimination of this latter group into four discrete clusters, three of which contained the above recognized species. The fourth cluster contained isolates from human and feline hosts, and should provisionally be considered a unique bacterial species, for which the name ‘Candidatus Helicobacter heilmannii’ is proposed.

Gram-negative bacteria were isolated from knots induced by Pseudomonas savastanoi in olive trees (Olea europaea L.). A total of nine endophytic bacterial strains were isolated, each from inside a different tree knot. Biochemical characterization indicated that all the strains belong to the family Enterobacteriaceae. Phylogenetic analyses of the 16S rRNA genes of these novel isolates revealed that they formed a homogeneous cluster within Erwinia species. DNA signatures of these isolates were identical to those described for the genus Erwinia. The strains formed a homogeneous group as shown by DNA–DNA hybridization analysis and numerical analysis of phenotypic data, clearly differentiated from all species of Erwinia with validly published names. The data provide strong evidence of the differentiation of these strains from the most closely related species. Therefore, these isolates represent a novel species, for which the name Erwinia toletana sp. nov. is proposed. The isolates are available at CFBP, CECT and ATCC. The G+C content is 52±0·5 mol%. The type strain is CFBP 6631T (=A37T=ATCC 700880T=CECT 5263T).

Two strains of curved bacteria, 7-1T and 7-2T, isolated from well water, were phylogenetically examined to determine their taxonomic position. Strain 7-1T is a Gram-negative, slightly curved rod. Analysis of the 16S rRNA gene sequence showed that strain 7-1T formed a cluster with [Aquaspirillum] delicatum and [Pseudomonas] lanceolata. It has some similar characteristics to [A.] delicatum and [P.] lanceolata, but has sufficient distance to separate it from other genera. DNA–DNA hybridization analysis, as well as chemotaxonomic and morphological studies, demonstrated that strain 7-1T, [A.] delicatum and [P.] lanceolata belong to a new genus, Curvibacter gen. nov. Strain 7-1T (=IAM 15033T=ATCC BAA-807T) is classified as the type strain of Curvibacter gracilis gen. nov., sp. nov., and [A.] delicatum and [P.] lanceolata are classified as Curvibacter delicatus comb. nov. and Curvibacter lanceolatus comb. nov., respectively. Strain 7-2T is a Gram-negative spirillum. Phylogenetic study based on the 16S rRNA gene sequences showed that it formed a cluster with the members of the genus Herbaspirillum, [Pseudomonas] huttiensis and [Aquaspirillum] autotrophicum. The classification is therefore proposed of strain 7-2T (=IAM 15032T=ATCC BAA-806T) as the type strain of Herbaspirillum putei sp. nov., and [P.] huttiensis and [A.] autotrophicum are transferred to the genus Herbaspirillum as Herbaspirillum huttiense comb. nov. and Herbaspirillum autotrophicum comb. nov., respectively.

Two Gram-negative, motile, non-spore-forming, bacteriochlorophyll a-containing slightly halophilic strains, SW-132T and SW-158, were isolated from sea water of the East Sea in Korea, and subjected to a polyphasic taxonomic study. The two isolates were characterized chemotaxonomically as having Q-10 as the predominant respiratory lipoquinone and major amounts of unsaturated fatty acids C18 : 1 ω7c and C17 : 1 ω6c. The DNA G+C contents of the two strains were in the range 66·8–65·9 mol%. The 16S rRNA gene sequences of strains SW-132T and SW-158 were 99·9 % (1 nt difference) similar and their mean level of DNA–DNA relatedness was 86 %. The 16S rRNA gene sequence analysis showed that strains SW-132T and SW-158 are phylogenetically closely related to Porphyrobacter species and Erythromicrobium ramosum. Similarities between the 16S rRNA gene sequences of strains SW-132T and SW-158 and the type strains of Porphyrobacter species and E. ramosum ranged from 97·8 to 99·0 %. DNA–DNA relatedness data indicated that strains SW-132T and SW-158 are members of a genomic species that is separate from the four Porphyrobacter species. On the basis of phenotypic and phylogenetic data and genetic distinctiveness, strains SW-132T (=KCTC 12229T=DSM 16220T) and SW-158 (=KCTC 12230) are classified as a novel Porphyrobacter species, for which the name Porphyrobacter donghaensis sp. nov. is proposed.

Phylogenetic 16S rRNA gene analysis was used to assign the bacterial leaf-nodulating endosymbionts of two tropical African Psychotria species to the genus Burkholderia. The microsymbionts of the different Psychotria hosts were recognized as distinct and novel species of Burkholderia on the basis of relatively low intersequence similarities and sufficiently large evolutionary distances when compared with each other and their closest validly named neighbours. The obligate endosymbiotic nature of the bacteria prevented their in vitro cultivation and the deposition of type strains to culture collections. Therefore, the provisional status Candidatus is assigned to the bacterial partners of Psychotria calva and Psychotria nigropunctata, with the proposal of the names ‘Candidatus Burkholderia calva’ and ‘Candidatus Burkholderia nigropunctata’, respectively.

Three mesophilic bacteria (strains AMX 26BT, UR374_02 and 12-3T) isolated respectively from an anaerobic digester, human urine and urban riverside soil were characterized. Cells were Gram-negative, motile, non-sporulating, straight to curved rods with one polar flagellum and had a strictly respiratory metabolism with O2 as the preferential terminal electron acceptor. Phylogenetic analysis based on 16S rRNA gene sequences revealed that all strains clustered within the Xanthomonadaceae branch of the Proteobacteria. Isolates AMX 26BT and UR374_02 exhibited 100 % 16S rRNA gene sequence similarity and both were related to strain 12-3T (99·6 % similarity). The closest relative of all the isolates was Pseudoxanthomonas broegbernensis DSM 12573T (similarity 97·1–97·5 %), and they were equidistantly related to Xanthomonas species (95·4–96·6 %), Stenotrophomonas species (95·3–96·1 %) and Pseudoxanthomonas taiwanensis ATCC BAA-4040T (95·3–95·4 %). Chemotaxonomic and biochemical data (branched-chain cellular fatty acid pattern without C13 : 0 iso 3-OH, ubiquinone with eight isoprenoid units, limited range of substrates used, ability to reduce nitrite but not nitrate with the production of N2O) supported their affiliation to the genus Pseudoxanthomonas. The results of DNA–DNA hybridization and/or phenotypic analysis allowed them to be differentiated from the two Pseudoxanthomonas species with validly published names and showed that strain 12-3T was genomically and phenotypically distinct from the other two isolates. On the basis of these results, two novel species of the genus Pseudoxanthomonas are proposed: Pseudoxanthomonas mexicana sp. nov., consisting of strains AMX 26BT (=ATCC 700993T=CIP 106674T=JCM 11524T) (type strain) and UR374_02 (=DSM 15133), and Pseudoxanthomonas japonensis sp. nov., consisting of strain 12-3T (=CCUG 48231T=CIP 107388T=JCM 11525T). The report of these two novel species leads to the emendation of the description of the genus Pseudoxanthomonas and the re-evaluation of the phenotype of P. broegbernensis DSM 12573T necessitates the emendation of its description.

Three Gram-negative, aerobic, rod-shaped bacterial strains were isolated, from the pollen of Japanese flowers, as producers of xylitol; these strains were subjected to a polyphasic taxonomic study. Phylogenetic analyses of the 16S rRNA gene sequences demonstrated that these three isolates formed a new cluster within a group of acetic acid bacteria in the α-Proteobacteria. The characteristics of the three isolates were as follows: (i) their predominant quinone was Q-10; (ii) their cellular fatty acid profile contained major amounts of 2-hydroxy acids and an unsaturated straight-chain acid (C18 : 1 ω7c); and (iii) their DNA G+C contents were in the range 51·9–52·3 mol%, which is around the lower limit of the reported range for the genera of acetic acid bacteria. The negligible or very weak productivity of acetic acid from ethanol and the osmophilic growth properties distinguished these strains from other acetic acid bacteria. The unique phylogenetic and phenotypic characteristics suggest that the three isolates should be classified within a novel genus and species with the proposed name Saccharibacter floricola gen. nov., sp. nov. The type strain is strain S-877T (=AJ 13480T=JCM 12116T=DSM 15669T).

Data on 72 non-pigmented bacterial strains that specifically induce nitrogen-fixing root nodules on the legume species Crotalaria glaucoides, Crotalaria perrottetii and Crotalaria podocarpa are reviewed. By SDS-PAGE analysis of total protein patterns and by 16S rRNA PCR-RFLP, these strains form a homogeneous group that is separate from other legume root-nodule-forming bacteria. The 16S rRNA gene-based phylogeny indicates that these bacteria belong to the genus Methylobacterium. They can grow on C1 compounds such as methanol, formate and formaldehyde but not methylamine as sole carbon source, and carry an mxaF gene, encoding methanol dehydrogenase, which supports their methylotrophic metabolism. Presence of a nodA nodulation gene, and ability to nodulate plants of Crotalaria species and to fix nitrogen are features that separate the strains currently included in this group from other members of the genus Methylobacterium. The present study includes additional genotypic and phenotypic characterization of this novel Methylobacterium species, i.e. nifH gene sequence, morphology, physiology, enzymic and carbon source assimilation tests and antibiotic resistance. The name Methylobacterium nodulans sp. nov. (type strain, ORS 2060T=CNCM I 2342T=LMG 21967T) is proposed for this group of root-nodule-forming bacteria.

A Gram-negative, non-motile, non-spore-forming, short rod-shaped bacterium (UST950701-009PT) was isolated from a marine biofilm in Hong Kong waters. Colonies are pink in colour, convex with a smooth surface and entire edge. Brown diffusible pigment is produced. Whitish colonies, with otherwise identical morphology, emerge from every culture upon ageing. The white colonies can be maintained as separate cultures (UST950701-009W) without turning pink. UST950701-009PT and UST950701-009W have identical 16S rRNA gene sequences and similar G+C (65·9–66·2 mol%) and fatty acid (86·22–88·52 % 18 : 1ω7c) contents. Phylogenetic analysis of the 16S rRNA gene sequence places UST950701-009PT within the Rhodobacter group of the α-subclass of the Proteobacteria. The nearest neighbours belong to the genus Loktanella, with similarity values ranging from 94·5 to 95·5 %. Data on G+C and fatty acid contents support the affiliation to the genus Loktanella. UST950701-009PT and -009W are heterotrophic, strictly aerobic and require NaCl for growth (2·0–14·0 %). Both grow in pH 5·0–10·0 and at 8–44 °C. Both are positive in oxidase, catalase and β-galactosidase tests, but they differ in the pattern of carbohydrate oxidation and assimilation. Molecular evidence together with phenotypic characteristics shows that UST950701-009PT constitutes a novel species within the genus Loktanella. The name Loktanella hongkongensis sp. nov. is proposed; the type strain is UST950701-009PT (=NRRL B-41039T=JCM 12479T) and a morphovar is UST950701-009W (=NRRL B-41040=JCM 12480).

DNA–DNA hybridization experiments and an evaluation of phenotypic characteristics, DNA base ratios and 16S rRNA gene sequences demonstrated that Wautersia eutropha (Davies 1969) Vaneechoutte et al. 2004 , the type species of the genus Wautersia, is a later synonym of Cupriavidus necator Makkar and Casida 1987 , the type species of the genus Cupriavidus. In conformity with Rules 15, 17, 23a and 37a(1) of the International Code of Nomenclature of Bacteria, the genus name Cupriavidus has priority over the genus name Wautersia, and all other members of the genus Wautersia are reclassified into Cupriavidus as Cupriavidus basilensis comb. nov. (type strain LMG 18990T=DSM 11853T), Cupriavidus campinensis comb. nov. (type strain LMG 19282T=CCUG 44526T), Cupriavidus gilardii comb. nov. (type strain LMG 5886T=CCUG 38401T), Cupriavidus metallidurans comb. nov. (type strain LMG 1195T=DSM 2839T), Cupriavidus oxalaticus comb. nov. (type strain LMG 2235T=CCUG 2086T=DSM 1105T), Cupriavidus pauculus comb. nov. (type strain LMG 3244T=CCUG 12507T), Cupriavidus respiraculi comb. nov. (type strain LMG 21510T=CCUG 46809T) and Cupriavidus taiwanensis comb. nov. (type strain LMG 19424T=CCUG 44338T).

A facultatively anaerobic, chemolithoautotrophic, sulfur-oxidizing bacterium, strain YK-1T, was isolated from an underground crude-oil storage cavity at Kuji in Iwate, Japan. The cells were motile, curved rods and had a single polar flagellum. Optimum growth occurred in a low-strength salt medium at pH 7·0 and 25 °C. It utilized sulfide, elemental sulfur, thiosulfate and hydrogen as the electron donors and nitrate as the electron acceptor under anaerobic conditions, but it did not use nitrite. Oxygen also served as the electron acceptor under the microaerobic condition (O2 in the head space 1 %). It did not grow on sugars, organic acids or hydrocarbons as carbon and energy sources. The DNA G+C content of strain YK-1T was 45 mol%. Phylogenetic analysis, based on the 16S rRNA gene sequence, showed that its closest relative was Thiomicrospira denitrificans in the ‘Epsilonproteobacteria’, albeit with low homology (90 %). On the basis of physiological and phylogenetic data, strain YK-1T should be classified into a novel genus and species, for which the name Sulfuricurvum kujiense gen. nov., sp. nov. is proposed. The type strain is YK-1T (=JCM 11577T=MBIC 06352T=ATCC BAA-921T).

Two denitrifying strains of heterotrophic, facultatively anaerobic, marine bacteria, designated DN34T and DN33, were isolated from sea-water samples collected in Nanwan Bay, Kenting National Park, Taiwan. They were Gram-negative. Cells in late exponential to early stationary phase of growth were predominantly straight or curved rods, but Y- or V-shaped forms were also observed. They were motile by means of one to several lateral or subpolar flagella. Both strains required NaCl for growth and exhibited optimal growth at about 30 °C, pH 8 and 3 % NaCl. They were capable of anaerobic growth by carrying out denitrifying metabolism using nitrate, nitrite or nitrous oxide as terminal electron acceptors or, alternatively, by fermenting glucose, mannose, sucrose or trehalose as substrates. Anaerobic fermentative growth on glucose resulted in formation of various organic acids, including formate, lactate, acetate, pyruvate and fumarate. The major cellular fatty acids were 2-OH-14 : 0, 3-OH-14 : 0 and 16 : 0. DN34T and DN33 had DNA G+C contents of 51·7 and 51·6 mol%, respectively. Physiological characterization, together with phylogenetic analysis based on 16S rRNA gene sequence analysis, revealed that the two denitrifying strains could be accommodated in a novel genus, for which the name Pseudovibrio gen. nov. is proposed. Pseudovibrio denitrificans sp. nov. is the type species, with DN34T (=BCRC 17323T=JCM 12308T) as the type strain.

A novel thermotolerant bacterium, designated strain I78T, was isolated from a self-temperature-recording in situ colonization system deployed in a hydrothermal diffusing flow (maximal temperature 78 °C) at the TOTO caldera in the Mariana Arc, Western Pacific. Cells were highly motile curved rods with a single polar flagellum. Growth was observed at 15–55 °C (optimum 35–40 °C; 60 min doubling time) and pH 5·0–8·0 (optimum pH 6·0). The isolate was a microaerobic chemolithomixotroph capable of using thiosulfate, elemental sulfur or sulfide as the sole energy source, and molecular oxygen as the sole electron acceptor. The isolate was able to grow chemolithoautotrophically with carbon dioxide. Various organic substrates such as complex proteinaceous compounds, carbohydrates, organic acids, amino acids and sugars could also support growth as the carbon source instead of carbon dioxide with sulfur oxidation. The G+C content of the genomic DNA was 43·8 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that the isolate belonged to the genus Thiomicrospira and was most closely related to Thiomicrospira crunogena strain TH-55T and Thiomicrospira sp. strain L-12, while DNA–DNA hybridization demonstrated that the novel isolate could be genetically differentiated from previously described strains of Thiomicrospira. On the basis of its physiological and molecular properties the isolate is representative of a novel Thiomicrospira species, for which the name Thiomicrospira thermophila sp. nov. is proposed (type strain, I78T=JCM 12397T=DSM 16397T).

Two Gram-negative, motile, non-spore-forming, rod-shaped organisms, strains SW-117T and SW-120T, were isolated from sea water of the Yellow Sea in Korea and subjected to a polyphasic taxonomic study. Strains SW-117T and SW-120T simultaneously contained both menaquinones (MK) and ubiquinones (Q) as isoprenoid quinones; the predominant menaquinone was MK-7 and the predominant ubiquinones were Q-7 and Q-8. The major fatty acid detected in the two strains was iso-C15 : 0. The DNA G+C content of strains SW-117T and SW-120T was 51 and 54 mol%, respectively. Phylogenetic analyses based on 16S rRNA gene sequences showed that strains SW-117T and SW-120T fall within the radiation of the cluster comprising Shewanella species. Strains SW-117T and SW-120T showed a 16S rRNA gene sequence similarity of 97·4 % and a DNA–DNA relatedness level of 10·1 %. Strains SW-117T and SW-120T exhibited 16S rRNA gene sequence similarity levels of 93·8–98·5 % and 92·4–97·0 %, respectively, to Shewanella species. Strain SW-117T exhibited DNA–DNA relatedness levels of 8·3–20·3 % to the type strains of six phylogenetically related Shewanella species. On the basis of phenotypic, phylogenetic and genetic data, strains SW-117T and SW-120T were classified in the genus Shewanella as two distinct novel species, for which the names Shewanella marisflavi sp. nov. (type strain, SW-117T=KCCM 41822T=JCM 12192T) and Shewanella aquimarina sp. nov. (type strain, SW-120T=KCCM 41821T=JCM 12193T) are proposed, respectively.

Phenotypic and phylogenetic studies were performed on four Campylobacter-like organisms recovered from three seals and a porpoise. Comparative 16S rRNA gene sequencing studies demonstrated that the organisms represent a hitherto unknown subline within the genus Campylobacter, associated with a subcluster containing Campylobacter jejuni, Campylobacter coli and Campylobacter lari. DNA–DNA hybridization studies confirmed that the bacteria belonged to a single species, for which the name Campylobacter insulaenigrae sp. nov. is proposed. The type strain of Campylobacter insulaenigrae sp. nov. is NCTC 12927T (=CCUG 48653T).

Three actinomycete strains isolated from soils and one strain isolated from a patient with lung nocardiosis in 1999 and 2001 in Japan have been provisionally assigned to the genus Nocardia on the basis of morphological criteria. These isolates were further investigated to determine their specific taxonomic status. Detailed chemotaxonomic characterization and 16S rRNA gene sequence analysis of these isolates confirmed that they belong to the genus Nocardia. The 16S rRNA gene sequences of the four strains were most similar to that of Nocardia farcinica. However, the sequence similarity values between these four strains and N. farcinica were <98·9 %. These four strains were susceptible to 5-fluorouracil, and they have the ability to decompose urea, which is a very characteristic trait. Furthermore, DNA–DNA relatedness data revealed that IFM 10311T, IFM 10312 and IFM 10313 comprise a single novel species of Nocardia, that IFM 10084T represents another novel species of Nocardia and that these two novel species could be distinguished from N. farcinica. The names Nocardia shimofusensis sp. nov. and Nocardia higoensis sp. nov. are proposed, with IFM 10311T (=NBRC 100134T=JCM 12122T=DSM 44733T) and IFM 10084T (=NBRC 100133T=JCM 12121T=DSM 44732T) as the respective type strains.

Two Gram-positive bacterial strains, CM2104T and CM2110, isolated from the inner part of abnormally spoiled oriental melon (Cucumis melo) in Korea, were subjected to a polyphasic taxonomic study. The cell-wall peptidoglycan of strains CM2104T and CM2110 contained meso-diaminopimelic acid as the diagnostic diamino acid. The predominant menaquinone was MK-8(H4). The major fatty acids detected in the two strains were iso-C16 : 0, C17 : 1 ω8c and C18 : 1 ω9c or C17 : 0. The DNA G+C content of the two strains was 73 mol%. Phylogenetic analysis based on 16S rRNA gene sequences showed that the strains formed a coherent cluster with a clade comprising two Janibacter species, Janibacter limosus and Janibacter terrae. Strains CM2104T and CM2110 exhibited a 16S rRNA gene sequence similarity value of 99·7 % and a mean DNA–DNA relatedness level of 89 %. Strains CM2104T and CM2110 showed 16S rRNA gene sequence similarity levels of 97·8–98·4 % to the type strains of J. limosus and J. terrae. DNA–DNA relatedness between strains CM2104T and CM2110 and the type strains of these two Janibacter species was 7–11 %. On the basis of the phenotypic and phylogenetic data and genomic distinctiveness, strains CM2104T and CM2110 should be placed within the genus Janibacter as members of a novel species, for which the name Janibacter melonis sp. nov. is proposed. The type strain is CM2104T (=KCTC 9987T=DSM 16063T=JCM 12321T).