- Volume 50, Issue 2, 2000

A Gram-negative bacterium, designated NS114T, was isolated from duplicate treatments of surface-sterilized Zea mays stems. The plants were grown in synthetic soil under greenhouse conditions and watered with fertilizer containing no nitrogen. Strain NS114T could not be isolated from plants watered with the standard level or 20% (w/v) of the standard level of nitrogen. Cells occurred as pairs in young cultures that attached to form angled arrangements in R2A broth and occasionally formed rounded, horseshoe arrangements in YM broth. Cell variation resulted in flocculent chains of coccoid cells in old cultures. Strain NS114T fermented glucose and sucrose. The G + C content was 48 mol%. Phylogenetic analysis of the 16S rRNA gene showed that the strain was a member of the domain Bacteria and branched from a point equidistant from an aquatic organism, Runella slithyformis and a marine isolate, 'Microscilla furvescens'. Phenotypic and genotypic analyses indicated that strain NS114T could not be assigned to any recognized genus; therefore a new genus and species, Dyadobacter fermentans gen. nov., sp. nov., is proposed, for which NS114T is the type strain.

beta-Haemolytic, catalase-positive, Gram-positive cocci that formed chains in broth media but did not react with Lancefield group antisera were isolated from skin lesions, spleen, liver and lungs of nine opossums, including eight from a research colony and one from a wildlife rehabilitation organization. The isolates had vigorous catalase activity that was retained on initial passage on non-blood-containing media, but this activity was lost in subsequent passages. The use of standard phenotypic tests did not lead to satisfactory identification of these organisms beyond the genus level, even if the aberrant catalase reaction was not considered. The 16S rRNA gene sequence of the isolates was most similar (96%) to Streptococcus dysgalactiae, but distinct from that species as 16S rRNA gene similarity of different strains of S. dysgalactiae was > 99%. Characterization of biochemical reactions and cell-wall fatty acid profiles also revealed significant differences between the opossum isolates and all other known Streptococcus spp., thus it is proposed as a new species with the name Streptococcus didelphis, sp. nov. The type strain is ATCC 700828T.

DNA base composition and DNA-DNA hybridization among the cyanobacterial genus Microcystis were determined using nine axenic Microcystis strains, including the three 'morphological' species of Microcystis aeruginosa, Microcystis viridis and Microcystis wesenbergii. These Microcystis species showed a similar DNA base composition (42.1-42.8 mol% G + C) and demonstrated more than 70% DNA relatedness, confirming their synonymy based on bacterial criteria.

A new anaerobic, thermophilic, syntrophic, fatty-acid-oxidizing bacterium designated strain TGB-C1T was isolated from granular sludge in a thermophilic upflow anaerobic sludge blanket (UASB) reactor. The cells were slightly curved rods and were weakly motile. Spore formation was not observed. The optimal temperature for growth was around 55 degrees C and growth occurred in the range 45 to 60 degrees C. The pH range for growth was 5.8-7.5, and the optimum pH was 6.5-7.0. Crotonate was the only substrate that allowed the strain to grow in pure culture. However, in co-culture with the thermophilic, hydrogenotrophic Methanobacterium thermoautotrophicum strain delta H, the isolate could syntrophically oxidize saturated fatty acids with 4-10 carbon atoms, including isobutyrate. During the degradation of isobutyrate by the co-culture, isobutyrate was isomerized to butyrate, which was then oxidized. The strain was not able to utilize sulfate, sulfite, thiosulfate, nitrate, fumarate or Fe(III) as electron acceptor. The DNA base composition was 51.0 mol%. 16S rDNA sequence analysis revealed that the strain belongs to the family Syntrophomonadaceae, but it was only distantly related to other known species of beta-oxidizing syntrophs. Hence, the name Syntrophothermus lipocalidus is proposed for TGB-C1T as a new species of a new genus.

The closer proximity of Frankia and Acidothermus cellulolyticus relative to the morphologically close Geodermatophilus found previously was confirmed by resequencing the rrs gene of Acidothermus cellulolyticus and the housekeeping gene, recA. The diagnostic sugar 2-O-methyl-D-mannose was detected only in Frankia, while hopanoid lipids were present at high levels in both Acidothermus and Frankia.

Previous studies have demonstrated that cellular fatty acid analysis is a useful tool for identifying unknown strains of rhizobia and establishing taxonomic relationships between the species. In this study, the fatty acid profiles of over 600 strains belonging to the genera Agrobacterium, Bradyrhizobium, Mesorhizobium, Rhizobium and Sinorhizobium were evaluated using the gaschromatography-based Sherlock Microbial Identification System (MIS). Data collected with the MIS showed that the three phylogenetically defined biovars of the genus Agrobacterium formed discrete clusters, whilst species belonging to the genus Mesorhizobium formed three subclusters which were easily distinguished. These three subclusters contained Mesorhizobium ciceri and Mesorhizobium mediterraneum, Mesorhizobium tianshanense fatty acid group I and Mesorhizobium plurifarium, and Mesorhizobium huakuii and Mesorhizobium loti. The genus Sinorhizobium was composed of an individual position for Sinorhizobium meliloti and a large cluster comprising Sinorhizobium fredii, Sinorhizobium saheli, Sinorhizobium terangae, Sinorhizobium kostiense and Sinorhizobium arboris. S. meliloti contained significantly higher levels of the fatty acid 19:0 cyclo omega 8 cis and clustered with Rhizobium sp. (Hedysarum coronarium). However, discrimination between the species of genera Sinorhizobium and Rhizobium was a function of the concentration of 16:0 3-OH. The genus Rhizobium contained a single cluster containing Rhizobium sp. (Hedysarum coronarium), Rhizobium gallicum, Rhizobium leguminosarum and Rhizobium etli, along with individual positions for Rhizobium giardinii, Rhizobium tropici, Rhizobium galegae and Rhizobium hainanense. R. tropici and R. hainanense exhibited similarity to Agrobacterium biovar 2, whilst R. galegae was similar to Agrobacterium biovar 1. R. giardinii appeared unique, with comparatively little similarity to the other species. Analysis of the genus Bradyrhizobium revealed large differences from the other genera studied. Two subgroups of Bradyrhizobium elkanii were detected and easily distinguished from Bradyrhizobium japonicum. Bradyrhizobium liaoningense and Bradyrhizobium sp. (Arachis hypogaea), a group isolated from Chinese peanut plants, showed similarities to B. japonicum, whilst a subgroup of M. tianshanense appeared identical to Bradyrhizobium sp. (Arachis hypogaea).

Chemotaxonomic and 16S rDNA sequence analyses of an isolate from the sputa and bronchial secretions of a patient with chronic lung disease clearly demonstrated that it belongs to the genus Nocardia. DNA-DNA hybridization data, as well as the biochemical characteristics of the isolate, indicate that it belongs to a new species that differs from previously described members of the genus Nocardia. The name Nocardia paucivorans sp. nov. is proposed for this isolate and is represented by strain IMMIB D-1632T (= DSM 44386T).

The genetic diversity of 55 xanthomonad strains isolated from passion fruit plants (Passiflora spp.) and identified as Xanthomonas campestris pv. passiflorae was initially assessed by randomly amplified polymorphic DNA (RAPD) analysis. The strains showed a high level of polymorphism with almost unique fingerprints. Fifteen clusters with a similarity of approximately 70% were identified, three of which were prevalent. There was a correlation between the clusters and the geographic origin of the strains. A representative strain of each cluster, together with the pathovar reference strain, were used to verify the relationships of these strains to 18 Xanthomonas species and Pseudomonas syringae pv. passiflorae. All Xanthomonas species yielded a unique RAPD profile and no consistent relatedness to the X. campestris pv. passiflorae strains was observed. Amplification products were also analysed by repetitive (rep) primers (BOX, ERIC and REP), RFLP of the 16S-23S rDNA intergenic spacer and SDS-PAGE of whole-cell proteins. All of these approaches generated profiles characteristic for each Xanthomonas species but the taxonomic position of the X. campestris pv. passiflorae strains could not be unequivocally assigned. Finally, DNA-DNA hybridization allowed a sound taxonomic allocation of the strains to Xanthomonas axonopodis pv. passiflorae.

Eight Gram-negative, aerobic, rod-shaped and peritrichously flagellated strains were isolated from flowers of the orchid tree (Bauhinia purpurea) and of plumbago (Plumbago auriculata), and from fermented glutinous rice, all collected in Indonesia. The enrichment culture approach for acetic acid bacteria was employed, involving use of sorbitol medium at pH 3.5. All isolates grew well at pH 3.0 and 30 degrees C. They did not oxidize ethanol to acetic acid except for one strain that oxidized ethanol weakly, and 0.35% acetic acid inhibited their growth completely. However, they oxidized acetate and lactate to carbon dioxide and water. The isolates grew well on mannitol agar and on glutamate agar, and assimilated ammonium sulfate for growth on vitamin-free glucose medium. The isolates produced acid from D-glucose, D-fructose, L-sorbose, dulcitol and glycerol. The quinone system was Q-10. DNA base composition ranged from 59.3 to 61.0 mol% G + C. Studies of DNA relatedness showed that the isolates constitute a single species. Phylogenetic analysis based on their 16S rRNA gene sequences indicated that the isolates are located in the acetic acid bacteria lineage, but distant from the genera Acetobacter, Gluconobacter, Acidomonas and Gluconacetobacter. On the basis of the above characteristics, the name Asaia bogorensis gen. nov., sp. nov. is proposed for these isolates. The type strain is isolate 71T (= NRIC 0311T = JCM 10569T).

The marine bacterium strain 2-40 was isolated from the salt marsh cord grass, Spartina alterniflora, in the Chesapeake Bay watershed, VA, USA. It is Gram-negative, requires sea salts and is a strict aerobe. It degrades numerous complex polysaccharides and synthesizes eumelanin. By 16S rDNA analysis, the isolate was shown to be a member of the gamma-subclass of the Proteobacteria, related to Microbulbifer hydrolyticus and to a cellulolytic nitrogen-fixing bacterium.

Phylogenetic positions of psychrophilic bacteria isolated from the Japan Trench were determined by sequencing analysis of PCR-amplified bacterial small subunit (16S) rRNA genes. Between surface and deep-sea psychrophiles, distinct positions clearly differed within the gamma-Proteobacteria. In phylogenetic analysis using neighbour-joining, maximum-parsimony and maximum-likelihood, strains from surface seawater were inferred to be located in the Halomonas aquamarina-meridiana clade within the family Halomonadaceae. Strains from deep seawater (5000-6000 m), however, formed a novel monophyletic clade within the Moraxella-Psychrobacter branch in the family Moraxellaceae, showing separation from terrestrial and Antarctic relatives. These deep-sea strains were also discriminated from other known Psychrobacter species in phenotype, e.g. limited growth in the absence of NaCl (optimum at about 3% NaCl), positive urease activity, acid production from xylose and arabinose, and the presence of multiple fimbriae. DNA relatedness values among six deep-sea strains were > 85% in DNA-DNA hybridization experiments and > 98% in aligned 16S rDNA sequences. From this evidence, a new species, Psychrobacter pacificensis, is proposed for these deep-sea psychrophiles; the type strain of Psychrobacter pacificensis is strain NIBH P2K6T (= IFO 16270T). Occurrence of psychrobacters in cold Japan Trench deep seawater and at the Antarctic sea surface suggests that deep-sea bacterial habitation and evolution have been mediated by global deep-ocean circulation linked to the sinking of cooled seawater in polar regions.

Withering syndrome is a fatal disease of wild and cultured abalone, Haliotis spp., that inhabit the west coast of North America. The aetiological agent of withering syndrome has recently been identified as a member of the family Rickettsiaceae in the order Rickettsiales. Using a combination of morphological, serological, life history and genomic (16S rDNA) characterization, we have identified this bacterium as a unique taxon and propose the provisional status of 'Candidatus Xenohaliotis californiensis'. The Gram-negative, obligate intracellular pleomorphic bacterium is found within membrane-bound vacuoles in the cytoplasm of abalone gastrointestinal epithelial cells. The bacterium is not cultivable on synthetic media or in fish cell lines (e.g. CHSE-214) and may be controlled by tetracyclines (oxytetracycline) but not by chloramphenicol, clarithromycin or sarafloxicin. Phylogenetic analysis based on the 16S rDNA of 'Candidatus Xenohaliotis californiensis' places it in the alpha-subclass of the class Proteobacteria but not to the four recognized subtaxa of the alpha-Proteobacteria (alpha-1, alpha-2, alpha-3 and alpha-4). The bacterium can be detected in tissue squashes stained with propidium iodide, microscopic examination of stained tissue sections, PCR or in situ hybridization. 'Candidatus Xenohaliotis californiensis' can be differentiated from other closely related alpha-Proteobacteria by its unique 16S rDNA sequence.

Haenam strains of Borrelia burgdorferi, which had been isolated from Ixodes granulatus and Apodemus agrarius in Haenam, Korea, were characterized by PCR-RFLP analysis of rrf (5S)-rrl (23S) intergenic spacer amplicons and by sequence analysis of the 16S rRNA gene (rDNA). The Msel and Dral restriction patterns of the 5S-23S intergenic spacer amplicons of Haenam strains differed from those of other B. burgdorferi sensu lato strains. Furthermore, in the phylogenetic tree based on the 16S rDNA sequences, Haenam strains formed a distinctive cluster, clearly separated from the other members of B. burgdorferi sensu lato. These results suggest that, apart from Borrelia garinii and Borrelia afzelii, other genotypes of B. burgdorferi sensu lato exist in Korea and the Haenam strain is a newly identified one.

Campylobacter-like organisms were isolated from the faeces of healthy individuals during a hygiene survey of abattoir workers. The strains, which exhibited characteristics of Campylobacter, being non-glucose-fermenting, oxidase- and catalase-positive, Gram-negative, motile rods, were identified to the genus level by a PCR assay. Nucleotide sequence analysis of the 16S rRNA gene, DNA homology experiments and determination of G + C content demonstrated that they constituted a previously undescribed species, whose nearest phylogenetic neighbours were Campylobacter hyointestinalis subsp. hyointestinalis, Campylobacter fetus and Campylobacter mucosalis. The name Campylobacter lanienae sp. nov. is proposed for this taxon and species-specific PCR primers were evaluated which will find use in the study of its epidemiology, prevalence and pathogenicity.

Two anaerobic acid-tolerant bacteria, CK58T and CK74T, were isolated from acidic beech litter and acidic peat-bog soil, respectively. Both bacteria were spore-forming, motile rods with peritrichous flagella. The capacity to sporulate decreased with prolonged cultivation. Cells of CK58T formed chains or aggregates and were linked by a connecting filament that consisted of a core and a surrounding sheath. Cellobiose, glucose, xylose, arabinose, maltose, mannose and salicin supported growth of CK58T. These substrates, as well as mannitol, lactose, sucrose, glycerol, melezitose, raffinose and rhamnose, supported growth of CK74T. Sorbitol, trehalose, H2/CO2, CO/CO2, vanillate, Casamino acids, peptone, and various purines and pyrimidines did not support the growth of either organism. Growth of CK58T and CK74T on glucose yielded butyrate, lactate, acetate, formate, H2 and CO2 as end products. Growth of CK58T and CK74T was observed at pH 3.7-7.1 and 3.6-6.9, respectively. CK58T and CK74T grew in nitrogen-free medium at pH 3.7 under an N2 atmosphere and reduced acetylene at rates approximating 1 nmol min-1 (mg protein)-1. CK58T and CK74T did not contain carbon monoxide dehydrogenase or cytochromes, produce methane, or dissimilate nitrate or sulfate. Thus, CK58T and CK74T were characterized as nonacetogenic, N2-fixing, fermentative chemo-organotrophs. The G + C contents of CK58T and CK74T were 31.4 and 30.7 mol%, respectively. CK58T and CK74T were phylogenetically most closely related to Clostridium pasteurianum. The 16S rRNA gene sequence similarity values of CK58T and CK74T to C. pasteurianum and each other did not exceed 96.5%, and it is proposed that strains CK58T and CK74T be named Clostridium akagii CK58T (DSM 12554T) and Clostridium acidisoli CK74T (DSM 12555T), respectively. These results suggest that previously uncharacterized clostridial species reside and might fix N2 in the annoxic microzones of acidic forest soil and litter.

The 16S-23S rDNA spacer regions of two Pectinatus species, two Zymophilus species and one Selenomonas species were cloned after PCR amplification. The results of PCR amplification showed that these species had two types of spacer regions which differ in molecular size (long and short). Only the long spacer regions in these bacteria contained one or two tRNA genes (alanine and/or isoleucine). The spacer regions in these bacteria had a relatively high level of homology. Homology was particularly high for bacteria belonging to the same genus. Interestingly, the order of the two tRNA genes present in the long spacer regions of Pectinatus and Selenomonas was the reverse of that which had been previously reported for other bacteria. The results of spacer homology analysis and the order of the tRNA genes suggest that the taxonomic classification of anaerobic bacteria isolated from the brewing process should be re-examined.

A polyphasic taxonomic study was performed on a group of isolates tentatively identified as Burkholderia cepacia, Ralstonia pickettii or Ralstonia paucula (formerly known as CDC group IVc-2). The isolates were mainly cultured from sputum of cystic fibrosis patients or from soil. SDS-PAGE of whole-cell proteins and AFLP fingerprinting distinguished at least five different species, and this was confirmed by DNA-DNA hybridizations. 16S rDNA sequence analysis of representative strains indicated that these organisms belong to the beta-subclass of the Proteobacteria, with the genera Burkholderia and Ralstonia as closest neighbours. Based on genotypic and phenotypic characteristics, the organisms were classified in a novel genus, Pandoraea. The DNA base composition of the members of the new genus is between 61.2 and 64.3 mol%. This novel genus includes four new species, Pandoraea apista (the type species) (type strain is LMG 16407T), Pandoraea pulmonicola (type strain is LMG 18106T), Pandoraea pnomenusa (type strain is LMG 18087T) and Pandoraea sputorum (type strain is LMG 18819T), and Pandoraea norimbergensis (Wittke et al. 1997) comb. nov. (type strain is LMG 18379T). The available clinical data indicate that at least some of these organisms may cause chronic infection in, and can be transmitted amongst, cystic fibrosis patients.

Two bacterial strains, KMM 227T and 231T, were isolated from seawater samples collected from the north-western Pacific Ocean at a depth of 4000-5000 m and were characterized using polyphasic taxonomy. Both were Gram-negative, psychrotolerant, heterotrophic, aerobic and required NaCl for growth (0.6-15.0%). The temperature for growth was 4-30 degrees C. Both strains were rod-shaped, with a single flagellum. However, strain KMM 231T revealed a single long fimbrium. Cellular fatty acids detected in the isolates were predominantly odd-numbered and iso-branched, with 15 and 17 carbons (ca. 70%). Also present were saturated and monounsaturated straight-chain fatty acids. Results of phylogenetic analyses, employing three tree-making methods, strongly indicated that the two strains formed a distinct lineage within a clade containing the genera Alteromonas, Colwellia and Pseudoalteromonas, in the gamma-Proteobacteria. The two strains shared 16S rDNA sequence similarity of 96.9% and genomic DNA relatedness of 27%; the latter was determined by dot-blot hybridization. The strains were differentiated by the presence of fimbria, production of chitinase, ability to grow on 15% NaCl and BIOLOG profiles. Given the polyphasic evidence accumulated in this study, it is proposed that the two deep-sea isolates be classified in the genus Idiomarina gen. nov., as Idiomarina abyssalis sp. nov. (type strain is KMM 227T) and Idiomarina zobellii sp. nov. (type strain is KMM 231T).

The 16S-23S rDNA internal transcribed spacer (ITS) of Roseobacter denitrificans, Roseobacter litoralis, Ruegeria algicola and strains of the recently described species Antarctobacter heliothermus and Roseovarius tolerans were analysed in order to examine DNA sequence variations and to draw conclusions about inter- and intraspecific relationships. A. heliothermus included four strains with an ITS fragment length of 1092 bp. Roseovarius tolerans was described on the basis of eight strains. Five of these harboured two ITS fragments of different lengths (959 and about 1100 bp), while the others had one fragment of either 1083 bp (two strains) or 1165 bp (one strain). ITS lengths of the related species Roseobacter denitrificans, Roseobacter litoralis and Ruegeria algicola were found to be 980, 984 and 1158 bp, respectively. Phylogenetic analyses of the DNA sequences allowed species affiliation of strains with sequence length differences of > 200 bp and recognition of relationships based on a well-supported ITS tree. The strains of A. heliothermus and Roseovarius tolerans each formed a monophyletic branch and they were separated from each other by Ruegeria algicola. This species was now clearly separated from Roseobacter denitrificans and Roseobacter litoralis, which corresponded to the new genus affiliation of Ruegeria algicola. These data were additionally supported by analyses of the structure, relative position and order of genes for tRNA(Ile) and tRNA(Ala) found within the ITS of each strain. Comparative DNA sequence analyses of ITS and 16S rDNA revealed limitations, on species and strain levels, with respect to the phylogenetic resolution of the 16S rDNA due to the limited number of informative (variable) sites, while ITS sequence analyses provided more variable and sufficiently conserved positions to discriminate between strains and to reconstruct their taxonomic relationships.