- Volume 49, Issue 3, 1999

Two novel hyperthermophilic, rod-shaped crenarchaeotes were isolated from an acidic hot spring in the Philippines. Cells were mostly straight or slightly curved rods 0.4-0.7 μm in width. Bent cells, branched cells, and cells bearing globular bodies were commonly observed. The isolates were heterotrophs and grew anaerobically and microaerobically. The addition of archaeal cell extract or a vitamin mixture to the medium significantly stimulated growth. The isolates grew over a temperature range of 60-92 °C, and optimally around 85 °C and grew over a pH range of 2.3-6.4, and optimally at pH 3.7-4.2. The isolates utilized glycogen, gelatin, beef extract, peptone, tryptone and yeast extract as carbon sources. They required sulfur, thiosulfate or sulfate as electron acceptors. The lipids mainly consisted of various cyclized glycerolbisdiphytanyl-glycerol tetraethers. The G+C content of the genomic DNAs was 43 mol%. The 16S rDNA contained two small introns. The comparison of the 16S rDNA exon sequences revealed that they represented an independent lineage in the family Thermoproteaceae. The two strains were included in a single species because of high levels of DNA-DNA relatedness. From these results, Caldivirga maquilingensis gen. nov., sp. nov. is proposed in the family Thermoproteaceae to accommodate these isolates. The type strain of C. maquilingensis is strain IC-167T (= JCM 10307T =MCC-UPLB 1200T = ANMR 0178T).

Phenotypic data indicate that gliding, yellow/orange-pigmented, agar-digest bacterial strains were members of the Cytophaga-Flavobacterium-Bacteroides (CFB) group. The strains were isolated from the surface of the marine benthic macroalga Fucus serratus l. and the surrounding seawater at three localities Danish waters. The bacteria were Gram-negative, flexirubin-negative, aerobic, catalase-positive and oxidase-negative and were psychrophilic and halophilic. All strains utilized d-fructose, l-fucose and α-ketobutyric acid and degraded alginic acid, carrageenan, starch and autoclaved yeast cells. Amplification with primers specific for repetitive extragenic palindromic elements by PCR divided the strains of this study into two groups. Both groups showed unique PCR amplification patterns compared to reference strains of the CFB group. Phylogenetic analysis of 16S rDNA sequences showed association of these organisms and [Cytophaga] lytica at the genus level. Hybridization of total chromosomal DNA revealed that the new strains and [Cytophaga] lytica ATCC 23178T were clearly distinct from each other and other previously described species of the CFB group. A new genus is described, Cellulophaga gen. nov. comprising two new species, Cellulophaga baltica gen. nov., sp. nov. (NN015840T = LMG 18535T) and Cellulophaga fucicola gen. nov., sp. nov. (NN015860T = LMG 18536T), as well as the emendation of [Cytophaga] lytica to Cellulophaga lytica gen. nov., comb. nov.

A phytoplasma was discovered in diseased specimens of field-grown hortensia (Hydrangea spp.) exhibiting typical phyllody symptoms. PCR amplification of DNA using phytoplasma specific primers detected phytoplasma DNA in all of the diseased plants examined. No phytoplasma DNA was found in healthy hortensia seedlings. RFLP patterns of amplified 16S rDNA differed from the patterns previously described for other phytoplasmas including six isolates of foreign hortensia phytoplasmas. Based on the RFLP, the Japanese Hydrangea phyllody (JHP) phytoplasma was classified as a representative of a new subgroup in the phytoplasma 16S rRNA group I (aster yellows, onion yellows, all of the previously reported hortensia phytoplasmas, and related phytoplasmas). A phylogenetic analysis of 16S rRNA gene sequences from this and other group I phytoplasmas identified the JHP phytoplasma as a member of a distinct sub-group (sub-group Id) in the phytoplasma clade of the class Mollicutes. The phylogenetic tree constructed from 16S rRNA gene sequences was consistent with the hypothesis that the JHP phytoplasma and its closest known relatives, the Australian grapevine yellows (AUSGY), Phormium yellow leaf (PYL), Stolbur of Capsicum annuum (STOL) and Vergilbungskrankheit of grapevine (VK) share a common ancestor. The unique properties of the DNA from the JHP phytoplasma clearly establish that it represents a new taxon, ‘Candidatus Phytoplasma japonicum’.

The phylogenetic positions of two strains of fermentative bacteria that had been isolated from the highest positive tubes inoculated with serial dilutions of estuarine mud in agar media with either glutamate or aspartate as substrate were determined by comparative sequence analysis of their 16S rRNA genes. The strain isolated with glutamate (glu 65) utilized several substrates, including a number of amino acids but no sugars. The degradation of certain substrates was enhanced by or dependent upon co-cultivation with a hydrogen-utilizing partner. In earlier work this strain was assigned to the new genus and species Acidaminobacter hydrogenoformans. On the basis of its 16S rRNA gene sequence Acidaminobacter hydrogenoformans has now been identified as a member of cluster XI of the Clostridium subphylum with Clostridium halophilum as its closest relative. The aspartate-fermenting strain asp 66T was a Gram-negative, rather aerotolerant anaerobe which utilized a wide range of substrates in a propionic fermentation and had the ability to fix molecular nitrogen. Strain asp 66T was shown to be a new member of the β-subclass of the Proteobacteria with Azoarcus sp. strain 6a3 and Rhodocyclus tenuis as its closest relatives. It is described as Propionibacter pelophilus gen nov., sp. nov., with the type strain asp 66T (= DSM 12018T).

A heterotrophic bacterial strain TL1T capable of aerobic denitrification was previously enriched in continuous culture from a landfill leachate treatment plant and isolated as a pure culture. The taxonomic position of this isolate within the β-subclass of the Proteobacteria was determined by 16S rDNA sequence analysis and by conventional taxonomy including substrate spectrum, quinone type (ubiquinone Q-8) and cellular fatty acid composition. Detection of the specific polyamine 2-hydroxyputrescine supports the membership of strain TL1T in the β-subclass of the Proteobacteria. The results of 16S rDNA sequencing showed that the strain clustered with, but was separate from, Thauera aromatica and Thauera selenatis. DNA-DNA hybridization experiments indicated that the new isolate represents a new species of the genus, for which the name Thauera mechernichensis is proposed; the type strain is DSM 12266T.

The genus Caulobacter is composed of prosthecate bacteria often specialized for oligotrophic environments. The taxonomy of Caulobacter has relied primarily upon morphological criteria: A strain that visually appeared to be a member of the Caulobacter has generally been called one without challenge. A polyphasic approach, comprising 16S rDNA sequencing, profiling restriction fragments of 16S-23S rDNA interspacer regions, lipid analysis, immunological profiling and salt tolerance characterizations, was used to clarify the taxonomy of 76 strains of the genera Caulobacter, Brevundimonas, Hyphomonas and Mycoplana. The described species of the genus Caulobacter formed a paraphyletic group with Caulobacter henricii, Caulobacter fusiformis, Caulobacter vibrioides and Mycoplana segnis (Caulobacter segnis comb, nov.) belonging to Caulobacter sensu stricto. Caulobacter bacteroides (Brevundimonas bacteroides comb, nov.), C. henricii subsp. aurantiacus (Brevundimonas aurantiaca comb, nov.), Caulobacter intermedius (Brevundimonas intermedia comb. nov.), Caulobacter subvibrioides (Brevundimonas subvibrioides comb. nov.), C. subvibrioides subsp. albus (Brevundimonas alba comb. nov.), Caulobacter variabilis (Brevundimonas variabilis comb. nov.) and Mycoplana bullata belong to the genus Brevundimonas. The halophilic species Caulobacter maris and Caulobacter halobacteroides are different from these two genera and form the genus Maricaulis gen. nov. with Maricaulis maris as the type specis Caulobacter leidyia was observed to cluster with species of the genus Sphingomonas. Caulobacter crescentus is synonymous with C.vibrioides and C. halobacteroides is synonymous with Maricaulis maris as determined by these analyses and DNA-DNA hybridization. Biomarkers discerning these different genera were determined. The necessary recombinations have been proposed and a description of Maricaulis is presented.

The taxonomic position of eight fluorescent Pseudomonas isolates, from two Lebanese spring waters, which were previously recognized by numerical analysis as members of a new subcluster (subcluster Vb) was examined. Excep for one strain, the new subcluster exhibited internal DNA hybridization values of 76-100%, and 9-53% hybridization was measured with the type or reference strains of other Pseudomonas species. The highest DNA binding value was found with Pseudomonas marginalis strains (37-53%). The G+C content of the DNA of the type strain was 58 mol%. A comparison of 1322 nt of the 16S rRNA gene sequence of the strain representing subcluster Vb (CFML 96-1951T) with the sequence of other strains of the genus Pseudomonas revealed that strain CFML 96-195T was part of the ‘Pseudomonas fluorescens intrageneric cluster’. On the basis of the results of phenotypic, DNA-DNA and phylogenetic analyses, a new Pseudomonas species, Pseudomonas libanensis sp. nov., is proposed for the seven strains of subcluster Vb. The type strain is P. libanensis CFML 96-195T and has been deposited in the Collection de I‘lnstitut Pasteur (Paris, France) as CIP 105460T. The P. libanensis strains are phenotypically and genotypically homogeneous and can be differentiated from most other fluorescent species by several phenotypic features. Differentiation of P. libanensis and Pseudomonas aeruginosa is based mainly on pyocyanin production; P. libanensis can be differentiated from P. fluorescens (all biovars) by β-aminobutyrate assimilation. The clinical significance of P. libanensis is unknown.

[Pseudomonas] echinoides DSM 1805T (= ATTC 14820T, DSM 50409T, ICBP NCIB 9420T) has been reinvestigated to clarify its taxonomic position. 16 sequence comparisons demonstrated that this species clusters phylogenetically with species of the genus Sphingomonas. Investigation fatty acid patterns, polar lipid profiles, polyamine patterns and quinc systems supported this delineation. Substrate utilization profiles and biochemical characteristics displayed no distinct overall similarity to an validly described species of the genus Sphingomonas. Therefore, the reclassification of [Pseudomonas] echinoides as Sphingomonas echinoides comb. nov. is proposed, based upon the estimated phylogenetic positon derived from 16S rRNA gene sequence data, chemotaxonomic data and previously published genomic DNA G+C content data.

A taxonomic characterization of twenty-one strains capable of degrading aromatic compounds under denitrifying conditions, isolated from ten different geographical locations, was performed on the basis of general morphological and physiological characteristics, cellular fatty acids, DNA base composition, small ribosomal (16S) subunit DNA sequences, whole-cell protein patterns and genomic DNA fragmentation analysis, in addition to DNA similarity estimations using hybridization methods. The collection of strains was subdivided into a number of different groups. A first group, consisting of four strains, could be assigned to the previously described species Azoarcus tolulyticus. A second group (five strains) had DNA which reannealed highly to that of strains of the first group, and it is considered to represent a genomova of A. tolulyticus. The third and fourth groups, composed of a total of five strains, represent a new species of Azoarcus, Azoarcus toluclasticus (group 3) and a genomovar of this species (group 4), respectively. Finally, the fifth group, with two strains, corresponds to another new species of the genus Azoarcus, Azoarcus toluvorans. In addition to these five groups, the collection includes five individual strains perhaps representing as many different new species. The above classification is partially consistent with the results of approaches other than DNA-DNA hybridization (electrophoretic patterns of whole-cell proteins and of the fragments obtained after digestion of total DNA with infrequently cutting restriction enzymes). On the other hand, no correlation of these groupings was found in terms of the cellular fatty acid composition. It is also unfortunate that no simple sets of easily determinable phenotypic properties could be defined as being characteristic of each of the groups.

Two strains of dissimilatory arsenate-reducing vibrio-shaped bacteria are assigned to the genus Sulfurospirillum. These two new species, Sulfurospirillum barnesii strain SES-3T and Sulfurospirillum arsenophilum strain MIT-13T, in addition to Sulfurospirillum sp. SM-5, two strains of Sulfurospirillum deleyianum, and Sulfurospirillum arcachonense, form a distinct clade within the ε subclass of the Proteobacteria based on 16S rRNA analysis.

The polyphenol oxidase (PPO) activities of the marine melanogenic strains MMB-1T and 2-40 were compared. Both contained a multifunctional PPO able to oxidize a wide range of substrates. In spite of this similarity, phylogenetic studies based on 16S rRNA sequences showed that these strains are not closely related. Strain 2-40 is not related to any previously described genus. On the basis of these studies and morphological and physiological characteristics, it is proposed that strain MMB-1T (= CECT 4803T = ATCC 700492T) represents a new species in the genus Marinomonas, Marinomonas mediterranea sp. nov.

16S rRNA gene sequences of three previously described amino-acid-fermenting anaerobes, Selenomonas acidaminovorans, ‘Selenomonas acidaminophila’ and Eubacterium acidaminophilum, were determined. All three were found to cluster within the Clostridium and related genera of the subphylum of the Gram-positive bacteria. The thermophile, S. acidaminovorans, formed an individual line of descent and was equidistantly placed between Dethiosulfovibrio peptidovorans and Anaerobaculum thermoterrenum (similarity of 85%), both of which also form single lines of descent. ‘S. acidaminophila’ was related to Clostridium quercicolum, a member of cluster IX, with a similarity of 90%, whereas E. acidaminophilum was closely related to Clostridium litorale (similarity of 96%) as a member of cluster XI. Based on the phylogenetic data presented in this report and the phenotypic descriptions of these bacteria published previously, it is recommended that S. acidaminovorans be transferred to a new genus, Thermanaerovibrio gen. nov., as Thermanaerovibrio acidaminovorans comb. nov. and ‘Selenomonas acidaminophila’ be transferred to a new genus, Anaeromusa gen. nov., as Anaeromusa acidaminophila comb. nov. Though the transfer of E. acidaminophilum to a new taxon is justified, this is not recommended until the taxonomic status of all the members of cluster XI has been reviewed.

A novel, asaccharolytic, amino-acid-degrading bacterium, designated strain GLU-3T, was isolated from an anaerobic lagoon of a dairy wastewater treatment plant. Strain GLU-3T stained Gram-negative and was an obligately anaerobic, non-spore-forming, slightly curved, rod-shaped bacterium (0.3 × 4.0–6.0 μm) which existed singly or in pairs. The DNA G+C content was 43 mol%. Optimum growth occurred at 35 °C and pH 7.5 on arginine with a generation time of 16 h. Good growth was obtained on arginine, histidine, threonine and glycine. Acetate was the end-product formed from all these substrates, but in addition, a trace of formate was detected from arginine and histidine, and ornithine was produced from arginine. Strain GLU-3T grew slowly on glutamate and produced acetate, carbon dioxide, formate, hydrogen and traces of propionate as the end-products. In syntrophic association with Methanobacterium formicicum, strain GLU-3T oxidized arginine, histidine and glutamate to give propionate as the major product; acetate, carbon dioxide and methane were also produced. Strain GLU-3T did not degrade alanine and the branched-chain amino acids valine, leucine and isoleucine either in pure culture or in association with M. formicicum. The nearest phylogenetic relative of strain GLU-3T was the thermophile Selenomonas acidaminovorans (similarity value of 89.5%). As strain GLU-3T is phylogenetically, physiologically and genotypically different from other amino-acid-degrading genera, it is proposed that it should be designated a new species of a new genus Aminomonas paucivorans gen. nov., sp. nov. (DSM 12260T).

A succinate-decarboxylating anaerobic bacterium, designated strain 19gly1 T was previously isolated from a mixed culture growing with glycolate. The almost complete sequence of the 16S rRNA gene (1495 nt) was determined for this strain. On the basis of 16S rRNA gene sequence homology, 19gly1T was identified as a member of the Sporomusa sub-branch of the 'low G+C Gram-positive bacteria. Phylogenetic analysis showed that strain 19gly1T was most closely related toSucciniclasticum ruminis. Phascolarctobacterium faecium and Acidaminococcus fermentans. The use of different algorithms, such as least-squares or neighbour-joining analyses of Jukes-Cantor pairwise distances, or maximum-parsimony or maximum-likelihood analyses of the aligned sequence data, revealed that strain 19gly1T grouped as the most deeply branching lineage of the strain 19gly1T-Succinclasticum-Acidaminococcus-Phascolarctobacterium cluster. The phenotypic characteristics of strain 19gly1T distinguish it from members of the genera Succiniclasticum, Phascolarctobacterium and Acidaminococcus. and the phylogenetic distances inferred from comparative analysis of the 16S rDNA sequences suggest that strain 19gly1T is a representative of a new genus. Accordingly, strain 19gly1T (= DSM 6222T) is proposed as the type strain of a new species within a new genus. Succinispira mobilis gen. nov., sp. nov.