- Volume 50, Issue 3, 2000

The morphologies, halotolerances, temperature requirements, pigment compositions and 16S rRNA gene sequences of five culture collection strains and six novel isolates of cyanobacteria with helical, tightly coiled trichomes were investigated. All strains were very similar morphologically and could be assigned to the genus Spirulina (or section Euspirulina sensu Geitler), according to traditional classification. However, the isolates showed significantly different requirements for salinity and temperature, which were in accordance with their respective environmental origins. The genetic divergence among the strains investigated was large. The results indicate the drastic underestimation of the physiological and phylogenetic diversity of these cyanobacteria by the current morphology-based classification and the clear need for new taxa. Three of the isolates originated from hypersaline waters and were similar with respect to their high halotolerance, broad euryhalinity and elevated temperature tolerance. By phylogenetic analyses, they were placed in a tight monophyletic cluster apart from all other cyanobacteria. Thus it is proposed to reclassify highly halotolerant cyanobacteria with tightly coiled trichomes in Halospirulina gen. nov., with the type species Halospirulina tapeticola sp. nov.

The hierarchic taxonomic framework described recently for the phylogenetic structure of the suborder Micrococcineae, class Actinobacteria, on the basis of 16S rDNA sequences and signature nucleotides was modified and extended. With the recent addition of novel taxa into the suborder, the phylogenetic coherence of some families was disrupted, leading to the emergence of novel lineages that, as judged by the depth of their branching points, were equivalent to those of described families. Bogoriellaceae fam. nov., Dermacoccaceae fam. nov., Rarobacteraceae fam. nov. and Sanguibacteraceae fam. nov. are proposed for these lineages. As a consequence of the restructuring process, some families have had to be emended, i.e. Dermatophilaceae, Cellulomonadaceae and Intrasporangiaceae.

A moderately thermophilic, organotrophic bacterium with vibrioid cells was isolated from a sample of a cyanobacterial mat from caldera Uzon, Kamchatka, Russia, and designated strain Z-9701T. Cells of strain Z-9701T were curved, Gram-negative rods, 0.5-0.7 x 2.5-5.0 microm in size, with tapering ends and with fast, wavy movement by means of lateral flagella located on the concave side of the cell. Colonies were small, white, irregular or round, 0.2 mm in diameter, and with even edges. Strain Z-9701T was an obligate anaerobe with a temperature optimum at 60-65 degrees C and a pH optimum at 7.3. It fermented glucose, fructose, mannose, N-acetyl-D-glucosamine, adonite, arginine, serine, peptone, yeast extract and Casamino acids. The fermentation products formed during growth on glucose were acetate, lactate, H2, CO2 and ethanol. Strain Z-9701T reduced elemental sulfur to H2S during organotrophic growth with glucose or peptides as energy and carbon sources. In the presence of S0, strain Z-9701T was capable of lithotrophic growth with molecular hydrogen as energy substrate and 0.1 g yeast extract l(-1) as carbon source. Sulfate, thiosulfate, nitrate, Fe(III) and sulfite were not reduced and did not stimulate growth. The G+C content of strain Z-9701T DNA was 54.6 mol%. The results of 16S rDNA sequence analyses revealed that strain Z-9701T belongs to the cluster within the Clostridium group formed by Thermanaerovibrio acidaminovorans, Dethiosulfovibrio peptidovorans, Anaerobaculum thermoterrenum and Aminobacterium colombiense, but the level of sequence similarity with the members of this cluster was not very high (87.6-92.2%). Among these organisms, Thermanaerovibrio acidaminovorans is phenotypically close to strain Z-9701T. However, the two organisms showed a relatively low level of similarity of their 16S rRNA sequences (92.2%) and of DNA-DNA hybridization (15 +/- 1%). Nevertheless, on the basis of the similar morphology and physiology of the new isolate and Thermanaerovibrio acidaminovorans, strain Z-9701T was placed in the genus Thermanaerovibrio and a new species, Thermanaerovibrio velox, proposed for it. The type strain is Z-9701T (= DSM 12556T).

A novel extremely halophilic archaeon, strain XF10T, was isolated from a salt lake in China. This organism was neutrophilic, non-motile and pleomorphic, and was rod, coccus or irregularly shaped. It required at least 1.5 M NaCl for growth and grew in a wide range of MgCl2 concentrations (0.005-0.5 M). Lipid extract of whole cells contained two glycolipids with the same chromatographic properties as two unidentified glycolipids found in the two described Natrinema species, Natrinema pellirubrum and Natrinema pallidum. Phylogenetic analysis based on 16S rDNA sequence comparison revealed that strain XF10T clustered with the two described Natrinema species and several other strains (strains T5.7, GSL-11 and Haloterrigena turkmenica JCM 9743) with more than 98.1% sequence similarities, suggesting that strain XF1OT belongs to the genus Natrinema. Comparative analysis of phenotypic properties and DNA-DNA hybridization between strain XF10T and the Natrinema species supported the conclusion that strain XF10T is a novel species within the genus Natrinema. The name Natrinema versiforme sp. nov. is proposed for this strain. The type strain is XF10T (=JCM 10478T=AS 1.2365T=ANMR 0149T).

In contrast to the current view that psychrophily combined with an absolute requirement for NaCl is connected with the Gram-negative cell wall type, psychrophilic and psychrotolerant, NaCl-requiring, Gram-positive bacteria have been isolated from tropical Atlantic, Arctic and Antarctic deep-sea sediments. Some of the isolates are even extremely psychrophilic, having maximum growth temperatures of 4 degrees C. On the basis of phenotypic characteristics, DNA base analyses, DNA-DNA hybridizations and partial and complete 16S rRNA gene sequence analyses, the strains from the three distinct geographical regions have been allocated to the obligately marine species Bacillus marinus. The distribution and origin of B. marinus are discussed and an emended description of the species is presented.

The generic position of two isolates from soils inside a gold mine cave in Kongju, Korea, was determined by 16S rDNA sequencing and chemotaxonomic characteristics. Phylogenetic analysis indicated that both of the isolates formed a clade with Lentzea albidocapillata and members of the genus Saccharothrix of the family Pseudonocardiaceae. The chemical composition of the isolates and of Lentzea albidocapillata was consistent with that of the genus Saccharothrix, which is characterized by a type III cell wall (the meso-isomer of diaminopimelic acid, and galactose and rhamnose as characteristic whole-cell sugars), MK-9(H4) as the major menaquinone, and a phospholipid type PII pattern (phosphatidylethanolamine as a diagnostic phospholipid). The combination of morphological features, chemotaxonomic characters and phylogenetic data supported the proposal that Lentzea albidocapillata, the only and type strain of the genus Lentzea, should be transferred to the genus Saccharothrix. On the basis of physiological properties, cellular fatty acid composition and DNA-DNA hybridization data, two new species within the genus Saccharothrix are proposed: Saccharothrix violacea sp. nov., type strain LM 036T (= IMSNU 50388T), and Saccharothrix albidocapillata comb. nov., type strain DSM 44073T (=IMSNU 21253T).

Nucleotide sequence analysis of the 16S-23S rRNA intergenic spacer regions of six type or reference strains belonging to the Mycoplasma mycoides cluster and of Mycoplasma putrefaciens suggested the presence of two subclusters. One subcluster comprised M. mycoides subsp. mycoides small colony (SC) type, M. mycoides subsp. mycoides large colony (LC) type and M. mycoides subsp. capri, whereas the second subcluster comprised Mycoplasma capricolum subsp. capricolum, M. capricolum subsp. capripneumoniae and Mycoplasma sp. bovine group 7. The type strains from M. mycoides subsp. mycoides SC and M. mycoides subsp. capri had identical spacer sequences. The existence of two subclusters was supported by predicted secondary structures of the analysed region. The nucleotide variations in the loop domains of the secondary structures might be a useful genetic marker to distinguish between the two subclusters. The secondary structure differences delineated the differences between the two subclusters more clearly than the nucleotide sequence alignments, which only showed a small number of differences, and some of these were common to both clusters. The data also provided evidence in favour of a reclassification of Mycoplasma sp. bovine group 7 as another subspecies of M. capricolum.

A polyphasic study was performed on 10 soil isolates of thermophilic denitrifying Bacillus strains from different geographical areas. The presence of two main characteristic bands following amplification of the internal transcribed spacer (ITS) region of rrn operons suggests a close relatedness to 'Bacillus thermodenitrificans'. The isolates cluster around two strains of 'B. thermodenitrificans' in riboprint and fatty acid analyses, though differences occur at the strain level. Subsequent DNA-DNA reassociation studies including the 10 isolates, 'B. thermodenitrificans' DSM 465T and DSM 466, and Bacillus stearothermophilus ATCC 12980T and Bacillus thermoleovorans ATCC 43513T revealed such a high level of genomic relatedness between the isolates and the DSM strains (> 73% similarity) that they must be considered strains of the same taxon. The degree of DNA-DNA similarity between the 12 strains of 'B. thermodenitrificans' and the type strains of the other two phylogenetically neighbouring Bacillus species was significantly lower (21-43% similarity). Based upon phylogenetic, chemotaxonomic and phenotypic evidence, the designation of B. thermodenitrificans sp. nov., nom. rev. is proposed. The type strain of B. thermodenitrificans is DSM 465T.

The type strain of Photobacterium histaminum, JCM 8968T (= ATCC 51805T), and that of Photobacterium damselae subsp. damselae, ATCC 33539T, exhibit 100% identity in their 16S rRNA sequence, more than 80% DNA-DNA homology and only one phenotypic difference. Also, like P. histaminum, P. damselae subsp. damselae was shown to excrete a large amount of histamine when cells were grown on medium containing excessive histidine under acidic conditions. Therefore, the name P. histaminum should be considered to be a later subjective synonym of P. damselae subsp. damselae.

Multilocus enzyme electrophoresis (MLEE) and numerical taxonomic methods were used to establish the degrees of relatedness among five Candida species commonly isolated from humans oral cavities. Of twenty enzymic systems assayed, five showed no enzymic activity (aspartate dehydrogenase, mannitol dehydrogenase, sorbitol dehydrogenase, glucosyl transferase and alpha-amylase). The obtained data revealed that some of these enzymes are capable of distinguishing strains of different species, but most of them could not organize all strains in their respective species-specific clusters. Numerical classification based on MLEE polymorphism must be regarded for surveys involving just one Candida species.

The molecular systematics of 337 strains of basidiomycetous yeasts and yeast-like fungi, representing 230 species in 18 anamorphic and 24 teleomorphic genera, was determined by sequence analysis of the D1/D2 region of the large-subunit rDNA. The data were compared with published sequences of other basidiomycetous fungi. The results demonstrated that the yeast species and genera are phylogenetically distributed among the Microbotryum, Sporidiobolus, Agaricostilbum and Erythrobasidium clades of the Urediniomycetes; the Tremellales, Trichosporonales ord. nov., Filobasidiales and Cystofilobasidiales clades of the Hymenomycetes; and the Ustilaginales, Microstromatales and Malasseziales clades of the Ustilaginomycetes. Genera such as Bensingtonia, Cryptococcus, Rhodotorula and Sporobolomyces are polyphyletic, i.e. they occur in two or more clades. In contrast, other genera, e.g. Bullera, Cystofilobasidium, Fellomyces, Filobasidiella, Filobasidium, Kondoa, Kurtzmanomyces, Leucosporidium, Rhodosporidium, Sporidiobolus and Udeniomyces, are monophyletic. The majority of the species can be identified using D1/D2 analyses, although the internal transcribed spacer region is required to distinguish closely related species. The intergenic spacer region is recommended for additional differentiation of species and strains.

The 18S rDNA nucleotide sequences of 25 Sporobolomyces species and five Sporidiobolus species were determined. Those of Sporobolomyces dimmenae JCM 8762T, Sporobolomyces ruber JCM 6884T, Sporobolomyces sasicola JCM 5979T and Sporobolomyces taupoensis JCM 8770T showed the presence of intron-like regions with lengths of 1586, 324, 322 and 293 nucleotides, respectively, which were presumed to be group I introns. A total of 63 18S rDNA nucleotide sequences was analysed, including 33 published reference sequences. Sporobolomyces species and the other basidiomycetes species were distributed throughout the phylogenetic tree. The resulting phylogeny indicated that Sporobolomyces is polyphyletic. Sporobolomyces species were mainly divided into four groups within the Urediniomycetes. The groups are designated as the Sporidiales, Agaricostilbum/Bensingtonia, Erythrobasidium and subbrunneus clusters. The last group, comprising four species, Sporobolomyces coprosmicola, Sporobolomyces dimmenae, Sporobolomyces linderae and Sporobolomyces subbrunneus, forms a new and distinct cluster in the phylogenetic tree in this study.

Strain delineation was performed by means of restriction endonuclease analysis (REA) of genomic DNA with the restriction enzyme HinfI followed by conventional electrophoresis. A total of 337 yeast isolates representing 21 Candida species and five non-Candida yeast species was evaluated. A survey of isolates showed that Candida albicans and non-albicans species could be divided into mutually exclusive groups, and that subgroups could be created. Individual REA patterns for 111 C. albicans isolates, four Candida krusei isolates and 35 Candida glabrata isolates varied greatly, whereas 11 Candida dubliniensis isolates, 48 Candida tropicalis isolates and 41 Candida guilliermondii isolates could be divided into two, nine and nine groups, respectively. REA of the 49 Candida parapsilosis isolates with HinfI, however, showed that 47 (95 %) of them belonged to one group. REA patterns of the other yeast isolates, representing 19 species, were also quite different at the species level. These results showed that REA with HinfI may be useful for the identification and strain delineation of common and emerging Candida species.