- Volume 52, Issue 4, 2002

Two thermophilic, strictly chemolithoautotrophic, microaerophilic, hydrogen-oxidizing members of the Bacteria designated strain EX-H1T and strain EX-H2T were isolated from two separate deep-sea hydrothermal vent sites at 9 degrees N 104 degrees W in the Pacific Ocean and Guaymas Basin. The motile 2-4-microm-long rods were Gram-negative and non-sporulating. The temperature range for growth was between 55 and 80 degrees C for EX- H1T (optimum at 73 degrees C) and 55-75 C for EX-H2T (optimum at 70 C). Both strains grew fastest at 2.5% (w/v) NaCl and at pH 6, although growth was observed from pH 4.7 to pH 7.5. EX-H1T and EX-H2T were able to use elemental sulfur, thiosulfate or hydrogen as an electron donor, and oxygen (2-3%, v/v) or nitrate as an electron acceptor. EX-H1T was also able to use elemental sulfur as an electron acceptor. EX-H1T and EX-H2T further differed in their genomic G+C content (38.5 and 37.4 mol%, respectively) and 16S rRNA sequences (4% difference). Maximum-likelihood analysis of the 16S rRNA phylogeny placed both isolates within the Aquificales as a distinct lineage and showed them to be only about 85% similar to Aquifex pyrophilus. On the basis of phenotypic and phylogenetic characteristics, it is proposed that EX-H1T and EX-H2T belong to a new genus within the Aquificales, namely Persephonella gen. nov. It is further proposed that EX-H1T be named Persephonella marina sp. nov., the type species of the genus, and that EX-H2T be named Persephonella guaymasensis sp. nov., a second species in this genus.

A novel, anaerobic, non-spore-forming, mobile, Gram-negative, thermophilic bacterium, strain TMOT, was isolated from a thermophilic sulfate-reducing bioreactor operated at 65 C with methanol as the sole substrate. The G+C content of the DNA of strain TMOT was 39.2 mol%. The optimum pH, NaCl concentration, and temperature for growth were 7.0, 1.0%, and 65 degrees C, respectively. Strain TMOT was able to degrade methanol to CO2 and H2 in syntrophic culture with Methanothermobacter thermautotrophicus AH or Thermodesulfovibrio yellowstonii. Thiosulfate, elemental sulfur, Fe(III) and anthraquinone-2,6-disulfonate were able to serve as electron acceptors during methanol degradation. In the presence of thiosulfate or elemental sulfur, methanol was converted to CO2 and partly to alanine. In pure culture, strain TMOT was also able to ferment methanol to acetate, CO2 and H2. However, this degradation occurred slower than in syntrophic cultures or in the presence of electron acceptors. Yeast extract was required for growth. Besides growing on methanol, strain TMOT grew by fermentation on a variety of carbohydrates including monomeric and oligomeric sugars, starch and xylan. Acetate, alanine, CO2, H2, and traces of ethanol, lactate and alpha-aminobutyrate were produced during glucose fermentation. Comparison of 16S rDNA genes revealed that strain TMOT is related to Thermotoga subterranea (98%) and Thermotoga elfii (98%). The type strain is TMOT (= DSM 14385T = ATCC BAA-301T). On the basis of the fact that these organisms differ physiologically from strain TMOT, it is proposed that strain TMOT be classified as a new species, within the genus Thermotoga, as Thermotoga lettingae.

During a survey of yeast ecology in a soft-drinks production facility, a dead wasp was removed from the sampling tap of an external sugar-syrup storage tank. A yeast isolated from the dead wasp was found to be similar, although not identical, in its physiological characteristics to Candida lactis-condensi and Candida stellata. Sequence analysis of the 26S rDNA D1/D2 variable domain revealed that this isolate was most closely related to C stellata, but differed sufficiently in its D1/D2 sequence to indicate that it belonged to a separate species. The yeast species has been named Candida davenportii sp. nov.; the type strain is NCYC 3013T (= CBS 9069T). C davenportii sp. nov. was osmotolerant, moderately preservative-resistant and able to grow in very acidic conditions, i.e. pH 14. This yeast grew well in fruit-containing soft drinks, cola-type beverages and a synthetic soft drink and is therefore a potential cause of spoilage of soft drinks and other sugary food products. Other related yeast species in the same taxonomic clade as C davenportii sp. nov. are also osmotolerant, growing in < 50% (w/v) sugar. Many of these species are associated with insects, specifically bees, bumblebees and leafcutter bees, and many have been reported as the causative agent of spoilage of sugary foods, such as condensed milk, fruit juices and concentrates. It is proposed that C davenportii sp. nov. and other closely related yeasts are primarily associated with Aculeates (bees and wasps). In turn, bees and wasps are attracted by sugary residues in foods such as fruit juices and concentrates, forming the source of infection of these yeasts and thus instigating spoilage.

16S rDNA was sequenced from 16 strains of the oral commensal Simonsiella and was used to assess relationships between Simonsiella strains and other members of the Neisseriaceae. In all analyses, Simonsiella strains grouped according to established species designations and the mammalian hosts from which they were isolated. The commensals from cats and dogs formed a monophyletic group. The monophyly of the genus Simonsiella, however, could be neither supported nor rejected; deep nodes in the trees were unstable depending on the phylogenetic method or on the particular sequences used in the analysis. Instabilities may be attributable to frequent gene transfer between Neisseria or other members of the Neisseriaceae and Simonsiella.

RNase P RNA gene (rnpB) sequences were PCR-amplified from different members of the Prochlorococcus group. Aligned nucleotide sequences revealed a variance of up to 27% for rnpB. Comparative secondary structure analysis showed that domains P12, P18 and P19 of these novel ribozyme sequences in particular are highly divergent. Thus, these regions in RNase P RNA might serve as potential targets for deoxyoligonucleotide primers for the identification of specific genotypes of Prochlorococcus and for probing field populations. Phylogenetic trees constructed from RNase P RNA sequences were similar to, but not fully congruent with, those derived previously using sequences of the 16S rRNA gene. However, the application of rnpB sequences allowed a better resolution within clades of very closely related genotypes. As is known from 16S rRNA-based phylogenetic trees, sequences from individual strains clustered according to their physiology and the conditions at the original site of isolation, rather than their geographical origin. All sequences obtained from high-light-adapted strains formed a single coherent clade, as did the four sequences from low-light-adapted strains that were previously isolated from the North Atlantic and the subtropical North Pacific. This suggests a remarkable genetic stability of Prochlorococcus genotypes that thrive under identical ecological conditions.

Prevotella nigrescens, Prevotella intermedia and Porphyromonas gingivalis are oral pathogens from the family Bacteroidaceae, regularly isolated from cases of gingivitis and periodontitis. In this study, the phylogenetic variability of these three bacterial species was investigated by means of 16S rRNA (rrs) gene sequence comparisons of a set of epidemiologically and geographically diverse isolates. For each of the three species, the rrs gene sequences of 11 clinical isolates as well as the corresponding type strains was determined. Comparison of all rrs sequences obtained with those of closely related species revealed a clear clustering of species, with only a little intraspecies variability but a clear difference in the rrs gene with respect to the next related taxon. The results indicate that the three species form stable, homogeneous genetic groups, which favours an rrs-based species identification of these oral pathogens. This is especially useful given the 7% sequence divergence between Prevotella intermedia and Prevotella nigrescens, since phenotypic distinction between the two Prevotella species is inconsistent or involves techniques not applicable in routine identification.

The examination of molecular phylogenies of cyanobacteria and other micro-organisms is increasing dramatically. The use of a single locus in these studies leaves the resulting phylogenies unconfirmed. In this study, the partial sequences of two loci containing segments of protein-encoding genes, the hetR and the phycocyanin locus (PC-IGS), were examined. Laboratory strains and natural populations of the heterocyst-forming cyanobacteria Anabaena, Aphanizomenon and Nodularia from the Baltic Sea were used, in total 41 sequences were determined and their phylogenies were analysed with maximum-likelihood methods. The hetR phylogenies suggested that the planktonic Aphanizomenon and Nodularia each comprise one species, while there were numerous Anabaena species present in the Baltic Sea. In the case of Nodularia, the PC-IGS phylogenies were incongruent with this and suggested that several lineages of Nodularia plankton species existed. In the hetR phylogeny, the floating and nodularin-producing strains of Nodularia were grouped together. For both the hetR and PC-IGS loci of cultured species of Nodularia their molecular phylogeny did not correspond well with the affiliation suggested by morphology. In sequences derived from species of Anabaena and Aphanizomenon the PC-IGS and hetR phylogenies were congruent, suggesting that Aphanizomenon sp. from the Baltic Sea is genetically distinct from both Aphanizomenon flos-aquae from lakes and Aphanizomenon sp. TR183 from the Baltic Sea. In both Nodularia and Anabaena/Aphanizomenon, the PC-IGS sequences showed a significant degree of either recombination events or selection, while none was detected within the hetR sequences. This is the first study comprising the phylogenies of multiple loci from all heterocystous cyanobacteria from the Baltic Sea and shows that earlier results using the PC-IGS locus should be interpreted cautiously in the absence of a confirmation using a second locus.

The organization of the rRNA genes of Anaplasma marginale, the type species of the genus Anaplasma, was identified to determine if the atypical rRNA gene arrangement identified in rickettsiae preceded divergence of the order Rickettsiales into the families Anaplasmataceae and Rickettsiaceae. The rRNA genes are encoded by two unlinked units, each present in a single copy per A. marginale genome. The 16S rRNA gene is separated from the linked 23S and 5S rRNA genes by a minimum of 100 kb. Similar to species belonging to the genus Rickettsia, the typical bacterial 16S-23S spacer region containing tRNA genes has been lost in A. marginale. In contrast, the fmt gene located upstream of the 23S rRNA gene in most Rickettsia spp. is not maintained in A. marginale, consistent with the fmt arrangement being a relatively late event in the evolution of rickettsial species.

The taxonomic position of the genera Saccharothrix and Nocardiopsis has evolved in recent years to accommodate an increasing number of actinomycete strains that cannot be clearly distinguished by morphological characters. More recently, the taxonomic reorganization of the genus Saccharothrix has determined the creation of new, related genera, increasing the complexity of the identification of this taxon. Nevertheless, today these genera can still only be identified by applying chemotaxonomic and molecular criteria, and no other tools are available for the rapid distinction of members of the two genera. Phylogenetic analysis based on 16S rDNA nucleotide sequences of reference strains has shown that both genera represent complete distinct lineages within the order Actinomycetales. Differences in the nucleotide sequences of the 16S rDNAs of reference strains were used to design two pairs of genus-specific primers to identify novel members of the genera Nocardiopsis and Saccharothrix by PCR amplification. The genus specificity of these primers was validated with reference strains as well as with wild-type isolates that exhibited morphological characteristics common to both genera. The diversity and taxonomic position of the isolates identified with these tools is also discussed.

Sixty-six yeast strains isolated from the nectar of various plant species in Central Europe were characterized by randomly amplified polymorphic DNA PCR (RAPD-PCR) and by sequencing of the variable D1/D2 domain of large-subunit (26S) rDNA. The usefulness of both methods for the determination and comparison of unknown ascomycetous and basidiomycetous yeast strains was compared and evaluated. The reproducibility of RAPD-PCR was shown to be low and the information obtained by this method was clearly not as precise as that obtained from sequence analysis. Numerous imponderables make RAPD-PCR analysis unreliable, at least as a means of identifying yeasts in ecological studies. The lack of standard protocols for RAPD-PCR analysis and the absence of a general database of banding patterns made it impossible to identify unknown yeast strains or to recognize new species. In contrast to RAPD-PCR, sequence analysis of the D1/D2 domain was found to be a fast and reliable method for the rapid identification of yeast species and was also shown to be an invaluable tool for the discovery of new species.