- Volume 53, Issue 1, 2003

Five strains of thermotolerant lactic acid bacteria (G 12, G 22, G 35T, G 43 and G 44) isolated from chicken faeces were characterized taxonomically. The strains were facultatively anaerobic, Gram-positive, catalase-negative, non-motile, non-spore-forming rods. They were heterofermentative lactobacilli that produced dl-lactic acid. Growth of the strains occurred at 45 °C but not at 15 °C. The optimum temperature for growth was 42 °C, as determined from the specific growth rate. The highest permissive temperatures for growth were 50 °C for strain G 35T and 48 °C for the other four strains. DNA G+C content of the strains was between 49 and 51 mol%. Complex fatty acid patterns of the strains showed the presence of C14 : 0, C16 : 0, sometimes C18 : 0, C18 : 1 and C19 : 0 cyclo in the cell walls. Phylogenetic analysis of the 16S rRNA gene sequences of the five strains placed them in the Lactobacillus casei/Pediococcus group, with Lactobacillus fermentum as their closest relative (about 95 % sequence similarity). DNA–DNA hybridization data indicated that the thermotolerant strains were not L. fermentum. Taken together, the findings of this study show that the five strains isolated from chicken faeces represent a novel species within the genus Lactobacillus, for which the name Lactobacillus thermotolerans is proposed (G 35T =DSM 14792T =JCM 11425T).

The taxonomic position of a thermophilic actinomycete strain isolated from soil was examined using a polyphasic approach. The strain, designated CH-M-1035T, was assigned to the genus Streptomyces on the basis of chemical and morphological criteria. It formed Rectiflexibiles aerial hyphae that carried long chains of rounded, smooth spores. The almost complete nucleotide sequence of the 16S rRNA gene of strain CH-M-1035T was determined and its comparison with the 16S rDNA sequences of previously studied streptomycetes confirmed the assignment of the novel strain to the genus Streptomyces. Strain CH-M-1035T clustered with species belonging to the Streptomyces thermodiastaticus clade in the 16S-rDNA-based phylogenetic tree. However, the phenotypic properties of strain CH-M-1035T differed from those of the recognized species within this clade. Therefore, it is proposed that strain CH-M-1035T be classified as a novel species within the genus Streptomyces, as Streptomyces mexicanus (type strain CH-M-1035T =DSM 41796T =BM-B-384T =NRRL B-24196T).

Two strains of Gram-variable, rod-shaped, endospore-forming, peritrichously flagellated, rod-shaped bacteria were isolated from a soil sample collected in Taejon City, Korea. The two strains (AM49T and AM141T) were found to be members of the genus Paenibacillus, on the basis of the results of phenotypic and phylogenetic analyses. Strains AM49T and AM141T were found to have a cell-wall peptidoglycan based on meso-diaminopimelic acid, MK-7 as their predominant menaquinone and anteiso-C15 : 0 as their major fatty acid. The DNA G+C contents of strains AM49T and AM141T were 48 and 47 mol%, respectively. The two strains formed distinct phylogenetic lineages within the radiation of the cluster comprising Paenibacillus spp. and a coherent cluster with Paenibacillus jamilae, Paenibacillus polymyxa, Paenibacillus azotofixans and Paenibacillus peoriae. The level of 16S rDNA similarity between strains AM49T and AM141T was 97·6 %, and 16S rDNA similarity values between strains AM49T and AM141T and the type strains of other Paenibacillus spp. ranged from 90·3 to 98·7 %. Levels of DNA–DNA similarity between the two strains and members of the genus Paenibacillus indicated that strains AM49T and AM141T were distinguishable from each other and from four phylogenetically related Paenibacillus spp. Therefore, on the basis of their phenotypic properties, phylogeny and genomic distinctiveness, it is proposed that strains AM49T and AM141T be placed in the genus Paenibacillus as two distinct novel species, Paenibacillus kribbensis (AM49T =KCTC 0766BPT =JCM 11465T) and Paenibacillus terrae (AM141T =KCCM 41557T =JCM 11466T).

A catalase-negative actinomycete, strain YIM 21741T, was isolated from a soil sample collected from a primeval forest at Xishuangbanna, Yunnan Province, China. Analysis of 16S rDNA showed the strain to be related to members of the genus Agromyces, with which it also shares morphological characteristics, e.g. branching hyphae breaking into diphtheroid and rod-like, irregular, non-motile fragments and a peptidoglycan containing the diagnostic amino acid 2,4-diamino-n-butyric acid. Whole-cell hydrolysates of strain YIM 21741T contained rhamnose and small quantities of glucose, galactose and mannose. The major menaquinone was MK-12, while MK-13 and MK-12 were minor components. Diagnostic phospholipids were phosphatidylglycerol and diphosphatidylglycerol. The G+C content of the DNA was 72·8 mol%. Physiological and biochemical characteristics reveal strain YIM 21741T to be different from all validly described species of the genus Agromyces. As DNA–DNA similarity values between this isolate and the phylogenetically neighbouring type strains of Agromyces bracchium and Agromyces luteolus are only moderate, the novel species Agromyces aurantiacus sp. nov. is proposed with strain YIM 21741T (=CCTCC 001012T =AS 4.1717T =DSM 14598T) as the type strain.

An actinomycete strain (YIM 90004T) was isolated from a saline soil sample from the Xinjiang Province, People's Republic of China. The strain displayed abundant aerial and substrate mycelia, and short spore chains were borne on the aerial mycelium. The spore chains were composed of non-motile, smooth-surfaced, rod-shaped spores. The cell wall of strain YIM 90004T contained mainly meso-diaminopimelic acid, alanine and glutamic acid (cell wall type III). Glucose, ribose, galactose, xylose and arabinose were the whole-cell sugars of the strain. The predominant phospholipids were phosphatidylinositol and phosphatidylglycerol; MK-10(H2) and MK-10(H4) were the predominant menaquinones. The DNA G+C content of strain YIM 90004T was 74·3 mol%. Chemotaxonomic properties and 16S rDNA analysis placed strain YIM 90004T in the genus Nocardiopsis. On the basis of phenotypic and phylogenetic analyses and DNA–DNA hybridization results, strain YIM 90004T (=CCRC 16285T =CCTCC AA99004T =DSM 44589T) is considered to represent a novel species of the genus Nocardiopsis, for which the name Nocardiopsis xinjiangensis is proposed.

Thirty-two strains of the novel species Starmerella meliponinorum sp. nov. were isolated from various substrates associated with three stingless bee species (tribe Meliponini) in Brazil and one in Costa Rica. The strains were found in garbage pellets (faecal material, discarded pollen or food, etc.), pollen provisions, adult bees, honey and propolis of Tetragonisca angustula, in honey from Melipona quadrifasciata and in adults of Melipona rufiventris and Trigona fulviventris. The sequence of the D1/D2 domains of the large-subunit rDNA showed that the novel species belongs to the Starmerella clade and is most closely related to Candida etchellsii, although the two differ in their sequences by 7 % base substitutions. S. meliponinorum is homothallic and assimilates few carbon sources. Nitrate is utilized as a sole nitrogen source. The type strain of S. meliponinorum is strain UFMG-01-J26.1T (=CBS 9117T).

A novel asexual ascomycetous yeast, Candida kunwiensis (SG99-26T =KCTC 17041T =CBS 9067T), was isolated from sweet potato (Ipomoea batatas) flowers in Korea and from the body surface of pollinating bumblebees in Germany. Comparative analysis of the D1/D2 domain of 26S rDNA of all available sequences for ascomycetous yeasts showed that the novel species was phylogenetically related to the genus Metschnikowia, but the sequence similarity was low. Morphologically and physiologically, C. kunwiensis in many ways resembles Metschnikowia pulcherrima, but can be distinguished from this species by its ability to assimilate lactic acid and its inability to produce pulcherrimin.

A 600 bp partial hsp60 gene sequence has been described previously as a novel genetic marker for species identification and phylogenetic studies within the genus Staphylococcus. In the present study, the 600 bp partial hsp60 gene sequences of 40 validly described Staphylococcus species and subspecies and four Macrococcus species were PCR-amplified and sequenced. Phylogenetic analysis revealed excellent concordance between the unrooted dendrograms based on partial hsp60 and 16S rRNA gene sequences. The genus Macrococcus is clearly separated from the genus Staphylococcus, but is closely related to the ‘sciuri group’, the only staphylococci that are cytochrome c oxidase-positive. The remaining Staphylococcus species clustered into five broad-based subdivisions, which corresponded to the ‘aureus group’, the ‘epidermidis group’, the ‘haemolyticus group’, the ‘saprophyticus group’ and the ‘intermedius group’. These results agreed remarkably well with the current taxonomy of this diverse family, which is based on classical phenotypic and biochemical testing. Furthermore, pairwise sequence comparisons indicated that the hsp60 gene is more divergent and more discriminatory than the 16S rRNA gene for species differentiation among strains of the genera Staphylococcus and Macrococcus. It is concluded that the hsp60 gene may be an efficient alternative target for taxonomic and phylogenetic studies on members of these genera.

Various bacterial species in the genus Pasteuria have great potential as biocontrol agents against plant-parasitic nematodes, although study of this important genus is hampered by the current inability to cultivate Pasteuria species outside their host. To aid in the study of this genus, an extensive 16S rRNA gene sequence phylogeny was constructed and this information was used to develop cultivation-independent methods for detection of Pasteuria in soils and nematodes. Thirty new clones of Pasteuria 16S rRNA genes were obtained directly from nematodes and soil samples. These were sequenced and used to construct an extensive phylogeny of this genus. These sequences were divided into two deeply branching clades within the low-G+C, Gram-positive division; some sequences appear to represent novel species within the genus Pasteuria. In addition, a surprising degree of 16S rRNA gene sequence diversity was observed within what had previously been designated a single strain of Pasteuria penetrans (P-20). PCR primers specific to Pasteuria 16S rRNA for detection of Pasteuria in soils were also designed and evaluated. Detection limits for soil DNA were 100–10 000 Pasteuria endospores (g soil)−1.

Polyphasic genotypic analysis of 25 Acidithiobacillus ferrooxidans strains isolated from ores and ore concentrates collected in different regions of the world showed considerable strain heterogeneity. Restriction patterns of the chromosomal DNA of these strains obtained by PFGE were specific for each strain. According to the degree of DNA relatedness, 17 of the 23 strains studied were divided into four genomovars. Six independent, considerably divergent strains could not be assigned to any of the genomovars. A comparison of nearly complete nucleotide sequences of the 16S rDNA of five representatives of the genomovars (including the type strain of A. ferrooxidans, ATCC 23270T) with those of species of the genus Acidithiobacillus available from GenBank showed that most of the A. ferrooxidans strains, together with the type strain and some other strains of the species Acidithiobacillus thiooxidans, comprised a monophyletic cluster. Within this major cluster, A. ferrooxidans strains fell into four phylogenetic groups that were equidistant from the phylogenetic group of A. thiooxidans strains. In general, the distribution of strains among the phylogenetic groups correlated with their distribution among the genomovars, except that the representatives of two different genomovars fell into one phylogenetic group. Thus, at least two levels of phylogenetic heterogeneity for A. ferrooxidans have been found. The phylogenetic heterogeneity of A. ferrooxidans strains, which are phenotypically indistinguishable, suggests the occurrence of microevolutionary processes in different econiches. This should be taken into account in the biohydrometallurgical applications of A. ferrooxidans strains.

A significant number of chimeric 16S rDNA sequences of diverse origin were identified in the public databases by partial treeing analysis. This suggests that chimeric sequences, representing phylogenetically novel non-existent organisms, are routinely being overlooked in molecular phylogenetic surveys despite a general awareness of PCR-generated artefacts amongst researchers.

Gregarines are thought to be deep-branching apicomplexans. Accordingly, a robust inference of gregarine phylogeny is crucial to any interpretation of apicomplexan evolution, but molecular sequences from gregarines are restricted to a small number of small-subunit (SSU) rDNA sequences from derived taxa. This work examines the usefulness of SSU rDNA and β-tubulin sequences for inferring gregarine phylogeny. SSU rRNA genes from Lecudina (Mingazzini) sp., Monocystis agilis Stein, Leidyana migrator Clopton and Gregarina polymorpha Dufour, as well as the β-tubulin gene from Leidyana migrator, were sequenced. The results of phylogenetic analyses of alveolate taxa using both genes were consistent with an early origin of gregarines and the putative ‘sister’ relationship between gregarines and Cryptosporidium, but neither phylogeny was strongly supported. In addition, two SSU rDNA sequences from unidentified marine eukaryotes were found to branch among the gregarines: one was a sequence derived from the haemolymph parasite of the giant clam, Tridacna crocea, and the other was a sequence misattributed to the foraminiferan Ammonium beccarii. In all of our analyses, the SSU rDNA sequence from Colpodella sp. clustered weakly with the apicomplexans, which is consistent with ultrastructural data. Altogether, the exact position of gregarines with respect to Cryptosporidium and other apicomplexans remains to be confirmed, but the congruence of SSU rDNA and β-tubulin trees with one another and with morphological data does suggest that further sampling of molecular data will eventually put gregarine diversity into a phylogenetic context.

Oxyrrhis marina and Perkinsus marinus are two alveolate species of key taxonomic position with respect to the divergence of apicomplexans and dinoflagellates. New sequences from Oxyrrhis, Perkinsus and a number of dinoflagellates were added to datasets of small-subunit (SSU) rRNA, actin, α-tubulin and β-tubulin sequences, as well as to a combined dataset of all three protein-coding genes, and phylogenetic trees were inferred. The parasitic Perkinsus marinus branches at the base of the dinoflagellate clade with high support in most of the individual gene trees and in the combined analysis, strongly confirming the position originally suggested in previous SSU rRNA and actin phylogenies. The SSU rRNA from Oxyrrhis marina is extremely divergent, and it typically branches with members of the Gonyaulacales, a dinoflagellate order where SSU rRNA sequences are also divergent. Conversely, none of the three protein-coding genes of Oxyrrhis is noticeably divergent and, in trees based on all three proteins individually and in combination, Oxyrrhis branches at the base of the dinoflagellate clade, typically with high bootstrap support. In some trees, Oxyrrhis and Perkinsus are sisters, but most analyses indicate that Perkinsus diverged prior to Oxyrrhis. Morphological characters have previously pointed to Oxyrrhis as an early branch in the dinoflagellate lineage; our data support this suggestion and significantly bolster the molecular data that support a relationship between Perkinsus and dinoflagellates. Together, these two organisms can be instrumental in reconstructing the early evolution of dinoflagellates and apicomplexans by helping to reveal aspects of the ancestors of both groups.