- Volume 51, Issue 3, 2001

The complete or nearly complete large-subunit rRNA (LSU rRNA) sequences were determined for representatives of several algal groups such as the chlorarachniophytes, cryptomonads, haptophytes, bacillariophytes, dictyochophytes and pelagophytes. Our aim was to study the phylogenetic position and relationships of the different groups of algae, and in particular to study the relationships among the different classes of heterokont algae. In LSU rRNA phylogenies, the chlorarachniophytes, cryptomonads and haptophytes seem to form independent evolutionary lineages, for which a specific relationship with any of the other eukaryotic taxa cannot be demonstrated. This is in accordance with phylogenies inferred on the basis of the small-subunit rRNA (SSU rRNA). Regarding the heterokont algae, which form a well-supported monophyletic lineage on the basis of LSU rRNA, resolution between the different classes could be improved by combining the SSU and LSU rRNA data. Based on a concatenated alignment of both molecules, the phaeophytes and the xanthophytes are sister taxa, as well as the pelagophytes and the dictyochophytes, and the chrysophytes and the eustigmatophytes. All these sister group relationships are highly supported by bootstrap analysis and by different methods of tree construction.

In order to reconstruct the evolution of euglenid flagellates, euglenozoan SSU rDNA data have been used to investigate phylogenetic relationships with a focus on osmotrophic taxa and especially on the Rhabdomonadales. The dataset consisting of the SSU rDNAs of osmotrophic, phagotrophic and phototrophic taxa was used in parsimony, maximum-likelihood and distance analyses. Five genera make up the Rhabdomonadales, all of them osmotrophic: Gyropaigne, Menoidium, Parmidium, Rhabdomonas and Rhabdospira. According to our analyses they form a strongly supported monophyletic assemblage which is characterized by a low sequence divergence compared to the euglenids in general. Closest relatives are the members of the osmotrophic genus Distigma. All primary osmotrophic species constitute a larger monophyletic group with the phototrophic euglenids and the phagotroph Peranema trichophorum. The combination of three rhabdomonadalian species Rhabdomonas gibba, Rhabdomonas spiralis and Rhabdospira spiralis with nearly identical SSU rDNA sequences is strongly recommended. The phagotroph Petalomonas cantuscygni branches at the bottom of the euglenid subtree with significantly weaker support. The inter-relationship of the three distinct euglenozoan taxa (euglenids, kinetoplastids and diplonemids) could not be convincingly resolved by this study.

This paper reports transmission electron microscopy (TEM) and immunofluorescence evidence for phragmoplast-mediated cytokinesis in the green alga, Trentepohlia (Ulvophyceae, Chlorophyta). This type of cytokinesis is normally found only in land plants and some charophycean green algae. Earlier TEM work documented a phragmoplast in another trentepohlialean alga, Cephaleuros. Numerous molecular studies have shown that both Trentepohlia and Cephaleuros are clearly not in the charophycean (streptophyte) lineage of green algae. Also, details of vegetative cytokinesis in Trentepohlia and Cephaleuros indicate differences from the processes and structures observed in the streptophytes. Parallel evolution could be the explanation for a phragmoplast-mediated cytokinesis in both the chlorophycean Trentepohliales and the streptophyte lineage of charophycean green algae and land plants.

'Isospora' lieberkuehni, an unusual isosporoid renal coccidium that parasitizes the European water frog was isolated from the edible frog, Rana kl. esculenta, in the Czech Republic. Sequencing of the small-subunit (SSU) rRNA gene showed that it belongs to the family Sarcocystidae, being closely related to a clade comprising members of the subfamily Toxoplasmatinae. The position within Sarcocystidae correlates with the mode of excystation via collapsible plates as postulated by previous authors. Phylogenetic, morphological and biological differences between 'Isospora' lieberkuehni and the other Stiedabody-lacking members of the genus Isospora justify separation of this coccidium on a generic level. Hyaloklossia Labbé, 1896 is the oldest available synonym and is herein re-erected. The original definition of the genus Hyaloklossia is emended based on recent observations.

Almost complete sequences of plastid SSU rDNA (16S rDNA) from 17 species belonging to the order Euglenales (sensu Nemeth, 1997; Shi et al., 1999) were determined and used to infer phylogenetic relationships between 10 species of Euglena, three of Phacus, and one of each of Colacium, Lepocinclis, Strombomonas, Trachelomonas and Eutreptia. The maximum-parsimony (MP), maximum-likelihood (ML) and distance analyses of the unambiguously aligned sequence fragments imply that the genus Euglena is not monophyletic. Parsimony and distance methods divide Euglenaceae into two sister groups. One comprises of representatives from the subgenera Phacus, Lepocinclis and Discoglena (sensu Zakryś, 1986), whereas the other includes members of Euglena and Calliglena subgenera (sensu Zakryś, 1986), intermixed with representatives of Colacium, Strombomonas and Trachelomonas. In all analyses subgenera Euglena--together with Euglena polymorpha (representative of the subgenus Calliglena)--and Discoglena--together with Phacus and Lepocinclis--form two well-defined clades. The data clearly indicate that a substantial revision of euglenoid systematics is very much required, nevertheless it must await while more information can be gathered, allowing resolution of outstanding relationships.

Phylogenetic analyses of 35 strains including 25 previously published sequences and 10 which have been newly sequenced, representing two species of Euglena, five species of Phacus and three species of Astasia, were carried out using the SSU rDNA. Parsimony, distance and maximum-likelihood inferred phylogenies support (1) monophyly of the euglenoids, (2) kinetoplastids as the sister group, (3) the phagotrophic Petalomonas cantuscygni Cann et Pennick anchoring the base of the euglenoid lineage, (4) evolution of phototrophy within the euglenoids from a single event, (5) multiple origins of osmotrophic euglenoids and (6) polyphyly of the genera Euglena Ehrenberg and Phacus Dujardin. Analyses also indicate that Lepocinclis Perty, Trachelomonas Ehrenberg and Astasia Dujardin are polyphyletic. In addition, the results suggest that neither the Euglenales nor the Eutreptiales form a monophyletic lineage, thus questioning currently available classifications. Concerning the phagotrophic mode of nutrition, the data suggest that the feeding apparatus arose multiple times.

The streptomycetes, producers of more than half of the 10,000 documented bioactive compounds, have offered over 50 years of interest to industry and academia. Despite this, their taxonomy remains somewhat confused and the definition of species is unresolved due to the variety of morphological, cultural, physiological and biochemical characteristics that are observed at both the inter- and the intraspecies level. This review addresses the current status of streptomycete taxonomy, highlighting the value of a polyphasic approach that utilizes genotypic and phenotypic traits for the delimitation of species within the genus.

The phylogenetic position and some taxonomically relevant characteristics of a small, low-virulence bacterial parasite of cats are described. A 16S rDNA analysis revealed that the organism was in the Mycoplasma clade and was most closely related to a parasite of pigs previously designated Eperythrozoon suis. As the organism has not been cultured in vitro and is maintained in serial passage in cats in vivo, Candidatus status is proposed for this novel taxon as 'Candidatus Mycoplasma haemominutum'.

Genotypic and phenotypic methods were applied to investigate differences between the closely related species Campylobacter hyoilei and Campylobacter coli. A unique DNA sequence from C. hyoilei was used to design a specific PCR assay that amplified a DNA product of 383 bp for all C. hyoilei strains, but not other Campylobacter species, including C. coli. The PCR assay could detect 100 fg pure C. hyoilei DNA, 2 x 10(2) c.f.u. ml(-1) using cultured cells and 8.3 x 10(3) c.f.u. 0.1 g(-1) in faeces. The C. hyoilei sequence utilized for specific detection and identification of this species showed similarities to sequences from bacteriophages Mu, P2 and 186, suggesting lysogination of the ancestral C. hyoilei genome. Activities of a set of 15 enzymes that participate in a variety of cellular functions, including biosynthesis, catabolism, energy generation, maintenance of redox balance and phosphate utilization, were tested using sets of strains of C. hyoilei and C. coli. Comparison of mean rates of enzyme activities revealed significant differences between species in the values determined for seven of these activities. Both the genetic and phenotypic data indicate that C. hyoilei is a unique Campylobacter species.

Strain EDIVT, an exopolysaccharide-producing bacterium, was subjected to polyphasic characterization. The bacterium produced copious amounts of an extracellular polysaccharide, forming slimy, viscous, intensely yellow-pigmented colonies on Czapek-Dox (CZD) agar. The culture fluids of the liquid version of CZD medium were highly viscous after cultivation for 5 d. Cells of strain EDIVT were Gram-negative, catalase-positive, oxidase-negative, nonspore-forming, rod-shaped and motile. Comparisons of 16S rDNA gene sequences demonstrated that EDIVT clusters phylogenetically with the species of the genus Sphingomonas sensu stricto. The G+C content of the DNA (64.5 mol%), the presence of ubiquinone Q-10, the presence of 2-hydroxymyristic acid (14:0 2-OH) as the major hydroxylated fatty acid, the absence of 3-hydroxy fatty acids and the detection of sym-homospermidine as the major component in the polyamine pattern, together with the presence of sphingoglycolipid, supported this delineation. 16S rDNA sequence analysis indicated that strain EDIVT is most closely related (99.4% similarity) to Sphingomonas trueperi LMG 2142T. DNA-DNA hybridization showed that the level of relatedness to S. trueperi is only 45.5%. Further differences were apparent in the cellular fatty acid profile, the polar lipid pattern, the Fourier-transform infrared spectrum and whole-cell proteins and in a number of biochemical characteristics. On the basis of the estimated phylogenetic position derived from 16S rDNA sequence data, DNA-DNA reassociation and phenotypic differences, strain EDIVT (= CIP 106154T = DSM 13101T) was recognized as a new species of Sphingomonas, for which the name Sphingomonas pituitosa sp. nov. is proposed. A component analysis of the exopolysaccharide (named PS-EDIV) suggested that it represents a novel type of sphingan composed of glucose, rhamnose and an unidentified sugar. Glucuronic acid, which is commonly found in sphingans, was absent. The mean molecular mass of PS-EDIV was approximately 3 x 10(6) Da.

Ten isolates each of two different bacterial species isolated from the surface of a smear-ripened cheese were found to exhibit many characteristics of the genus Corynebacterium. The isolates were Gram-positive, catalase-positive, non-spore-forming rods that did not undergo a rod/coccus transformation when grown on complex media. Chemotaxonomic investigation revealed that the strains belonged unambiguously to the genus Corynebacterium. Their cell walls contained arabinose, galactose and short-chain mycolic acids (C22 to C36) and their peptidoglycan contained meso-diaminopimelic acid. The G+C content of the DNA was 51-60 mol%. MK-9 (H2) was the principal menaquinone. The 16S rDNA sequences of four isolates of each bacterium were determined and aligned with those of other members of the coryneform group. Phylogenetic analysis showed that the strains represented two new sublines within the genus Corynebacterium; Corynebacterium variabile and Corynebacterium ammoniagenes were their nearest known phylogenetic neighbours. Corynebacterium variabile and Corynebacterium ammoniagenes showed the highest levels of sequence homology with the isolates; however, DNA-DNA hydridization studies indicated that the Corynebacterium strains isolated from the cheese smear did not belong to either Corynebacterium variabile or Corynebacterium ammoniagenes (26 and 46% chromosomal similarity, respectively). On the basis of the phylogenetic and phenotypic distinctiveness of the unknown isolates, it is proposed that the bacteria be classified as two new Corynebacterium species, for which the names Corynebacterium mooreparkense sp. nov. and Corynebacterium casei sp. nov. are proposed. Type strains have been deposited in culture collections as Corynebacterium mooreparkense LMG S-19265T (= NCIMB 30131T) and Corynebacterium casei LMG S-19264T (= NCIMB 30130T).

Three strains of a previously undescribed Actinomyces-like bacterium were isolated from human clinical specimens. Phenotypic studies indicated that the strains were members of the genus Actinomyces and were presumptively identified as Actinomyces turicensis. Comparative 16S rRNA gene sequencing studies showed that although the bacterium is phylogenetically closely related to Actinomyces turicensis, it nevertheless constitutes a new sub-line within the genus Actinomyces. Based on phenotypic and molecular chemical and molecular genetic evidence, it is proposed that the unknown Actinomyces-like bacterium from human clinical specimens be classified as Actinomyces funkei sp. nov. The type strain of Actinomyces funkei is CCUG 42773T (= CIP 106713T).

An unknown Gram-positive rod-shaped bacterium was isolated from skin scrapings from the infected head of a sheep and subjected to a polyphasic taxonomic analysis. Chemical analysis revealed the presence of straight-chain and monounsaturated fatty acids and short-chain (C32-C36) mycolic acids consistent with the genus Corynebacterium. Comparative 16S rRNA gene sequencing confirmed that the unknown rod was a member of the genus Corynebacterium, with the organism forming a distinct sub-line and displaying greater than 3% sequence divergence with established species. The unknown Corynebacterium isolate was readily distinguished from recognized species of the genus by biochemical tests and electrophoretic analysis of whole-cell proteins. Based on phylogenetic and phenotypic evidence, it is proposed that the unknown bacterium from a sheep be classified in the genus Corynebacterium, as Corynebacterium capitovis sp. nov. The type strain of Corynebacterium capitovis is CCUG 39779T (= CIP 106739T).

The genus Synechococcus (cyanobacteria), while containing morphologically similar isolates, is polyphyletic and organisms presently classified as such require reclassification into several independent genera. Studies based on analysis of 16S rRNA gene sequences have shown that members of the genus Synechococcus are affiliated to three of seven deeply branching cyanobacterial lineages. In addition, some strains do not appear to be associated with any of these lineages and may represent novel clades. In this report, a cyanobacterial phylogeny based on 16S rDNA sequences, including 14 newly sequenced Synechococcus isolates, is presented. One newly sequenced Synechococcus strain (PCC 7902) did not have any close relatives amongst cyanobacterial isolates currently contained in 16S rDNA sequence databases and was only loosely affiliated to a cyanobacterial lineage in which no other Synechococcus strains were found. Three hot-spring Synechococcus isolates, including two that were newly sequenced in this study (PCC 6716 and PCC 6717), formed an additional cyanobacterial lineage. These results indicated that Synechococcus species are affiliated to five of eight deeply branching cyanobacterial lineages. Part of the phycocyanin (PC) gene sequence (cpc), including the intergenic spacer (IGS) between cpcB and cpcA and the corresponding flanking regions (cpcBA-IGS), was used to investigate relationships between closely related Synechococcus isolates. Previously described PCR primers did not amplify this region from the majority of strains under investigation, so a new set of primers was designed that allowed amplification and sequencing of the cpcBA-IGS and flanking regions from 38 Synechococcus species. Phylogenetic analysis of this region was largely consistent with that obtained from 16S rDNA sequence analysis and revealed a relationship between the primary PC DNA sequence and the phycobilin content of cells.

Genomic DNA homologies were examined from six Microcystis (cyanobacteria) strains, including five different species, Microcystis aeruginosa, Microcystis ichthyoblabe, Microcystis novacekii, Microcystis viridis and Microcystis wesenbergii. All DNA-DNA reassociation values between two strains of M. aeruginosa and the other four species exceeded 70%, which is considered high enough for them to be classified within the same bacterial species. It is proposed to unify these five species into M. aeruginosa under the Rules of the Bacteriological Code and NIES843T (= IAM M-247T) is proposed as the type strain. Two other species, Microcystis flos-aquae and Microcystis pseudofilamentosa, should be regarded as morphological variations of this unified M. aeruginosa. The current taxonomy of cyanobacteria depends too much upon morphological characteristics and must be reviewed by means of bacteriological methods as well as traditional botanical methods.

An anthracycline-producing actinomycete (strain SF2303) was compared with two other anthracycline producers, Actinomadura carminata IFO 15903T and Nonomuraea roseoviolacea IFO 14098T, using morphological, physiological, chemotaxonomic and molecular-genetic criteria. The morphological and cultural characteristics of these three strains are similar. The physiological properties of strain SF2303 and N. roseoviolacea IFO 14098T are very similar, but are different from those of A. carminata IFO 15903T in the utilization of some sugars and the reduction of nitrate. Their chemotaxonomic properties [cell wall chemotype, IIIB; major menaquinone, MK-9 (III, VIIl-H4); phospholipid type, PIV; cellular fatty acids 10M-17:0/17:1 and iso-16:0 as major components and 2-hydroxy fatty acids as minor components; mycolic acid, absent] were identical and indicated that these three strains belong to the family Streptosporangiaceae. On the basis of 16S rDNA sequences and phylogenetic analysis, they were all included in the cluster formed by species of Nonomuraea. The levels of DNA relatedness between strain SF2303 and N. roseoviolacea IFO 14098T ranged from 71 to 78%; however, the levels of relatedness between the two strains and A. carminata IFO 15903T were lower (49-60%). Therefore, strain SF2303 was identified as a member of the species N. roseoviolacea and it is proposed that Actinomadura carminata Gauze et al. 1973 should be transferred to a new subspecies of the genus Nonomuraea Zhang et al. 1998 as N. roseoviolacea subsp. carminata comb. nov.

Cell-wall-less uncultivated parasitic bacteria that attach to the surface of host erythrocytes currently are classified in the order Rickettsiales, family Anaplasmataceae, in the genera Haemobartonella and Eperythrozoon. Recently 16S rRNA gene sequences have been determined for four of these species: Haemobartonella felis and Haemobartonella muris and Eperythrozoon suis and Eperythrozoon wenyonii. Phylogenetic analysis of these sequence data shows that these haemotrophic bacteria are closely related to species in the genus Mycoplasma (class Mollicutes). These haemotrophic bacteria form a new phylogenetic cluster within the so-called pneumoniae group of Mycoplasma and share properties with one another as well as with other members of the pneumoniae group. These studies clearly indicate that the classification of these taxa should be changed to reflect their phylogenetic affiliation and the following is proposed: (i) that Haemobartonella felis and Haemobartonella muris should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemofelis' and 'Candidatus Mycoplasma haemomuris' and (ii) that Eperythrozoon suis and Eperythrozoon wenyonii should be transferred to the genus Mycoplasma as 'Candidatus Mycoplasma haemosuis' and 'Candidatus Mycoplasma wenyonii'. The former Haemobartonella and Eperythrozoon species described here represent a new group of parasitic mycoplasmas that possess a pathogenic capacity previously unrecognized among the mollicutes. These haemotrophic mycoplasmas have been given the trivial name haemoplasmas. These results call into question the affiliation of the remaining officially named species of Haemobartonella and Eperythrozoon which should be considered species of uncertain affiliation pending the resolution of their phylogenetic status.