Physiological and biochemical contributions to the taxonomy of the genus Prototheca

16 strains of the genus Prototheca do not produce extracellular amylolytic enzymes. The base composition of their DNA shows rather continuous values from 62% to 78% GC (guanine + cytosine). Their assignment to four species and their possible relationship with Chlorella protothecoides are discussed.Abbreviations Used GC guanine + cytosine - SSC saline sodium citrate     

Physiological and biochemical contributions to the taxonomy of the genus prototheca

Five physiological and biochemical characters, which had proved to be valuable for the taxonomy of the genus Chlorella, were studied in the genus Prototheca. There is no hydrogenase activity and no liquefaction of gelatin. Most strains are very acidtolerant (limit of growth at pH 2.0 or 2.5) and very salt-tolerant (limit of growth at 4 or 5% NaCl). Two strains grow well at 38°C. The 16 strains, which were previously assigned to seven taxa, fall into four different groups. Our results tend to support the assumption that Prototheca might be related to Chlorella protothecoides.     

Taxonomic implications of Prototheca and Chlorella cell wall polysaccharide characterization

Summary Four polysaccharide fractions were obtained by acid and alkaline degradation of purified cell walls of Prototheca spp. and Chlorella spp. These fractions were further hydrolyzed and the component sugars identified. Five Prototheca strains and two Chlorella protothecoides strains have essentially similar polysaccharide compositions, which significantly differ from those of C. vulgaris and C. pyrenoidosa. This emphasizes the close affinity of C. protothecoides to Prototheca spp. not shared by other Chlorella spp.     

MOLECULAR PHYLOGENY AND PHENOTYPIC VARIATION IN THE HETEROTROPHIC GREEN ALGAL GENUS PROTOTHECA (TREBOUXIOPHYCEAE,CHLOROPHYTA)1

Species of the heterotrophic green microalgal genus Prototheca and related taxa were phylogenetically analyzed based on the nuclear small subunit (SSU) and the 5′ end of the large subunit (LSU) rRNA gene (rDNA) sequences. We propose restricting the genus Prototheca to the four species: P. moriformis Krüger, P. stagnora (Cooke) Pore, P. ulmea Pore, and P. zopfii Krüger. The main diagnostic feature of these taxa is the absence of growth on trehalose.Of these, it was suggested that P. moriformis should be merged into P. zopfii; P. moriformis and three varieties of P. zopfii constituted a paraphyletic assemblage with estimated short evolutionary distances. The trehalose‐assimilating strains (Prototheca wickerhamii Tubaki et Soneda strains and Auxenochlorella protothecoides (Krüger) Kalina et Pun?ochá?ová SAG 211‐7a), together with an invertebrate pathogen Helicosporidium sp., diverged before the radiation of the four species of Prototheca in the SSU rDNA and composite (SSU rDNA plus LSU rDNA) analyses. Comparison between the results from physiological data in this work (fermentative pattern) and those described earlier (growth requirements) lead us to propose a hypothesis that the phenotypic variation, which did not represent diagnostic characters for species delimitation, may reflect the history of genetic diversification within the genus Prototheca as inferred from rDNA sequence characters.     

Molecular genetic evidence for the transfer of Oerskovia species into the genus Cellulomonas

A close genetic relationship among strains of Oerskovia turbata, O. xanthineolytica and various coryneforms is indicated by DNA-DNA reassociation studies. O. xanthineolytica shares high homology values (over 60%) with Cellulomonas cartae, Nocardia cellulans, Brevibacterium fermentans and Corynebacterium manihot. O. turbata and other cellulomonads show lower DNA homology values (20–25%) which are still high enough, however, to indicate a relationship at the genus level. Based on these data and supported by comparative analysis of the ribosomal 16 S RNA and the similarity of peptidoglycan types, the transfer of Oerskovia species into the genus Cellulomonas is justified.This paper is respectively dedicated to our teacher and mentor, Professor Dr. O. Kandler, on the occasion of his 60th birthday.     

The use of <Emphasis Type="Italic">gyrB</Emphasis> sequence analysis in the phylogeny of the genus <Emphasis Type="Italic">Amycolatopsis</Emphasis>

Partial gyrB sequences (>1 kb) were obtained from 34 type strains of the genus Amycolatopsis. Phylogenetic trees were constructed to determine the effectiveness of using this gene to predict taxonomic relationships within the genus. The use of gyrB sequence analysis as an alternative to DNA–DNA hybridization was also assessed for distinguishing closely related species. The gyrB based phylogeny mostly confirmed the conventional 16S rRNA gene-based phylogeny and thus provides additional support for certain of these 16S rRNA gene-based phylogenetic groupings. Although pairwise gyrB sequence similarity cannot be used to predict the DNA relatedness between type strains, the gyrB genetic distance can be used as a means to assess quickly whether an isolate is likely to represent a new species in the genus Amycolatopsis. In particular a genetic distance of >0.02 between two Amycolatopsis strains (based on a 315 bp variable region of the gyrB gene) is proposed to provide a good indication that they belong to different species (and that polyphasic taxonomic characterization of the unknown strain is worth undertaking). Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users. The GenBank accession numbers for the gyrB gene sequences obtained in this study are shown in Table 1.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

Four species of the unicellular green alga Chlorella, C. vulgaris, C. luteoviridis, C. minutissima, and C. zofingiensis, were characterized with respect to DNA similarities as determined by quantitative DNA hybridization procedures. In contrast to previous DNA hybridization procedures. In contrast to previous results, C. vulgaris turned out to be a homogeneous species with the exception of strain 211-11c of the Göttingen collection, which was shown to belong to C. kessleri. Similary, C. luteoviridis and C. minutissima represent well defined species in terms of phenotypic and genotypic features. Whitin C. zofingiensis on strain is clearly different with respect to DNA base composition and DNA hybridization data even though it shares phenotypic characteristics with the other strains of C. zofingiensis.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

The genetic relationships of nine strains of Chlorella saccharophila were determined by DNA hybridization techniques. Four strains are closely related to the type strain 211-9a and one strain seems to be moderately related, whereas the taxonomic position of the remaining three strains is not clear. C. saccharophila, like C. sorokiniana, is another species of Chlorella containing strains which are heterogeneous in their overall DNA base sequence and partly also in morphological, biochemical and physiological characters.     

Physiological and biochemical contributions to the taxonomy of the genus Prototheca

Prototheca zopfii (12 strains) is able to use glucose, fructose, propanol, glycerol, and acetate as sources of carbon for growth. One of the strains is biochemically (utilization also of galactose and mannose), and two strains are morphologically slightly different.Two strains can be identified as P. wikerhamii. They exhibit good growth with glucose, fructose, galactose, trehalose, propanol, glycerol, acetate, and glutamate as sources of carbon. P. spec. 263-2 grows only with glucose and acatate. P. zopfii and P. wickerhamii are able to use urea, glycine, and glutamate as sources of nitrogen. P. spec. 263-2, on the other hand, cannot utilize these organic nitrogen compounds for growth.Four strains of Chlorella protothecoides are able to use glucose, fructose, galactose, and acetate as sources of carbon for growth in the dark. Three of them utilize also mannose, trehalose, and glutamate. Two strains can grow with glycerol, and one is able to use lactose. — Urea and glycine can serve as sources of nitrogen for the four strains of C. protothecoides. Glutamate supports growth of three strains, and one strain is able to use nicotinamide.     

Two Phosphate- and Potassium-solubilizing Bacteria Isolated from Tianmu Mountain, Zhejiang, China

Summary Two phosphate- and potassium-solubilizing strains (KNP413 and KNP414) were isolated from the soil of Tianmu Mountain, Zhejiang Province (China) and they were phenotypically and phylogenetically characterized. Both isolates effectively dissolved mineral phosphate and potassium, while strain KNP414 showed higher dissolution capacity even than Bacillus mucilaginosus AS1.153, the inoculant of potassium fertilizer widely used in China. When grown on Aleksandrov medium, both strains were rod-shaped spore-formers with a large capsule, and they formed slimy and translucent colonies. The DNA G+C contents were 57.7 mol% for strain KNP413 and 56.1 mol% for strain KNP414. Strain KNP413 shared a 16S rRNA gene sequence similarity of more than 99.1% with strain KNP414 and Bacillus mucilaginosus strains HSCC 1605 and YNUC0001, and a 94.6% similarity with Bacillus mucilaginosus VKM B-1480D, the type strain of Bacillus mucilaginosus. Strains KNP413 and KNP414 together with other Bacillus mucilaginosus were clustered with Paenibacillus strains in a group. The use of a specific PCR primer PAEN515F designed for differentiating the genus Paenibacillus from other members of the Bacillaceae showed that strains KNP413 and KNP414 had the same amplified 16S rRNA gene fragment (0.9-kb) as members of the genus Paenibacillus. In conclusion, phosphate- and potassium-solubilizing strains KNP413 and KNP414 should be integrated into the same species different from strain VKM B-1480D and they might be transferred to the genus of Paenibacillus, i.e. Paenibacillus mucilaginosus.The GenBank accession numbers of the 16S rRNA gene sequences are AY646227 for KNP413 and AY646228 for KNP414.     

Physiological and biochemical contributions to the taxonomy of the genus Chlorella

Starch hydrolysis, i.e., the production of extracellular amylolytic enzymes, was found to be a specific character for most species of the genus Chlorella. C. fusca var. vacuolata, C. spec. 211-30, and C. spec. 211-11r hydrolyse starch, whereas C. vulgaris, C. fusca var. rubescens, C. zofingiensis, C. fusca var. fusca, C. minutissima, C. homosphaera, C. kessleri, C. luteoviridis, and C. protothecoides are unable to hydrolyse starch. Only C. sorokiniana and C. saccharophila appear heterogenous; within C. sorokiniana, 7 strains hydrolyse starch and 9 do not; and within C. saccharophila, 6 strains exhibit amylolytic activity and 2 do not. — A key for the identification, according to 9 easily determined physiological and biochemical characters, of the Chlorella species is presented.     

Evaluation of the susceptibility of Prototheca zopfii to milk pasteurization

Protothecosis has been reported in humans (gastroenteritis, bursitis, etc.) and in many other animal species. Bovine mastitis represents the main form of occurrence of protothecosis in cattle. Milk as well as dairy products, when contaminated with Prototheca spp., represent a potential means of transmission of this zoonosis. The purpose of this study was to evaluate the susceptibility of forty Prototheca zopfii strains isolated from milk from intramammary infections in dairy cows and also from bulk milk tanks of dairy farms, to the different ratios of temperature/time employed in the thermal treatment of milk: 72–75 °C/1 5 seconds, 72–75 °C/20 seconds and 62–65 °C/30 minutes. The samples were subjected to these different temperature/time ratios. The evaluation of the thermal susceptibility of the P. zopfii strains showed that 34 strains were resistant in at least one of the tests. The results point out the need to consider the importance of mastitis caused by Prototheca spp. asrepresenting a public health risk. This revised version was published online in June 2006 with corrections to the Cover Date.     

Comparison of 16S rDNA analysis and rep-PCR genomic fingerprinting for molecular identification of Yersinia pseudotuberculosis

16S rDNA sequence analysis and repetitive element sequence-based PCR (rep-PCR) genomic fingerprinting were evaluated on 11 type strains of the genus Yersinia and 17 recognized serotype strains of Y. pseudotuberculosis to investigate their genetic relatedness and to establish the value of techniques for the identification of Y. pseudotuberculosis. A phylogenetic tree constructed from 16S rDNA sequences showed that the type strains of Yersinia species formed distinct clusters with the exception of Y. pestis and Y. pseudotuberculosis. Moreover, Y. pestis NCTC 5923^T was found to be closely related to Y. pseudotuberculosis serotypes 1b, 3, and 7. Dendrograms generated from REP-PCR, and ERIC-PCR data revealed that members of the genus Yersinia differed from each other with the degree of similarity 62% and 58%, respectively. However, the BOX-PCR results showed that Y. pestis 5923^T clustered with the Y. pseudotuberculosis group with a degree of similarity 74%. According to these findings, 16S rDNA sequence analysis was unable to reliably discriminate Y. pseudotuberculosis from Y. pestis. However, REP-PCR and especially ERIC-PCR provided an effective means of differentiating between members of the taxa. This revised version was published online in June 2006 with corrections to the Cover Date.     

Species-specific ability ofChlorella strains (Chlorophyceae) to form stable symbioses withHydra viridis

46 strains ofChlorella, identified by physiological and biochemical characters, were examined for their ability to form stable symbioses with aposymbioticHydra viridis. It was found to be a species-specific characteristic. Among the 15 taxa studied, onlyC. saccharophila var.ellipsoidea, C. saccharophila var.saccharophila, C. fusca var.vacuolata, C. kessleri, C. luteoviridis, andC. protothecoides formed stable symbioses withHydra viridis. Among the 11 known physiological and biochemical characters of theseChlorella species, only acid tolerance seems to be correlated with symbiosis: All symbiotic species are capable of growing at or below pH 4.0.     

Deoxyribonucleic acid reassociation in the taxonomy of the genus Chlorella

DNA hybridization techniques showed Chlorella fusca var. vacuolata and C. kessleri to be homogeneous species with DNA homologies of 90–100% C. fusca var. fusca and var. rubescens, however, have only about 15% DNA homology with C. fusca var. vacuolata and should no longer be regarded as varieties. A good correlation was found so far between biochemical and physiological characters used in the taxonomy of Chlorella and DNA relatedness. Mutant strains of Chlorella were tested for DNA homologies to prove the reliability of the taxonomical interpretation.     

Genetic variability and taxonomic revision of the genus Auxenochlorella (Shihira et Krauss) Kalina et Puncocharova (Trebouxiophyceae,Chlorophyta)

The monotypic genus Auxenochlorella with its type species A. protothecoides is so far only known from specific habitats such as the sap of several tree species. Several varieties were described according to physiological performances in culture on different organic substrates. However, two strains designated as Auxenochlorella were isolated from other habitats (an endosymbiont of Hydra viridis and an aquatic strain from an acidic volcano stream). We studied those isolates and compared them with six strains of Auxenochlorella belonging to different varieties. The integrative approach used in this study revealed that all strains showed similar morphology but differed in their SSU and ITS rDNA sequences. The Hydra endosymbiont formed a sister taxon to A. protothecoides, which included the varieties protothecoides, galactophila, and communis. The variety acidicola is not closely related to Auxenochlorella and represented its own lineage within the Trebouxiophyceae. In view of these results, we propose a new species of Auxenochlorella, A. symbiontica, for the Hydra symbiont, and a new genus Pumiliosphaera, with its type species, P. acidophila, for acidophilic strain. These results are supported by several compensatory base changes in the conserved region of ITS‐2 and ITS‐2 DNA barcodes.     

Identification of a unicellular,non-pigmented alga that mediates growth inhibition in anuran tadpoles: a new species of the genus Prototheca (Chlorophyceae: Chlorococcales)

A non-pigmented, unicellular alga isolated from the faeces of British anuran tadpoles and which is associated with growth inhibition in these tadpoles, was described and identified using cytological, ultrastructural, nutrient assimilation and immunological studies. The alga possessed all the distinctive morphological features of the genus Prototheca, it grew weakly on Prototheca Isolation Medium (PIM), it required thiamine for continued growth and replication, and it could assimilate the five major substrates used to speciate the protothecans. All of these characteristics, together with previous nucleic acid hybridisation studies, indicated that the microorganism belonged to the genus Prototheca. There are currently five species recognised as valid (Pore, 1985 & 1986): Prototheca zopfii Kruger, 1884, P. wickerhamii Tubaki & Soneda, 1959, P. moriformis Kruger, 1884, P. stagnora Cooke, 1968 and P. ulmea Pore, 1986.The immunology showed that the new species was related to two of the protothecans, but overall it showed that the alga was antigenically distinct from the other protothecans tested in the immunoassay. This, together with its inability to grow strongly on PIM, its ability to assimilate a wide rage of carbon substrates and its ability to mediate growth inhibition in anuran tadpoles, indicated a new species of Prototheca. We therefore propose the name Prototheca richardsi sp. n.     

Capnocytophaga: New genus of gram-negative gliding bacteria. IV. DNA base composition and sequence homology

Isolates of Gram-negative fermentative gliding bacteria which are prominantly cultivated from the subgingival sulcus in association with periodontal lesions have been the subject of a collaborative taxonomic study. Thirty-five oral strains, isolated from various states of periodontal health and disease, were examined for DNA base composition and patterns of DNA sequence homology. The phentotypically similar organism, Bacteroides ochraceus ATCC 27872, as well as two representatives of gliding bacteria in the family Cytophagaceae, Myxococcus fulvus and Sporocytophaga myxococcoides, were included in these comparisons. Mol-percent guanine and cytosine (% G+C) was determined by thermal denaturation. Relatedness was also assessed by interspecific reassociation of DNA measured by the use of a single-strand specific S1 endonuclease. DNA purified from oral gliders, B. ochraceus ATCC27872 and S. myxococcoides contained 33–41% G+C as compared with 67% in DNA from M. fulvus. Three homology groups (designated as 25, 4 and 27) were delineated by DNA homology. Homology at the 77% level was demonstrated between B. ochraceus ATCC 27872 and the oral reference strain 25. Homology group 4 comprised four strains, all of which were isolated from cases of rapidly advancing periodontal disease. The relatively high degree of genetic divergence, observed as intergroup homology levels of less than 25%, supports the naming of three species of Capnocytophaga, C. ochracea, C. sputigena and C. gingivalis by Leadbetter et al. (1979) corresponding to DNA homology groups 25, 4 and 27, respectively.     

Deoxyribonucleic acid reassociation and interspecies relationships of the genusChlorella (Chlorophyceae)

Phylogenetic relationships within the genusChlorella were studied by means of DNA/DNA hybridization under both optimal and relaxed reassociation conditions as well as by determination of the thermal stability of hybrid DNA duplexes. The results indicate a relationship betweenC. fusca var.fusca, C. fusca var.rubescens, C. fusca var.vacuolata, and the genusScenedesmus. In addition, the strains endosymbiotic withParamecium bursaria seem to be related with theC. vulgaris/sorokiniana group. The relations between most other species, however, could not be sufficiently resolved by the above methods. This implies considerable phylogenetic divergency within the genusChlorella.