- Volume 51, Issue 2, 2001

A polyphasic taxonomic study was performed on 23 strains isolated from cystic fibrosis (CF) patients in the USA. These strains were tentatively identified as Burkholderia cepacia, Burkholderia vietnamiensis and Burkholderia or Ralstonia sp. using biochemical tests and 16S rDNA-based PCR assays. Visual comparison of protein profiles indicated that they belonged to a single new group ('group 13'). The polyphasic taxonomic data showed that 18 of these strains represent a new member of the B. cepacia complex, referred to in this report as B. cepacia genomovar VI, whereas the other five strains belonged to Burkholderia multivorans. By means of biochemical tests, B. cepacia genomovar VI strains can be separated from B. cepacia genomovars I and III, Burkholderia stabilis, B. vietnamiensis and Burkholderia gladioli, but not from B. multivorans. Separation of B. cepacia genomovar VI and B. multivorans is possible using AFLP (amplified fragment length polymorphism) fingerprinting and DNA-DNA hybridizations. Retrospective analysis of epidemiological and genotypic data suggests that strains of B. cepacia genomovar VI have been involved in chronic colonization of CF patients and have been spread from person to person.

A polyphasic taxonomic study was performed on Sphingomonas sp. strain RW1T. The organism was isolated from water of the River Elbe and has been known as a potent metabolizer of dibenzo-p-dioxin and its relatives. TLC of a mild alkaline hydrolysate of extractable cellular lipids of strain RW1T and type strains of 21 Sphingomonas species gave a spot of sphingoglycolipid (SGL)-1 (glucuronosyl ceramide), which is characteristic of sphingomonads. In addition, strain RW1T and type strains of three Sphingomonas species (Sphingomonas yanoikuyae, Sphingomonas terrae and Sphingomonas macrogoltabidus) showed a second spot of SGL (SGL-1') identified as galacturonosyl ceramide. The presence of SGL-1 in cellular lipids suggested that strain RW1T is a member of the genus Sphingomonas. DNA-DNA reassociation rates between strain RW1T and each type strain of 14 Sphingomonas species including Sphingomonas paucimobilis, type species for the genus, revealed that strain RW1T is independent from these species. Results of phylogenetic analysis of 16S rDNA sequences of strain RW1T and type strains of 21 named Sphingomonas species verified that strain RW1T belongs to the genus Sphingomonas. Strain RW1T could be differentiated from named species of the genus by phenotypic characteristics and has been assigned to a new species, Sphingomonas wittichii sp. nov. The type strain is DSM 6014T (= JCM 10273T = EY 4224T). DNA G+C content is 67 mol %.

Two anaerobic, thermophilic, Gram-positive, non-spore forming bacteria with an array of polysaccharide-degrading enzymes were isolated from the leachate of a waste pile from a canning factory in Hoopeston, East Central Illinois, USA. The results of 16S rDNA sequence homology indicated that their closest relatives belong to the saccharolytic, thermophilic and anaerobic genera of Thermoanaerobacterium and Thermoanaerobacter. Although, the evolutionary distances between these bacteria and their closest relatives are greater than 11%, there is no defining phenotypic characteristic for the creation of a new genus. It is proposed that these bacteria should be placed in the genus Thermoanaerobacterium, which requires emendment of the genus description with regard to the reduction of thiosulfate to sulfur, because neither isolate is capable of this reduction. Thermoanaerobacterium polysaccharolyticum reduces thiosulfate to sulfide, whereas Thermoanaerobacterium zeae is unable to reduce thiosulfate. The cells of both isolates are rod-shaped and exist as single cells or sometimes in pairs. Cells are motile by means of flagella. Growth occurs between 45 and 72 degrees C, with optimum temperature of 65-68 degrees C at pH 6.8. The pH range for growth is from 4 to 8 at a temperature of 65 degrees C. Both organisms ferment glucose, arabinose, maltose, mannose, rhamnose, sucrose, trehalose, xylose, cellobiose, raffinose, melibiose and melezitose. The major end products of fermentation with glucose are ethanol and CO2, with lesser amounts of acetate, formate, lactate and hydrogen. The DNA G+C contents of Thermoanaerobacterium polysaccharolyticum sp. nov. and Thermoanaerobacterium zeae sp. nov. are 46 and 42 mol%, respectively. The type strains are KMTHCJT (= ATCC BAA-17T = DSM 13641T) and mel2T (= ATCC BAA-16T = DSM 13642T), respectively.

A novel hyperthermophilic, heterotrophic, rod-shaped archaeon was isolated from a terrestrial hot spring at Oguni-cho, Kumamoto Prefecture, Japan. The new isolate, strain TE7T, grew under aerobic, microaerobic and anaerobic conditions. Isolate TE7T grew optimally at 90-94 degrees C and pH 7.0-7.5 (adjusted at 25 degrees C) under atmospheric air with vigorous shaking. Strain TE7T cells were motile rods 2-10 microm in length and covered with a surface-layer lattice. Cell yields at 90 degrees C under aerobic conditions were twice that under anaerobic conditions. Under aerobic conditions, growth was inhibited by elemental sulfur, but thiosulfate stimulated growth. Under anaerobic conditions, no growth was observed in the presence of nitrate and nitrite, but elemental sulfur, thiosulfate, L-cystine and oxidized glutathione stimulated growth. The 16S rDNA sequence of TE7T exhibited a close relationship to the sequences of Pyrobaculum aerophilum and Thermoproteus neutrophilus, which belong to the cluster of the genus Pyrobaculum. DNA-DNA hybridization analysis showed a low level of DNA similarity between TE7T and previously described Pyrobaculum species. As TE7T is phenotypically and phylogenetically different from the other members of this genus, it is described as a new species named Pyrobaculum oguniense (type strain TE7T = JCM 10595T = DSM 13380T).

The phylogenetic relationships of Xylella fastidiosa strains isolated from different hosts, including citrus trees, coffee, grapevine, plum and pear, were inferred by sequence analysis of the 16S rDNA and 16S-23S intergenic spacer region. A high level of similarity (97.1-100%) was found in the 16S rDNA of the Xylella fastidiosa strains. The 16S-23S region showed a higher level of variation, with similarity values ranging from 79.8 to 100%. Two tRNAs (tRNA(Ala) and tRNA(Ile)) were encountered within the spacer sequence. The phylogenetic trees, constructed using the neighbour-joining method, showed that the citrus, coffee, peach and plum strains were closely related and separate from grapevine strains. The pear strain remained isolated from all the other Xylella strains in both analyses and produced values of less than 20% in DNA-DNA hybridization experiments with a citrus strain. These results show that this strain does not belong to the Xylella fastidiosa genomic species.

The generic position of two aerobic, Gram-positive, non-acid-alcohol-fast actinomycetes was established following the isolation of their PCR-amplified 16S rRNA genes and alignment of the resultant sequences with the corresponding sequences from representatives of the families Actinosynnemataceae and Pseudonocardiaceae. The assignment of the organisms to the genus Saccharopolyspora was strongly supported by chemotaxonomic and morphological data. The strains were distinguished both from one another and from representatives of validly described Saccharopolyspora species on the basis of a number of phenotypic properties. It is proposed that the organisms, strains 07T (= AS4.1520T = IFO 16345T = JCM 10665T) and 216T (= AS4.1511T = IFO 16346T = JCM 10664T), be classified in the genus Saccharopolyspora as Saccharopolyspora flava sp. nov. and Saccharopolyspora thermophila sp. nov., respectively.

Four strains of strictly anaerobic, sulfur- and thiosulfate-reducing bacteria, SR12T, SR13, SR15T and WS100T, were isolated from 'Thiodendron' sulfur mats obtained from different saline environments. All isolates were motile, Gram-negative, non-spore-forming curved rods with pointed or rounded ends. The sizes of cells varied from 0.9 x 3-5 microm for strains SR12T, SR13 and SR15T to 0.9 x 4.8 microm for strain WS100T. All strains could form long spiral filamentous cells up to 70-110 microm during the early stage of growth. All strains were motile by a tumbling movement and possessed lateral flagella arranged at the concave side of cells. Incomplete cross-septa were distinctive features of all strains. Growth occurred at temperatures of 10-40 degrees C with an optimum at 28 degrees C. The pH limits for growth were 5.5 to 8.0, with optimal growth at pH 6.5-7.0. All isolates were obligately anaerobic and slightly halophilic and grew in media containing 0.5-5% NaCl with an optimum at 2% NaCl. All strains were chemoorganoheterotrophic, having a fermentative type of metabolism and utilized proteins, peptides, amino acids and some organic acids, but not sugars, fatty acids or alcohols. Some organic substrates (isoleucine, valine, alanine, glutamate) were utilized only by strain SR12T in the presence of sulfur or thiosulfate. Fermentation of citrate yielded mainly acetate, CO2 and H2. Sulfur and thiosulfate were reduced to hydrogen sulfide during the fermentation of organic substances, which increased cell yields and growth rates. Sulfate, sulfite, fumarate, nitrate, Fe2O3, MnO2, DMSO and elemental selenium were not used as electron acceptors by these strains. The G+C contents of the DNA were 51 mol% for strains SR12T, SR13 and SR15T and 52 mol% for strain WS100T. Based on morphological, physiological and phylogenetic similarities, all four isolates could be assigned to three new species of the genus Dethiosulfovibrio, named Dethiosulfovibrio russensis (type strain DSM 12538T), Dethiosulfovibrio marinus (type strain DSM 12537T) and Dethiosulfovibrio acidaminovorans (type strain DSM 12590T).

In this report, placement of Rickettsia felis in the spotted fever group (SFG) rather than the typhus group (TG) of Rickettsia is proposed. The organism, which was first observed in cat fleas (Ctenocephalides felis) by electron microscopy, has not yet been reported to have been cultivated reproducibly, thereby limiting the standard rickettsial typing by serological means. To overcome this challenge, several genes were selected as targets to be utilized for the classification of R. felis. DNA from cat fleas naturally infected with R. felis was amplified by PCR utilizing primer sets specific for the 190 kDa surface antigen (rOmpA) and 17 kDa antigen genes. The entire 5,513 bp rompA gene was sequenced, characterized and found to have several unique features when compared to the rompA genes of other Rickettsia. Phylogenetic analysis of the partial sequence of the 17 kDa antigen gene indicated that R. felis is less divergent from the SFG rickettsiae than from the TG rickettsiae. The data corroborate results from previous reports that analysed the citrate synthase, 16S rRNA, rompB (135 kDa surface antigen), metK, ftsY, polA and dnaE genes that placed R. felis as a member of the SFG. The organism is passed trans-stadially and transovarially, and infection in the cat flea has been observed in the midgut, tracheal matrix, muscle, hypodermis, ovaries and testes.

Facultatively psychrophilic alkaliphilic strains were isolated from seawater obtained off the coast of Rumoi, Hokkaido, Japan. They were Gram-negative, aerobic straight rods with polar flagella. The isolates were catalase- and oxidase-positive and able to grow at 4 degrees C, but not at 40 degrees C. They produced acid from D-glucose under aerobic conditions. The isolates reduced nitrate to nitrite and hydrolysed casein and gelatin, but not starch or DNA. NaCl was required for growth at pH 10 but was not required at neutral pH. The major isoprenoid quinone was ubiquinone-9 (Q-9) and the DNA G+C content was 62.3-63.2 mol%. The whole-cell fatty acids mainly consisted of C16:0, C16:1(9c) and C18:1(9c), with 3-OH C10:0 and 3-OH C12:0 as the hydroxyl fatty acids. A larger amount of trans-unsaturated fatty acid, C16:1(9t) was observed when the cells were grown at pH 7 compared to when cells were grown at pH 10. Phylogenetic analysis based on 16S rRNA gene sequencing indicated that the bacteria are members of the genus Pseudomonas. Analysis of DNA-DNA relatedness data with several close phylogenetic neighbours revealed a low level of hybridization (less than 61%). On the basis of phenotypic characteristics, phylogenetic analysis and DNA-DNA relatedness data, it is concluded that these isolates represent a separate new species. Accordingly, the name Pseudomonas alcaliphila is proposed. The type strain is AL15-21T (= JCM 10630T = IAM 14884T).

Actinomycete strain YIM 90002T (= CCTCC 99003T = CCRC 16284T) was isolated from a soil sample collected from a salt lake in the west of China. The aerial mycelium of this organism is well developed but not fragmented and, at maturity, forms short chains of spores. Spores in short chains are oval- to rod-shaped and have wrinkled surfaces. Substrate mycelium is branched with non-fragmenting hyphae and forms single oval to round spores borne on sporophores or dichotomously branching sporophores. Single spores have wrinkled surfaces. Single spores and spores in short chains are non-motile. Strain YIM 90002T contains meso-diaminopimelic acid, DD-diaminopimelic acid, glycine, lysine and aspartic acid in its cell wall and has glucose, galactose, ribose, xylose, arabinose and mannose as whole-cell sugars (no diagnostic sugars). The phospholipids are phosphatidylglycerol, phosphatidylinositol and phosphatidylethanolamine. The major menaquinones are MK-9(H6), MK-10(H2) and MK-10(H4). Phylogenetic data indicate that this strain belongs to the family Nocardiopsaceae. The morphological and physiological characteristics and chemotaxonomic and phylogenetic data for this strain differ from those of previously described actinomycetes. Therefore, a new genus, Streptimonospora, is proposed for this organism; the type species of the genus is Streptimonospora salina gen. nov., sp. nov., and the type strain of S. salina is strain YIM 90002T.

Strains DCB-MT and DCB-F were isolated from anaerobic 3-chlorobenzoate (3CB)-mineralizing cultures enriched from marine sediments. The isolates are large, Gram-negative rods with a collar girdling each cell. The isolates are obligate anaerobes capable of reductive dechlorination of 3CB to benzoate. Growth by chlororespiration in strain DCB-MT yielded 1.7 g protein mol(-1) 3CB dechlorinated with lactate as the electron donor. Strain DCB-MT also used fumarate, sulfate, sulfite, thiosulfate and nitrate as physiological electron acceptors for growth, but grew poorly on sulfate and nitrate. Reductive dechlorination was inhibited completely by sulfite and thiosulfate but not by sulfate. Both strains were incapable of growth at NaCl concentrations below 0.32% (w/v). They grew well at sea-water salt concentrations; however, the optimum growth rate was achieved at a NaCl concentration half that of sea water. The 16S rDNA sequence analysis shows strains DCB-MT and DCB-F to be 99% similar to each other and 93% similar to their closest relative, Desulfomonile tiedjei strain DCB-1T. Strain DCB-MT can also be distinguished from strain DCB-1T by its inability to use acetate for growth on 3CB and by its requirement for NaCl. The morphology, physiology and 16S rDNA sequences of DCB-MT and DCB-F suggest that these strains represent a new, marine-adapted species of the genus Desulfomonile, designated Desulfomonile limimaris sp. nov. The type strain is strain DCB-MT (= ATCC 700979T).

To resolve relationships between members of the family Thermomonosporaceae, phylogenetic analyses using three sets of nucleotide sequences from 16S rDNA, 23S rDNA and the 16S-23S internal transcribed spacer (ITS) were carried out. Nearly all species of the family were included in this study. On the basis of congruous phylogenetic results and chemotaxonomic data, the following proposals are made. First, Actinomadura libanotica, Actinomadura aurantiaca, Actinomadura glomerata and Actinomadura longicatena are transferred to the genus Actinocorallia as Actinocorallia libanotica comb. nov., Actinocorallia aurantiaca comb. nov., Actinocorallia glomerata comb. nov. and Actinocorallia longicatena comb. nov., respectively. All the species of this genus are phylogenetically coherent and of phospholipid type PII (presence of phosphatidylethanolamine), distinguishing them from other Actinomadura species that are of phospholipid type PI (absence of diagnostic phospholipids). Second, Excellospora viridilutea is transferred to the genus Actinomadura as Actinomadura viridilutea comb. nov. As a result of the proposed transfers, the family Thermomonosporaceae now contains four genera Thermomonospora, Actinomadura, Actinocorallia and Spirillospora. The genus Actinocorallia and family Thermomonosporaceae are redescribed.

The 16S rRNA gene (16S rDNA) is currently the most widely used gene for estimating the evolutionary history of prokaryotes. To date, there are more than 30,000 16S rDNA sequences available from the core databases, GenBank, EMBL and DDBJ. This great number may cause a dilemma when composing datasets for phylogenetic analysis, since the choice and number of reference organisms are known to affect the resulting tree topology. A group of sequences appearing monophyletic in one dataset may not be so in another. This can be especially problematic when establishing the relationships of distantly related sequences at the division (phylum) level. In this study, a multiple-outgroup approach to resolving division-level phylogenetic relationships is suggested using 16S rDNA data. The approach is illustrated by two case studies concerning the monophyly of two recently proposed bacterial divisions, OP9 and OP10.

The taxonomic positions of five enteroadherent bacterial pig isolates, showing phenotypic characteristics most similar to those of Enterococcus durans and Enterococcus hirae, were investigated in a polyphasic study that included 16S rDNA sequence analysis, DNA-DNA hybridizations, DNA base-ratio determinations, whole-cell protein fingerprinting, D11344-primed PCR typing and an extensive examination of phenotypic properties. The results demonstrated that the organisms represent a new species in the Enterococcus faecium species group, for which the name Enterococcus villorum sp. nov. is proposed. The type strain is LMG 12287T (= CCM 4887T).

Four bacterial strains were isolated from the fly larvae of an obligate parasitic fly, Wohlfahrtia magnifica (Diptera: Sarcophagidae). These isolates were characterized by a polyphasic approach and represent a new lineage of gamma-Proteobacteria as their closest relative is Xylella fastidiosa (87.1% 16S rDNA similarity). The four strains are identical at the 16S rDNA level, the level of similarity between them, based on DNA-DNA hybridization, is high (97.8-102.5%) and they are similar in their physiological and biochemical characteristics, although they differ in their utilization of different sole carbon sources. All produce chitinase. They are obligately aerobic: no growth is detected under anaerobic conditions, even in the presence of NO3- as terminal electron acceptor. Their predominant respiratory quinone is Q-8. The G+C content of their DNA is 42 mol%. Their cell membrane contains phosphatidylglycerol, diphosphatidylglycerol, phosphatidylethanolamine, phosphatidylserine and two unknown polar lipids. Their main fatty acids are C18:1, C16:0 and C14:0. To accommodate these bacteria, a new genus, Schineria gen. nov., with the type species Schineria larvae sp. nov., is proposed.

Mycoplasmas were isolated from the respiratory tracts of prairie voles (Microtus ochrogaster). This paper presents biochemical, serological and molecular genetic characterizations of those organisms and proposes a new species, Mycoplasma microti sp. nov. The type strain of Mycoplasma microti is strain IL371T (ATCC 700935T).

Biochemical, serological and molecular genetic studies were performed on seven mycoplasma isolates that were recovered from the upper respiratory tract of clinically ill desert tortoises. The isolates were serologically related to each other but serologically distinct from previously described species. Unique mycoplasma species-specific 16S rRNA nucleotide sequences were found in the proposed type strain. The name Mycoplasma agassizii is proposed for these isolates. The type strain is PS6T (= ATCC 700616T) which caused upper respiratory tract disease (URTD) in experimentally infected tortoises.