Isolation and characterization of a mercury-resistant broad-host-range plasmid from Pseudomonas cepacia

Abstract The sequence of 1383 nucleotides of the DNA encoding 16S rDNA was determined for strains of human intestinal spirochaetes, comprising an unnamed isolate and " Brachyspira aalborgi " NCTC 11492. A phylogenetic tree was inferred from aligned sequence comparisons between the intestinal spirochaetes, representatives of the Spirochaetales and Escherichia coli . The type strain of Brachyspira aalborgi , though related to the Serpulina spp. at approx. 96.5% sequence similarity was distinct and separated from the unnamed human intestinal isolate, HIS Oman, N26. The latter formed a separated and novel lineage that bisected the Spirochaetales.     

Polymerase chain reaction for identification of human and porcine spirochaetes recovered from cases of intestinal spirochaetosis

Abstract A polymerase chain reaction (PCR) amplification of 16S rDNA was developed to identify spirochaetes recovered from cases of intestinal spirochaetosis in humans and pigs; these bacteria belong to a distinct genetic group of spirochaetes, with the proposed name ' Anguillina coll '. The PCR incorporated a universal eubacterial 16S rDNA sequencing primer (1492r), and a 21-base forward primer designed to include a nucleotide sequence specific for ' A. coli '. The PCR was used to correctly identify DNA extracted from 43 isolates of ' A. coli ' from humans and pigs, whilst no product was produced from Escherichia coli , or from other intestinal spirochaetes, including 38 isolates of Serpulina spp., and one each of Treponema succinifaciens and Brachyspira aalborgi . The amplification provided a rapid and simple means of identifying DNA from isolates of ' A. coli ', and could be used on boiled whole ' A. coli ' cells, with a detection limit equivalent to 2.5 × 10² cells. The reaction was used to detect and identify these spirochaetes from selective agar plates inoculated with stool specimens from infected pigs.     

Phylogenetic evidence for novel and genetically different intestinal spirochetes resembling Brachyspira aalborgi in the mucosa of the human colon as revealed by 16S rDNA analysis

Intestinal spirochetes (Brachyspira spp.) are causative agents of intestinal disorders in animals and humans. Phylogenetic analysis of cloned 16S rRNA genes from biopsies of the intestinal mucosa of the colon from two Swedish 60-years old adults without clinical symptoms revealed the presence of intestinal spirochetes. Seventeen clones from two individuals and 11 reference strains were analyzed and the intestinal spirochetes could be divided into two lineages, the Brachyspira aalborgi and the Brachyspira hyodysenteriae lineages. All of the clones grouped in the B. aalborgi lineage. Moreover, the B. aalborgi lineage could be divided into three distinct phylogenetic clusters as confirmed by bootstrap and signature nucleotide analysis. The first cluster comprised 6 clones and the type strain B. aalborgi NCTC 11492T. The cluster 1 showed a 16S rRNA gene similarity of 99.4-99.9%. This cluster also harbored the only other strain of B. aalborgi isolated so far, namely strain W1, which was subjected to phylogenetic analysis in this work. The second cluster harbored 9 clones with a 98.7 to 99.5% range of 16S rDNA similarity to the B. aalborgi cluster 1. Two clones branched distinct and early of the B. aalborgi line forming the third cluster and was found to be 98.7% similar to cluster 1 and 98.3-99.1% to cluster 2. Interestingly, this shows that considerable variation of intestinal spirochetes can be found as constituents of the colonic microbiota in humans, genetically resembling B. aalborgi. The presented data aid significantly to the diagnostic and taxonomic work on these organisms.     

Spathaspora arborariae sp. nov., a d-xylose-fermenting yeast species isolated from rotting wood in Brazil

Four strains of a new yeast species were isolated from rotting wood from two sites in an Atlantic Rain Forest and a Cerrado ecosystem in Brazil. The analysis of the sequences of the D1/D2 domains of the large-subunit rRNA gene showed that this species belongs to the Spathaspora clade. The new species ferments d -xylose efficiently and is related to Candida jeffriesii and Spathaspora passalidarum , both of which also ferment d -xylose. Similar to S. passalidarum , the new species produces unconjugated asci with a single greatly elongated ascospore with curved ends. The type strain of Spathaspora arborariae sp. nov. is UFMG-HM19.1A^T (=CBS11463^T=NRRL Y-48658^T).     

Bacillus tianmuensis sp. nov., isolated from soil in Tianmu Mountain national natural reserve, Hangzhou, China

In a project aiming to isolate strains with the ability to produce nonribosomal peptides, a Gram-negative, endospore-forming, rod-shaped strain, designated B5^T, was isolated from a soil sample collected from Tianmu Mountain national natural reserve in Hangzhou, China. Strain B5^T contained meso -diaminopimelic acid in the cell wall peptidoglycan. The major cellular fatty acids were anteiso-C_15:0 and iso-C_15:0. The DNA G+C content was 42.5 mol%. The phylogenetic analysis based on 16S rRNA gene sequence indicated that strain B5^T fell within the genus Bacillus , with highest sequence similarity values to Bacillus barbaricus DSM 14730^T (96.4%) and Bacillus macauensis JCM 13285^T (95.5%). The isolate, however, could be distinguished from Bacillus strains with validly published names by low 16S rRNA gene sequence similarity values, distinct phenotypic and chemotaxonomic characteristics. On the basis of these polyphasic evidences, it is demonstrated that the isolate B5^T represents a novel species of the genus Bacillus , for which the name Bacillus tianmuensis sp. nov. is proposed. The type strain is B5^T (=DSM 22111^T=CGMCC 1.8879^T).     

Description of Oxalicibacterium horti sp. nov. and Oxalicibacterium faecigallinarum sp. nov., new aerobic, yellow-pigmented, oxalotrophic bacteria

Three strains of aerobic, Gram-negative, rod-shaped, non-spore-forming, yellow-pigmented bacteria (OD1^T, YOx^T and NS13), which were isolated in previous studies by enrichment in a mineral medium with potassium oxalate as the sole carbon source, were characterized. On the basis of 16S rRNA gene sequence similarity, strains OD1^T, YOx^T and NS13 belong to the Betaproteobacteria , most closely related to Oxalicibacterium flavum TA17^T (97.2–99.7% sequence similarity). The major whole-cell fatty acids were C_16:0, C_16:1ω7c and C_17:0 cyclo. The results of DNA–DNA hybridization and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains OD1^T and YOx^T from O. flavum TA17^T and from each other. Therefore, it is concluded that the strains OD1^T and YOx^T represent novel species within the genus Oxalicibacterium , for which the names Oxalicibacterium horti sp. nov. (type strain OD1^T=DSM 21640^T=NBRC 13594^T) and Oxalicibacterium faecigallinarum sp. nov. (type strain YOx^T=DSM 21641^T=CCM 2767^T) are proposed.     

PCR detection of Brachyspira aalborgi and Brachyspira pilosicoli in human faeces

Previously-developed PCR protocols specific for the 16S rRNA gene of the intestinal spirochaetes Brachyspira aalborgi and Brachyspira pilosicoli were adapted for the detection of these species in human faeces, following DNA extraction and purification using mini-prep columns. The limits of detection in seeded faeces for B. aalborgi and B. pilosicoli respectively were 2x10(2) and 7x10(3) cells per PCR reaction, equivalent to 5x10(4) and 1x10(5) cells per g of faeces. The PCR techniques were applied to faecal samples from two patients with histological evidence of intestinal spirochaetosis. In the first patient, in whom B. aalborgi had been identified by 16S rDNA PCR from colonic biopsies, a positive amplification for B. aalborgi only was obtained from the faeces. The organism could not be isolated from these faeces. In the second patient, both colonic biopsies and faeces were PCR positive for B. pilosicoli only, and B. pilosicoli was isolated from the faeces. These new faecal PCR protocols should be valuable for future studies on the epidemiology of intestinal spirochaete infections in human populations, particularly as it is not currently possible to isolate B. aalborgi from faeces.     

Comparative 16S rDNA sequence analysis of some alkaliphilic bacilli and the establishment of a sixth rRNA group within the genus Bacillus

Abstract Comparative sequence analysis of the 16S rDNA of 14 alkaliphilic or alkalitolerant, Gram-positive, aerobic, endo-spore forming bacterial strains was performed. Bacillus alcalophilus DSM 485^T and Bacillus cohnii DSM 6307^T were included to represent the two validly described alkaliphiles assigned to the genus Bacillus . The majority of isolates (8 strains) clustered with B. alcalophilus DSM 485^T forming a distinct phylogenetic group (rRNA group 6) within the radiation of the genus Bacillus and related taxa. Bacillus cohnii DSM 6307^T and two of the isolates, DSM 8719 and DSM 8723, grouped with B. fastidiosus and B. megaterium and are allocated to rRNA group 1. The remaining two strains DSM 8720 and DSM 8721 show an equidistant relationship to both groups.     

Three anamorphic yeast species Candida sanitii sp. nov., Candida sekii sp. nov. and Candida suwanaritii, three novel yeasts in the Saturnispora clade isolated in Thailand

Nine strains of three novel anamorphic yeast species were obtained from samples collected in Thailand including six strains (RV96, RV152, R14, RS9, RS58 and EA1) obtained from estuarine waters collected from two mangrove forests, one strain (ST84) from insect frass and two strains (SR16 and UB13) from forest soils. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analysis of the D1/D2 domain of the large subunit rRNA gene, the nine strains were found to represent three novel Candida species in the Saturnispora clade. Five strains (RV96, RV152, R14, RS9 and RS58) were assigned as a single novel species, which was named Candida sanitii sp. nov. The type strain is RV152^T (BCC 25967^T=NBRC 103864^T=CBS 10864^T). Strain EA1 was named as Candida suwanaritii sp. nov. The type strain is EA1^T (BCC 29900^T=NBRC 104877^T=CBS 11021^T). Three strains (ST84, SR16 and UB13) represented another novel species, for which Candida sekii sp. nov. is proposed. The type strain is ST84^T (BCC 8320^T=NBRC 105671^T=CBS 10931^T).     

Candida siamensis sp. nov., an anamorphic yeast species in the Saturnispora clade isolated in Thailand

Three strains of a novel anamorphic yeast species were isolated from natural samples collected in Thailand, including strain EF10 isolated from sediment in a mangrove forest, strain ST-473 from rotten wood, and strain ST-479 from a wild mushroom ( Hygrophorus sp.). Analysis of the D1/D2 domains of the large-subunit rRNA gene sequence revealed that the sequence of strain EF10 differed from the other two strains (ST-473 and ST-479), which had identical sequences, by only one nucleotide (nt) substitution in 535 nt. The closest species in terms of pairwise sequence similarity was Saturnispora mendoncae , with 32 nt substitutions and seven gaps in 535 nt. Phylogenetic analysis demonstrated that the three strains form a cluster with S. mendoncae and the other eight species in the Saturnispora clade. Phenotypic characteristics of the three strains were the same including proliferation by multilateral budding, absence of ascospores and negative diazonium blue B and urease reactions. The major ubiquinone was Q-7. On this basis, the three strains were assigned to a single novel species of the genus Candida , for which the name Candida siamensis sp. nov. is proposed. The type strain is EF10^T (=BCC 29901^T=NBRC 104878^T=CBS 11022^T).     

Three new species of bipolar budding yeasts of the genus Hanseniaspora and its anamorph Kloeckera isolated in Thailand

In the course of a survey of yeast biodiversity in the natural substrates in Thailand, eight strains were found to represent three hitherto undescribed species of Hanseniaspora/Kloeckera . They were isolated from insect frass, flower, lichen, rotted fruit and rotted wood. Based on the morphological and physiological characteristics, and sequences of D1/D2 domain, six strains represent a single species of the genus Hanseniaspora , described as Hanseniaspora thailandica sp. nov. (type BCC 14938^T=NBRC 104216^T=CBS 10841^T), and another strain as Hanseniaspora singularis sp. nov. (type BCC 15001^T=NBRC 104214^T=CBS 10840^T). A further strain, which belongs to Kloeckera and does not produce ascospores, is described as Kloeckera hatyaiensis sp. nov. (type BCC 14939^T=NBRC 104215^T=CBS 10842^T). Strains belonging to H. thailandica sp. nov. differed by 17–19 nucleotide substitutions from Hanseniaspora meyeri , the closest species. DNA reassociation between the two taxa showed 30–48% relatedness. Kloeckera hatyaiensis sp. nov. and H. singularis sp. nov. differed by eight and 16 nucleotide substitutions with one gap from the nearest species, Hanseniaspora clermontiae and Hanseniaspora valbyensis , respectively.     

Characterization and species recognition of Lactobacillus plantarum strains by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene

Twenty-one strains, labelled Lactobacillus plantarum or Lact. plantarum -like, and isolated from different natural sources, were characterized by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene using Hin dIII and Eco RI cleaved chromosomal DNA, together with Lact. plantarum ATCC 14917^T, Lact. pentosus ATCC 8041^T, Lact. plantarum ATCC 10776 and Lact. plantarum ATCC 8014. The fermentation patterns on API 50CH were recorded at 30°C and 37°C for all strains. The phenotypes were heterogeneous, and the ability to ferment 17 of the 49 carbohydrates varied. The fermentation of some carbohydrates, for example D-raffinose and D-arabitol, was temperature-dependent. Strains having identical API profiles were separated by the plasmid profile. All strains but one (affiliated to Lact. casei ) had identical 16S ribosomal DNA sequences ( Lact. plantarum/Lact. pentosus ). The RFLP study resulted in identical ribopatterns for 17 of the strains, including the type strain of Lact. plantarum (pattern A1). Four strains had related fragment patterns to that of Lact. plantarum sensu stricto; three of these strains had more than 60% DNA: DNA homology to the type strain of Lact. plantarum , and one had less than 50% DNA: DNA homology to Lact. plantarum ATCC 14917^T. Two strains had fragment patterns similar to the type strain of Lact. pentosus , and they had more than 80% DNA: DNA homology to Lact. pentosus ATCC 8041^T. One of the Lact. pentosus strains shared one band with the A1 pattern. The ribopatterns of Lact. plantarum were homogeneous (identical for 85% of the strains), irrespective of phenotype and source of isolation. RFLP of the 16S rRNA genes using Eco RI and Hin dIII might be used for species recognition of Lact. plantarum , but seems less suitable for strain typing.     

Candida ratchasimensis sp. nov. and Candida khaoyaiensis sp. nov., two anamorphic yeast species isolated from flowers in Thailand

Two yeast strains of the genus Candida were isolated from wild flowers collected in Khao Yai National Park, Nakhonratchasima Province, Thailand. Based on morphological, physiological and chemotaxonomic characteristics and sequence analysis of the D1/D2 domain of the 26S rRNA gene, strains BCC 7722^T (=NBRC 102563^T=CBS 10611^T) and BCC 7729^T (=NBRC 102565^T=CBS 10839^T) were found to represent two distinct novel Candida species, for which the names Candida ratchasimensis sp. nov. and Candida khaoyaiensis sp. nov. are proposed, respectively. In the phylogenetic tree constructed according to the neighbour-joining method based on sequences of the D1/D2 domain of the 26S rRNA gene, strains BCC 7722^T (GenBank accession no. AY228492 ) and BCC 7729^T (accession no. DQ400367 ) constituted a cluster with Candida cellae that was connected with a clade with Starmerella meliponinorum and Candida etschellsii . Within the D1/D2 domain, C. ratchasimensis and C. khaoyaiensis differ from C. cellae in 25 nucleotide substitutions with five gaps and 29 nucleotide substitutions with one gap, respectively.     

Spirochaeta smaragdinae sp. nov., a new mesophilic strictly anaerobic spirochete from an oil field

An obligately anaerobic spirochete designated strain SEBR 4228^T (T = type strain) was isolated from an oil field of Congo, Central Africa. The strain grew optimally with a sodium chloride concentration of 5% (sodium chloride concentration growth range 1.0–10%) at 37°C (growth temperature range 20–40°C) and pH of 7.0–7.2 (pH growth range pH 5.5–8.0). Strain SEBR 4228^T grew on carbohydrates (glucose, fructose, ribose, d -xylose, galactose, mannitol and mannose), glycerol, fumarate, peptides and yeast extract. Yeast extract was required for growth and could not be replaced by vitamins. It reduced thiosulfate and sulfur, to H₂S. Glucose was oxidised to lactate, acetate, CO₂ and H₂S in the presence of thiosulfate but in its absence lactate, ethanol, CO₂ and H₂ were produced. Fumarate was fermented to acetate and succinate. The G+C content of strain SEBR 4228^T was 50%. Strain SEBR 4228^T was spiral shaped measuring 5–30 by 0.3–0.5 μm and was motile with a corkscrew-like motion. Electron microscopy revealed the presence of periplasmic flagella in a 1-2-1 arrangement. Strain SEBR 4228^T possessed features typical of the members of the genus Spirochaeta . 16S rRNA sequence analysis revealed that it was closely related to Spirochaeta bajacaliforniensis (similarity 98.6%). The lack of DNA homology with S. bajacaliforniensis (38%), together with other phenotypic differences, indicated that strain SEBR 4228^T is a new species, which we have designated Spirochaeta smaragdinae . The type strain is SEBR 4228^T (= DSM 11293).     

Vanderwaltozyma verrucispora sp. nov., a new ascomycetous yeast species

A new yeast species, Vanderwaltozyma verrucispora , is proposed in this study based on two strains isolated from partially decayed leaves in Japan and one strain from soil in Taiwan. The species is characterized by the fermentation of glucose and galactose, formation of one to four spheroidal to ellipsoidal ascospores with warty surfaces in each ascus, and assimilation of a few carbon and nitrogen compounds. Genus assignment and distinction of the species from the other two recognized species of Vanderwaltozyma is based on the morphological and physiological characteristics, and phylogenetic analysis of nucleotide sequences of the D1/D2 domains of the large subunit (LSU) rRNA gene. From these comparisons, the name V. verrucispora sp. nov. is proposed. Sequence analysis of the D1/D2 domains of the LSU rRNA gene reveals that the phylogenetically closest relative of V. verrucispora is Vanderwaltozyma yarrowii . The type strain of the new species, which was isolated from a partially decayed leaf in Kagoshima Prefecture, Japan, is NBRC 1884^T (=CBS 10887^T=BCRC 23141^T).     

Torulaspora quercuum sp. nov. and Candida pseudohumilis sp. nov., novel yeasts from human and forest habitats

Strains XZ-46A, XZ-105, XZ-129 and XZ-281^T isolated from the oral cavities of healthy Tibetan volunteers were revealed to represent two novel ascomycetous yeast species by molecular taxonomic characterizations. Strain XZ-281^T was most closely related to Candida humilis , but differed from the type strain of the species by eight (1.2%) substitutions in the 26S rRNA gene D1/D2 domain and by >100 (>20%) mismatches in the internal transcribed spacer (ITS) region. Strains XZ-46A, XZ-105 and XZ-129 had identical or similar D1/D2 and ITS sequences with each other and with strain 17YF^T isolated from a leaf of an oak tree ( Quercus sp.). The closest relative of this group was Torulaspora microellipsoides . They differed from the type strain of the species by five (0.9%) substitutions in the D1/D2 domain and >70 (>15%) mismatches in the ITS region. A sexual state was observed in strain 17YF^T, but not in the other four oral strains. An anamorphic name Candida pseudohumilis sp. nov. is proposed for strain XZ-281^T (=AS 2.3956^T=CBS 11404^T) and a teleomorphic name Torulaspora quercuum sp. nov. is proposed for strain 17YF^T (=AS 2.3768^T=CBS 11403^T) and the other three oral strains.     

Wickerhamomyces edaphicus sp. nov. and Pichia jaroonii sp. nov., two ascomycetous yeast species isolated from forest soil in Thailand

Four yeast strains were isolated from soil in a mixed deciduous forest in Amphoe Wang Nam Khiao, Nakhon Ratchasima province, Thailand. On the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the large-subunit (LSU) rRNA gene, small-subunit rRNA gene and internal transcribed spacer (ITS) region, the three strains (S-29, S-63 and S-80) were found to represent a single species of the genus Wickerhamomyces , which were named Wickerhamomyces edaphicus sp. nov. The type strain is S-29^T (BCC 21231^T=NBRC 101969^T=CBS 10408^T). Strain S-75 represented a novel species of the genus Pichia on the basis of morphological, biochemical, physiological and chemotaxonomic characteristics, and the sequence analyses of the D1/D2 domain of the LSU rRNA gene, for which the name Pichia jaroonii sp. nov. is proposed. The type strain is S-75^T (BCC 23061^T, NRBC 102180^T=CBS 10930^T).     

Candida carvajalis sp. nov., an ascomycetous yeast species from the Ecuadorian Amazon jungle

In the course of a yeast biodiversity survey of different ecological habitats found in Ecuador, two yeast strains (CLQCA 20-011^T and CLQCA20-014) were isolated from samples of rotten wood and fallen leaf debris collected at separate sites in the central region of the Ecuadorian Amazonia. These strains were found to represent a novel yeast species based on the sequences of their D1/D2 domain of the large-subunit (LSU) rRNA gene and their physiological characteristics. Phylogenetic analysis based on LSU D1/D2 sequences revealed this novel species to be most closely related to Candida asparagi, Candida fructus, Candida musae and two as yet undescribed Candida species, with the six yeast taxa collectively forming a distinct species group within the Clavispora clade. The species name of Candida carvajalis sp. nov. is proposed to accommodate these strains, with CLQCA 20-011^T (NCYC 3509^T, CBS 11361^T) designated as the type strain.     

Assignment of Actinomyces pyogenes-like (CDC coryneform group E) bacteria to the genus Actinomyces as Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov.

In a previous study the authors reported the characterization of some facultatively anaerobic, Gram-positive, non-sporeforming rods which were found in mixed cultures from various infectious processes, including patients with otitis, empyema, perianal abscesses and decubitus ulcers. Phenotypically these organisms closely resembled Actinomyces pyogenes although their precise taxonomic position remained unknown. In the present investigation the authors have determined the 16S rRNA gene sequences of some representative strains of the Actinomyces pyogenes -like bacteria and report the results of a comparative sequence analysis. On the basis of the results of the present and earlier findings two new Actinomyces species, Actinomyces radingae sp. nov. and Actinomyces turicensis sp. nov. are proposed. The type strains are DSM 9169^T and DSM 9168^T, respectively.     

Isolation and characterization of Ferroplasma thermophilum sp. nov., a novel extremely acidophilic, moderately thermophilic archaeon and its role in bioleaching of chalcopyrite

Aims:  To isolate Ferroplasma thermophilum L1^T from a low pH environment and to understand its role in bioleaching of chalcopyrite.
Methods and Results:  Using serial dilution method, a moderately thermophilic and acidophilic ferrous iron-oxidizing archaeon, named L1^T, was isolated from a chalcopyrite-leaching bioreactor. The morphological, biochemical and physiological characteristics of strain L1^T and its role in bioleaching of chalcopyrite were studied. Strain L1^T was a nonmotile coccus that lacked cell wall. Strain L1^T had a temperature optimum of 45°C and the optimum pH for growth was 1·0. Strain L1^T was capable of chemomixotrophic growth on ferrous iron and yeast extract. Results of fatty acid analysis, DNA–DNA hybridization, G+C content, and analysis based on 16S rRNA gene sequence indicated that strain L1^T should be grouped in the genus Ferroplasma , and represented a new species, Ferroplasma thermophilum . Ferroplasma thermophilum in combination with Acidithiobacillus caldus and Leptospirillum ferriphilum could improve the copper dissolution in bioleaching of chalcopyrite.
Conclusions:  A novel extremely acidophilic, moderately thermophilic archaeon isolated from a bioleaching reactor has been identified as F. thermophilum that played an important role in bioleaching of chalcopyrite at low pH.
Significance and Impact of the Study:  This study contributes to understand the characteristics of F. thermophilum L1^T and its role in bioleaching of sulfide ores.