Characterization of Two Novel Biovar of <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis> Isolated from Root Nodules of <Emphasis Type="Italic">Vicia faba</Emphasis>

A total of eight strains of bacteria were isolated from the root nodule of Vicia faba on the selective media of Rhizobium. Two of these strains produced phenotypically distinct mucoid colonies (one slow growing and the other fast growing) and were examined using a polyphasic approach for taxonomic identification. The two strains (MTCC 7405 and MTCC 7406) turned out to be new strains of biovar 1 Agrobacterium rather than Rhizobium, as they showed growth on alkaline medium as well as on 2% NaCl and neither catabolized lactose as the carbon source nor oxidized Tween-80. The distinctness between the two strains was marked with respect to their growth on dextrose and the production of lysine dihydrolase, ornithine decarboxylase and DNA G + C content. 16S rDNA sequencing and their comparison with the 16S rDNA sequences of previously described agrobacteria as well as rhizobia strains confirmed the novelty of the two strains. Both of the strains clustered with strains of Agrobacterium tumefaciens in the 16S rDNA-based phylogenetic tree. The phenotypic and biochemical properties of the two strains differed from those of the recognized biovar of A. tumefaciens. It is proposed that the strains MTCC 7405 and MTCC 7406 be classified as novel biovar of the species A. tumefaciens (Type strains MTCC 7405 = DQ383275 and MTCC 7406 = DQ383276).

IS<Emphasis Type="Italic">Rm31</Emphasis>, a new insertion sequence of the IS<Emphasis Type="Italic">66</Emphasis> family in<Emphasis Type="Italic"> Sinorhizobium meliloti</Emphasis>

Sinorhizobium meliloti natural populations show a high level of genetic polymorphism possibly due to the presence of mobile genetic elements such as insertion sequences (IS), transposons, and bacterial mobile introns. The analysis of the DNA sequence polymorphism of the nod region of S. meliloti pSymA megaplasmid in an Italian isolate led to the discovery of a new insertion sequence, ISRm31. ISRm31 is 2,803 bp long and has 22-bp-long terminal inverted repeat sequences, 8-bp direct repeat sequences generated by transposition, and three ORFs (A, B, C) coding for proteins of 124, 115, and 541 amino acids, respectively. ORF A and ORF C are significantly similar to members of the transposase family. Amino acid and nucleotide sequences indicate that ISRm31 is a member of the IS66 family. ISRm31 sequences were found in 30.5% of the Italian strains analyzed, and were also present in several collection strains of the Rhizobiaceae family, including S. meliloti strain 1021. Alignment of targets sites in the genome of strains carrying ISRm31 suggested that ISRm31 inserts randomly into S. meliloti genomes. Moreover, analysis of ISRm31 insertion sites revealed DNA sequences not present in the recently sequenced S. meliloti strain 1021 genome. In fact, ISRm31 was in some cases linked to DNA fragments homologous to sequences found in other rhizobia species.     

Identification of iron-reducing<Emphasis Type="Italic"> Thermus</Emphasis> strains as<Emphasis Type="Italic"> Thermus scotoductus</Emphasis>

Thermus strain SA-01, previously isolated from a deep (3.2 km) South African gold mine, is closely related to Thermus strains NMX2 A.1 and VI-7 (previously isolated from thermal springs in New Mexico, USA, and Portugal, respectively). Thermus strains SA-01 and NMX2 A.1 have also been shown previously to grow using nitrate, Fe(III), Mn(IV) or S^O as terminal electron acceptors and to be capable of reducing Cr(VI), U(VI), Co(III), and the quinone-containing compound anthraquinone-2,6-disulfonate. The objectives of this study were to determine the phylogenetic positions of the three known metal-reducing Thermus strains and to determine the phylogenetic significance of metal reduction within the genus Thermus. Phylogenetic analyses of 16S rDNA sequences, BOX PCR genomic fingerprinting, and DNA–DNA reassociation analyses indicated that these strains belong to the previously described genospecies T. scotoductus. The morphologies and lipid fatty acid profiles of these metal-reducing strains are consistent with their identification as T. scotoductus; however, the T. scotoductus strains tested in this study evinced a wide intraspecies variability in some other phenotypic traits, e.g., carbon substrate utilization and pigmentation. Iron reduction occurred in all strains of T. scotoductus tested except the mixotrophic, sulfur-oxidizing strain IT-7254. Thermus strains belonging to other species did not reduce Fe(III) to Fe(II) or reduced it only poorly.Communicated by J. Wiegel     

Molecular Characterization of <Emphasis Type="Italic">Kluyveromyces marxianus</Emphasis> Strains Isolated from <Emphasis Type="Italic">Agave fourcroydes</Emphasis> (Lem.) in Yucatan,Mexico

The molecular characterization of 14 strains of Kluyveromyces marxianus isolated from Agave fourcroydes (Lem.) in Yucatan, Mexico, was performed by AP-PCR analysis, PCR-RFLP of 5.8S-ITS, and complete NTS regions. A sequence analysis of the D1/D2 domain of the 26S rDNA was also carried out in six selected strains. The AP-PCR approach had the highest discrimination power for the molecular characterization of new henequen K. marxianus strains. PCR-RFLP of 5.8S-ITS regions did not reveal polymorphisms in this group of strains. The restriction enzyme digestion analysis of NTS region enables the separation among strains which coincides with ascospore shape groups. The molecular tools used in this article may be useful to confirm a preliminary screen of yeasts isolated from henequen without the use of growth characteristics or morpho-physiological tests.     

Characterization of the genome of <Emphasis Type="Italic">Saccharomyces</Emphasis> yeasts from red berry wines

Using restriction analysis of noncoding rDNA regions, multiplex PCR, and molecular karyotyping, we have examined Saccharomyces strains isolated from red berry wine materials in Russia, Belarus, and Ukraine. According to the molecular analysis, all strains belong to the species S. cerevisiae. A correlation was revealed between microsatellite fingerprints of the strains and the source of their isolation. The strains isolated from juices and from the surface of different berries showed distinct PCR profiles. The genome compositions of interspecific Saccharomyces hybrids of natural and laboratory origin were studied.     

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.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

Selective enrichment,isolation and molecular detection of <Emphasis Type="Italic">Salinibacter</Emphasis> and related extremely halophilic <Emphasis Type="Italic">Bacteria</Emphasis> from hypersaline environments

Salinibacter is a genus of red, extremely halophilic Bacteria. Thus far the genus is represented by a single species, Salinibacter ruber, strains of which have been isolated from saltern crystallizer ponds in Spain and on the Balearic Islands. Both with respect to its growth conditions and its physiology, Salinibacter resembles the halophilic Archaea of the order Halobacteriales. We have designed selective enrichment and isolation techniques to obtain Salinibacter and related red extremely halophilic Bacteria from different hypersaline environments, based on their resistance to anisomycin and bacitracin, two antibiotics that are potent inhibitors of the halophilic Archaea. Using direct plating on media containing bacitracin, we found Salinibacter-like organisms in numbers between 1.4×10³ and 1.4×10⁶ml⁻¹ in brines collected from the crystallizer ponds of the salterns in Eilat, Israel, being equivalent to 1.8–18% of the total colony counts obtained on identical media without bacitracin. A number of strains from Eilat were subjected to a preliminary characterization, and they proved similar to the type strain of S. ruber. We also report here the isolation and molecular detection of Salinibacter-like organisms from an evaporite crust on the bottom of salt pools at the Badwater site in Death Valley, CA. These isolates and environmental 16S rRNA gene sequences differ in a number of properties from S. ruber, and they may represent a new species of Salinibacter or a new related genus. Guest Editor: John M. Melack Saline Waters and their Biota     

The karyotype and 5S rRNA genes from Spanish individuals of the bat species <Emphasis Type="Italic">Rhinolophus</Emphasis><Emphasis Type="Italic">hipposideros</Emphasis> (Rhinolophidae; Chiroptera)

The karyotype of individuals of the species Rhinolophus hipposideros from Spain present a chromosome number of 2n = 54 (NFa = 62). The described karyotype for these specimens is very similar to another previously described in individual from Bulgaria. However, the presence of one additional pair of autosomal acrocentric chromosomes in the Bulgarian karyotype and the differences in X chromosome morphology indicated that we have described a new karyotype variant in this species. In addition, we have analyzed several clones of 1.4 and 1 kb of a PstI repeated DNA sequence from the genome of R. hipposideros. The repeated sequence included a region with high identity with the 5S rDNA genes and flanking regions, with no homology with GenBank sequences. Search for polymerase III regulatory elements demonstrated the presence of type I promoter elements (A-box, Intermediate Element and C-box) in the 5S rDNA region. In addition, upstream regulatory elements, as a D-box and Sp1 binding sequences, were present in flanking regions. All data indicated that the cloned repeated sequences are the functional rDNA genes from this species. Finally, FISH demonstrated the presence of rDNA in nine chromosome pairs, which is surprising as most mammals have only one carrier chromosome pair.     

Characterization of a novel South American population of the astaxanthin producing yeast <Emphasis Type="Italic">Xanthophyllomyces dendrorhous</Emphasis> (<Emphasis Type="Italic">Phaffia rhodozyma</Emphasis>)

A novel population of the biotechnologically important yeast Xanthophyllomyces dendrorhous, the sexual stage of Phaffia rhodozyma, has been recently isolated for the first time in the southern Hemisphere (Patagonia, Argentina). The aim of the present work was to phenotypically and genotypically characterize two representative strains of this new population, and assess such strains as a potential biotechnological source of astaxanthin, fatty acids and extracellular enzymes. Minor variations were found in physiological tests. PCR fingerprinting studies (MSP-PCR) showed the main differences between X. dendrorhous Patagonian and Type strains. Patagonian strains accumulated a xanthophyll-like pigment, which was identified as astaxanthin. These strains showed low fatty acids content (mainly polyunsaturated fatty acids) and, of a total of six extracellular enzymes tested, only produced amylase. Genetic differences between Patagonian and collection X. dendrorhous strains could be explained by geographic isolation and habitat specificity.     

New species in the genus <Emphasis Type="Italic">Francisella</Emphasis> (Gammaproteobacteria; Francisellaceae); <Emphasis Type="Italic">Francisella piscicida</Emphasis> sp. nov. isolated from cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella strain, GM2212, previously isolated from moribund farmed Atlantic cod (Gadus morhua) in Norway, is closely related to Francisella philomiragia among Francisella spp. according to its complete 16S rDNA, 16S-23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and hypothetical lipoprotein (LpnB) sequences. A comparison between GM2212 and the type strain of Francisella philomiragia were performed by DNA–DNA hybridization and fatty acid analysis. The DNA–DNA hybridization showed a 70% similarity. The fatty acid analysis showed only minor differences between the Francisella isolates. Due to the inconclusive result from the DNA–DNA hybridisation, major emphasis concerning the status of this isolate is made on previously published molecular, phenotypic and biochemical characters. All characteristics taken together support the establishment of GM2212 as a novel species, for which the name Francisella piscicida sp. nov. is proposed (=CNCM I-3511^T = DSM 18777^T = LMG registration number not yet available).     

Molecular characterization of the 5S ribosomal gene of the <Emphasis Type="Italic">Bradysia hygida</Emphasis>(Diptera:Sciaridae)

The rRNA genes are amongst the most extensively studied eukaryotic genes. They contain both highly conserved and rapidly evolving regions. The aim of this work was to clone and to sequence the Bradysia hygida 5S rDNA gene. A positive clone was sequenced and its 346 bp sequence was analyzed against the GenBank database. Sequence analysis revealed that the B. hygida 5S (Bh5S) rDNA gene is 120 bp long and is 87% identical to the aphid Acyrthosiphon magnoliae 5S rDNA gene. The Bh5S rDNA gene presents two unusual features: a GG pair at the 5' end of the gene sequence and the localization of the polyT signal immediately after the 3' end of the gene. In situ5S hybridization experiments revealed that the Bh5S rDNA gene is localized in the autosomal A chromosome     

Discrimination among <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> H serotypes,serovars and strains based on 16S rRNA, <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">aroE</Emphasis> gene sequence analyses

Our aim was to investigate the capability of each of three genes, 16S rRNA, gyrB and aroE, to discriminate, first, among Bacillus thuringiensis H serotypes; second, among B. thuringiensis serovars from the same H serotype; and third, among B. thuringiensis strains from the same serovar. The 16S rRNA, gyrB and aroE genes were amplified from 21 B. thuringiensis H serotypes and their nucleotide sequences determined. Additional strains from four B. cereus sensu lato species were included for comparison purposes. These sequences were pair-wise compared and phylogenetic relationships were revealed. Each of the three genes under study could discriminate among B. thuringiensis H serotypes. The gyrB and aroE genes showed a discriminatory power among B. thuringiensis H serotypes up to nine fold greater than that of the 16S rRNA gene. The gyrB gene was retained for subsequent analyses to discriminate B. thuringiensis serovars from the same H serotype and to discriminate strains from same serovar. A total of 42 B. thuringiensis strains, which encompassed 25 serovars from 12 H serotypes, were analyzed. The gyrB gene nucleotide sequences were different enough as to be sufficient to discriminate among B. thuringiensis serovars from the same H serotype and among B. thuringiensis strains from the same serovar. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Characterization of <Emphasis Type="Italic">Francisella</Emphasis> sp., GM2212, the first <Emphasis Type="Italic">Francisella</Emphasis> isolate from marine fish,Atlantic cod (<Emphasis Type="Italic">Gadus morhua</Emphasis>)

A Francisella sp., isolate GM2212^T, previously isolated from diseased farmed Atlantic cod Gadus morhua in Norway is characterized. The complete 16S rDNA, 16S–23S intergenic spacer, 23S rDNA, 23S–5S intergenic spacer, 5S rDNA, FopA, lipoprotein TUL4 (LpnA), malate dehydrogenase and a hypothetical lipoprotein (LpnB) is sequenced and compared with Francisella tularensis and Francisella philomiragia. All these sequences support a close relationship between GM2212^T and F. philomiragia. The bacterium grows at 10–25°C with an optimum at about 20°C, a temperature range clearly different from F. tularensis and F. philomiragia. GM2212^T is catalase-positive, indole positive, oxidase-negative, do not produce H₂S in Triple Sugar Iron agar, and does not hydrolyze gelatin, is resistant to erythromycin and susceptible to ceftazidime, the latter five characteristics separating it from F. philomiragia. Cysteine enhances growth. Acid is produced from d-glucose, maltose, sucrose (weak) but not from lactose or glycerol. GM2212^T grows on both MacConkey agar and in nutrient broth (6% NaCl). The bacterium is resistant to trimethoprim-sulfamethoxazole, penicillines, cefuroxime and erythromycin; but is susceptible to ceftazidime, tetracycline, gentamicin, ciprofloxacin. Based on the molecular and phenotypical characteristics, we suggest that this GM2212 isolate, may represent a new species of Francisella. Isolate GM2212^T (=CNCM I-3481^T = CNCM I-3511^T = DSM 18777^T).     

Phylogenetic Studies of <Emphasis Type="Italic">Gordonia</Emphasis> Species Based on <Emphasis Type="Italic">gyrB</Emphasis> and <Emphasis Type="Italic">secA1</Emphasis> Gene Analyses

Phylogenetic relations within the genus Gordonia were analyzed using partial gyrB and secA1 gene sequences of 23 type species in comparison with those of 16S rRNA gene. The gyrB and secA1 phylogenies showed agreement with that constructed using 16S rRNA gene sequences. The degrees of divergence of the gyrB and secA1 genes were approximately 3.4 and 1.7 times greater, respectively, than that of 16S rRNA gene. The gyrB gene showed more discriminatory power than either the secA1 or 16S rRNA gene, facilitating clear differentiation of any two Gordonia species using gyrB gene analysis. Our data indicate that gyrB and secA1 gene sequences are useful as markers for phylogenetic study and identification at the species level of the genus Gordonia.     

Identification of the alga known as <Emphasis Type="Italic">Nannochloropsis</Emphasis> Z-1 isolated from a prawn farm in Hainan,China as <Emphasis Type="Italic">Chlorella</Emphasis>

An alga known as “Nannochloropsis”, isolated from a prawn farm in Hainan, China, has been critically investigated and identified as Chlorella, a member of the Chlorophyceae based on fatty acid composition, ultrastructure, and 18S rDNA. Cells of this alga were spherical, measured by 1–6 μm in diameter and were enclosed in thin walls of approximately 0.04 μm thickness. They contained several small mitochondria, two to three thylakoids and had no vacuoles. There were many pyrenoids in the algal cells and their thylakoid lamellae were sparse and not translucent. Many lipid droplets were present in the cytoplasm. The total lipid content of this alga was 3% per gram dry weight and its major fatty acids were C_16:0, C_18:0, C_18:1, C_18:2, C_18:3 and C_20:0. Eicosapentaenoic acid (C_20:5, EPA) was not detected. The length of its 18S rDNA sequence was 1,712 bp. 18S rDNA sequence analyses indicated that this alga was a species of Chlorella.     

Molecular divergence of the soil yeasts <Emphasis Type="Italic">Williopsis</Emphasis> sensu stricto

Fifty-three strains of Saturn-spored yeasts were analyzed by means of restriction analysis of the amplified fragment of rDNA comprising the 5.8S rRNA gene and the internal transcribed spacers ITS1 and ITS2. The use of endonucleases HaeIII and MspI enabled clear differentiation of yeast species Williopsis mucosa, W. salicorniae, Zygowilliopsis californica, and Komagataea pratensis and the Williopsis sensu stricto complex. The minisatellite primer M13 was proposed for differentiation between sibling species of Williopsis sensu stricto, which have identical restriction profiles. PCR with primer M13 enabled reidentification of a number of collection strains, species identification of Saturn-spored isolates from the Far East, and detection of three strains affiliated to novel taxa. The latter have unique PCR profiles and differ in the nucleotide sequences of ITS1 and ITS2 fragments of rDNA. Possible variations in the results obtained with different molecular methods are discussed.Translated from Mikrobiologiya, Vol. 73, No. 6, 2004, pp. 768–776.Original Russian Text Copyright © 2004 by Naumova, Gazdiev, Naumov.     

Molecular Phylogenetic Relationships Between Prostanoid-Containing Okinawan Soft Coral (<Emphasis Type="Italic">Clavularia viridis</Emphasis>) and Nonprostanoid-Containing <Emphasis Type="Italic">Clavularia</Emphasis> Species Based on Ribosomal ITS Sequence

To study phylogenetic relationships among Okinawan soft corals of the genus Clavularia, the ribosomal internal transcribed spacer sequences of host corals and the 18S rDNA sequences of symbiotic algae were analyzed. The molecular phylogenetic trees of hosts showed that a prostanoid-containing species, Clavularia viridis, is deeply diverged from other species of Clavularia which do not biosynthesize the prostanoids as the main secondary metabolites. Comparison of their trees suggested poor phylogenetic concordance between hosts and symbionts.     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.