Megaplasmids in Thermus oshimai isolates from two widely separated geographical areas: restriction fragment profiling and DNA homology

Megaplasmid DNA was detected in ten isolates belonging to the recently described thermophilic eubacterial species Thermus oshimai and isolated from hot springs in Portugal (eight isolates) and Iceland (two isolates). The estimated size of the large plasmids purified from T. oshimai SPS-18 from S. Pedro do Sul, Portugal, and from isolate JK-91 from Hveragerdhi-Hengill, Iceland, was 214 and 275 kb, respectively. No sequence homologous to isolate SPS-18 megaplasmid is present in chromosomal DNA as indicated by Southern hybridization analysis. Overall examination of the HindIII fragment profiles of megaplasmid DNAs purified from isolates from the same geographical area gave similar but not always identical restriction profiles on agarose gels. Restriction fragment length polymorphism (RFLP) was higher for megaplasmids present in isolates purified from the Portuguese and Icelandic isolates than for megaplasmids from the same hot spring. Megaplasmid RFLP correlated with previous results obtained on the polymorphism of macrorestriction patterns of whole genomic DNA and with the RFLP of co-resident small plasmid DNA that was found in one half of the isolates examined. The 16-kb HindIII–HindIII fragment from isolate SPS-18 megaplasmid showed DNA–DNA homology with restriction fragments of similar size generated by the large plasmids present in all the other isolates, even in those from hot springs of widely separated geographical areas. This suggests a high degree of sequence conservation in T. oshimai megaplasmids. Received: 12 May 1997 / Accepted: 17 July 1997     

Comparative genomic analysis of isolates belonging to the six species of the genus Thermus using pulsed-field gel electrophoresis and ribotyping

Fifty isolates belonging to the six validly described species of the genus Thermus (T. aquaticus, T. filiformis, T. thermophilus, T. scotoductus, T. brockianus, and T. oshimai) isolated from hot springs of different geographical areas were compared using macrorestriction analysis of genomic DNA and ribotyping. With the exception of presumed clones, the macrorestriction patterns of isolates obtained with EcoRI or NdeI were distinct. However, isolates belonging to the same species exhibited similar profiles particularly when they were isolated from the same hot spring. The estimated genomic size of strains of the Thermus spp. varied between approximately 1.8 and 2.5 Mbp. Ribotyping with BamHI and HindIII produced 30 and 35 distinct ribotypes, respectively. In spite of the variability of the hybridization patterns produced, the ribotypes obtained for isolates belonging to the same species also shared, in general, several fragments of identical size, and these fragments were similar when isolates originated from the same spring. Received: 7 October 1996 / Accepted: 10 March 1997     

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     

Differences Among Helicobacter pylori Strains Isolated from Three Different Populations and Demonstrated by Restriction Enzyme Analysis of an Internal Fragment of the Conserved Gene hpaA

Background. Our goal was to test the idea that Helicobacter pylori genotypes vary from one population to another. Methods. Analysis of Sau3A and Hinf I restriction fragment–length polymorphism (RFLP) in a 375-bp polymerase chain reaction amplicon of hpaA was used to compare 31 H. pylori isolates from a relatively small and genetically homogeneous population (Goteborg, Sweden) with those of large, genetically heterogeneous populations located in two different countries (50 isolates from Houston, TX, and 69 isolates from Minas Gerais, a state in the southeastern region of Brazil). Results. Five different Sau3A and three different Hinf I restriction patterns were found; different combinations of these comprise 10 different RFLP types, I through X. The RFLP types found in the United States and Brazil collections were very similar, except for two Brazil isolates belonging to type VIII and five Brazil isolates belonging to type X, neither type found in the United States. The overall profile of H. pylori isolates from Sweden was remarkably different, with 18 of 31 (58%) having a new Sau3A restriction pattern, termed gS; 10 of these 18 isolates had Hinf I restriction pattern E (RFLP type VIII), and 8 had Hinf I restriction pattern F (RFLP type IX). No isolates from Sweden belonged to RFLP type III or type X. Conclusions. RFLP typing of a 375-bp polymerase chain reaction–amplified DNA fragment of H. pylori hpaA revealed that H. pylori genotypes can and do vary from one population to another. We conclude that the unique RFLP profile shown by the group of H. pylori isolates from Goteborg is the result of a cohort effect in this relatively small, stable, genetically homogeneous population. Also, the overall similarity between RFLP profiles of the H. pylori isolates from Texas and Minas Gerais coincides with the fact that although geographically distanced, these populations are similar in being large, dynamic, and genetically heterogeneous.     

Polyphasic analysis of Thermus isolates from geothermal areas in Iceland

Genetic relationships and diversity of 101 Thermus isolates from different geothermal regions in Iceland were investigated by using multilocus enzyme electrophoresis (MLEE) and small subunit ribosomal rRNA (SSU rRNA) sequence analysis. Ten polymorphic enzymes were used and seven distinct and genetically highly divergent lineages of Thermus were observed. Six of seven lineages could be assigned to species whose names have been validated. The most diverse lineage was Thermus scotoductus. In contrast to the other lineages, this lineage was divided into very distinct genetic sublineages that may represent subspecies with different habitat preferences. The least diverse lineage was Thermus brockianus. Phenotypic and physiological analysis was carried out on a subset of the isolates. No relationship was found between growth on specific single carbon source to the grouping obtained by the isoenzyme analysis. The response to various salts was distinguishing in a few cases. No relationship was found between temperature at the isolation site and the different lineages, but pH indicated a relation to specific lineages.     

Evidence that two genomic species of Rhizobium are associated with Medicago truncatula

Seventy-three isolates of rhizobia sampled from root nodules of Medicago truncatula were analyzed by restriction fragment length polymorphism (RFLP) of DNA regions amplified by the polymerase chain reaction (PCR) targeting the symbiotic plasmid (nifD-K, nodD1, and nodD2 genes) and the chromosome (16S rDNA plus intergenic spacer). Two genotypic groups were found, regardless of the DNA region targeted. These two groups were given the status of genomic species based on results of DNA/DNA hybridization. Received: 1 August 1995 / Accepted: 13 October 1995     

Some properties of arctic rhizobia

Forty-eight strains of Rhizobium isolated from the root nodules of three species of legumes indigenous to the high tundra (Astragalus alpinus, Oxytropis maydelliana andOxytropis arctobia) are phenotypically heterogenous with respect to intrinsic antibiotic resistance, expression of nitrogenase activityex planta and plasmid content. All of the strains possess a 250–300 kb plasmid and are homologous to each other on the genomic DNA level but have little DNA homology with selected reference strains of well characterized species of rhizobia. The arctic rhizobia have an optimum growth temperature of 23°C and can grow slowly at 5°C. The DNA from four of the isolates, which were selected for detailed investigation, have sequences homologous tonif andnod genes fromRhizobium trifolii.     

Restriction Fragment Length Polymorphism Analyses of the Plasmid DNAs in Strains of Xanthomonas campestris pv. vesicatoria from Different Geographic Areas

Restriction fragment length polymorphisms (RFLPs) of plasmid DNAs in Xanthomonas campestris pv. vesicatoria were analysed using 77 strains from the United states, Argentina, Australia, Taiwan, and Korea. One or more plasmids were detected in all tested strains, irrespective of geographic origin, host plant from which isolated, or chemical resistance. All Korean strains contained a few plasmids of similar high molecular weight, whereas some small plasmids occured only in strains from the United States, Argentina, and Taiwan. After digesting total plasmid DNAs with each of four restriction endonucleases, 18 fragments with sizes from about 1 to 23 kb were visualized. Seventy-seven strains of diverse geographic origins, with different levels of resistance to streptomycin and copper, were classified into the 14 RFLP groups based on the restriction endonuclease digestion patterns of their plasmid DNAs. Strains belonging to each group shared DNA fragments of identical size, suggesting the possible presence of similar plasmids in these strains. A 5.8-kb EcoRI plasmid DNA probe prepared from the United States strain 81-23 hybridized to EcoRI plasmid digests from all tested strains. Other plasmid DNA fragments of the strain81-2,3 used as probes had no homology to plasmid DNA fragments from several strains around the world. The variation in hybridization profiles of plasmid DNA was very similar to the results obtained by RFLP analysis of plasmid DNA digested by four restriction enzymes. Most of the Korean strains tested were highly sensitive to streptomycin and copper, whereas most strains from other geographic areas showed a high level of resistance to one or two of the chemicals. Cluster analysis of genetic distance between the strains based on the data obtained generated the dendrograms that separated all Korean strains from the other strains, suggesting that plasmid DNA of the Korean strains may be genetically very different from those of the others.     

Identification of Ochratoxigenic Aspergillus Section Nigri Isolated from Grapes by ITS‐5.8S rDNA Sequencing Analysis and In Silico RFLP

To characterize Aspergillus section Nigri strains involved in the ochratoxin A (OTA) contamination of Tunisian wine and table grapes, a total of 33 strains were analysed. A molecular characterization of the isolates was performed by the amplification of internal transcribed spacer (ITS1‐5.8S rDNA‐ITS2) region combined with amplicon sequencing. Analysis of similarity between the obtained sequences and those deposited in the GenBank database was performed. Twelve strains were confirmed to belong to the Aspergillus carbonarius species. Strains belonging to the Aspergillus niger aggregate group were classified by in silico RFLP assay into two patterns N and T, corresponding to A. niger and Aspergillus tubingensis. Among the 21 OTA producing isolates analysed, 13 showed the T‐type pattern and 8 showed the N‐type pattern. The presented method showed to be a reliable alternative to the classic RFLP method. Our findings unambiguously revealed that multiple aspergilli species isolated from wine and table grape in Tunisia are able to produce OTA.     

Genomic Analysis of Borrelia japonica Sp. Nov. Isolated from Ixodes ovatus in Japan

Genetic characteristics of 12 Borrelia isolates from the tick, Ixodes ovatus, I. persulcatus, and the rodent, Apodemus speciosus ainu, in Japan were compared to members of the three genospecies of Borrelia burgdorferi sensu lato; B. burgdorferi sensu stricto, B. garinii and group VS461. The methods used in this study were the quantitative microplate DNA hybridization assay and restriction fragment length polymorphism (RFLP) analyses of the flagellin structural genes and the 16S rRNA genes. The six isolates from I. persulcatus and A. speciosus ainu were identified as genospecies B. garinii using RFLP analysis of the flagellin and 16S rRNA genes. In contrast, RFLP analysis of the six isolates from I. ovatus indicated that they were different from the three reported genospecies. DNA homology studies confirmed the RFLP results. The six isolates from I. ovatus had DNA homologies ranging from 85 to 99%, whereas DNA relatedness of the I. ovatus isolate with strains belonging to the three genospecies was 50 to 64%. These results suggest that the strains isolated from I. ovatus in Japan differ from the three genospecies and should be classified as a new genospecies of B. burgdorferi sensu lato. We propose that strains isolated from I. ovatus should be classified as B. japonica sp. nov.     

Genomic studies on killer yeasts belonging to the genusPichia

Twenty-four species belonging to the genusPichia were investigated using restriction fragment length polymorphism (RFLP) and Southern blot hybridization of their genomic DNA.Saccharomyces cerevisiae, Kluyveromyces lactis, Williopsis mrakii andCandida albicans were also included in this study. The RFLP patterns were obtained from digestion of yeast DNA with several restriction endonuclease enzymes, and showed various bands with different mobility; in most isolates, the more deeply stained bands were species-specific. This observation was confirmed by the results obtained from Southern blot hybridization of theEcoRI andXhoI RFLP patterns withP. anomala UCSC 25F DNA, digested with the same enzymes, used as probes. These bands are likely to be ribosomal DNA as shown by hybridization of digested DNA from unrelated yeast species (S. cerevisiae, K. lactis andC. albicans). However, one hybridized band, located at 3.9–4.1 Kb, seems to be peculiar to thePichia species. Our study confirms the usefulness of molecular tools in studying genetic relatedness among yeasts.     

Isolation and characterization of Thermus thermophilus Gy1211 from a deep-sea hydrothermal vent

We examined a single, non-spore-forming, aerobic, thermophilic strain that was isolated from a deep-sea hydrothermal vent in the Guaymas Basin at a depth of 2000 m and initially placed in a phenetic group with Thermus scotoductus (X-1). We identified this deep-sea isolate as a new strain belonging to Thermus thermophilus using several parameters. DNA–DNA hybridization under stringent conditions showed 74% similarity between the deep-sea isolate and T. thermophilus HB-8^T (T = type strain). Phenotypic characteristics, such as the utilization of carbon sources, hydrolysis of different compounds, and antibiotic sensitivity were identical in the two strains. The polar lipids composition showed that strain Gy1211 belonged to the genus Thermus. The fatty acids composition indicated that this strain was related to the marine T. thermophilus strain isolated from the Azores. The new isolate T. thermophilus strain Gy1211 grew optimally at 75°C, pH 8.0, and 2% NaCl. A hydrostatic pressure of 20 MPa, similar to the in situ hydrostatic pressure of the deep-sea vent from which the strain was isolated, had no effect on growth. Strain HB-8^T, however, showed slower growth under these conditions. Received: November 26, 1997 / Accepted: May 20, 1999     

Thermus sediminis sp. nov., a thiosulfate-oxidizing and arsenate-reducing organism isolated from Little Hot Creek in the Long Valley Caldera,California

Thermus species are widespread in natural and artificial thermal environments. Two new yellow-pigmented strains, L198^T and L423, isolated from Little Hot Creek, a geothermal spring in eastern California, were identified as novel organisms belonging to the genus Thermus. Cells are Gram-negative, rod-shaped, and non-motile. Growth was observed at temperatures from 45 to 75 °C and at salinities of 0–2.0% added NaCl. Both strains grow heterotrophically or chemolithotrophically by oxidation of thiosulfate to sulfate. L198^T and L423 grow by aerobic respiration or anaerobic respiration with arsenate as the terminal electron acceptor. Values for 16S rRNA gene identity (≤ 97.01%), digital DNA–DNA hybridization (≤ 32.7%), OrthoANI (≤ 87.5%), and genome-to-genome distance (0.13) values to all Thermus genomes were less than established criteria for microbial species. The predominant respiratory quinone was menaquinone-8 and the major cellular fatty acids were iso-C_15:0, iso-C_17:0 and anteiso-C_15:0. One unidentified phospholipid (PL1) and one unidentified glycolipid (GL1) dominated the polar lipid pattern. The new strains could be differentiated from related taxa by β-galactosidase and β-glucosidase activity and the presence of hydroxy fatty acids. Based on phylogenetic, genomic, phenotypic, and chemotaxonomic evidence, the novel species Thermus sediminis sp. nov. is proposed, with the type strain L198^T (= CGMCC 1.13590^T = KCTC XXX).

     

Genetic relationship of Tricholoma matsutake and T. nauseosum from the Northern Hemisphere based on analyses of ribosomal DNA spacer regions

The genetic relationship among Tricholoma matsutake and T. nauseosum strains collected from various parts of the Northern Hemisphere was investigated using sequence analysis of the rDNA ITS region and PCR-RFLP analysis of the rDNA IGS-1 region. ITS sequence similarity between T. matsutake and T. nauseosum ranged between 98.1% and 100%. The strains of T. matsutake from coniferous forests and those from broad-leaved forests showed more than 99.8% similarity in their ITS sequences. Three distinct RFLP types were detected when IGS-1 regions were digested with Cfr13I. RFLP patterns showed no variability among the strains of T. nauseosum and those of T. matsutake from broad-leaved forests. This pattern corresponded to the dominant RFLP type in the Japanese population of T. matsutake. Thus, strains belonging to this RFLP type are widely distributed throughout East Asia and Europe and associated with many tree species of Pinaceae and Fagaceae. The result suggests that T. matsutake in coniferous and broad-leaved forests and T. nauseosum should be treated as the same species genetically.     

In vitro construction of bacteriophage λ and plasmid DNA molecules containing DNA fragments from bacteriophage T4

Restriction endonucleases EcoRI and HindIII generated fragments of T4 cytosine-containing DNA were inserted into bacteriophage vector λgtSu_III and plasmid vectors pMB9 and pBR313. Resulting clones were screened for hybridization with ³²P labeled T4 tRNA. Recombinant bacteriophages and plasmids were isolated which contained a T4 fragment coding for T4 RNA species 1 and 2 and T4 tRNA^Arg. Selected λ-T4 hybrid bacteriophages were grown to high titer and their DNA analyzed by gel electrophoresis.     

The large plasmids found in enterohemorrhagic and enteropathogenic Escherichia coli constitute a related series of transfer-defective Inc F-IIA replicons

Forty-six of 52 (88.5%) enterohemorrhagic Escherichia coli (EHEC) strains screened carried a “common” plasmid of about 90 kb which encoded sequences homologous to the Inc F-IIA replicon. A similarly high incidence of Inc F-IIA plasmid-containing strains was observed in other groups of diarrheagenic E. coli, but not in random environmental coliform isolates. Enteropathogenic E. coli (EPEC) contain plasmids of similar properties and share a 23-kb DNA fragment with plasmids from EHEC. The common region encodes the F-IIA replication region and sequences homologous to the transfer operon of the Inc F-II plasmid R1. Sequence homology varied between plasmids isolated from different EHEC/EPEC strains with >80% showing homology to the regions encoding the rep and par genes. Only 5% of plasmids from EHEC strains had intact sequences homologous to the DNA between these two regions, including the oriT site. Some plasmids with an apparently intact tra operon still failed to plaque F-pilus-specific phages. This is consistent with observations that the large plasmids of EHEC and EPEC are phenotypically nonconjugative. These results suggest that the large plasmids of EHEC/EPEC constitute a family of transfer-deficient Inc F-IIA plasmids with varying degrees of deletion in tra function. The evolutionary ramifications of this finding are considered.     

The nickel resistance determinant cloned from the enterobacterium Klebsiella oxytoca: conjugational transfer,expression, regulation and DNA homologies to various nickel-resistant bacteria

Klebsiella oxytoca strain CCUG 15788, isolated from a mineral oil emulsion tank in Göteborg, Sweden, was found to be nickel-resistant (tolerating 10 mm NiCl₂ in non-complexing mineral-gluconate media; inducible resistance). The nickel resistance determinants were transferred by helper-assisted conjugation to various strains of Escherichia coli and Citrobacter freundii and expressed to between 5 and 10 mm NiCl₂. A 4.3 kb HindIII fragment was cloned from the genomic DNA of K. oxytoca. Ligated into the vector pSUP202, the fragment caused constitutive nickel resistance (of up to 3 or 10 mm Ni²⁺) in various E. coli strains. After cloning into the broad host range vector pVDZ'2 the fragment even expressed low nickel resistance in the transconjugant of Alcaligenes eutrophus AE104. With the 4.3 kb HindIII fragment as a biotinylated DNA probe it was shown by DNA-DNA hybridization that the nickel resistance determinant resides on the chromosome of K. oxytoca and not on its circular plasmid pKO1 (160 kb) or linear plasmid pKO2 (50 kb). Nickel resistance strongly correlated with the presence of the 4.3 kb HindIII fragment in the transconjugants. No homologies were detected when the nickel resistance determinants of other well-known nickel-resistant bacteria, such as A. eutrophus CH34 or A. denitrificans 4a-2, were used as target DNA. Among the 60 strains examined, positive signals only appeared with the 3.1 kb DNA fragment from A. xylosoxydans 31A and the genomic DNA of two enterobacterial strains (5-1 and 5–5) isolated from nickel-rich soil in New Caledonia.     

Genetic structure of natural populations of Nitrobacter in an aquatic environment

Nitrification has been essentially studied as a chemical process and the studies of the microbial population have been slightly worked out. This work constitutes a first attempt to study the diversity of Nitrobacter strains with the aim of working out the genetic structure of natural populations. The Nitrobacter population structure was compared between freshwater and sediments of the same lake. Nitrobacter isolates were identified and characterized by DNA/DNA hybridization, restriction pattern of rRNA genes, PCR/RFLP analysis of the ribosomal intergenic spacer and plasmid patterns. It was shown that this lake population was split into 3 subpopulations: a specific freshwater subpopulation, a specific sediment subpopulation and a nonspecific one. A 60 MDa plasmid was detected in 3,3% of freshwater isolates. A 37 MDa plasmid was detected in 59% of sediments isolates. This 37 MDa plasmid was not associated with a genomic group, but associated with a localization: the sediments. These results suggest that this 37 MDa plasmid could be implicated in the adaptation to sediment environment.     

Conserved Plasmid Hydrogen-Uptake (hup)-Specific Sequences within Hup+Rhizobium leguminosarum Strains

Thirteen Rhizobium leguminosarum strains previously reported as H₂-uptake hydrogenase positive (Hup⁺) or negative (Hup⁻) were analyzed for the presence and conservation of DNA sequences homologous to cloned Bradyrhizobium japonicum hup-specific DNA from cosmid pHU1 (M. A. Cantrell, R. A. Haugland, and H. J. Evans, Proc. Natl. Acad. Sci. USA 80:181-185, 1983). The Hup phenotype of these strains was reexamined by determining hydrogenase activity induced in bacteroids from pea nodules. Five strains, including H₂ oxidation-ATP synthesis-coupled and -uncoupled strains, induced significant rates of H₂-uptake hydrogenase activity and contained DNA sequences homologous to three probe DNA fragments (5.9-kilobase [kb] HindIII, 2.9-kb EcoRI, and 5.0-kb EcoRI) from pHU1. The pattern of genomic DNA HindIII and EcoRI fragments with significant homology to each of the three probes was identical in all five strains regardless of the H₂-dependent ATP generation trait. The restriction fragments containing the homology totalled about 22 kb of DNA common to the five strains. In all instances the putative hup sequences were located on a plasmid that also contained nif genes. The molecular sizes of the identified hup-sym plasmids ranged between 184 and 212 megadaltons. No common DNA sequences homologous to B. japonicum hup DNA were found in genomic DNA from any of the eight remaining strains showing no significant hydrogenase activity in pea bacteroids. These results suggest that the identified DNA region contains genes essential for hydrogenase activity in R. leguminosarum and that its organization is highly conserved within Hup⁺ strains in this symbiotic species.     

Wide host range cloning vectors: a cosmid clone bank of an Agrobacterium Ti plasmid

Plasmids from two virulent strains of Agrobacterium rhizogenes belonging to biotypes 1 and 2 are compared for DNA homology with the nopaline Ti plasmid from Agrobacterium tumefaciens C58 by means of Southern blot hybridizations. We find that both A. rhizogenes plasmids share strong sequence homology with regions of the Ti plasmid that affect oncogenicity of A. tumefaciens C58. The biotype 1 plasmid shows an additional region of homology at approximately the position of the genes responsible for conjugative transfer of pTiC58. Neither A. rhizogenes plasmid shows any detectable homology with the T-DNA of A. tumefaciens C58. Possible analogies between hairy root and crown gall induction are discussed on the basis of the results presented.