The occurrence of ice nucleation activity in pathogenic bacterial species

We tested the nucleation activity (INA) of 122 strains of plant pathogenic bacteria (12 varieties and 15 subspecies) stored in collection of the University of Göttingen (GSPB). The strains are isolates from diverse host plants and different geographic regions. One-hundred and seven isolates belong to the Pseudomonads, nine to the genus Erwinia and six to the Xanthomonas. The INA was analysed by ?3°, ?5°, ?7° and ?9°C. The observed value of INA cells ranged from non-detectable to a maximum concentration of ice nuclei in a range from ?7.85 at ?5°C to ?2.63 at ?3°C in 1.82 × 10³ cfu to 3.3 × 10³ cfu per ml. The data indicated that 71 (58.2%) of the 122 strains had INA cells, and 51 (41.8%) were inactive. The highest amount of strains with INA cells we found in Pseudomonads (69). In comparison only one strain was active at Erwinia and at Xanthomonas, 46 strains were isolated from the genus Phaseolus vulgaris and 6 from the genus Beta vulgaris. The other isolates with ice active cells belonged to the 13 other plant species. The 51 inactive pathovars were isolated from 21 different culture plants. The pathogens under test were isolated in 16 different countries, mainly in Germany and USA.     

A novel method for identifying Chlamydomonas reinhardtii (Chlorophyta) and closely related species from nature

Here, we introduce a new method for efficiently sampling Chlamydomonas reinhardtii and closely related species using a colony PCR-based screen with novel primer sets designed to specifically detect these important model microalgae. To demonstrate the utility of our new method, we collected 130 soil samples from a wide range of habitats in Ontario, Canada and identified 33 candidate algae, which were barcoded by sequencing a region of the rbcL plastid gene. For select isolates, 18S rRNA gene and YPT4 nuclear markers were also sequenced. Based on phylogenetic and haplotype network analyses of these three loci, seven novel isolates were identified as C. reinhardtii, and one additional isolate appeared to be more closely related to C. reinhardtii than any other known species. All seven new C. reinhardtii strains were interfertile with previously collected C. reinhardtii field isolates, validating the effectiveness of our molecular screen.     

Molecular evidence for host-adapted races of the fungal endophyte Epichloë bromicola after presumed host shifts

Abstract.— Host shifts of plant‐feeding insects and parasites promote adaptational changes that may result in the formation of host races, an assumed intermediate stage in sympatric speciation. Here, we report on genetically differentiated and host‐adapted races of the fungal endophyte Epichloë bromicola, which presumably emerged after a shift from the grass Bromus erectus to other Bromus hosts. Fungi of the genus Epichloë (Ascomycota) and related anamorphs of Neotyphodium are widespread endophytes of cool‐season grasses. Sexually reproducing strains sterilize the host by formation of external fruiting structures (stromata), whereas asexual strains are asymptomatic and transmitted via seeds. In E. bromicola, strains infecting B. erectus are sexual, and strains from two woodland species, B. benekenii and B. ramosus, are asexual and seed transmitted. Analyses of amplified fragment length polymorphism fingerprinting and of intron sequences of the tub2 and tef1 genes of 26 isolates from the three Bromus hosts collected at natural sites in Switzerland and nearby France demonstrated that isolates are genetically differentiated according to their host, indicating that E. bromicola does not form a single, randomly mating population. Phylogenetic analyses of sequence data did not unambiguously resolve the exact origin of asexual E. bromicola strains, but it is likely they arose from within sexual populations on B. erectus. Incongruence of trees derived from different genes may have resulted from recombination at some time in the recent history of host strains. Reciprocal inoculations of host plant seedlings showed that asexual isolates from B. benekenii and B. ramosus were incapable of infecting B. erectus, whereas the sexual isolates from B. erectus retained the assumed ancestral trait of broad compatibility with Bromus host seedlings. Because all isolates were interfertile in experimental crosses, asexual strains may not be considered independent biological species. We suggest that isolates infecting B. benekenii and B. ramosus represent long‐standing host races or incipient species that emerged after host shifts and that may evolve through host‐mediated reproductive isolation toward independent species.     

Ruminococcus hydrogenotrophicus sp. nov., a new H2/CO2-utilizing acetogenic bacterium isolated from human feces

A new H₂/CO₂-utilizing acetogenic bacterium was isolated from the feces of a non-methane-excreting human subject. The two strains S5a33 and S5a36 were strictly anaerobic, gram-positive, non-sporulating coccobacilli. The isolates grew autotrophically by metabolizing H₂/CO₂ to form acetate as sole metabolite and were also able to grow heterotrophically on a variety of organic compounds. The major end product of glucose and fructose fermentation was acetate; the strains also formed ethanol, lactate and, to a lesser extent, isobutyrate and isovalerate. The G+C content of DNA of strain S5a33 was 45.2 mol%. 16S rRNA gene sequencing demonstrated that the two acetogenic isolates were phylogenetically identical and represent a new subline within Clostridium cluster XIVa. Based on phenotypic and phylogenetic considerations, a new species, Ruminococcus hydrogenotrophicus, is proposed. The type strain of R. hydrogenotrophicus is S5a33 (DSM 10507). Furthermore, H₂/CO₂ acetogenesis appeared to be a common property of most of the species phylogenetically closely related to strain S5a33 (Clostridium coccoides, Ruminococcus hansenii, and Ruminococcus productus). Received: 11 April 1996 / Accepted: 11 June 1996     

Halomonas magadii sp. nov., a new member of the genus Halomonas, isolated from a soda lake of the East African Rift Valley

A number of novel alkaliphilic organotrophic bacteria have been isolated from several saline and alkaline East African soda lakes. The new isolates grow at pH values between 7.0 and 11.0, with pH optima for growth between 9.0 and 10.0. Growth occurs at total salts concentration between 0% and 20% (w/v) with optimum at 0%–7% (w/v). Phylogenetic analyses based on 16S rDNA sequence comparison indicate that these isolates are related (>96% similarity) to members of the Halomonadaceae within the γ-3 subdivision of the Proteobacteria. These analyses indicate that existing species within the Halomonadaceae fell within three main groups, one group comprising the type species of Halomonas, Halomonas elongata, and a number of other known species including one soda lake isolate. A second group constituting most of the remaining known species of Halomonas and related Chromohalobacter spp. includes 3 soda lake isolates with high DNA–DNA homologies. The third group included Halomonas halodenitrificans, Halomonas desiderata, Halomonas cupida, and 13 soda lake isolates. Phenotypic comparisons indicated that the majority of soda lake strains shared similar morphological, phenotypic, and chemotaxonomic properties to known strains of Halomonas but grew under alkaline conditions. The 3 soda lake isolates with high DNA–DNA homologies were, however, significantly different in antibiotic sensitivity pattern and in the utilization of several substrates, were unable to reduce nitrite, and showed low DNA–DNA homologies with known halomonads in the same group. We propose that these isolates comprise a new species of the genus Halomonas that we name Halomonas magadii sp. nov. The type strain is strain 21 MI (NCIMB 13595). Received: July 20, 1999 / Accepted: October 29, 1999     

A comparative study on the phylogenetic diversity of culturable actinobacteria isolated from five marine sponge species

A cultivation-based approach was employed to compare the culturable actinobacterial diversity associated with five marine sponge species (Craniella australiensis, Halichondria rugosa, Reniochalina sp., Sponge sp., and Stelletta tenuis). The phylogenetic affiliation of the actinobacterial isolates was assessed by 16S rDNA-RFLP analysis. A total of 181 actinobacterial strains were isolated using five different culture media (denoted as M1–M5). The type of medium exhibited significant effects on the number of actinobacteria recovered, with the highest number of isolates on M3 (63 isolates) and the lowest on M1 (12 isolates). The genera isolated were also different, with the recovery of three genera on M2 and M3, and only a single genus on M1. The number of actinobacteria isolated from the five sponge species was significantly different, with a count of 83, 36, 30, 17, and 15 isolates from S. tenuis, H. rugosa, Sponge sp., Reniochalina sp., and C. australiensis, respectively. M3 was the best isolation medium for recovery of actinobacteria from S. tenuis, H. rugosa, and Sponge sp., while no specific medium preference was observed for the recovery of actinobacteria from Reniochalina sp., and C. australiensis. The RFLP fingerprinting of 16S rDNA genes digested with HhaI revealed six different patterns, in which 16 representative 16S rDNAs were fully sequenced. Phylogenetic analysis indicated that 12 strains belong to the group Streptomyces, three strains belong to Pseudonocardia, and one strain belongs to Nocardia. Two strains C14 (from C. australiensis) and N13 (from Sponge sp.) have only 96.26% and 96.27% similarity to earlier published sequences, and are therefore potential candidates for new species. The highest diversity of three actinobacteria genera was obtained from Sponge sp., though the number of isolates was low. Two genera of actinobacteria, Streptomyces, and Pseudonocardia, were isolated from both S. tenuis and C. australiensis. Only the genus of Streptomyces was isolated from H. rugosa and Reniochalina sp. Sponge species have been demonstrated here to vary as sources of culturable actinobacterial diversity, and the methods for sampling such diversity presented may be useful for improved sampling of such diversity.     

EVIDENCE FOR GENETIC DIFFERENTIATION BETWEEN CHOKE-INDUCING AND ASYMPTOMATIC STRAINS OF THE EPICHLOË GRASS ENDOPHYTE FROM BRACHYPODIUM SYLVATICUM

Life cycle and breeding system variation in Epichloë grass endophytes (choke disease) is tightly linked to the degree of stroma formation. It is not known whether this variation results from differences in host resistance, fungal virulence, or environmental conditions. We found genetic differentiation between 173 asymptomatic (NS) and 93 stromata-forming (S) Epichloë strains isolated from one grass species, Brachypodium sylvaticum, based on 13 presumed allozyme loci, of which six were variable. The fungal strains originated from 10 sites in Switzerland, three sites of which were represented by both NS and S subpopulations. In total, 19 allozyme genotypes, that were nonrandomly distributed among S and NS were detected. Genetic variation measured as G_ST between S and NS strains isolated from the same site ranged from 0.73 to 0.98. Clonality, measured as linkage disequilibrium at one site, was significant in the NS subpopulation (P ? 0.001), but not in the S subpopulation (P = 0.21), implying asexual reproduction by NS strains as well as successful horizontal transmission of S strains. Since all seeds are usually infected vegetatively, horizontal transmission implies the occurrence of multiple host infections. Altogether, these results provide indirect evidence that NS and S strains do not belong to one panmictic population and that differentiation patterns of stroma formation found in nature are due to genetic differences among fungi in associations with their host plants. We discuss the direction of evolution of disease expression in this system. The distribution of genetic variability suggests that the asymptomatic strains were derived from stromata-forming populations.     

Genotypic Characterization of Phage-Typed Indigenous Soybean Bradyrhizobia and Their Host Range Symbiotic Effectiveness

Analysis of genetic diversity among indigenous rhizobia and its symbiotic effectiveness with soybean cultivar is important for development of knowledge about rhizobial ecology. In India, little is known about the genetic resources and diversity of rhizobia nodulating soybean. Indigenous bradyrhizobia isolated from root nodules of soybean plants, collected from traditional cultivating regions of two states (Madhya Pradesh and Uttar Pradesh) of India, were screened for bacteriophage sensitivity to identify successful broad host range symbiotic effectivity. Of 172 rhizobial isolates, 91 showed sensitivities to eight lytic phages and form ten groups on the basis of sensitivity patterns. The genetic diversity of 23 isolates belonging to different phage groups was assessed along with that of strains USDA123 and USDA94 by the restriction fragment length polymorphism (RFLP) analysis of 16S rDNA, intergenic spacer (IGS) (16S–23S rDNA), and DnaK regions. RFLP analysis of 16S rDNA formed 5 groups, whereas 19 and 9 groups were revealed by IGS and the DnaK genes, respectively. The IGS regions showed many amplified polymorphic bands. Nine isolates which revealed high RFLP polymorphism in the abovementioned regions (16S rRNA, IGS, DnaK) were used for 16S rRNA sequence analyses. The results indicate that taxonomically, all isolates were related to Rhizobium etli, Bradyrhizobium spp., and Bradyrhizobium yuanmingense. The doubling time of isolates varied from 9 h (MPSR155) to 16.2 h (MPSR068) in YM broth. Five isolates which did not show cross infectivity with isolated phage strains were studied for symbiotic efficiency. All isolates showed broad host range symbiotic effectiveness forming effective nodules on Vigna mungo, Vigna radiata, Vigna unguiculata, and Cajanus cajan. The present study provides information on genetic diversity and host range symbiosis of indigenous soybean rhizobia typed by different phages.     

Identification of a Vibrio strain producing antimicrobial agents in the excretory organs of Nautilus pompilius (Cephalopoda: Nautiloidea)

The aim of the present study was to identify and characterize bacteria producing antimicrobial compounds in the excretory organs of Nautilus pompilius. Culture-dependent and culture-independent complementary approaches were used for bacterial identification such as: culture on selective media, Gram staining, CARD-FISH, direct DNA extraction from host tissue, PCR amplification and sequencing of the bacterial 16S rRNA gene. Results show presence of three bacterial groups: γ-Proteobacteria with three clusters (Pseudomonadales, Vibrionales, Alteromonadales), β-Proteobacteria and spirochetes. In order to screen for active strains, antimicrobial activity was tested by diffusion agar assay against Micrococcus luteus, Escherichia coli, Vibrio harveyi, and Candida albicans. Nautilus isolates showed antimicrobial activities against both Gram-positive and Gram-negative reference strains. Most of the active strains were phylogenetically related to environmental Vibrionaceae. These strains were always abundant in N. pompilius PA but were absent from Nautilus macromphalus from other geographical areas. Therefore, we suggest that antimicrobial active Vibrionaceae infect N. pompilius by environmental transmission.     

Host range and gene contents of Bacillus thuringiensis strains toxic towards Spodoptera exigua

Thirty-five strains of the entomopathogenic bacterium Bacillus thuringiensisactive on Spodoptera exigua, were characterized by means of serological identification and determination of crygene contents by PCR. The insecticidal activity of these 35 strains was further confirmed against S. exiguaand tested against two other species of the same genus: S. littoralisand S. frugiperda. The results indicate that serovars aizawai, thuringiensis, and kurstakiwere the most frequent within S. exigua-active strains and that serovar aizawaihad the highest number of strains exhibiting toxicity against the three species bioassayed. The presence in crygenes as determined by PCR suggests a non random distribution of some crygenes among serovars. Genes cry1C, cry1D, and cry1E, which are known to code for proteins toxic against Spodopteraspecies, were very common within S. exigua-active strains, specially in those belonging to serovar aizawai. However, some strains harbouring one or more of these genes were not toxic to S. littoralisor S. frugiperda; and some strains lacking all of the Spodoptera-active genes were found to be toxic to all three species. This suggests differences in the expression levels among strains bearing toxic genes and the involvement of other genes toxic to Spodopteraspecies. Since strains sharing the same crygenes exhibited different host ranges, the results indicate the need to perform toxicity bioassays in addition to other tests (serological identification and PCR) in order to determine the insecticidal activity of B. thuringiensisstrains.     

PHYLOGENETIC POSITION OF SYMBIODINIUM (DINOPHYCEAE) ISOLATES FROM TRIDACNIDS (BIVALVIA), CARDIIDS (BIVALVIA), A SPONGE (PORIFERA), A SOFT CORAL (ANTHOZOA), AND A FREE-LIVING STRAIN

The genus Symbiodinium is the commonly observed symbiotic dinoflagellate (zooxanthellae) that forms mutual associations with various marine invertebrates. Numerous studies have revealed that the genus is comprised of a group of diverse taxa, and information on the phylogenetic relationships among the genus’ members is increasing. In this study, small subunit (SSU) ribosomal RNA (ssrRNA) gene sequences were determined for 15 more Symbiodinium strains from 12 relatively unstudied host taxa (Indo-Pacific tridacnids, cardiids, sponge, and soft coral), 1 hitherto unreported free-living Symbiodinium strain, and 4 other Symbiodinium strains from four other host taxa (Indo-Pacific zoanthid, foraminifer, jellyfish, and mid-Pacific hard coral). Their respective phylogenetic positions were inferred, and strains that are either closely related to or distinct from previously reported Symbiodinium taxa were revealed. The cultured Symbiodinium strains isolated from individuals of six species of tridacnids and three species of cardiids all had identical ssrRNA gene sequences, are closely related to S. microadriaticum Freudenthal, and are indistinguishable from the RFLP Type A strain previously reported. However, the ssrRNA gene sequences of clam symbionts that were obtained via gene cloning were different from those of the cultured isolates and represent strains that are close to the RFLP Type C strains. The Symbiodinium-like dinoflagellate from the Indo-Pacific sponge Haliclona koremella De Laubenfels is distinct from any of the Symbiodinium taxa studied and may be similar to the symbiont previously isolated from the stony coral Montipora patula Quelch. The isolates from the soft coral Sarcophyton glaucum Quoy et Gaimard and from the zoanthid Zoanthus sp. are both very closely related to S. pilosum Trench et Blank. The free-living Symbiodinium isolate is very closely related to the symbiont isolated from the Indo-Pacific foraminifer Amphisorus hemprichii Ehrenberg, which in turn is distinct from the Red Sea strain isolated from a similar host. Theisolate from Cassiopeia sp. is different from S. microadriaticum F., the type species harbored by Cassiopeia xamachana Bigelow, and is instead very closely related to S. pulchrorum Trench isolated from a sea anemone. The symbiont from the stony coral M. verrucosa Lamarck is a sister taxon to the symbionts isolated from the foraminifera Marginopora kudakajimensis Gudmundsson and Sorites orbiculus Forskål. These data suggest that polymorphic symbioses extend from cnidarians to some bivalve, foraminifer, and jellyfish host species.     

Patterns of aphid resistance in the genus Vicia

The performance of three aphid species, Aphis fubae, Acyrthosiphon pisum and Megoura viciae, was quantified on 22 taxonomically-selected host plant species from the genus Vicia (vetches), using intrinsic rate of increase (r_m) k standard errors. A. pisum proved to be the most successful aphid species, both in terms of highest potential population increase and widest host range; M. viciae was least successful, with most restricted host range and low r_m values on all but the closest relatives of Vicia faba. A wide range of antibiotic resistance was found within the genus, and host resistance to the three aphid species was found to be broadly related to classification patterns based on taxonomy and life-form. Analysis of the components of r_m revealed that nymphal survival rates were the most significant factor limiting host colonisation, not pre-reproductive period which is the most important factor in cultivars of V. faba. The consequences of aphid polyphagy on performance are discussed.     

Bacterial Diversity in a Nonsaline Alkaline Environment: Heterotrophic Aerobic Populations

Heterotrophic populations were isolated and characterized from an alkaline groundwater environment generated by active serpentinization, which results in a Ca(OH)₂-enriched, extremely diluted groundwater with pH 11.4. One hundred eighty-five strains were isolated in different media at different pH values during two sampling periods. To assess the degree of diversity present in the environment and to select representative strains for further characterization of the populations, we screened the isolates by using random amplified polymorphic DNA-PCR profiles and grouped them based on similarities determined by fatty acid methyl ester analysis. Phenotypic characterization, determinations of G+C content, phylogenetic analyses by direct sequencing of 16S rRNA genes, and determinations of pH tolerance were performed with the selected isolates. Although 38 different populations were identified and characterized, the vast majority of the isolates were gram positive with high G+C contents and were affiliated with three distinct groups, namely, strains closely related to the species Dietzia natrolimnae (32% of the isolates), to Frigoribacterium/Clavibacter lineages (29% of the isolates), and to the type strain of Microbacterium kitamiense (20% of the isolates). Other isolates were phylogenetically related to strains of the genera Agrococcus, Leifsonia, Kytococcus, Janibacter, Kocuria, Rothia, Nesterenkonia, Citrococcus, Micrococcus, Actinomyces, Rhodococcus, Bacillus, and Staphylococcus. Only five isolates were gram negative: one was related to the Sphingobacteria lineage and the other four were related to the α-Proteobacteria lineage. Despite the pH of the environment, the vast majority of the populations were alkali tolerant, and only two strains were able to grow at pH 11.     

Spiroplasma species in the Costa Rican highlands: implications for biogeography and biodiversity

More than 1,000 Spiroplasma isolates have been obtained from horse flies and deer flies (Diptera:Tabanidae) in the United States and Canada. However, the spiroplasma biota of Central America is poorly known. In August of 1995 and 1998, 13 isolates were obtained in 14 attempts from horse flies of a single species, Poeciloderas quadripunctatus, taken in the Costa Rican highlands (1,100–2,000 m). The majority of the “isolates” proved to be mixtures of two or more Spiroplasma species, but after filter cloning, single strains emerged that were designated as representatives of the 13 accessions. Six distinct spiroplasma serogroups were identified from these isolations. Three of the strains are putative new species with no serological relationship to any other Spiroplasma species. A fourth strain is a putative new species that may be distantly related to S. helicoides, a southeastern U.S. species. These four strains are accorded herein status as representatives of new serogroups: strain BARC 4886 (group XXXV); strain BARC 4900 (group XXXVI); strain BARC 4908 (group XXXVII); and GSU5450 (group XXXVIII). A fifth Spiroplasma species was very closely related to S. lineolae, known previously only from the Georgia (U.S.) coast. The sixth was most closely related to subgroup VIII-3, known from Texas and the southeastern U.S. Discovery of six spiroplasma species in only 13 attempted isolations reflects diversity seldom equaled in southeast Georgia, and never elsewhere in the U.S. These results are consistent with a hypothesis that spiroplasma diversity increases from north (Nova Scotia) to south (Georgia and Costa Rica). The discovery of significant affinity between some spiroplasmas of the southeastern U.S. and the Costa Rican highlands was unexpected, but may reflect a climatically complex Pleistocene history.

Isolation and Characterization of Endophytic Colonizing Bacteria from Agronomic Crops and Prairie Plants

Endophytic bacteria reside within plant hosts without causing disease symptoms. In this study, 853 endophytic strains were isolated from aerial tissues of four agronomic crop species and 27 prairie plant species. We determined several phenotypic properties and found approximately equal numbers of gram-negative and gram-positive isolates. In a greenhouse study, 28 of 86 prairie plant endophytes were found to colonize their original hosts at 42 days postinoculation at levels of 3.5 to 7.7 log₁₀ CFU/g (fresh weight). More comprehensive colonization studies were conducted with 373 corn and sorghum endophytes. In growth room studies, none of the isolates displayed pathogenicity, and 69 of the strains were recovered from corn or sorghum seedlings at levels of 8.3 log₁₀ CFU/plant or higher. Host range greenhouse studies demonstrated that 26 of 29 endophytes were recoverable from at least one host other than corn and sorghum at levels of up to 5.8 log₁₀ CFU/g (fresh weight). Long-range dent corn greenhouse studies and field trials with 17 wild-type strains and 14 antibiotic-resistant mutants demonstrated bacterial persistence at significant average colonization levels ranging between 3.4 and 6.1 log₁₀ CFU/g (fresh weight) up to 78 days postinoculation. Three prairie and three agronomic endophytes exhibiting the most promising levels of colonization and an ability to persist were identified as Cellulomonas, Clavibacter, Curtobacterium, and Microbacterium isolates by 16S rRNA gene sequence, fatty acid, and carbon source utilization analyses. This study defines for the first time the endophytic nature of Microbacterium testaceum. These microorganisms may be useful for biocontrol and other applications.     

Natural variability in the mycelial form of emmonsia crescens

A range of variations in colony characteristics and microscopical morphology of Emmonsia crescens Emmons & Jellison 1960 have been studied. A total of 13 strains of the mycelial stage of the fungus were examined in detail. The isolates originated from the lungs of 8 species of wild mammals collected in Czechoslovakia, Bulgaria, France and U.S.S.R. According to their colony appearance the strains were classified into 3 groups as follows: powdered, floccose and fluffy. The microscopic structure of the colonies was relatively unique, differences were found in the size of aleuries and the type of their walls. The morphological variability of E. crescens was not markedly influenced by host species or geographic origin of strains.     

Phylogenetic diversity of isolates belonging to genera Geobacillus and Aeribacillus isolated from different geothermal regions of Turkey

The phylogenetic diversity of 31 thermophilic bacilli belonging to genera Geobacillus and Aeribacillus were investigated which were isolated from various geothermal sites of Turkey. Twenty-seven of these isolates were found to be belonged within the genus Geobacillus, whereas 4 of them were identified as Aeribacillus pallidus. The comparative 16S rRNA gene sequence analyses revealed that the A. pallidus isolates displayed sequence similarity values from 98.0 to 99.6% to their closest relative. Furthermore, Geobacillus isolates showed sequence similarity values from 88.9 to 99.8% with the reference type strains. According to the phylogenetic analysis, isolates belonging to genus Geobacillus were diverged into nine clusters and among these isolates, 19 of them were identified as strains related to G. caldoproteolyticus, G. thermodenitrificans, G. stearothermophilus, G. thermoglucosidasius and G. toebii with the most abundant 13 isolates from G. caldoproteolyticus. Four of the Geobacillus isolates were named as unidentified mix group, as they found to be genetically very homogenous like their closely related type species: G. thermoleovorans, G. vulcani, G. lituanicus, G. kaustophilus, G. caldovelox, G. caldotenax, and G. uralicus. Moreover, the sequence comparisons of E173a, E265, C161ab and A142 isolates demonstrated that they represented novel species among genus Geobacillus as they shared lower than 96.7% sequence similarity to all the described type species. The AluI-, HaeIII- and TaqI-ARDRA results were in congruence with the 16S rRNA gene sequence analyses. By ARDRA results, the isolates were able to be differentiated and clustered, the discriminative restriction fragments of these isolates and type species were determined and the novelty of E173, E265, C161ab and A142 isolates could be displayed. Some differentiating phenotypic characters and the ability of amylase, glucosidase and protease production of these bacilli were also studied and biotechnologically valuable thermostable enzyme producing isolates were introduced in order to use in further studies.     

Evolutional and Geographical Relationships of <Emphasis Type="Italic">Bartonella grahamii</Emphasis> Isolates from Wild Rodents by Multi-locus Sequencing Analysis

To clarify the relationship between Bartonella grahamii strains and both the rodent host species and the geographic location of the rodent habitat, we have investigated 31 B. grahamii strains from ten rodent host species from Asia (Japan and China), North America (Canada and the USA), and Europe (Russia and the UK). On the basis of multi-locus sequencing analysis of 16S rRNA, ftsZ, gltA, groEL, ribC, and rpoB, the strains were classified into two large groups, an Asian group and an American/European group. In addition, the strains examined were clearly clustered according to the geographic locations where the rodents had been captured. In the phylogenetic analysis based on gltA, the Japanese strains were divided into two subgroups: one close to strains from China, and the other related to strains from Far Eastern Russia. Thus, these observations suggest that the B. grahamii strains distributed in Japanese rodents originated from two different geographic regions. In the American/European group, B. grahamii from the North American continent showed an ancestral lineage and strict host specificity; by contrast, European strains showed low host specificity. The phylogenetic analysis and host specificity of B. grahamii raise the possibility that B. grahamii strains originating in the North American continent were distributed to European countries by adapting to various rodent hosts. An erratum to this article can be found at     

Isolation of bacteria of the genus <Emphasis Type="Italic">Variovorax</Emphasis> from the <Emphasis Type="Italic">Thioploca</Emphasis> mats of Lake Baikal

Three strains of gram-negative bacteria were isolated from the mats of colorless sulfur bacteria Thioploca (Lake Baikal). The cells of new strains are motile with peritrichous flagella. Bacteria are aerobic, obligate chemoorganoheterotrophs growing within the pH range of 3.0–8.8 with the optimum at 8.3 and within the temperature range of 5–42°C with the optimum at 28°C. The cells contained menaquinones MK-8 H2 as the major component, as well as MK-7 H2 (less than 15%), while the content of ubiquinone Q8 was at least an order of magnitude lower. The G+C content of DNA in the new strains varied from 67.4 to 69.9 mol %. The level of DNA-DNA hybridization between the strains ranged from 80 to 94%, indicating that all the isolates belonged to one species. Analysis of the 16S rRNA gene nucleotide sequences of the type strain (Gen-Bank HQ400611) revealed close homologues among the known species of the genus Variovorax: 98% resemblance with the type strains of the species V. paradoxus, V. soli, V. ginsengisoli, and V. boronicumulans and 96% similarity with the type strain of V. dokdonensis. However, since the isolates differed significantly in the composition of fatty acids and isoprenoid quinones from the nearest neighbors in the phylogenetic tree, they cannot be related implicitly to the known species.