Genotypic and phenotypic polymorphism of environmental strains of the moderately thermophilic bacterium <Emphasis Type="Italic">Sulfobacillus sibiricus</Emphasis>

Five cultures of moderately thermophilic spore-forming acidophilic chemolithotrophic bacteria were isolated from the zones of spontaneous heating of pyrrhotite-containing pyrite-arsenopyrite gold-arsenic sulfide ores in an operating open pit (strains B1, B2, B3, OFO, and SSO). Analysis of the chromosomal DNA structure revealed the differences between these cultures at the strain level (apart from B3 and SSO, which had identical restriction profiles). All the strains had a similar G+C DNA molar content (47.4–48.3%). The level of DNA reassociation was 85 to 95%. The similarity between the DNA of the type strain Sulfobacillus sibiricus N1 isolated from arsenopyrite ore concentrate and that of these strains (83–93%) indicates that they belong to the same species. The strains had similar values of pH and temperature optimal for growth on ferrous iron (1.6–2.0 and 45–55°C, respectively). They were mixotrophs; Fe(II), S^o, and sulfide minerals along with organic compounds were used as energy sources and electron donors. However, the kinetic parameters of growth and substrate oxidation varied from strain to strain. Genetic variety of the strains from diverse ecosystems and environments is possibly the result of the different rates of microevolution processes.     

Sulfobacillus sibiricus sp. nov., a new moderately thermophilic bacterium

In the course of pilot industrial testing of a biohydrometallurgical technology for processing goldarsenic concentrate obtained from the Nezhdaninskoe ore deposit (East Siberia, Sakha, Yakutiya), a new gram-positive rod-shaped spore-forming moderately thermophilic bacterium (designated as strain N1) oxidizing Fe2+, S0, and sulfide minerals in the presence of yeast extract (0.02%) was isolated from a dense pulp. Physiologically, strain N1 differs from previously described species of the genus Sulfobacillus in having a somewhat higher optimal growth temperature (55 degrees C). Unlike the type strain of S. thermosulfidooxidans, strain N1 could grow on medium with 1 mM thiosulfate or sodium tetrathionate as a source of energy only within several passages and failed to grow, in the absence of an inorganic energy source, on media with sucrose, fructose, glucose, reduced glutathione, alanine, cysteine, sorbitol, sodium acetate, or pyruvate. The G + C content of the DNA of strain N1 was 48.2 mol %. The strain showed 42% homology after DNA-DNA hybridization with the type strain of S. thermosulfidooxidans and 10% homology with the type strain of S. acidophilus. The isolate differed from previously studied strains of S. thermosulfidooxidans in the structure of its chromosomal DNA (determined by the method of pulsed-field gel electrophoresis) which remained stable as growth conditions were changed. According to the results of the 16S rRNA gene analysis, the new strain forms a single cluster with the bacteria of the species Sulfobacillus thermosulfidooxidans (sequence similarity of 97.9-98.6%). Based on these genetic and physiological features, strain N1 is described as a new species Sulfobacillus sibiricus sp. nov.     

Ferrous iron oxidation in moderately thermophilic acidophile Sulfobacillus sibiricus N1(T)

The iron-oxidizing system of a moderately thermophilic, extremely acidophilic, gram-positive mixotroph, Sulfobacillus sibiricus N1(T), was studied by spectroscopic, high-performance liquid chromatography and inhibitory analyses. Hemes B, A, and O were detected in membranes of S. sibiricus N1(T). It is proposed that the electron transport chain from Fe2(+) to O? is terminated by 2 physiological oxidases: aa?-type cytochrome, which dominates in the early-exponential phase of growth, and bo?-type cytochrome, whose role in iron oxidation becomes more prominent upon growth of the culture. Both oxidases were sensitive to cyanide and azide. Cytochrome aa? was more sensitive to cyanide and azide, with K(i) values of 4.1 and 2.5 μmol·L?1, respectively, compared with K(i) values for cytochrome bo?, which were 9.5 μmol·L?1 for cyanide and 7.0 μmol·L?1 for azide. This is the first evidence for the participation of a bo?-type oxidase in ferrous iron oxidation. The respiratory chain of the mixotroph contains, in addition to the 2 terminal oxidases, a membrane-bound cytochrome b???.     

Genotypic and phenotypic correlationships among some strains ofLactobacillus helveticus

Summary Evidence for the presence of extrachromosomal elements inLactobacillus helveticus ATCC 15009 and the absence of plasmid DNA in two other strains ofL. helveticus is reported. These three strains did not show any difference in regard to lactose metabolism, proteolytic activity, and antibiotic resistance or in N-acetyl-D-glucosamine fermentation. The only difference found is a higher resistance to arsenate forL. helveticus ATCC 15009, suggesting linkage of this resistance to plasmids present in this strain.     

Growth of moderately thermophilic iron-oxidizing bacterium strain TI-1 in synthetic medium

The moderately thermophilic iron-oxidizing bacterium strain TI-1, which lacks enzyme systems involved in CO₂ fixation, grows at 45°C in Fe²⁺ medium supplemented with yeast extract to give a maximum cell growth of 1.0 × 10⁸ cells per ml, but does not grow in Fe²⁺ medium without yeast extract. To elucidate the physiology of the strain, a synthetic medium was developed. It was found that the best synthetic medium was Fe²⁺-6AA, containing Fe²⁺, salts, and the following six l-amino acids: alanine, aspartic acid, glutamic acid, arginine, serine, and histidine. In this medium, strain TI-1 showed a maximum cell growth of 10 × 10⁸ cells/ml. The six amino acids in the Fe²⁺-6AA medium were used not only as a carbon source but also as a source of nitrogen. Inorganic nitrogen sources, such as ammonium ion, hydrazine, hydroxylamine, nitrite, and nitrate, were not used as a sole source of nitrogen, but rather strongly inhibited the utilization of the six amino acids at 1 mM. In the Fe²⁺ (10 mM)-6AA medium supplemented with 21 mM Fe³⁺, reduction of Fe³⁺ to Fe²⁺ that was dependent on the added amino acids was observed, suggesting another role of the amino acids in the growth of strain TI-1. Washed, intact cells of strain TI-1 had the activity to reduce Fe³⁺ to Fe²⁺.     

Isolation and characterization of a moderately thermophilic nitrite-oxidizing bacterium from a geothermal spring

Geothermal environments are a suitable habitat for nitrifying microorganisms. Conventional and molecular techniques indicated that chemolithoautotrophic nitrite-oxidizing bacteria affiliated with the genus Nitrospira are widespread in environments with elevated temperatures up to 55 °C in Asia, Europe, and Australia. However, until now, no thermophilic pure cultures of Nitrospira were available, and the physiology of these bacteria was mostly uncharacterized. Here, we report on the isolation and characterization of a novel thermophilic Nitrospira strain from a microbial mat of the terrestrial geothermal spring Gorjachinsk (pH 8.6; temperature 48 °C) from the Baikal rift zone (Russia). Based on phenotypic properties, chemotaxonomic data, and 16S rRNA gene phylogeny, the isolate was assigned to the genus Nitrospira as a representative of a novel species, for which the name Nitrospira calida is proposed. A highly similar 16S rRNA gene sequence (99.6% similarity) was detected in a Garga spring enrichment grown at 46 °C, whereas three further thermophilic Nitrospira enrichments from the Garga spring and from a Kamchatka Peninsula (Russia) terrestrial hot spring could be clearly distinguished from N. calida (93.6-96.1% 16S rRNA gene sequence similarity). The findings confirmed that Nitrospira drive nitrite oxidation in moderate thermophilic habitats and also indicated an unexpected diversity of heat-adapted Nitrospira in geothermal hot springs.     

Genotypic and phenotypic diversity of Lactobacillus rossiae strains isolated from sourdough

AIM: To characterize the genetic and phenotypic diversity of 33 strains of Lactobacillus rossiae. METHODS AND RESULTS: Genotypic identification was carried out by partial 16S rRNA gene sequence analysis. Genetic diversity was evaluated by RAPD-PCR analysis. Phenotypic diversity was evaluated through fermentative profile by Biolog system, proteinase and peptidase activities using synthetic substrates, and acidification capacity and amino acid profile during sourdough fermentation. The genetic analyses excluded clonal relatedness among the strains used. A large phenotypic diversity was found. It mainly concerned the capacity to use carbon sources available in sourdough during fermentation, the quotient of fermentation and the peptidase activities, especially towards proline containing synthetic substrates. The free amino acid profiles differed either for the total concentration or for the type of amino acids. With a few exceptions, proteinase activity towards wheat albumins and globulins was weak. CONCLUSIONS: Overall, no relationships between genetic and physiological analyses were found, and the strains examined showed a marked genetic and phenotypic heterogeneity. L. rossiae strains had interesting properties for application in sourdough fermentation. Although some strains combined several technological traits, the association of more strains seemed to be a requisite to get optimal sourdough characteristics. SIGNIFICANCE AND IMPACT OF THE STUDY: It represents the first characterization of the diversity within the L. rossiae species. Besides, it may represent an example of computerized analysis of genotypic and phenotypic information to select strains for improving sourdough characteristics.     

Genotypic and phenotypic heterogeneity of Streptococcus thermophilus strains isolated from dairy products

AIMS: Forty strains of Streptococcus thermophilus isolated from dairy products were identified and typed by a polyphasic approach which included phenotypic and genotypic criteria. METHODS AND RESULTS: Strains were identified by sugar fermentation pattern and species-specific PCR. Phenotypic diversity was evaluated by a chemometric model taking into account some biochemical characteristics (e.g. acidifying and peptidase activities) of technological interest. Genotypic diversity was evidenced by PCR fingerprinting. The overall phenotypic and genotypic information was elaborated on a multivariate statistical basis by principal components analysis and cluster analysis, respectively. When acidifying and peptidase activities were considered, PCA indicated that most of the strains isolated from Pecorino Toscano cheese were separable from the others. Similarly, most of the starter culture strains tended to separate from the cheese isolates. CONCLUSIONS: A wide strain heterogeneity among Strep. thermophilus strains isolated from dairy products was observed. SIGNIFICANCE AND IMPACT OF THE STUDY: A computerized analysis of genotypic and phenotypic information could be applied successfully to differentiate and characterize reliably and rapidly isolates occurring in different dairy products and to comprehend the technological role of specific Strep. thermophilus strains in dairy technology.     

Genotypic and phenotypic diversity of phlD-containing Pseudomonas strains isolated from the rhizosphere of wheat

Production of 2,4-diacetylphloroglucinol (2,4-DAPG) in the rhizosphere by strains of fluorescent Pseudomonas spp. results in the suppression of root diseases caused by certain fungal plant pathogens. In this study, fluorescent Pseudomonas strains containing phlD, which is directly involved in the biosynthesis of 2,4-DAPG, were isolated from the rhizosphere of wheat grown in soils from wheat-growing regions of the United States and The Netherlands. To assess the genotypic and phenotypic diversity present in this collection, 138 isolates were compared to 4 previously described 2, 4-DAPG producers. Thirteen distinct genotypes, one of which represented over 30% of the isolates, were differentiated by whole-cell BOX-PCR. Representatives of this group were isolated from eight different soils taken from four different geographic locations. ERIC-PCR gave similar results overall, differentiating 15 distinct genotypes among all of the isolates. In most cases, a single genotype predominated among isolates obtained from each soil. Thirty isolates, representing all of the distinct genotypes and geographic locations, were further characterized. Restriction analysis of amplified 16S rRNA gene sequences revealed only three distinct phylogenetic groups, one of which accounted for 87% of the isolates. Phenotypic analyses based on carbon source utilization profiles revealed that all of the strains utilized 49 substrates and were unable to grow on 12 others. Individually, strains could utilize about two-thirds of the 95 substrates present in Biolog SF-N plates. Multivariate analyses of utilization profiles revealed phenotypic groupings consistent with those defined by the genotypic analyses.     

Purification and characterization of chitinase B from moderately thermophilic bacterium Ralstonia sp. A-471

Chitinase B was purified from a culture medium of Ralstonia sp. A-471 by precipitation with (NH4)2SO4 and column chromatography with DEAE-Toyopearl 650 M and Sephacryl S-200. The purified enzyme was homogeneous on SDS-PAGE. The molecular weight was 45,000 by SDS-PAGE. The optimum pH was 5.0 and stable pH was from 5.0 to 10.0. In the early stage of the reaction, chitinase B produced beta-anomer of (GlcNAc)2 from the substrate (GlcNAc)6, whereas (GlcNAc)4 produced almost at equilibrium, indicating that the enzyme predominantly hydrolyzes the second glycosidic linkage from the nonreducing end of (GlcNAc)6.     

Stabilization mechanism of cytochrome c552 from a moderately thermophilic bacterium, Hydrogenophilus thermoluteolus

Cytochrome c(552) (PH c(552)) from moderately thermophilic Hydrogenophilus thermoluteolus exhibits stability intermediate between those of cytochrome c(552) (HT c(552)) from thermophilic Hydrogenobacter thermophilus and cytochrome c(551) (PA c(551)) from mesophilic Pseudomonas aeruginosa. To understand the mechanism of stabilization of PH c(552), we introduced mutations into PH c(552) at five sites, which, in HT c(552), are occupied by the amino acids responsible for stability higher than the less stable PA c(551). When PH c(552) Val-78 was mutated to Ile, as found in HT c(552), the resulting variant showed increased stability. Mutation of Ala-7, Met-13, and Tyr-34 to the corresponding residues in PA c(551) (Phe, Val, and Phe, respectively) resulted in destabilization. We also found that PH c(552) Lys-43 contributed to stability through the formation of an attractive electrostatic interaction with Asp-39. These results suggest that the intermediate stability of PH c(552) is due to the amino acids at these five sites.     

Structural polymorphism of the RecA protein from the thermophilic bacterium Thermus aquaticus.

The Escherichia coli RecA protein has served as a model for understanding protein-catalyzed homologous recombination, both in vitro and in vivo. Although RecA proteins have now been sequenced from over 60 different bacteria, almost all of our structural knowledge about RecA has come from studies of the E. coli protein. We have used electron microscopy and image analysis to examine three different structures formed by the RecA protein from the thermophilic bacterium Thermus aquaticus. This protein has previously been shown to catalyze an in vitro strand exchange reaction at an optimal temperature of about 60 degrees C. We show that the active filament formed by the T. aquaticus RecA on DNA in the presence of a nucleotide cofactor is extremely similar to the filament formed by the E. coli protein, including the extension of DNA to a 5.1-A rise per base pair within this filament. This parameter appears highly conserved through evolution, as it has been observed for the eukaryotic RecA analogs as well. We have also characterized bundles of filaments formed by the T. aquaticus RecA in the absence of both DNA and nucleotide cofactor, as well as hexameric rings of the protein formed under all conditions examined. The bundles display a very large plasticity of mass within the RecA filament, as well as showing a polymorphism in filament-filament contacts that may be important to understanding mutations that affect surface residues on the RecA filament.     

Genotypic and phenotypic relatedness of 80 strains of Branhamella catarrhalis of worldwide origin

Abstract 80 clinical Branhamella catarrhalis strains of worldwide origin were examined for genotypic relatedness and phenotypic characteristics. Using a quantitative bacterial dot method for DNA-DNA hybridization the strains were found to form a homogeneous group with ΔT_m-values ranging from 0.0–2.3°C. In Minibact-N, an identification kit for oxidase positive, Gram-negative diplococci using eight phenotypic characteristics, all isolates were correctly identified and also demonstrated complete homogeneity except for β-lactamase production. Type strains representing the genera Branhamella, Moraxella and Neisseria were also examined for comparison. B. catarrhalis strain NCTC 4103-known to be atypical-had a ΔT_m-value of 5.7°C and produced γ-glutamylaminopeptidase, in contrast to all other B. catarrhalis strains. In GN MicroPlate^TM, a kit which tests utilizable carbon sources, B. catarrhalis strains were found to be able to utilize up to 16 to 95 carbon sources.     

Degradation of phenanthrene and anthracene by Nocardia otitidiscaviarum strain TSH1, a moderately thermophilic bacterium

Aims:  The metabolism of phenanthrene and anthracene by a moderate thermophilic Nocardia otitidiscaviarum strain TSH1 was examined.
Methods and Results:  When strain TSH1 was grown in the presence of anthracene, four metabolites were identified as 1,2-dihydroxy-1,2-dihydroanthracene, 3-(2-carboxyvinyl)naphthalene-2-carboxylic acid, 2,3-dihydroxynaphthalene and benzoic acid using gas chromatography-mass spectrometry (GC-MS), reverse phase-high performance liquid chromatography (RP-HPLC) and thin-layer chromatography (TLC). Degradation studies with phenanthrene revealed 2,2'-diphenic acid, phthalic acid, 4-hydroxyphenylacetic acid, o -hydroxyphenylacetic acid, benzoic acid, a phenanthrene dihydrodiol, 4-[1-hydroxy(2-naphthyl)]-2-oxobut-3-enoic acid and 1-hydroxy-2-naphthoic acid (1H2NA), as detectable metabolites.
Conclusions:  Strain TSH1 initiates phenanthrene degradation via dioxygenation at the C-3 and C-4 or at C-9 and C-10 ring positions. Ortho -cleavage of the 9,10-diol leads to formation of 2,2'-diphenic acid. The 3,4-diol ring is cleaved to form 1H2NA which can subsequently be degraded through o -phthalic acid pathway. Benzoate does not fit in the previously published pathways from mesophiles. Anthracene metabolism seems to start with a dioxygenation at the 1 and 2 positions and ortho -cleavage of the resulting diol. The pathway proceeds probably through 2,3-dicarboxynaphthalene and 2,3-dihydroxynaphthalene. Degradation of 2,3-dihydroxynaphthalene to benzoate and transformation of the later to catechol is a possible route for the further degradation of anthracene.
Significance and Impact of the Study:  For the first time, metabolism of phenanthrene and anthracene in a thermophilic Nocardia strain was investigated.     

Activity of the Enzymes of Carbon Metabolism in Sulfobacillus sibiricus under Various Conditions of Cultivation

The thermoacidophilic iron-oxidizing chemolithotroph Sulfobacillus sibiricus N1^T is characterized by steady growth and amplified cell yield when grown in vigorously aerated medium containing Fe²⁺, glucose, and yeast extract as energy sources. In this case, carbon dioxide, glucose, and yeast extract are used as carbon sources. Glucose is assimilated through the fructose-bisphosphate pathway and the pentose-phosphate pathway. The glyoxylate bypass does not function in S. sibiricus, and the tricarboxylic acid cycle is disrupted at the level of 2-oxoglutarate dehydrogenase. The presence of ribulose-bisphosphate carboxylase indicates that carbon dioxide fixation proceeds through the Calvin cycle. The activity of ribulose-bisphosphate carboxylase is highest in autotrophically grown cells. The cells also contain pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase.     

A new iron oxidase from a moderately thermophilic iron oxidizing bacterium strain TI-1.

Iron oxidase was purified from plasma membranes of a moderately thermophilic iron oxidizing bacterium strain TI-1 in an electrophoretically homogeneous state. Spectrum analyses of purified enzyme showed the existence of cytochrome a, but not cytochrome b and c types. Iron oxidase was composed of five subunits with apparent molecular masses of 46 kDa (alpha), 28 kDa (beta), 24 kDa (gamma), 20 kDa (delta), and 17 kDa (epsilon). As the molecular mass of a native enzyme was estimated to be 263 kDa in the presence of 0.1% n-dodecyl-beta-D-maltopyranoside (DM), a native iron oxidase purified from strain TI-1 seems to be a homodimeric enzyme (alpha beta gamma delta epsilon)(2). Optimum pH and temperature for iron oxidation were pH 3.0 and 45 degrees C, respectively. The K(m) of iron oxidase for Fe(2+) was 1.06 mM and V(max) for O(2) uptake was 13.8 micromol x mg(-1) x min(-1). The activity was strongly inhibited by cyanide and azide. Purified enzyme from strain TI-1 is a new iron oxidase in which electrons of Fe(2+) were transferred to haem a and then to the molecular oxygen.     

Genotypic and phenotypic characterization of industrial autochthonous Saccharomyces cerevisiae for the selection of well-adapted bioethanol-producing strains

In northwestern Argentina, sugarcane-derived industrial fermentation is being extensively used for bioethanol production, where highly adaptive native strains compete with the baker's yeast Saccharomyces cerevisiae traditionally used as starter culture. Yeast populations of 10 distilleries from Tucumán (Argentina) were genotypic and phenotypic characterized to select well-adapted bioethanol-producing autochthonous strains to be used as starter cultures for the industrial production of bioethanol fuel. From the 192 isolates, 69.8% were identified as S. cerevisiae, 25.5% as non-Saccharomyces, and 4.7% as Saccharomyces sp. wild yeasts. The majority of S. cerevisiae isolates (68.5%) were non-flocculating yeasts, while the flocculating strains were all obtained from the only continuous fermentation process included in the study. Simple Sequence Repeat analysis revealed a high genetic diversity among S. cerevisiae genotypes, where all of them were very different from the original baker's strain used as starter. Among these, 38 strains multi-tolerant to stress by ethanol (8%), temperature (42.5 °C) and pH (2.0) were obtained. No major differences were found among these strains in terms of ethanol production and residual sugars in batch fermentation experiments with cell recycling. However, only 10 autochthonous strains maintained their viability (more than 80%) throughout five consecutive cycles of sugarcane-based fermentations. In summary, 10 autochthonous isolates were found to be superior to baker's yeast used as starter culture (S. cerevisiae Calsa) in terms of optimal technological, physiological and ecological properties. The knowledge generated on the indigenous yeast populations in industrial fermentation processes of bioethanol-producing distilleries allowed the selection of well-adapted bioethanol-producing strains.     

Genotypic and phenotypic characterization of two Swedish isolates and two prototypic strains of Coxiella burnetii

Two Swedish isolates of Coxiella burnetii and the two prototype strains of the species, Nine Mile and Priscilla, were characterized with regard to their multiplication and cytopathic effect on BGM cells and by PCR-based amplification of repetitive element DNA and the C. burnetii -specific plasmids QpH1 and QpRS. Moreover, 1330 bp of each 16S rRNA gene were sequence-determined. All four strains multiplied at virtually the same rate and displayed the same type of vacuoles in the BGM cells. Genetic homogeneity was observed inasmuch as the 16S rDNA sequences were identical and the strains showed identical PCR amplification patterns using primers specific to enterobacterial repetitive intragenic consensus DNA sequences. The two Swedish strains and the Priscilla strain also showed identical patterns after PCR amplification of repetitive extragenic palindromic DNA sequences, whereas the Nine Mile strain demonstrated a similar, but not identical pattern. Thus, the investigated strains demonstrated very similar phenotypic and genotypic characteristics. This finding is discussed in view of the very rare occurrence of domestic Q fever in Sweden.     

Activity of the enzymes of carbon metabolism in Sulfobacillus sibiricus under various conditions of cultivation

The thermoacidophilic iron-oxidizing chemolithotroph Sulfobacillus sibiricus N1T is characterized by steady growth and amplified cell yield when grown in vigorously aerated medium containing Fe2+, glucose, and yeast extract as energy sources. In this case, carbon dioxide, glucose, and yeast extract are used as carbon sources. Glucose is assimilated through the fructose-bisphosphate pathway and the pentose-phosphate pathway. Glyoxylate bypass does not function in S. sibiricus, and the tricarboxylic acid cycle is disrupted at the level of 2-oxoglutarate dehydrogenase. The presence of ribulose-bisphosphate carboxylase indicates that carbon dioxide fixation proceeds through the Calvin cycle. The activity of ribulose-bisphosphate carboxylase is highest in autotrophically grown cells. The cells also contain pyruvate carboxylase, phosphoenolpyruvate carboxylase, phosphoenolpyruvate carboxykinase, and phosphoenolpyruvate carboxytransphosphorylase.