Differences in Composition and Mucosal Adhesion of Bifidobacteria Isolated from Healthy Adults and Healthy Seniors

Fifty-one Bifidobacterium strains were isolated from the feces of healthy adults (30–40 years old) and seniors (older than 70 years of age). B. adolescentis, B. breve, B. infantis, and B. longum were isolated from the healthy adults and B. adolescentis and B. longum from elderly subjects. The tested bacteria bound, in vitro, to intestinal mucus in a strain dependent manner. The strains isolated from healthy adults, and especially B. adolescentis, bound better to intestinal mucus than those isolated from seniors. These results indicate that the mucosal adhesive properties of the human Bifidobacterium flora were reduced with the aging of the host. This shift to a Bifidobacterium flora with reduced adhesive abilities may explain the decrease in bifidobacteria levels in the intestinal microflora of aging people. Received: 7 February 2001 / Accepted: 3 April 2001     

Simultaneous nitrification and denitrification by diverse <Emphasis Type="Italic">Diaphorobacter</Emphasis> sp.

Eight bacterial isolates closely related to Diaphorobacter sp. were isolated from activated biomass surviving on wastewater laden with dyes and nitro-substituted chemicals and were identified by 16S rDNA sequence analysis. The isolates showed sequence similarity of 99–100% to other Diaphorobacter strains such as ZY 2006b, F2, NA5, PCA039, D. nitroreducens KSP4, and KSP3 and 98–99% sequence homology to D. nitroreducens NA10B (type strain JCM 11421). Neighbor-joining tree revealed that all the eight strains formed tight cluster together and also showed close clustering with other Diaphorobacter strains. Isolates demonstrated the ability to perform simultaneous nitrification and denitrification under aerobic conditions. Strains HPC 805, 815, 821, and 856 gave highest chemical oxygen demand removal (85–93%) and ammonia removal (92–96%), which correlated well with higher growth rates of the cultures. Simultaneously, complete removal of nitrate supplied in the medium in presence of ammonium and acetate (electron donor) was observed in addition to aerobic nitrite release from ammonium. Thus, the above strains showed ability to perform partial nitrification followed by further aerobic removal of common intermediate nitrite, which indicated their potential application in treatment systems for treatment of high-nitrogen-containing wastewaters.     

<Emphasis Type="Italic">Salinarchaeum laminariae</Emphasis> gen. nov., sp. nov.: a new member of the family <Emphasis Type="Italic">Halobacteriaceae</Emphasis> isolated from salted brown alga <Emphasis Type="Italic">Laminaria</Emphasis>

Halophilic archaeal strains R26^T and R22 were isolated from the brown alga Laminaria produced at Dalian, Liaoning Province, China. Cells from the two strains were pleomorphic rods and Gram negative, and colonies were red pigmented. Strains R26^T and R22 were able to grow at 20–50°C (optimum 37°C) in 1.4–5.1 M NaCl (optimum 3.1–4.3 M) at pH 5.5–9.5 (optimum pH 8.0–8.5) and neither strain required Mg²⁺ for growth. Cells lyse in distilled water and the minimum NaCl concentration required to prevent cell lysis was 8% (w/v) for strain R26^T and 12% (w/v) for strain R22. The major polar lipids of the two strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester and minor phosphatidylglycerol sulfate; glycolipids were not detected. Phylogenetic analyses based on 16S rRNA genes and rpoB′ genes revealed that strains R26^T and R22 formed a distinct clade with the closest relative, Natronoarchaeum mannanilyticum. The DNA G+C content of strains R26^T and R22 was 65.8 and 66.4 mol%, respectively. The DNA–DNA hybridization value between strains R26^T and R22 was 89%. The phenotypic, chemotaxonomic and phylogenetic properties suggest that the strains R26^T and R22 represent a novel species in a new genus within the family Halobacteriaceae, for which the name Salinarchaeum laminariae gen. nov., sp. nov. is proposed. The type strain is R26^T (type strain R26^T = CGMCC 1.10590^T = JCM 17267^T, reference strain R22 = CGMCC 1.10589).     

Biodegradation of malathion by <Emphasis Type="Italic">Brevibacillus</Emphasis> sp. strain KB2 and <Emphasis Type="Italic">Bacillus cereus</Emphasis> strain PU

We report here the degradation of a pesticide, malathion, by Brevibacillus sp. strain KB2 and Bacillus cereus strain PU, isolated from soil samples collected from malathion contaminated field and an army firing range respectively. Both the strains were cultured in the presence of malathion under aerobic and energy-limiting conditions. Both strains grew well in the medium having malathion concentration up to 0.15%. Reverse phase HPLC–UV analysis indicated that Strain KB2 was able to degrade 72.20% of malaoxon (an analogue of malathion) and 36.22% of malathion, while strain PU degraded 87.40% of malaoxon and 49.31% of malathion, after 7 days of incubation. The metabolites mal-monocarboxylic acid and mal-dicarboxylic acid were identified by Gas chromatography/mass spectrometry. The factors affecting biodegradation efficiency were investigated and effect of malathion concentration on degradation rate was also determined. The strain was analyzed for carboxylesterase activity and maximum activity 210 ± 2.5 U ml⁻¹ and 270 U ± 2.7 ml⁻¹ was observed for strains KB2 and PU, respectively. Cloning and sequencing of putative malathion degrading carboxylesterase gene was done using primers based PCR approach.     

Toxicity of a <Emphasis Type="Italic">Bacillus thuringiensis israelensis</Emphasis>-like strain against <Emphasis Type="Italic">Spodoptera frugiperda</Emphasis>

Bacillus thuringiensis (Bt) Berliner is a promising agent for microbial control of agriculturally and medically important insects. This study aimed at searching for Bt strains encoding Cry proteins that act more efficiently against fall armyworm. Thirty Bt strains were isolated from soil samples in Pernambuco State and evaluated through bioassays. Among these, strain I4A7 was the most efficient against the fall armyworm, Spodoptera frugiperda (J. E. Smith, 1797) (Lepidoptera: Noctuidae), and thus it was characterized by biochemical sodium dodecyl sulfate–polyacrylamide gel electrophoresis (SDS–PAGE) and molecular (polymerase chain reaction (PCR) and sequencing reaction) methods. The protein pattern of this strain on a SDS–PAGE was similar to that of B. thuringiensis israelensis (Bti). Moreover, I4A7 cry DNA sequence showed high identity (99–100%) to genes cry4Aa, 4Ba, 10Aa, 11Aa, cyt1Aa and cyt2B from Bti. The toxicity of the newly isolated Bti-like strain upon S. frugiperda should be considered as this strain might be used in combination with other Bt strains, such as B. thuringiensis var. kurstaki (Btk). Handling Editor: Helen Roy.     

Characterization of Clinical Isolates of <Emphasis Type="Italic">Gordonia</Emphasis> Species in Japanese Clinical Samples During 1998–2008

During 1998–2008, there were 31 strains of Gordonia species isolated from clinical specimens in our laboratory. Our identification of the 31 strains of Gordonia species showed that major pathogenic Gordonia species in Japan were classifiable, respectively into 14 and 13 strains of Gordonia sputi and Gordonia bronchialis. The four remaining strains were identified as three Gordonia species: G. aichiensis (2 strains), and G. terrae (1 strain), and G. otitidis (1 strain). Results of drug susceptibility tests for these 31 strains of Gordonia isolates are reported herein.     

<Emphasis Type="Italic">Ectothiorhodospira magna</Emphasis> sp. nov., a new large alkaliphilic purple sulfur bacterium

Two strains of purple sulfur bacteria of the family Ectothiorhodospiraceae were isolated from moderately saline steppe lakes (with pH above 9.0) of the Transbaikal region (strain B7-7) and Mongolia (strain M10). The cells of the novel strains were spiral-shaped, 2.0–3.2 × 9.6–20.0 μm, motile due to a polar tuft of flagella. Photosynthetic pigments were represented by bacteriochlorophyll a and carotenoids of the spirilloxanthin series. Photosynthetic membranes were represented by long strands of lamellae distributed throughout the whole cell; unlike most Ectothiorhodospiraceae species, the membranes were not packed into regular stacks. Bacteria were capable of weak growth on sulfide and slow grow on hydrogen under photoautotrophic conditions. The best growth was noted on sulfide in the presence of acetate and bicarbonate. Thiosulfate did not stimulate phototrophic growth, even in the presence of organic substrates. The new isolates were alkaliphiles growing at a pH optimum of 9–10. Growth was possible within a salinity range of 0–80 g/l NaCl, with an optimum at 5–15 g/l NaCl. The morphology, the structure of the photosynthetic apparatus (strands of lamellae), and the physiology of the new strains were similar to those of Thiorhodospira sibirica. However, analysis of the 16S rRNA gene sequences demonstrated that the studied isolates were closely related to the type strain Ectothiorhodospira shaposhnikovii (99% similarity) of the family Ectothiorhodospiraceae, whereas the level of similarity between the new strains and Thiorhodospira sibirica was only 94–95%. According to the results of DNA-DNA hybridization, the DNA-DNA homology level between the tested strains was almost 100%; the similarity between the new isolates and the type strain Ectothiorhodospira shaposhnikovii was only 58%. The isolates differed from other representatives of the genus Ectothiorhodospira in the structure of the gene encoding the key enzyme of autotrophic CO₂ fixation, ribulose-1,5-bisphosphate carboxylase (RuBisCo), which was similar to the RuBisCo genes of members of another family of sulfur bacteria, Chromatiaceae. The new isolates of purple bacteria were described as a new species of the genus Ectothiorhodospira, Ect. magna sp. nov. with the type strain B7-7^T (= VKM B-2537 = DSM 22250).     

Evolutionary Diversification Indicated by Compensatory Base Changes in ITS2 Secondary Structures in a Complex Fungal Species, <Emphasis Type="Italic">Rhizoctonia solani</Emphasis>

The rRNA cistron (18S–ITS1–5.8S–ITS2–28S) is used widely for phylogenetic analyses. Recent studies show that compensatory base changes (CBC) in the secondary structure of ITS2 correlate with genetic incompatibility between organisms. Rhizoctonia solani consists of genetically incompatible strain groups (anastomosis groups, AG) distinguished by lack of anastomosis between hyphae of strains. Phylogenetic analysis of internal transcribed spacer (ITS) sequences shows a strong correlation with AG determination. In this study, ITS sequences were reannotated according to the flanking 5.8S and 28S regions which interact during ribogenesis. One or two CBCs were detected between the ITS2 secondary structure of AG-3 potato strains as compared to AG-3 tobacco strains, and between these two strains and all other AGs. When a binucleate Rhizoctonia species related to Ceratobasidiaceae was compared to the AGs of R. solani, which were multinucleate (3–21 nuclei per cell), 1–3 CBCs were detected. The CBCs in potato strains of AG-3 distinguish them from AG-3 tobacco strains and other AGs yielding further evidence that the potato strains of AG-3 originally described as R. solani are a species distinct from other AGs. The ITS1–5.8S–ITS2 sequences were analyzed by direct sequencing of PCR products from 497 strains of AG-3 isolated from potato. The same 10 and 4 positions in ITS1 and ITS2, respectively, contained variability in 425 strains (86%). Nine different unambiguous ITS sequences (haplotypes) could be detected in a single strain by sequencing cloned PCR products indicating that concerted evolution had not homogenized the rRNA cistrons in many AG-3 strains. Importantly, the sequence variability did not affect the secondary structure of ITS2 and CBCs in AG-3. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Molecular sequencing and analysis of soluble methane monooxygenase gene clusters from methanotroph <Emphasis Type="Italic">Methylomonas</Emphasis> sp<Emphasis Type="Italic">.</Emphasis> GYJ3

Summary A methanotroph Methylomonas sp. GYJ3 was isolated, whose sMMO genes and 16S rDNA were sequenced and analysed, demonstrating that the bacterium might be a type I methanotroph (γ-Proteobacteria) and was closer to Methylomonas sp. KSWIII/KSPIII. This result was consistent with the result previously determined by biochemistry and morphological taxonomy. Sequence comparison among six open reading frames and the deduced amino acid sequences of the sMMO genes from six strains revealed that the strain GYJ3 had highly conserved regions in MMOX with other strains, amounting to 78–99% homology at protein level and 71–97% homology at DNA level. Highly conserved sequences lay in two iron-binding regions. Furthermore, scanning electron microscopy of the strain GYJ3 showed rod shapes with a slightly bent configuration on the even surfaces and with plump bodies.     

Copper-resistant bacteria from industrial effluents and their role in remediation of heavy metals in wastewater

Six copper-resistant bacterial strains were isolated from wastewater of tanneries of Kasur and Rohi Nala. Two strains tolerated copper at 380 mg/L, four up to 400 mg/L. Three strains were identified as members of the genusSalmonella; one strain was identified asStreptococcus pyrogenes, one asVagococcus fluvialis and the last was identified asEscherichia coli. The pH and temperature optimum for two of them were 7.0 and 30 °C, respectively; four strains had corresponding optima at 7.5 and 37 °C, respectively. All bacterial isola-tes showed resistance against Ag⁺ (280–350 mg/L), Co²⁺ (200–420), Cr^VI (280–400), Cd²⁺ (250–350), Hg²⁺ (110–200), Mn²⁺ (300–380), Pb²⁺ (300–400), Sn²⁺ (480–520) and Zn²⁺ (300–450). Largesized plasmids (>20 kb), were detected in all of the strains. After the isolates were cured of plasmids with ethidium bromide, the efficiency of curing was estimated in the range of 60–90%. Reference strain ofE. coli was transformed with the plasmids of the bacterial isolates which grew in Luria-Bertani medium containing 100 mg/L Cu²⁺. The capability to adsorb and afterwards accumulate Cu²⁺ inside their cells was assayed by atomic absorption spectrophotometer; all bacterial cells had the ability to adsorb 50–80 % of the Cu²⁺ and accumulate 30–45 % Cu²⁺ inside them after 1 d of incubation.     

Screening for marine nanoplanktic microalgae from Greek coastal lagoons (Ionian Sea) for use in mariculture

Mediterranean mariculture uses imported strains of marine phytoplankton, raising questions of ecological risk and ability to adapt to local conditions for mass culture outdoors. In this context, we report here on the mass-culture potential and chemical composition of six strains of Prasinophyceae (five strains of Tetraselmis sp. and one Pyramimonas sp.) isolated from a Greek coastal lagoon. Proximate composition had a pattern of 10–20% ash, 35–65% protein, 6–10% lipids, and 25–45% other organics including carbohydrates. The amino acid profiles were typical for the marine representatives of the class. All strains had a high PUFA content with dominant the ω3 fraction in four of them. The fatty acid profiles indicated a Tetraselmis strain with high EPA (14%) and a Pyramimonas strain with high DHA (6%). These strains might be a good alternative for the common commercial strains used in Mediterranean aquaculture.     

<Emphasis Type="Italic">Singulispaera mucilagenosa</Emphasis> sp. nov., a novel acid-tolerant representative of the order <Emphasis Type="Italic">Planctomycetales</Emphasis>

Two novel strains of budding bacteria, Z-0071^T and Z-0072, were isolated from dystrophic humified waters formed by xylotrophic fungi in the course of spruce wood degradation. The cells of both strains are coccoid (0.95–1.80 μm), nonmotile, single or arranged in pairs. The cells have a complex system of intracellular membranes and are covered with fimbriae and surrounded by a mucous capsule up to 0.3 μm thick. Both strains are aerobic organoheterotrophic, mesophilic, and acid-tolerant microorganisms that are able to grow under microaerobic conditions. They utilize N-acetyl-glucosamine, carbohydrates, and lactate as growth substrates. The strains grow in a pH range of 4.0–7.5 with an optimum at 6.0–6.5. The temperature range for growth is 4–30°C, with an optimum at 25–28°C. Strains Z-0071^T and Z-0072, inhabitants of dystrophic low-mineral waters, are NaCl-sensitive: the NaCl content in the media above 0.5 g/l inhibited growth. The main fatty acids of strains Z-0071^T and Z-0072 are C_16:0, C_18:1ω9c, and C_{18:2ω9c, 12c}. The DNA G + C base content is 51.2–51.7 mol %. The sequences of the 16S rRNA gene fragments (1310 bp) of strains Z-0071^T and Z-0072 were found to be identical. The obtained sequences showed a 94.3% similarity with the sequences of the type strain of the most closely related species Singulisphaera acidiphila MOB10≅T. The phenotypic and phylogenetic properties of strains Z-0071^T and Z-0072 support classification of these strains within the genus Singulisphaera as a new species Singulisphaera mucilagenosa sp. nov., with the type strain Z-0071^T (VKM B-2626).     

Isolation and characterization of homocholine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strains A9 and B9b

Soil isolates, identified as Pseudomonas sp. strain A9 and Pseudomonas sp. strain B9b (based on the phenotypic features and phylogenetic analysis) were found to degrade homocholine aerobically. Morphological characterization using the optical microscope under light and phase contrast conditions showed that cells of strain A9 formed short rods measuring approximately 0.5–1 × 1.5–2.0 μm in size while those of B9b formed long rods of 0.5–1 × 2.5–3.0 μm during the early growth phase on both nutrient broth and basal-homocholine (basal-HC) media. Strain A9 was able to grow on basal-HC medium at a wide range of temperatures (4–41°C) whereas strain B9b was not able to grow at either 4 or 41°C. Comparative 16S rRNA sequencing studies indicated that strain A9 fell into the Pseudomonas putida subclade whereas strain B9b located in Pseudomonas fulva subclade. Washed cells of strains A9 and B9b degraded homocholine completely within 6 h with concomitant formation of several metabolites. Analysis of the metabolites by capillary electrophoresis, fast atom bombardment–mass spectrometry, and gas chromatography–mass spectrometry, showed trimethylamine (TMA) as the major metabolite beside β-alanine betaine and trimethylaminopropionaldehyde. Therefore, the possible degradation pathway of homocholine in the isolated strains is through successive oxidation of the alcohol group (–OH) to aldehyde (–CHO) and acid (–COOH), and thereafter the cleavage of β-alanine betaine C–N bonds yielding trimethylamine and an alkyl chain.     

Rapid biodegradation and decolorization of Direct Orange 39 (Orange TGLL) by an isolated bacterium <Emphasis Type="Italic">Pseudomonas aeruginosa</Emphasis> strain BCH

A newly isolated novel bacterium from sediments contaminated with dyestuff was identified as Pseudomonas aeruginosa strain BCH by 16S rRNA gene sequence analysis. The bacterium was extraordinarily active and operative over a wide rage of temperature (10–60°C) and salinity (5–6%), for decolorization of Direct Orange 39 (Orange TGLL) at optimum pH 7. This strain was capable of decolorizing Direct Orange 39; 50 mg l⁻¹ within 45 ± 5 min, with 93.06% decolorization, while maximally it could decolorize 1.5 g l⁻¹ of dye within 48 h with 60% decolorization. Analytical studies as, UV–Vis spectroscopy, FTIR, HPLC were employed to confirm the biodegradation of dye and formation of new metabolites. Induction in the activities of lignin peroxidases, DCIP reductase as well as tyrosinase was observed, indicating the significant role of these enzymes in biodegradation of Direct Orange 39. Toxicity studies with Phaseolus mungo and Triticum aestivum revealed the non-toxic nature of degraded metabolites.     

Nicotine degradation by two novel bacterial isolates of <Emphasis Type="Italic">Acinetobacter</Emphasis> sp. TW and <Emphasis Type="Italic">Sphingomonas</Emphasis> sp. TY and their responses in the presence of neonicotinoid insecticides

Two novel nicotine-degrading bacterial strains were isolated from tobacco waste and identified as Acinetobacter sp. TW and Sphingomonas sp. TY based on morphology, physiological and biochemical tests, Biolog analysis and 16S rDNA sequencing. The 16S rDNA sequences have been deposited in GenBank under the accession numbers FJ753401 for TW and FJ754274 for TY. The best culture conditions for nicotine degradation were 25–37°C and pH 7.0–8.0 for strain TW and 25–30°C and pH 6.0–7.0 for strain TY. Under the best conditions, the cell growth and nicotine-degradation kinetics of the two isolates were assessed, and 1.0 g/l nicotine was completely degraded within 12 and 18 h for TW and TY, respectively. Moreover, the presence of four widely-used commercial neonicotinoid insecticides in the medium had no effects on nicotine degradation by TW; among the four tested neonicotinoids, only thiamethoxam significantly delayed nicotine degradation by TY. TW and TY were also able to degrade selected neonicotinoids. This is the first report of nicotine degradation by Acinetobacter sp. and Sphingomonas sp. This study showed that these two newly isolated bacteria may be suitable for the disposal of tobacco waste and the reduction of nicotine in tobacco leaves.     

<Emphasis Type="Italic">Marinobacter zhanjiangensis</Emphasis> sp. nov., a marine bacterium isolated from sea water of a tidal flat of the South China Sea

A novel Gram-negative, catalase- and oxidase-positive, non-sporulating, rod-shaped, aerobic bacterium, designated strain JSM 078120^T, was isolated from sea water collected from a tidal flat of Naozhou Island, South China Sea. Growth occurred with 1–15% (w/v) total salts (optimum, 2–4%), at pH 6.0–10.0 (optimum, pH 7.5) and at 4–35°C (optimum, 25–30°C). The major cellular fatty acids were C_18:1 ω9c, C_16:0, C_12:0 3-OH and C_16:1 ω7c. The predominant respiratory quinone was ubiquinone Q-9, and the genomic DNA G + C content was 60.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences indicated that strain JSM 078120^T should be assigned to the genus Marinobacter, being related most closely to the type strains of Marinobacter segnicrescens (sequence similarity 98.2%), Marinobacter bryozoorum (97.9%) and Marinobacter gudaonensis (97.6%). The sequence similarities between the novel isolate and the type strains of other recognized Marinobacter species ranged from 96.7 (with Marinobacter salsuginis) to 93.3% (with Marinobacter litoralis). The levels of DNA–DNA relatedness between strain JSM 078120^T and the type strains of M. segnicrescens, M. bryozoorum and M. gudaonensis were 25.3, 20.6 and 18.8%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 078120^T represents a novel species of the genus Marinobacter, for which the name Marinobacter zhanjiangensis sp. nov. is proposed. The type strain is JSM 078120^T (= CCTCC AB 208029^T = DSM 21077^T = KCTC 22280^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 078120^T is FJ425903.     

<Emphasis Type="Italic">Rhizobium sphaerophysae</Emphasis> sp. nov., a novel species isolated from root nodules of <Emphasis Type="Italic">Sphaerophysa salsula</Emphasis> in China

Four gram-negative, aerobic, motile, non-spore, forming rods with a wide pH and temperature range for growth (pH 7.0–11.0, optimum pH 8.0; 20–45°C, optimum 28°C) strains were isolated from root nodules of Sphaerophysa salsula and characterized by means of a polyphasic approach. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the four strains formed a new lineage related to the genus Rhizobium and the sequence similarities between the isolate and the most related type strain Rhizobium giardinii was 96.5%. These strains also formed a distinctive group from the reference strains for defined Rhizobium species based on housekeeping gene sequences (atpD and recA), BOX-PCR fingerprinting, phenotypic features and symbiotic properties. The representative strain CCNWGS0238^T has DNA-DNA relatedness of less than 33.4% with the most closely related species R. giardinii. It is therefore proposed as a new species, Rhizobium sphaerophysae sp. nov., with isolate CCNWGS0238^T (=ACCC17498^T = HAMBI3074^T) as the type strain.     

<Emphasis Type="Italic">Virgibacillus litoralis</Emphasis> sp. nov., a moderately halophilic bacterium isolated from saline soil

A Gram-positive, moderately halophilic, endospore-forming, catalase- and oxidase-positive, motile, rod-shaped, aerobic bacterium, designated strain JSM 089168^T, was isolated from saline soil collected from Naozhou Island, Leizhou Bay, South China Sea. The organism was able to grow with 2–25% (w/v) total salts (optimum, 5–10%), at pH 6.0–10.0 (optimum, pH 8.0) and 10–45°C (optimum, 30°C). meso-Diaminopimelic acid was present in the cell-wall peptidoglycan. The strain contained MK-7 as the predominant menaquinone, and diphosphatidylglycerol and phosphatidylglycerol as the major polar lipids. The major cellular fatty acids were anteiso-C_15:0, iso-C_15:0 and anteiso-C_17:0, and the DNA G + C content was 40.2 mol%. Phylogenetic analysis based on 16S rRNA gene sequence comparisons revealed that strain JSM 089168^T should be assigned to the genus Virgibacillus, being related most closely to the type strains of Virgibacillus carmonensis (sequence similarity 97.6%), Virgibacillus necropolis (97.3%) and Virgibacillus halodenitrificans (97.1%). Levels of DNA–DNA relatedness between strain JSM 089168^T and the type strains of V. carmonensis, V. necropolis and V. halodenitrificans were 20.4, 14.3 and 12.0%, respectively. The combination of phylogenetic analysis, DNA–DNA relatedness, phenotypic characteristics and chemotaxonomic data supported the view that strain JSM 089168^T represents a novel species of the genus Virgibacillus, for which the name Virgibacillus litoralis sp. nov. is proposed. The type strain is JSM 089168^T (=DSM 21085^T =KCTC 13228^T). The GenBank/EMBL/DDBJ accession number for the 16S rRNA gene sequence of strain JSM 089168^T is FJ425909.     

Molecular and biological characterization of native <Emphasis Type="Italic">Bacillus thuringiensis</Emphasis> strains for controlling tomato leafminer (<Emphasis Type="Italic">Tuta absoluta</Emphasis> Meyrick) (Lepidoptera: Gelechiidae) in Colombia

Twenty-eight soil samples were obtained from open fields and greenhouses used for tomato cultivation in various regions of Colombia. For functional characterization, 99 Bacillus thuringiensis (Bt) strains were isolated and characterized by abundance and morphology of microscopic crystals, SDS–PAGE of protein extracts and M-PCR analyses of genes of the cry1 family, as well as for their insecticidal activity against Tuta absoluta second instar larvae. Native Bt strains had amorphous (5%), bi-pyramidal (27%), square (8%), spherical (38%) and triangular (22%) crystal forms. Based on the presence of 1–4 different crystal forms, 18 different profiles were established. The SDS–PAGE analyses of protein extracts established ten different strain groups based on their protein band weight and potential biological activity. The M-PCR technique identified 35 native Bt strains based on the presence of the 6 genes cry1Aa, cry1Ab, cry1Ac, cry1B, cry1C and cry1D, whose frequency of occurrence was 76, 26, 21, 35, 32 and 8.8%, respectively. Thirteen different PCR profiles were found in native Bt strains. Several gene combinations tended to co-occur with elevated frequency, such as the pairs cry1Ac/cry1C, cry1Ab/cry1Ac and cry1Ab/cry1B, for which Pearson correlation coefficients were 0.69, 0.52 and 0.54, respectively. Native strains ZBUJTL39 and ZCUJTL11 had up to three times higher biological activity against T. absoluta second instar larvae than the reference strain Bt var. kurstaki HD1, with an LD₅₀ of 2.4 μg/ml (P < 0.05) for native Bt strain ZCUJTL11. This study suggests a high biodiversity of native Bt strains from tomato growing regions in Colombia, which has important implications for designing biological control strategies for T. absoluta.     

<Emphasis Type="Italic">Oceanobacillus manasiensis</Emphasis> sp. nov., a moderately halophilic bacterium isolated from the salt lakes of Xinjiang,China

Three Gram reaction positive, rod-shaped, moderately motile halophilic bacterial strains, designated YD3-56^T, YD16, and YH29, were isolated from the sediments of Manasi and Aiding salt lakes in the Xinjiang region of China, respectively. The strains grew optimally at 30–37°C, pH 8–11, in the presence of 5–10% (w/v) NaCl. Phylogenetic analysis based on 16S rRNA gene sequences revealed that the strains were closely related to members of the genus Oceanobacillus, exhibiting 99.1–99.2% similarity to O. kapialis KCTC 13177^T, 99.2–99.3% to O. picturae KCTC 3821^T, and 94.2–96% sequence similarity to other described Oceanobacillus species. SDS-PAGE of whole cell proteins preparations demonstrated that the strains exhibited high similarity to each other, but distinguished from O. kapialis KCTC 13177^T and O. picturae KCTC 3821^T (75%). DNA-DNA hybridization revealed that the similarity between the representative strain YD3-56^T and O. kapialis KCTC 13177^T was 35.3%, and the similarity between YD3-56^T and O. picturae KCTC 3821^T was 22.3%. Chemotaxonomic analysis of the strains showed menaquinone-7 was the predominant respiratory quinine. Major cellular fatty acids were anteiso-C_15:0 and anteiso-C_17:0. The polar lipid pattern for strain YD3-56^T predominantly contained phosphatidylcholine, and trace to moderate amounts of phosphatidyl ethanolamine and hydroxy-phosphatidyl ethanolamine. The diamino acid in murein was meso-diaminopimelic acid. The DNA G+C content of the strains was 39.7–40.1 mol%. On the basis of these results, the three strains should be classified as a novel species of the genus Oceanobacillus, for which the name Oceanobacillus manasiensis sp. nov. has been proposed, with the type strain as YD3-56^T (=CGMCC 1.9105^T =NBRC 105903^T).