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Groundwater sampling is a critical step in subsurface microbial ecology. Here, we compared two different sampling methods: commonly used disposable bailers (unimproved sampler) and an impro...

While meteoric water recharge is known to stimulate biogenic methane formation in shale and coal seams, the underlying mechanisms are currently unresolved. To this end, we conducted fieldwork in the Tempoku Coal Field, Japan. Pore water in core samples and well water in boreholes were analyzed for dissolved components and isotopic compositions. The hydraulic gradient was determined using values of borehole hydraulic head. Cl− concentrations (38 mg L−1 to 16,600 mg L−1), δ18O(H2O) values (−10.5‰ to −2.4‰), and δD(H2O) values (−71.6‰ to −17.8‰) increased with depth (

A novel mesophilic sulfate-reducing bacterium, strain HN2T, was isolated from groundwater sampled from the subsurface siliceous mudstone of the Wakkanai Formation located in Horonobe, Hokkaido, Japan. The bacterium was Gram-negative and vibrio-shaped, and its motility was conferred by a single polar flagellum. Cells had desulfoviridin. Catalase and oxidase activities were not detected. It grew in the temperature range of 25–40 °C (optimum, 35 °C) and pH range of 6.3–8.1 (optimum, pH 7.2–7.6). It used sulfate, thiosulfate, dimethyl sulfoxide, anthraquinone-2,6-disulfonate, Fe3+, and manganese oxide, but not elemental sulfur, nitrite, nitrate, or fumarate as electron acceptors. The strain showed weak growth with sulfite as the electron acceptor. Fermentative growth with pyruvate, lactate and cysteine was observed in the absence of sulfate, but not with malate or fumarate. NaCl was not required, but the strain tolerated up to 40 g l–1. Strain HN2T did not require vitamins. The major cellular fatty acids were iso-C15 : 0 (23.8 %), C18 : 1 ω9t (18.4 %), C18 : 0 (15.0 %), C16 : 0 (14.5 %), and anteiso-C17 :0 (10.1 %). The major respiratory quinone was menaquinone MK-6(H2). The G+C content of the genomic DNA was 56.7 mol%. Based on 16S rRNA gene sequence analysis, the closest phylogenetic relative of strain HN2T is Desulfovibrio psychrotolerans JS1T (97.0 %). Digital DNA–DNA hybridization (dDDH) and average nucleotide identity (ANI) values of the strains HN2T and D. psychrotolerans JS1T were 22.2 and 79.8 %, respectively. Based on the phenotypic and molecular genetic evidence, we propose a novel species, D. subterraneus sp. nov. with the type strain HN2T (=DSM 101010T=NBRC 112213T).

A microbial consortium associated with coal from the coal-bearing Soya Formation in the Tempoku Coalfield (northern Hokkaido, Japan) was cultivated with reaction solutions of lignite and hydrogen peroxide (H2O2) (i.e., chemically solubilized lignite) to evaluate in situ biogenic methane generation. Column experiments with H2O2 flowing through pulverized lignite achieved maximum concentrations of dissolved organic carbon, acetic acid, and formic acid of 6330, 612, and 1810 mg/L, respectively. Cultivation experiments using the above reaction solution as a substrate for methanogens produced nearly 6 cm3 CH4 per g lignite with a maximum rate of 0.14 cm3 per g per day. Pyrosequencing analysis of the microbial consortium after cultivation confirmed the presence of methanogens, such as Methanoregulaceae and Methanosarcinaceae, as the dominant archaea in the culture. The observed production of biogenic methane from a reaction solution of lignite and H2O2 under the simulated conditions indicates the potential for successful in situ microbially enhanced coalbed methane generation using H2O2.

Future world energy consumption is projected to increase along with the high economic growth of emerging countries. This will make it necessary to expand the development of unconventional resources such as shale oil and gas and coalbed methane. The production of secondary methane gas using microorganisms at oil fields and coal mines is another area of great interest in the development of unconventional resources. This paper describes a feasibility study involving a new method to convert unused organic matter contained in sedimentary rock in northern Hokkaido into biogenic methane. The new method, subsurface cultivation and gasification (SCG), is proposed for the production of biogenic methane gas in the subsurface environment. Our approach uses hydrogen peroxide (H2O2) to decompose organic matter rapidly into usable substrates for methanogens. Two wells were drilled through several lignite seams in the Soya coal-bearing formations in the Tempoku coalfield in northern Hokkaido, Japan, and lignite cores were recovered. Batch tests using a 1 wt% H2O2 solution were performed on the lignite to estimate the amount of low-molecular-weight organic acids as substrates for methanogens. Oxidative decomposition of the lignite produced a high yield of low-molecular-weight organic acids. Moreover, it was confirmed that methanogens converted the H2O2 reaction solution containing organic acids into methane. H2O2 would be useful for effective SCG at lignite seams, and conversion of organic matter from lignite into biogenic methane with the help of microorganisms is expected to be highly profitable. The results reveal that using SCG to produce biogenic methane in this coalfield shows great commercial promise.

Geobacter spp., regarded as strict anaerobes, have been reported to grow under aerobic conditions. To elucidate the role of fatty acids in aerobiosis of Geobacter spp., we studied the effect of aerobiosis on fatty acid composition and turnover in G. bemidjiensis BemT. G. bemidjiensis BemT was grown under the following different culture conditions: anaerobic culture for 4 days (type 1) and type 1 culture followed by 2-day anaerobic (type 2) or aerobic culture (anaerobic-to-aerobic shift; type 3). The mean cell weight of the type 3 culture was approximately 2.5-fold greater than that of type 1 and 2 cultures. The fatty acid methyl ester and hydrocarbon fraction contained hexadecanoic (16:0), 9-cis-hexadecenoic [16:1(9c)], tetradecanoic (14:0), tetradecenoic [14:1(7c)] acids, hentriacontanonaene, and hopanoids, but not long-chain polyunsaturated fatty acids. The type 3 culture contained higher levels of 14:0 and 14:1(7c) and lower levels of 16:0 and 16:1(9c) compared with type 1 and 2 cultures. The weight ratio of extracted lipid per dry cell was lower in the type 3 culture than in the type 1 and 2 cultures. We concluded that anaerobically-grown G. bemidjiensis BemT followed by aerobiosis were enhanced in growth, fatty acid turnover, and de novo fatty acid synthesis.

Since the beginning of the 21st century, various works have sought to enhance coalbed methane (CBM) production by stimulating indigenous microbes in coal seams. This concept of microbially enhanced coalbed methane (MECBM) generation relies greatly on the solubilization of the coal, which determines the rate of microbial conversion of the coal into biogenic methane. This study examined the potential of using dilute hydrogen peroxide (H2O2) (0.3%) to solubilize lignite at common subsurface temperatures (10–50 °C) via batch experiments at a solid/liquid ratio of 1 g/150 mL. Dissolved organic carbon (maximum 450 mg C/L) and organic acid (formic, acetic, oxalic, malonic, and succinic acid, maximum total of 145 mg C/L) concentrations reached their maximum levels within periods of 1.2 days (at 50 °C) to 45 days (at 10 °C). H2O2 consumption was a first-order reaction with respect to its concentration, and the linear (r = 0.998) Arrhenius plot for the rate constants yielded its activation energy to be 73 kJ/mol. If the methanogens convert acetic and formic acids to methane, under the current experimental conditions they can produce about 6.0 m3 of methane per ton of lignite. Dilute H2O2 would thus be useful for effective MECBM production at lignite seams. The aromatic-C content of the lignite markedly decreased by about 30% during the reaction, implying high reactivity of catechol structures in the lignite with H2O2.

We have characterized a member of the profilin (PROF) family protein as a common antigen in three pathogens-Babesia bovis (B. bovis), Babesia bigemina (B. bigemina), and Babesia microti (B. microti)-and evaluated its immunogenic and cross-protective properties against a challenge infection with B. microti in BALB/c mice. The recombinant PROF proteins of B. bovis, B. bigemina, and B. microti were successfully expressed in Escherichia coli (E. coli) as soluble GST fusion proteins (rBboPROF, rBbigPROF, and rBmPROF, respectively), and they were found to be antigenic. On probing with mouse anti-rPROF serum, green fluorescence was observed on the parasites' cytosols by confocal laser microscopy. Immunization regimes in BALB/c mice using rPROFs induced cross-protective immunity against B. microti infection based on high levels of cytokines and immunoglobulin (IgG) titers, a reduction in peak parasitemia levels, and earlier clearance of the parasite as compared with control mice. The findings of the present study indicate that PROF is a common antigen among bovine and murine Babesia parasites, and it might be used as a common vaccine candidate against babesiosis.

Kushiro Coalfield is one of the largest coalfields in Japan, and Kushiro Coal Mine (KCM) is currently the only underground coal mine in the country. At KCM, coalbed methane (CBM) has been extracted from the Eocene Harutori Formation and water-dissolved gas from the Paleocene Shiomi Formation. This study investigates the origin of these gases and their formation environments. A positive correlation between δD(CH4) of the sampled gas and δD(H2O) of the groundwater, following δD(CH4) = δD(H2O) − 180 ± 20, indicates that CBM and water-dissolved gas in KCM are classified as secondary biogenic methane and were formed within in situ groundwater. The lower δ13C(CO2) values (less than −6.9‰) from KCM compared with representative CBM of biogenic origin from other coalfields imply the contribution of oxidants (e.g., SO42−) in the biodegradation of sedimentary organic matter (SOM) to CO2 in KCM. Conversely, high δ13C(CaCO3) values (>12.5‰) of calcite deposited in the coal seams of KCM imply negligible contributions of oxidants for SOM biodegradation when the calcite was formed. The methanogenic conditions at the time of calcite formation may have arisen when the rate of groundwater flow in the coal seams was higher than at present.

The effect of oxidative weathering on the composition of sedimentary organic matter (SOM) of lignite, subbituminous coal, quartzose arenite, siliceous mudstone, siliceous shale and diatomaceous shale was investigated in Hokkaido, Japan, in a subarctic humid climate. For 15 samples in total, a humic substance extraction method was applied to divide SOM into lipid-C, humin-C and alkali soluble-C. The alkali soluble-C was further divided into fulvic acid (FA)-C, humic acid (HA)-C and a smaller molecular size fraction (SMSF)-C, which comprised organic molecules able to pass through a dialysis membrane (

application/pdf, Oocyst stage of Toxoplasma gondii is characterized by a durable wall that confers a strong protection to this protozoan parasite in face of harsh environmental conditions. Thus, it is considered the key for transmission of T. gondii. Analysis of oocyst wall composition is mandatory therefore; the aim of this study was to identify novel T. gondii oocyst wall proteins and test their use in detection of these oocysts in environmental samples. Five candidates of novel T. gondii oocyst wall proteins (TgOWPs) were identified and named TgOWP8 through TgOWP12. Recombinant protein of TgOWP8 was expressed in E. coli using glutathione S-transferase as fusion protein. Polyclonal antibody was produced and validated by indirect immunofluorescence antibody assay (IFA). For detection by IFA, we used different methods for fixation and permeabilization of oocysts to improve the antigen-antibody detection. Specificity to wall of T. gondii oocyst was confirmed and revealed absence of cross reactivity with bradyzoite cyst wall and tachyzoites. Although some TgOWPs were identified previously, our study represents a continuation of molecular investigations of oocyst wall proteins as an essential structure for the longevity and infectivity of this stage and also provided new trial to improve T. gondii oocysts detection. c 2017 The Authors

Bacterial utilization of humic acids (HAs) was examined under aerobic conditions using Pseudomonas spp. from diatomite from a depth of 250 m below ground level in the Horonobe Underground Research Laboratory. HA decolorization and bacterial aggregation were observed during cultivation when an auxiliary carbon source was added. High-performance size-exclusion chromatography showed that high-molecular-weight HAs were produced. Fourier transform infrared spectroscopy indicated that carboxyl groups and polysaccharide-related substances in HAs were eliminated, while aliphatic structural units and amide groups were added to HAs. These results suggested that Pseudomonas spp. utilize and alter the molecular structure of HAs under aerobic conditions caused by the construction of underground facilities.

A methanogenic organism from the domain Archaea , designated strain T10T, was isolated from groundwater sampled from a deep diatomaceous shale formation located in Horonobe, Hokkaido, Japan. The strain utilized H2/CO2 and formate as substrates for methanogenesis. Cells were strictly anaerobic, Gram-negative-staining, flagellated, irregular coccoids, 0.7–1.6 µm in diameter, and occurred singly. The strain grew at 25–45 °C (optimum 37–42 °C), at pH 5.8–8.2 (optimum pH 6.7–6.8) and in the presence of 0–1.3 M NaCl (optimum 0.1–0.2 M NaCl). The G+C content of the genomic DNA was 62.9 mol%. 16S rRNA gene sequencing revealed that, although the strain is a member of the genus Methanoculleus , it clearly differed from all described species of this genus (95.5–98.3 % sequence similarity). Values for DNA–DNA hybridization with type strains of closely related Methanoculleus species were less than 50 %. Phenotypic and phylogenetic features of strain T10T clearly indicate that it represents a novel species of the genus Methanoculleus , for which the name Methanoculleus horonobensis sp. nov. is proposed. The type strain is T10T ( = DSM 21626T = JCM 15517T).

Host cell invasion by apicomplexan parasites driven by gliding motility and empowered by actin-based movement is essential for parasite survival and pathogenicity. The parasites share a conserved invasion process: actin-based motility led by the coordination of adhesin-cytoskeleton via aldolase. A number of studies of host cell invasion in the Plasmodium species and Toxoplasma gondii have been performed. However, the mechanisms of host cell invasion by Babesia species have not yet been studied. Here, we show that Babesia gibsoni aldolase (BgALD) forms a complex with B. gibsoni thrombospondin-related anonymous protein (BgTRAP) and B. gibsoni actin (BgACT), depending on tryptophan-734 (W-734) in BgTRAP. In addition, actin polymerization is mediated by BgALD. Moreover, cytochalasin D, which disrupts actin polymerization, suppressed B. gibsoni parasite growth and inhibited the host cell invasion by parasites, indicating that actin dynamics are essential for erythrocyte invasion by B. gibsoni. This study is the first molecular approach to determine the invasion mechanisms of Babesia species.

Geobacter spp., regarded as strict anaerobes, have been reported to grow under aerobic conditions. To elucidate the role of fatty acids in aerobiosis of Geobacter spp., we studied the effect of aerobiosis on fatty acid composition and turnover in G. bemidjiensis Bem

Decomposition of humic substances (HSs) is a slow and cryptic but non-negligible component of carbon cycling in sediments. Aerobic decomposition of HSs by microorganisms in the surface environment has been well documented; however, the mechanism of anaerobic microbial decomposition of HSs is not completely understood. Moreover, no microorganisms capable of anaerobic decomposition of HSs have been isolated. Here, we report the anaerobic decomposition of humic acids (HAs) by the anaerobic bacterium Clostridium sp. HSAI-1 isolated from the deep terrestrial subsurface. The use of 14C-labelled polycatechol as an HA analogue demonstrated that the bacterium decomposed this substance up to 7.4% over 14 days. The decomposition of commercial and natural HAs by the bacterium yielded lower molecular mass fractions, as determined using high-performance size-exclusion chromatography. Fourier transform infrared spectroscopy revealed the removal of carboxyl groups and polysaccharide-related substances, as well as the generation of aliphatic components, amide and aromatic groups. Therefore, our results suggest that Clostridium sp. HSAI-1 anaerobically decomposes and transforms HSs. This study improves our understanding of the anaerobic decomposition of HSs in the hidden carbon cycling in the Earth’s subsurface.

The apicoplast housekeeping machinery, specifically apicoplast DNA replication, transcription, and translation, was targeted by ciprofloxacin, thiostrepton, and rifampin, respectively, in the in vitro cultures of four Babesia species. Furthermore, the in vivo effect of thiostrepton on the growth cycle of Babesia microti in BALB/c mice was evaluated. The drugs caused significant inhibition of growth from an initial parasitemia of 1% for Babesia bovis , with 50% inhibitory concentrations (IC 50 s) of 8.3, 11.5, 12, and 126.6 μM for ciprofloxacin, thiostrepton, rifampin, and clindamycin, respectively. The IC 50 s for the inhibition of Babesia bigemina growth were 15.8 μM for ciprofloxacin, 8.2 μM for thiostrepton, 8.3 μM for rifampin, and 206 μM for clindamycin. The IC 50 s for Babesia caballi were 2.7 μM for ciprofloxacin, 2.7 μM for thiostrepton, 4.7 μM for rifampin, and 4.7 μM for clindamycin. The IC 50 s for the inhibition of Babesia equi growth were 2.5 μM for ciprofloxacin, 6.4 μM for thiostrepton, 4.1 μM for rifampin, and 27.2 μM for clindamycin. Furthermore, an inhibitory effect was revealed for cultures with an initial parasitemia of either 10 or 7% for Babesia bovis or Babesia bigemina , respectively. The three inhibitors caused immediate death of Babesia bovis and Babesia equi . The inhibitory effects of ciprofloxacin, thiostrepton, and rifampin were confirmed by reverse transcription-PCR. Thiostrepton at a dose of 500 mg/kg of body weight resulted in 77.5% inhibition of Babesia microti growth in BALB/c mice. These results implicate the apicoplast as a potential chemotherapeutic target for babesiosis.

application/pdf, Here we report on an epidemiological study regarding the molecular prevalence of different genotypes of Theileria orientalis present among domestic cattle and water buffalo populations bred in Thailand. A phylogenetic analysis based on the parasitic gene encoding a major piroplasm surface protein revealed the presence of 5 genotypes (Types 1, 3, 5, 7, and N-3) in cattle and 7 genotypes (Types 1, 3, 4, 5, 7, N-2, and N-3) in water buffaloes. Types 4, 7, and N-3 of T. orientalis were reported for the first time in water buffaloes. The previously reported C and Thai types from Thailand clustered as types 7 and 6, respectively, in the present analysis. Great similarities were observed among nucleotide sequences of isolates of the same genotype from cattle and water buffaloes, and, therefore, water buffaloes were considered to serve as a reservoir for these genotypes of T. orientalis in Thailand. In conclusion, T. orientalis parasites circulating in Thailand are more diverse in their genetic characters than previously anticipated.

application/pdf, Theileria orientalis is a benign bovine protozoan parasite that occasionally causes serious economic loss in the livestock industry. We report the findings of a molecular epidemiological survey of T. orientalis in 94 Vietnamese yellow cattle, 43 water buffaloes, 21 sheep, 21 goats and 85 blood-sucking ticks of cattle in the Thua Thien Hue province of Vietnam. The major piroplasm surface protein (MPSP) gene of T. orientalis was detected using polymerase chain reaction from 13 cattle (13.8%), 11 water buffaloes (25.6%), 1 sheep (4.8%) and 9 ticks (10.6%). Phylogenetic analysis using MPSP gene sequences showed the presence of seven genotypes, four previously categorized genotypes (Types 1, 3, 5 and 7) and three new genotypes (Types N-1, N-2 and N-3).

The Toxoplasma gondii deoxyribose phosphate aldolase-like (TgDPA) gene is expressed predominantly in bradyzoites. This finding allowed us to infer that TgDPA is important in the tachyzoite-to-bradyzoite development or maintenance of cyst structure although the function of this gene is still unknown. We conducted yeast two-hybrid screening to identify proteins interacting with TgDPA, and the actin depolymerizing factor (TgADF) gene was obtained. Co-immunoprecipitation and a GST pull-down assay demonstrated that TgDPA interacts with TgADF. To reveal the significance of the protein-protein interaction between TgDPA and TgADF, actin polymerization and disassembly kinetics were examined. Addition of GST-TgDPA to TgADF lowered the extent of actin polymerization and enhanced the filamentous actin disassembly. These results demonstrated that this is the novel protein-protein interaction in T. gondii, and that TgDPA can enhance the activity of TgADF. This phenomenon might play an important role in T. gondii bradyzoites by affecting the actin turnover.

For the last couple of years, a method that permits the collection of precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high-throughput manner was established. We applied this novel method, 'tss-seq', to elucidate the transcriptome of tachyzoites of the Toxoplasma gondii, which resulted in the identification of 124,000 TSSs, and they were clustered into 10,000 transcription regions (TRs) with a statistics-based analysis. The TRs and annotated ORFs were paired, resulting in the identification of 30% of the TRs and 40% of the ORFs without their counterparts, which predicted undiscovered genes and stage-specific transcriptions, respectively. The massive data for TSSs make it possible to execute the first systematic analysis of the T. gondii core promoter structure, and the information showed that T. gondii utilized an initiator-like motif for their transcription in the major and novel motif, the downstream thymidine cluster, which was similar to the Y patch observed in plants. This encyclopaedic analysis also suggested that the TATA box, and the other well-known core promoter elements were hardly utilized.

When pDHA4, a vector carrying all five pfaA – pfaE genes responsible for docosahexaenoic acid (DHA; 22:6) biosynthesis in Moritella marina MP-1, was coexpressed in Escherichia coli with the individual pfaA – pfaD genes for eicosapentaenoic acid (EPA; 20:5) biosynthesis from Shewanella pneumatophori SCRC-2738, both polyunsaturated fatty acids were synthesized only in the recombinant carrying pfaB for EPA synthesis. Escherichia coli coexpressing a deleted construct comprising pfaA, pfaC, pfaD and pfaE for EPA and pfaB for DHA produced EPA and DHA. Both EPA and DHA were detected in bacteria that inherently contained pfa genes for DHA. These results suggest that PfaB is the key enzyme determining the final product in EPA or DHA biosynthesis.

We developed a sandwich ELISA for the detection of circulating Toxoplasma gondii MIC10 antigens. In T. gondii culture supernatant, MIC10 was detected in a growth dependent manner. Mice were infected with a lethal dose of either a virulent RH strain, an avirulent Beverley strain or a sub-lethal dose of a PLK strain of T. gondii. MIC10 appeared 2 days after infection and increased gradually in the sera of RH-infected mice. A detectable but significantly lower amount of MIC10 was observed in the sera of mice infected intraperitoneally with Beverley tachyzoites. In contrast, the MIC10 antigen in mice sera following oral infection with Beverley cysts was below detectable levels during the course of the experiment. In sera of PLK-infected mice, MIC10 was predominantly observed between late acute and early chronic phase. Our data show that the kinetics of circulating MIC10 differs depending on the strain and route of infection.

Toxoplasma gondii, unlike its mammalian host, utilizes a type II fatty acid biosynthesis pathway in which the steps of fatty acid biosynthesis are catalyzed by independent enzymes. Due to this difference, the enzymes of this pathway are good targets for the development of new therapeutic drugs directed against toxoplasmosis. In this report, we show by using reverse transcription-polymerase chain reaction analysis that beta-Hydroxyacyl-acyl carrier protein dehydratase (TgFABZ) is expressed both in tachyzoites and bradyzoites. Indirect immunofluorescence antibody test further shows the localization of TgFABZ protein in the apicoplast of both tachyzoites and bradyzoites. Enzyme dynamic analysis shows that the purified recombinant TgFABZ protein is soluble and active. The Km value of the enzyme for its substrate analog crotonoyl-CoA was estimated to be 82.57 +/- 10 microM.

Trace elements were extracted from natural hijiki by heating with water (25-95 °C) or commercial hijiki by soaking, washing, and rinsing with water at room temperature. Eleven elements (As, B, Cd, Ce, Cu, Fe, Li, Sn, Tl, Y and Zn) were determined with ICP-MS in the acid-digested and diluted solutions of commercial/natural hijiki or in the water extracts of natural hijiki. Extractions were effective in removing metals in the range 55-65 °C. The traditional procedure of soaking and washing commercial hijiki was inadequate to sufficiently lower trace elements. The iron concentration in commercial hijiki was high, indicating a possible contribution from the iron cauldron used in producing commercial hijiki. Results showed unsuitability of the commercial production process for reducing trace elements sufficiently. To lower trace elements, a procedure involving two steps was proposed for preparing consumable hijiki. Hijiki may be recommended as a supplement for deficiencies of B, Fe, Cu and Zn.

A methanogenic archaeon, strain HC-2T, was isolated from a deep diatomaceous shale formation. The strain grew on methanol, monomethylamine, dimethylamine, trimethylamine and dimethylsulphide, but not on acetate, H2/CO2, formate, 2-propanol, 2-butanol or cyclopentanol. Cells were Gram-stain-negative, non-motile, and coccus-like, 0.9–1.4 µm in diameter, and occurred singly, in pairs, or as aggregates. The strain grew at 10–40 °C (optimum 35 °C), pH 5.9–7.4 (optimum pH 6.6–6.8) and in 0–0.6 M NaCl (optimum 0.1–0.2 M). The genomic DNA G+C content was 41.5 mol% and the 16S rRNA gene sequence was closely related to those of Methanosarcina lacustris DSM 13486T (99.1 %) and Methanosarcina siciliae DSM 3028T (98.3 %). Values for DNA–DNA hybridization with these strains were less than 30 %. The phenotypic and phylogenetic features of HC-2T indicate that it represents a novel species of the genus Methanosarcina , for which the name Methanosarcina subterranea sp. nov. is proposed. The type strain is HC-2T ( = DSM 22503T = JCM 15540T = NBRC 102578T).

Degradation of n-alkanes in diesel oil by Pseudomonas aeruginosa strain WatG (WatG) was verified in soil microcosms. The total petroleum hydrocarbon (TPH) degradation level in two bioaugmentation samples was 51% and 46% for 1 week in unsterilized and sterilized soil microcosms, respectively. The TPH degradation in the biostimulation was of control level (15%). The TPH degradation in aeration-limited samples was clearly reduced when compared with that in aeration-unlimited ones under both sterilized and unsterilized conditions. Addition of WatG into soil microcosms was accompanied by dirhamnolipid production only in the presence of diesel oil. These findings suggest that degradation of n-alkanes in diesel oil in soil microcosms would be facilitated by bioaugmentation of WatG, with production of dirhamnolipid, and also by participation of biostimulated indigenous soil bacteria.

Bacteria possessing high capacity to degrade gasoline, kerosene, diesel oil, and lubricating oil were screened from several areas of Hokkaido, Japan. Among isolates, two strains, WatG and HokM, which were identified as new strains of Pseudomonas aeruginosa and Serratia marcescens species, respectively, showed relatively high capacity and wide spectrum to degrade the hydrocarbons in gasoline, kerosene, diesel, and lubricating oil. About 90-95% of excess amount of total diesel oil and kerosene added to mineral salts media as a sole carbon source could be degraded by WatG within 2 and 3 weeks, respectively. The same amount of lubricating oil was 60% degraded within 2 weeks. Strain HokM was more capable than WatG in degrading aromatic compounds in gasoline. This strain could also degrade kerosene, diesel, and lubricating oil with a capacity of 50-60%. Thus, these two isolates have potential to be useful for bioremediation of sites highly contaminated with petroleum hydrocarbons.

We evaluated the microbial communities in acetate-rich production waters from separators of a high-temperature gas-petroleum reservoir in Higashi-Niigata, Japan. Bacterial and archaeal 16S rRNA gene libraries constructed from these waters were dominated by Acetobacterium-, Methanofollis-, and Methanosarcina-related sequences. The libraries constructed from enrichment cultures of the production waters were dominated by sequences related to the Acetobacterium- and Methanofollis-related sequences.

A 40-kbp DNA fragment was isolated from the cosmid library of Vibrio marinus strain MP-1. Among the 22 putative open reading frames (ORFs) in this fragment, ORFs 8, 9, 10 and 11 had high homology with ORFs 5, 6, 7 and 8 of the eicosapentaenoic acid biosynthesis gene cluster, respectively. Then, we speculate that these ORFs are responsible for docosahexaenoic acid biosynthesis in this bacterium.

A 9-hexadecenoic acid cis-trans isomerase (9-isomerase) that catalyzed the cis-to-trans isomerization of the double bond of free 9-cis-hexadecenoic acid [16:1(9c)] was purified to homogeneity from an extract of Pseudomonas sp. strain E-3 and characterized. Electrophoresis of the purified enzyme on both incompletely denaturing and denaturing polyacrylamide gels yielded a single band of a protein with a molecular mass of 80 kDa, suggesting that the isomerase is a monomeric protein of 80 kDa. The 9-isomerase, assayed with 16:1(9c) as a substrate, had a specific activity of 22.8 μmol h–1 (mg protein)–1 and a K m of 117.6 mM. The optimal pH and temperature for catalysis were approximately pH 7–8 and 30° C, respectively. The 9-isomerase catalyzed the cis-to-trans conversion of a double bond at positions 9, 10, or 11, but not that of a double bond at position 6 or 7 of cis-mono-unsaturated fatty acids with carbon chain lengths of 14, 15, 16, and 17. Octadecenoic acids with a double bond at position 9 or 11 were not susceptible to isomerization. These results suggest that 9-isomerase has a strict specificity for both the position of the double bond and the chain length of the fatty acid. The enzyme catalyzed the cis-to-trans isomerization of fatty acids in a free form, and in the presence of a membrane fraction it was also able to isomerize 16:1(9c) esterified to phosphatidylethanolamine. The 9-isomerase was strongly inhibited by catecholic antioxidants such as α-tocopherol and nordihydroguaiaretic acid, but was not inhibited by 1,10-phenanthroline or EDTA or under anoxic conditions. Based on these results, the possible mechanism of catalysis by this enzyme is discussed.

A gene encoding a Babesia bovis protein that shares significant degree of similarity to other apicomplexan thrombospondin-related anonymous proteins (TRAPs) was found in the genomic database and designated as BbTRAP2. Recombinant protein containing a conserved region of BbTRAP2 was produced in E. coli. A high antigenicity of recombinant BbTRAP2 (rBbTRAP2) was observed with field B. bovis-infected bovine sera collected from geographically different regions of the world. Moreover, antiserum against rBbTRAP2 specifically reacted with the authentic protein by Western blot analysis and an indirect fluorescent antibody test. Three bands corresponding to 104-, 76-, and 44-kDa proteins were identified in the parasite lysates and two bands of 76- and 44-kDa proteins were detected in the supernatant of cultivated parasites, indicating that BbTRAP2 was proteolytically processed and shed into the culture. Apical and surface localizations of BbTRAP2 were observed in the intracellular and extracellular parasites, respectively, by confocal laser microscopic examination. Moreover, native BbTRAP2 was precipitated by bovine erythrocytes, suggesting its role in the attachment to erythrocytes. Furthermore, the specific antibody to rBbTRAP2 inhibited the growth of B. bovis in a concentration-dependent manner. Consistently, pre-incubation of the free merozoites with the antibody to rBbTRAP2 resulted in an inhibition of the parasite invasion into host erythrocytes. Interestingly, the antibody to rBbTRAP2 was the most inhibitive for the parasite’s growth as compared to those of a set of antisera produced against different recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), rhoptry-associated protein 1 C-terminal (BbRAP-1CT), and spherical body protein 1 (BbSBP-1). These results suggest that BbTRAP2 might be a potential candidate for development of a subunit vaccine against B. bovis infection.

application/pdf, The nutritional and pharmaceutical values of long-chain polyunsaturated fatty acids (LC-PUFAs) such as arachidonic, eicosapentaenoic and docosahexaenoic acids have been well recognized. These LC-PUFAs are physiologically important compounds in bacteria and eukaryotes. Although little is known about the biosynthetic mechanisms and functions of LC-PUFAs in bacteria compared to those in higher organisms, a combination of genetic, bioinformatic, and molecular biological approaches to LC-PUFA-producing bacteria and some eukaryotes have revealed the notably diverse organization of the pfa genes encoding a polyunsaturated fatty acid synthase complex (PUFA synthase), the LC-PUFA biosynthetic processes, and tertiary structures of the domains of this enzyme. In bacteria, LC-PUFAs appear to take part in specific functions facilitating individual membrane proteins rather than in the adjustment of the physical fluidity of the whole cell membrane. Very long chain polyunsaturated hydrocarbons (LC-HCs) such as hentriacontanonaene are considered to be closely related to LC-PUFAs in their biosynthesis and function. The possible role of LC-HCs in strictly anaerobic bacteria under aerobic and anaerobic environments and the evolutionary relationships of anaerobic and aerobic bacteria carrying pfa-like genes are also discussed.

The intracellular protozoan parasite Toxoplasma gondii is capable of invading any nucleated cell and replicates within a parasitophorous vacuole (PV). This microenvironment is modified by secretory proteins from organelles named rhoptries and dense granules. In this report, we identify a novel dense granule protein, which we refer to as GRA22. GRA22 has no significant homology to any other known proteins. GRA22 possesses a signal peptide at the N-terminal end which is responsible for dense granule and PV localization. The RH strain GRA22 contains 12 copies of tandem repeats consisting each of 21 amino acids located between the 42nd and 293rd amino acid residues from a full length of 624 amino acids. On the other hand, ME49 strain GRA22 has 10 copies of tandem repeats. The Neospora caninum GRA22 ortholog completely lacks this repetitive sequence. GRA22 knock out parasites show a similar growth rate as the parental strain. However, the timing of egress is earlier than that of the parental strain. These results suggest that GRA22 is involved in regulating parasite egress in T. gondii.

The effect of inhibitors of histone deacetylase (HDAC) on Apicomplexa has been previously reported with the discovery of apicidin, a cyclic tetrapeptide having broad-spectrum antiparasitic activity. In the current study, we expressed Babesia bovis (B. bovis) recombinant-HDAC 3 (rBbHDAC3) as a GST-fusion protein in Escherichia coli (E. coli) and found that it was antigenic. An antiserum against the recombinant protein was generated in mice. The mice serum demonstrated the presence of HDAC in B. bovis by a Western blot assay. The murine anti-rBbHDAC3 reacted with B. bovis, Babesia bigemina (B. bigemina), Theileria equi (T. equi), and Babeisa caballi (B. caballi) merozoites in the indirect fluorescent antibody test (IFAT). Furthermore, the HDAC-enzymatic activity of the rBbHDAC3 protein was evaluated by a colorimetric assay. The enzymatic activity of rBbHDAC3 was inhibited by 100 ng/ml of apicidin, and the inhibitory effect of apicidin was dose-dependent. The inhibition of BbHDAC3 by apicidin was confirmed by Western blot, IFAT, and reverse transcription-polymerase chain reaction (RT-PCR). Finally, apicidin potentially inhibited the in vitro growth of Babesia parasites. The lower IC50 values of apicidin against apicomplexan parasites than those of mammalian cells point to HDAC as an excellent drug target. The findings of the present study indicate that BbHDAC3 is a potential target for apicidin and might be a promising target for the development of novel anti-babesial drugs.

A recombinant C-terminal antigen derived from Babesia caballi 48-kDa rhoptry protein (rBc48/CT) was made for the development of a serologically diagnostic test. Antiserum raised against the rBc48/CT reacted specifically with the corresponding native protein by Western blotting and the indirect fluorescent antibody test (IFAT). Next, an indirect enzyme-linked immunosorbent assay (Bc48/CT-ELISA) and an immunochromatographic test based on the Bc48/CT (Bc48/CT-ICT) were constructed and employed for the detection of an antibody to B. caballi in a variety of equine sera. The results of Bc48/CT-ELISA and Bc48/CT-ICT were highly concordant with those of IFAT and ELISA, with full-length protein of Bc48 used as the reference tests. Our results demonstrate the success of Bc48/CT as antigen for the serological diagnosis of B. caballi infection in horses.

We conducted a molecular epidemiological study on Babesia bovis in Mongolia. Three hundred blood samples collected from cattle grazed in seven different districts were initially screened using a previously established diagnostic polymerase chain reaction (PCR) assay for the detection of B. bovis-specific DNA. Positive samples were then used to amplify and sequence the hyper-variable regions of three B. bovis genes encoding the merozoite surface antigen (MSA)-1, MSA-2b, and MSA-2c. The diagnostic PCR assay detected B. bovis among cattle populations of all districts surveyed (4.4–26.0%). Sequences of each of the three genes were highly homologous among the Mongolian isolates, and found in a single phylogenetic cluster. In particular, a separate branch was formed only by the Mongolian isolates in the MSA-2b gene-based phylogenetic tree. Our findings indicate that effective preventative and control strategies are essential to control B. bovis infection in Mongolian cattle populations, and suggest that a careful approach must be adopted when using immunization techniques.

Five Babesia bovis recombinant proteins, including merozoite surface antigen 2c (BbMSA-2c), C-terminal rhoptry-associated protein 1 (BbRAP-1/CT), truncated thrombospondin-related anonymous protein (BbTRAP-T), spherical body protein 1 (BbSBP-1), and spherical body protein 4 (BbSBP-4), were evaluated as diagnostic antigens to detect the infection in cattle. The recombinant proteins were highly antigenic when tested with experimentally B. bovis -infected bovine serum in Western blot analysis. Furthermore, five antisera that had been raised against each of the recombinant proteins reacted specifically with the corresponding authentic protein, as determined in Western blot analysis. Next, enzyme-linked immunosorbent assays (ELISAs) using these recombinant proteins were evaluated for diagnostic use, and the sensitivity and specificity of each protein were demonstrated with a series of serum samples from experimentally B. bovis -infected cattle. Furthermore, a total of 669 field serum samples collected from cattle in regions of B. bovis endemicity in seven countries were tested with the ELISAs, and the results were compared to those of an indirect fluorescent antibody test (IFAT), as a reference. Among five recombinant antigens, recombinant BbSBP-4 (rBbSBP-4) had the highest concordance rate (85.3%) and kappa value (0.705), indicating its reliability in the detection of specific antibodies to B. bovis in cattle, even in different geographical regions. Overall, we have successfully developed an ELISA based on rBbSBP-4 as a new serological antigen for a practical and sensitive test which will be applicable for epidemiologic survey and control programs in the future.

application/pdf, Theileria orientalis is a benign protozoan species that is widely distributed in Japan, yet sometimes causes serious economic losses in the livestock industry. In this study, we conducted a molecular survey based on genes encoding the major piroplasm surface protein (MPSP) and p23 for T. orientalis detected in cattle grazing in southern areas of Japan, consisting of 2 farms in Kumamoto prefecture (Aso and Kuma districts) and 3 farms in Okinawa prefecture (Ishigaki, Iriomote, and Yonaguni Islands). High prevalence rates of T. orientalis infection were shown in all the cattle populations using the diagnostic MPSP- and p23-PCR assays. Phylogenetic analyses revealed 4 MPSP genotypes and 3 p23 genotypes. Furthermore, MPSP genotype-specific PCR methods were developed in this study and wide distributions of 5-district genotypes of T. orientalis were observed for the examined farms. Our results indicate that at least 5 types of T. orientalis exist in Kumamoto and Okinawa prefectures of Japan and that genotype-specific PCR assays are highly applicable for the quarantine of transported cattle and for epidemiological surveys of bovine theileriosis in Japan.

Bovine babesiosis is a tick-transmitted hemoprotozoan disease that is mainly caused by Babesia bovis and Babesia bigemina and is characterized by significant morbidity and mortality worldwide. The disease is widespread in the northeastern region of Thailand, where an increasingly large part of the livestock is composed of water buffaloes. The present study was therefore conducted to investigate the epidemiological distribution of B. bovis and B. bigemina in water buffaloes in the northeastern region of Thailand. A total of 305 buffalo blood samples were randomly collected from five provinces and simultaneously analyzed by the nested PCR (nPCR) assay, ELISA, and IFAT techniques. The overall prevalence of B. bovis and B. bigemina was 11.2% and 3.6% by nPCR, 14.7% and 5.9% by ELISA, and 16.8% and 5.6% by IFAT, respectively. The high concordance between the molecular and the serological detection tests revealed the specificity and sensitivity of the diagnostic assays used for the detection of infection as well as the endemic stability status of the parasites in the surveyed areas. Statistically significant differences in the prevalence of the two infections were observed on the basis of age and location but not gender. Our data provide valuable information regarding the epidemiology of B. bovis and B. bigemina infection in water buffaloes in the northeastern region of Thailand which will likely be very beneficial for management and control programs of this disease.