The Journal of General and Applied Microbiology
Online ISSN : 1349-8037
Print ISSN : 0022-1260
ISSN-L : 0022-1260
Advance online publication
Showing 1-25 articles out of 25 articles from Advance online publication
  • Takumi Horiike, Osamu Otsuka, Yasuhiro Tanaka, Takeshi Terahara, Chiak ...
    Article ID: 2018.11.003
    Published: 2019
    [Advance publication] Released: March 29, 2019

    Tellurium (Te) has been increasingly used as a semiconductor material in copious amounts, with a concomitant increase in its discharge from industrial effluents and mining wastewater into the environment. However, soluble Te, such as tellurate (VI) and tellurite (IV), is toxic to organisms. Thus, highly efficient technologies need to be developed for a double-benefit detoxification and recovery of soluble Te from industrial and mining wastewater. Since industrial wastewater contains high concentrations of salt, salt-tolerant microorganisms that metabolize rare metals such as Te have been the subject of focus for the effective detoxification and recovery of Te. In the present study, a total of 52 salt-tolerant tellurate-reducing microorganisms were isolated from marine environmental samples. Of these, 18 strains achieved greater than, or equal to, 50% removal of water-soluble Te from a medium containing 0.4 mM tellurate after 72 h incubation. The 18 isolated strains belonged to 13 species of the following 9 genera: Sulfitobacter, Ruegeria, Hoeflea, Alteromonas, Marinobacter, Pseudoalteromonas, Shewanella, Idiomarina, and Vibrio. No microorganism has been reported to reduce tellurate and tellurite from six of the aforementioned genera, namely, Sulfitobacter, Ruegeria, Alteromonas, Marinobacter, Idiomarina, and Vibrio. Especially, one of the isolates Sulfitobacter sp. strain TK39B, removed 82% (w/w) of soluble Te with a 4% NaCl tolerance. These results showed that salt-tolerant tellurate-reducing bacteria that can be used in the detoxification and recovery of Te are widely present in the marine environment.

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  • Liutengzi Cai, Mishuai Zhang, Tianci Shao, You He, Jingyi Li, Bingjie ...
    Article ID: 2018.11.002
    Published: 2019
    [Advance publication] Released: March 22, 2019

    In this study, a mutant xylanase of high thermostability was obtained by site-directed mutagenesis. The homologous 3D structure of xylanase was successfully modeled and the mutation sites were predicted using bioinformatics software. Two amino acids of XynZF-2 were respectively substituted by cysteines (T205C and A52C) and a disulfide bridge was introduced into the C-terminal of XynZF-2. The mutant gene xynZFTA was cloned into pPIC9K and expressed in P. pastoris. The optimum temperature of the variant XynZFTA was improved from 45°C to 60°C, and XynZFTA retained greater than 90.0% activity (XynZF-2 retained only 50.0% activity) after treatment at 50°C for 5 min. The optimum pH of mutant xylanase was similar to XynZF-2 (pH = 5.0). The pH stability span (5.0~7.0) of the mutant xylanase was increased to 3.0~9.0. Overall, the results implied that the introduction of a disulfide bridge in the C-terminal structure improved the thermostability and pH stability of XynZF-2.

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  • Naoyuki Tanaka, Tomoyuki Hatano, Soshi Saito, Yukari Wakabayashi, Tets ...
    Article ID: 2018.11.001
    Published: 2019
    [Advance publication] Released: March 15, 2019

    Many organisms produce endogenous hydrogen sulfide (H2S) as a by-product of protein, peptide, or L-cysteine degradation. Recent reports concerning mammalian cells have demonstrated that H2S acts as a signaling molecule playing important roles in various biological processes. In contrast to mammals, bacterial H2S signaling remains unclear. In this work, we demonstrate that Escherichia coli generates H2S through the assimilation of inorganic sulfur, without L-cysteine degradation. Comparison of phenotypes and genomes between laboratory E. coli K-12 strains revealed a major contribution of CRP (a protein that controls the expression of numerous genes involved in glycolysis) to H2S generation. We found that H2S was produced by cells growing in a synthetic minimal medium containing thiosulfate as a sole inorganic sulfur source, but not in a medium only containing sulfate. Furthermore, E. coli generated H2S in a CRP-dependent manner as a response to glucose starvation. These results indicate that CRP plays a key role in the generation of H2S coupled to thiosulfate assimilation, whose molecular mechanisms remains to be elucidated. Here, we propose a potential biological role of the H2S as a signaling mediator for a cross-talk between carbon and sulfur metabolism in E. coli.

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  • Saori Watahiki, Nobutada Kimura, Atsushi Yamazoe, Takamasa Miura, Yuji ...
    Article ID: 2018.10.003
    Published: 2019
    [Advance publication] Released: March 08, 2019

    Bioremediation may affect the ecological system around bioremediation sites. However, little is known about how microbial community structures change over time after the initial injection of degraders. In this study, we have assessed the ecological impact of bioaugmentation using metagenomic and metatranscriptomic approaches to remove trichlorinated ethylene/cis-dichloroethylene (TCE/cDCE) by Rhodococcus jostii strain RHA1 as an aerobic chemical compound degrader. Metagenomic analysis showed that the number of organisms belonging to the genus Rhodococcus, including strain RHA1, increased from 0.1% to 76.6% of the total microbial community on day 0 at the injection site. Subsequently, the populations of strain RHA1 and other TCE/cDCE-degrading bacteria gradually decreased over time, whereas the populations of the anaerobic dechlorinators Geobacter and Dehalococcoides increased at later stages. Metatranscriptomic analysis revealed a high expression of aromatic compound-degrading genes (bphA1-A4) in strain RHA1 after RHA1 injection. From these results, we concluded that the key dechlorinators of TCE/cDCE were mainly aerobic bacteria, such as RHA1, until day 1, after which the key dechlorinators changed to anaerobic bacteria, such as Geobacter and Dehalococcocides, after day 6 at the injection well. Based on the α-diversity, the richness levels of the microbial community were increased after injection of strain RHA1, and the microbial community composition had not been restored to that of the original composition during the 19 days after treatment. These results provide insights into the assessment of the ecological impact and bioaugmentation process of RHA1 at bioremediation sites.

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  • Yoichi Noda, Seisuke Arai, Ikuo Wada, Koji Yoda
    Article ID: 2018.10.002
    Published: 2019
    [Advance publication] Released: March 06, 2019

    Incorporation of membrane and secretory proteins into COPII vesicles are facilitated either by the direct interaction of cargo proteins with COPII coat proteins, or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in the yeast Saccharomyces cerevisiae. The ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that the efficient incorporation of Mnn4, a type II membrane protein required for mannosyl phosphate transfer to glycoprotein-linked oligosaccharides, into COPII vesicles is also dependent on the function of Svp26. We show that Mnn4 is localized to the Golgi. In addition to Mnn4, Mnn6 is known to be also required for the transfer of mannosyl phosphate to the glycans. We show, by indirect immunofluorescence, that Mnn6 localizes to the ER. As in the case with Svp26, deletion of the MNN6 gene results in the accumulation of Mnn4 in ER. In vitro COPII vesicle budding assays show that Svp26 and Mnn6 facilitate the incorporation of Mnn4 into COPII vesicles. In contrast to Svp26, which is itself efficiently captured into the COPII vesicles, Mnn6 was not incorporated into the COPII vesicles. Mnn4 and Mnn6 have the DXD motif which is often found in the many glycosyltransferases and functions to coordinate a divalent cation essential for the reaction. Alcian blue dye binding assay shows that substitution of the first D in this motif present in Mnn4 by A impairs the Mnn4 function. In contrast, amino acid substitutions in DXD motifs present in Mnn6 did not affect the function of Mnn6. These results suggest that Mnn4 may be directly involved in the mannosyl phosphate transfer reaction.

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  • Jirayut Euanorasetr, Bungonsiri Intra, Nutthanit Thunmrongsiri, Jitra ...
    Article ID: 2018.10.001
    Published: 2019
    [Advance publication] Released: February 28, 2019

    Spirotetronate compounds are polyketide secondary metabolites with diverse biological functions, such as antibacterial, antitumor and antiviral activities. Three pure spirotetronate compounds (2EPS-A, -B, -C) isolated from Actinomadura strain 2EPS showed inhibitory activity against dengue virus serotype 2 (DENV-2). 2EPS-A, -B and -C demonstrated the LC50 values of 11.6, 27.5 and 12.0 μg/ml, respectively, in a test of cytotoxicity to Vero cells. The least cytotoxic, 2EPS-B, was further analyzed for its impact on viral propagation in a cell-based replication assay. At a concentration of 6.25 μg/ml, it could reduce the DENV-2 infection in Vero cells by about 94% when cells infected with DENV-2 were exposed to 2EPS-B, whereas direct treatment of DENV-2 with 2EPS-B at the same concentration prior to subsequent infection to Vero cell yielded no inhibition. 2EPS-A, -B an -C showed strong DENV-2 NS2B-NS3 protease inhibition in an in vitro assay, with IC50 values of 1.94 ± 0.18, 1.47 ± 0.15 and 2.51 ± 0.21 μg/ml, respectively. Therefore, the spirotetronate compounds appear to prevent viral replication and viral assembly by inhibition of the viral protease.

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  • Nho-Eul Song, Chan-Mi Lee, Sang-Ho Baik
    Article ID: 2018.09.004
    Published: 2019
    [Advance publication] Released: February 15, 2019

    Biogenic amines (BAs) are widely present in nearly all fermented foods and beverages, and excess consumption can cause adverse health effects. To prepare BA-free Korean black raspberry wine (BRW), four autochthonous starter yeast strains without hazardous BA synthesis activity were selected and their physiological and biochemical properties were examined. The selected strains were identified as Saccharomyces cerevisiae based on 26S rDNA sequencing and microsatellite analysis. Molecular fingerprinting revealed that isolates were quite different from commercial wine yeast S. cerevisiae (52.4% similarity), but genetically relevant to commercial beer yeasts. The four S. cerevisiae strains produced over 10% ethanol during BRW fermentation. In addition, the fermented BRW with these strains showed higher levels of total flavonoids and similar antioxidant activity compared to the control sample. Potentially hazardous BAs that commonly occur in black raspberry extract (BRE) such as cadaverine, histamine, and spermidine were also not detected in the fermented BRW. Thus, we suggest that our strains are promising fermentation tools to ensure high quality and enhanced functionality in the production of BA-free BRW.

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  • Jia-fu Huang, Dan-feng Zhang, Bo Leng, Zhi-chao Lin, Yu-tian Pan
    Article ID: 2018.06.002
    Published: 2019
    [Advance publication] Released: February 12, 2019

    In the present study, the conditions for Azotobacter chroococcum fermentation using Agaricus bisporus wastewater as the culture medium were optimized. We analyzed the total number of living A. chroococcum in the fermentation broth, using multispectral imaging flow cytometry. Single-factor experiments were carried out, where a Plackett-Burman design was used to screen out three factors from the original six processing factors wastewater solubility, initial pH, inoculum size, liquid volume, culture temperature, and rotation speed that affected the total number of viable A. chroococcum. The Box-Behnken response surface method was used to optimize the interactions between the three main factors and to predict the optimal fermentation conditions. Factors significantly affecting the total number of viable A. chroococcum, including rotation speed, wastewater solubility, and culture temperature, were investigated. The optimum conditions for A. chroococcum fermentation in A. bisporus wastewater were a rotation speed of 200 rpm, a solubility of 0.25%, a culture temperature of 26°C, an initial pH of 6.8, a 5% inoculation volume, a culture time of 48 h, and a liquid volume of 120 mL in a 250 mL flask. Under these conditions, the concentration of total viable bacteria reached 4.29 ± 0.02 ✕ 107 Obj/mL A. bisporus wastewater can be used for the cultivation of A. chroococcum.

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  • Jun-ichi Matsuoka, Fumiko Ishizuna, Tetsuhiro Ogawa, Makoto Hidaka, Lo ...
    Article ID: 2018.09.003
    Published: 2019
    [Advance publication] Released: February 05, 2019

    Azorhizobium caulinodans, a kind of rhizobia, has a reb operon encoding pathogenic R-body components, whose expression is usually repressed by a transcription factor PraR. Mutation on praR induced a high expression of reb operon and the formation of aberrant nodules, in which both morphologically normal and shrunken host cells were observed. Histochemical GUS analyses of praR mutant expressing reb operon-uidA fusion revealed that the bacterial cells within the normal host cells highly expressed the reb operon, but rarely produced R-bodies. On the other hand, the bacterial cells within the shrunken host cells frequently produced R-bodies but rarely expressed the reb operon. This suggests that R-body production is not only regulated at the transcriptional level, but by other regulatory mechanisms as well.

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  • Yuuki Tanabe, Seisuke Arai, Ikuo Wada, Hiroyuki Adachi, Takashi Kamaku ...
    Article ID: 2018.09.001
    Published: 2019
    [Advance publication] Released: January 31, 2019

    After being translocated into the ER lumen, membrane and secretory proteins are transported from the ER to the early Golgi by COPII vesicles. Incorporation of these cargo proteins into COPII vesicles are facilitated either by direct interaction of cargo proteins with COPII coat proteins or by ER exit adaptor proteins which mediate the interaction of cargo proteins with COPII coat proteins. Svp26 is one of the ER exit adaptor proteins in yeast Saccharomyces cerevisiae. ER exit of several type II membrane proteins have been reported to be facilitated by Svp26. We demonstrate here that efficient incorporation of Mnt2 and Mnt3 into COPII vesicles is also dependent on the function of Svp26. Mnt2 and Mnt3 are Golgi-localized α-1,3-mannosyltransferases with type II membrane topology involved in protein O-glycosylation. Immunoisolation of the yeast Golgi subcompartments quantitatively showed that Mnt2 and Mnt3 are more abundant in the early Golgi fraction than in the late Golgi fraction. Subcellular fractionation and fluorescence microscopy showed that deletion of the SVP26 gene results in the accumulation of Mnt2 and Mnt3 in ER. Using an in vitro COPII vesicle formation assay, we further demonstrate that Svp26 facilitates incorporation of Mnt2 and Mnt3 into COPII vesicles. Finally, we showed that Mnt2 and Mnt3 were co-immunoprecipitated with Svp26 from digitonin-solubilized membranes. These results indicate that Svp26 functions as an ER exit adaptor protein of Mnt2 and Mnt3.

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  • Saki Goto, Naoyuki Suzuki, Ken'ichiro Matsumoto, Seiichi Taguchi, Kenj ...
    Article ID: 2018.09.002
    Published: 2019
    [Advance publication] Released: January 31, 2019

    For enhancing the lactate (LA) fraction of poly(lactate-co-3-hydroxybutyrate)s [P(LA-co-3HB)s], an exogenous D-lactate dehydrogenase gene (ldhD) was introduced into Escherichia coli. Recombinant strains of E. coli DH5α, LS5218, and XL1-Blue harboring the ldhD gene from Lactobacillus acetotolerans HT, together with polyhydroxyalkanoate (PHA)-biosynthetic genes containing a lactate-polymerizing enzyme (modified PHA synthase) gene, accumulated the P(LA-co-3HB) copolymer from glucose under microaerobic conditions (100 strokes/min). The LA fraction of copolymers synthesized in the strains of DH5α, LS5218, and XL1-Blue were 19.8, 15.7, and 28.5 mol%, respectively, which were higher than those of the strains without the ldhD gene (<6.7 mol% of LA units). Introduction of the exogenous ldhD gene into E. coli strains resulted in an enhanced LA fraction in P(LA-co-3HB)s.

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  • Jed O. Eberly, Michael L. Mayo, Matthew R. Carr, Fiona H. Crocker, Kar ...
    Article ID: 2018.08.001
    Published: 2019
    [Advance publication] Released: January 29, 2019

    Explosives such as hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) are common contaminants found in soil and groundwater at military facilities worldwide, but large-scale monitoring of these contaminants at low concentrations is difficult. Biosensors that incorporate aptamers μ with high affinity and specificity for a target are a novel way of detecting these compounds. This work describes novel riboswitch-based biosensors for detecting RDX. The performance of the RDX riboswitch was characterized in Escherichia coli using a range of RDX concentrations from 0–44 μmol l–1. Fluorescence was induced at RDX concentrations as low as 0.44 μmol l–1. The presence of 4.4 μmol l–1 RDX induced an 8-fold increase in fluorescence and higher concentrations did not induce a statistically significant increase in response.

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  • Shazwana Sha'arani, Hirofumi Hara, Hiroya Araie, Iwane Suzuki, Megat J ...
    Article ID: 2018.08.003
    Published: 2019
    [Advance publication] Released: January 28, 2019

    This study gives the first picture of whole RNA-Sequencing analysis of a PCB-degrading microbe, Rhodococcus jostii RHA1. Genes that were highly expressed in biphenyl-grown cells, compared with pyruvate-grown cells, were chosen based on the Reads Per Kilobase Million (RPKM) value and were summarized based on the criteria of RPKM ≥100 and fold change ≥2.0. Consequently, 266 total genes were identified as genes expressed particularly for the degradation of biphenyl. After comparison with previous microarray data that identified highly-expressed genes, based on a fold change ≥2.0 and p-value ≤0.05, 62 highly-expressed genes from biphenyl-grown cells were determined from both analytical platforms. As these 62 genes involve known PCB degradation genes, such as bph, etb, and ebd, the genes identified in this study can be considered as essential genes for PCB/biphenyl degradation. In the 62 genes, eleven genes encoding hypothetical proteins were highly expressed in the biphenyl-grown cells. Meanwhile, we identified several highly-expressed unannotated DNA regions on the opposite strand. In order to verify the encoded proteins, two regions were cloned into an expression vector. A protein was successfully obtained from one region at approximately 25 kDa from the unannotated strand. Thus, the genome sequence with transcriptomic analysis gives new insight, considering re-annotation of the genome of R. jostii RHA1, and provides a clearer picture of PCB/biphenyl degradation in this strain.

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  • Min Shen, Zhifeng Yin, Dan Xia, Qingxin Zhao, Yijun Kang
    Article ID: 2018.08.002
    Published: 2019
    [Advance publication] Released: January 25, 2019

    We created a combined system using duckweed and bacteria to enhance the efficiency of ammonium nitrogen (NH4+-N) and total nitrogen (TN) removal from aquaculture wastewater. Heterotrophic nitrifying bacterium was isolated from a sediment sample at an intensive land-based aquaculture farm. It was identified as Acinetobacter sp. strain A6 based on 16S rRNA gene sequence (accession number MF767879). The NH4+-N removal efficiency of the strain and duckweed in culture media and sampled aquaculture wastewater at 15°C was over 99% without any accumulation of nitrite or nitrate. This was significantly higher than strain A6 or duckweed alone. Interestingly, the presence of NO3- increased NH4+-N removal rate by 35.17%. Strain A6 and duckweed had mutual growth promoting-effects despite the presence of heavy metals and antibiotics stresses. In addition, strain A6 colonized abundantly and possibly formed biofilms in the inner leaves of duckweed, and possessed indoleacetic acid (IAA)- and siderophore-producing characteristics. The mutual growth promotion between strain A6 and duckweed may be the reason for their synergistic action of N removal.

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  • Moushree Pal Roy, Vaskar Das, Amal Patra
    Article ID: 2018.07.004
    Published: 2018
    [Advance publication] Released: December 19, 2018

    An extracellular L-asparaginase was isolated and purified from Bacillus megaterium MG1 to apparent homogeneity. The purification procedure involved a combination of ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration techniques, resulting in a purification factor of 31.52 fold with a specific activity of 215 U mg-1. The molecular mass of the purified enzyme was approximately 47 kDa on SDS-PAGE and 185 kDa on native PAGE gel as well as in gel filtration column chromatography, revealing that the enzyme was a homotetramer. The Km and Vmax values of the purified enzyme were calculated to be 2.0 ⅹ 10-4 M and 1.198 mM s-1. Maximum enzyme activity was observed over a wide range of temperature and pH values with an optimum temperature of 37°C and pH 8.5. SDS and metal ions such as Fe2+, Cu2+, Mg2+, Co2+, Mn2+, and Ca2+ decreased the enzyme activity remarkably, whereas the addition of Na+ and K+ led to an increase in activity. The insensitivity of the protein in the presence of EDTA suggested that the enzyme might not essentially be a metalloprotein. Its marked stability and activity in organic solvents and reducing agents suggest that this asparaginase is highly suitable as a biotechnological tool with industrial applications.

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  • Chunji Li, Bingxue Li, Ning Zhang, Qifan Wang, Wenjing Wang, Hongtao Z ...
    Article ID: 2018.07.002
    Published: 2018
    [Advance publication] Released: December 13, 2018

    Carotenoids are ubiquitous constituents of living organisms. These structurally diverse pigments have received considerable attention due to their biotechnological applications and potential beneficial effects on human health. In this study, we characterized an over-producing β-carotene mutant of Sporidiobolus pararoseus, obtained by ultraviolet mutagenesis, named MuY9. We compared the transcriptome between the wild-type and MuY9. A total of 348 differential expressed genes (DEGs) were found, and only one DEG crtYB is involved in carotenoid biosynthesis. The overproduction of β-carotene in MuY9 should be attributed to the up-regulation of crtYB. Functional identification of crtYB was performed using heterologous complementation in Escherichia coli. Our findings indicate that the enzymatic conversions of geranylgeranyl pyrophosphate to phytoene, as well as lycopene to β-carotene, are catalyzed by this CrtYB. Furthermore, our insights into the crtYB gene should facilitate a more detailed understanding of the carotenogenic pathway in S. pararoseus, and advance the development of the genetic engineering for the bio-production of carotenoids.

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  • Chunji Li, Bingxue Li, Ning Zhang, Na Wei, Qifan Wang, Wenjing Wang, Y ...
    Article ID: 2018.07.001
    Published: 2018
    [Advance publication] Released: November 29, 2018

    Carotenoids represent a diverse class of aliphatic C40 molecules with a variety of applications in the food and pharmaceutical industries. Sporidiobolus pararoseus NGR produces various carotenoids, including torulene, torularhodin and β-carotene. Salt stress significantly increases the torulene accumulation of S. pararoseus NGR. However, little is known, about the molecular mechanisms underlying the increased torulene biosynthesis. In this work, we investigated the effects of NaCl treatment on the contents of carotenoids (both qualitatively and quantitatively) and transcriptome. A total of 12.3 Gb of clean bases were generated in six cDNA libraries. These bases were de novo assembled into 9,533 unigenes with an average length of 1,654 nt and N50 of 2,371 nt. Transcriptome analysis revealed that of 3,849 differential expressed genes (DEGs) in response to salt stress, 2,019 were up-regulated, and 1,830 were down-regulated. Among these DEGs, we identified three carotenogenic genes crtE, crtYB, and crtI. In addition, fourteen candidate genes were predicted to participate in the conversion from torulene to torularhodin. Our findings should provide insights into the mechanisms of carotenoid biosynthesis and salt-tolerance of S. pararoseus NGR.

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  • Gumpanat Mahipant, Junichi Kato, Naoya Kataoka, Alisa S. Vangnai
    Article ID: 2018.06.001
    Published: 2018
    [Advance publication] Released: November 27, 2018

    Given their applicability in genetic engineering, undomesticated Bacillus strains are extensively used as non-natural hosts for chemical production due to their high tolerance of toxic substrates or products. However, they are difficult to genomically modify due to their low transformation efficiencies. In this study, the Bacillus-E. coli shuttle vector pHY300PLK, which is widely used in gram-positive bacteria, was adopted for genome integration in organic solvent-tolerant Bacillus isolates. The Bacillus-replicative vector was used to deliver homologous recombinant DNA and propagate itself inside the host cell, increasing the likelihood of genome integration of the recombinant DNA. Then, the unintegrated vectors were cured by cell cultivation in antibiotic-free medium with facilitation of nickel ions. The developed protocol was successfully demonstrated and validated by the disruption of amyE gene in B. subtilis 168. With an improved clonal selection protocol, the probability of clonal selection of the amyE::cat genome-integrated mutants was increased up to 42.0 ± 10.2%. Genome integration in undomesticated, organic solvent tolerant Bacillus strains was also successfully demonstrated with amyE as well as proB gene creating the gene-disrupted mutants with the corresponding phenotype and genotype. Not only was this technique effectively applied to several strains of undomesticated B. subtilis, but it was also successfully applied to B. cereus. This study validates the possibility of the application of Bacillus-replicative vector as well as the developed protocol in a variety of genome modification of undomesticated Bacillus species.

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  • Mana Yanagiba, Kazuo Masaki, Hideyuki Shinmori, Takafumi Naganuma
    Article ID: 2018.05.006
    Published: 2018
    [Advance publication] Released: November 21, 2018

    The yeast Lipomyces accumulates triacylglycerols (TAGs) as intracellular fat globules, and these TAGs can be used as source materials for biodiesel production. In this study, we aimed to use this yeast to produce lipids from renewable resources. Using plate culture and micrograph methods, strains with a high lipid-accumulation ability were screened from 15,408 types of systems combining renewable resources, strains, and culture temperatures. The lipid-accumulation ability of the strains was estimated from the fat globule volume, which was calculated using a micrograph. The reliability of this method was examined, and strains with a high lipid-accumulation ability were identified for each renewable resource. Seventy-seven Lipomyces strains (7 deposit, 68 wild-type, 2 mutants) with a high lipid-accumulation ability were selected. A few strains possessed the ability to accumulate large amounts of TAGs from more than four different renewable resources. We found that strains with a high lipid-accumulation ability could efficiently convert consumed carbon sources into TAGs, which could be easily recovered from the fat globules of these strains through physical disruption.

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  • Kristian Daly, Jennifer Kelly, Andrew W. Moran, Robert Bristow, Iain S ...
    Article ID: 2018.07.003
    Published: 2018
    [Advance publication] Released: November 09, 2018

    Fish production is increasingly important to global food security. A major factor in maintaining health, productivity and welfare of farmed fish is the establishment and promotion of a stable and beneficial intestinal microbiota. Understanding the effects of factors such as host and environment on gut microbial community structure is essential for developing strategies for stimulating the establishment of a health-promoting gut-microbiota. We compared intestinal microbiota of common carp and rainbow trout, two fish with different dietary habits, sourced from various farm locations. There were distinct differences in the gut microbiota of carp and trout intestine. The microbiota of carp was dominated by Fusobacteriia and Gammaproteobacteria, while the trout microbiota consisted predominantly of Mollicutes and Betaproteobacteria. The majority of bacterial sequences clustered into a relatively low number of operational taxonomic units (OTUs) revealing a comparatively simple microbiota, with Cetobacterium, Aeromonas and Mycoplasma being highly abundant. Within each species, fish from different facilities were found to have markedly similar predominant bacterial populations despite distinctly different rearing environments, demonstrating intra-species uniformity and significant influence of host selectivity. This study demonstrates that in fish the host species imparts substantial impact in shaping the community structure of the intestinal microbiota.

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  • Md. Shahbaz Anwar, Ashutosh Paliwal, Nazia Firdous, Amit Verma, Ashish ...
    Article ID: 2018.05.007
    Published: 2018
    [Advance publication] Released: October 31, 2018

    Numerous microbes reside in the rhizosphere having plant growth promoting activity, and enhancing the property by increasing plant yield. Plant growth promoting rhizobacteria (PGPR) has gradually increased in agriculture and offers an attractive way to replace chemical fertilizers, pesticides and supplements. Soil was collected from the rhizosphere of an agricultural farm and the psychrotrophic bacterial strains STA3 (KY888133) and RM2 (KY888134) were successfully isolated, and screened on the basis of phosphate solubilization. Further characterization was carried out by morphological, biochemical, and 16S rDNA characterization methods. The unique nature of psychrotrophic Pentoea ananatis and a suitable combination with Pseudomonas fluorescens regarding plant growth promotion activity has not been studied before to our knowledge. An assessment of various parameters of plant growth promoting activity, such as IAA, phosphate solubilization, bio-control activity, HCN and siderophore production, has been carried out. Both strains were found to be positive in various parameters except HCN and Biocontrol activity, which were positive only for the strain RM2. Also, shelf life and efficacy was determined before and after formulation. A great consistency was observed in all the cultures, even after 70 days of storage under bio-formulation at room temperature, while in the case of the co-culture CPP-2, the cfu ml-1 was greater, followed by RM2 and STA3. Moreover, the growth indices of the pea plant were found to be better in the co-culture CPP-2 compared with individual strains, followed by RM2 and STA3. Thus, the study suggests that the co-culture CPP-2 has a great potential for plant growth promotion as compared with individual strains followed by RM2 and STA3.

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  • Ronilo Jose D. Flores, Takao Ohashi, Kanae Sakai, Tohru Gonoi, Hiroko ...
    Article ID: 2018.05.003
    Published: 2018
    [Advance publication] Released: October 10, 2018

    Pseudozyma antarctica and Malassezia furfur are basidiomycetous yeasts under the subphylum Ustilaginomycotina. P. antarctica is a commensal organism found in certain plant species, while M. furfur is associated with several skin diseases of animals including humans. N-linked glycans of P. antarctica and M. furfur were prepared, digested with glycosidases, and structurally analyzed using high performance liquid chromatography (HPLC) and mass spectrometry (MS). Analyses revealed the presence of neutral N-linked glycans ranging in length from Man3GlcNAc2-PA to Man9GlcNAc2-PA. The two species shared the most abundant neutral N-linked glycan: Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M8A). The second and third most abundant neutral N-linked glycans for P. antarctica were Manα1-2Manα1-6(Manα1-2Manα1-3)Manα1-6(Manα1-2Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M9A) and Manα1-6(Manα1-3)Manα1-6(Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M5A), respectively. In the case of M. furfur, Manα1-2Manα1-6(Manα1-3)Manα1-6(Manα1-2Manα1-3)Manβ1-4GlcNAcβ1-4GlcNAc (M7A) was the second most abundant, while both M8A and M9A were tied for the third most abundant. The presence of putative galactose residues in the hypermannosylated neutral N-linked glycans is also discussed. This report is the first to analyze the neutral N-linked glycans of P. antarctica and M. furfur.

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  • Tao Su, Meiru Si, Yunfeng Zhao, Shumin Yao, Chengchuan Che, Yan Liu, C ...
    Article ID: 2018.05.005
    Published: 2018
    [Advance publication] Released: September 25, 2018

    Alkyl hydroperoxidase reductase AhpD, which is functionally equivalent to the bacterial flavin-containing disulfide reductase AhpF, acts as a proton donor for the organic peroxide-scavenging alkyl hydroperoxidase AhpC. Although AhpD has long been demonstrated in Mycobacterium tuberculosis, its physiological and biochemical functions remain largely unknown in other actinobacteria, including Corynebacterium glutamicum, Streptomyces, Mycobacterium smegmatis. Here, we report that C. glutamicum AhpD contributed to regenerate a variety of thiol-dependent peroxidase in the decomposition of peroxide by linking a dihydrolipoamide dehydrogenase (Lpd)/dihydrolipoamide succinyltransferase (SucB)/NADH system through the cyclization of their own active site dithiol to the oxidized disulphide. The CXXC motif of AhpD was essential to maintain the peroxides reduction activity of thiol-dependent peroxidase. ΔahpDahpD2 mutants exhibited significantly decreased resistance to adverse stress conditions and obviously increased the accumulation of reactive oxygen species (ROS). The physiological roles of AhpD in resistance to adverse stresses, were corroborated by their induced expression under various stresses and their direct regulation under the stress-responsive ECF-sigma factor SigH. C. glutamicum AhpDs were disulfide oxidoreductases behaving like thioredoxin (Trx) in regenerating thiol-dependent peroxidase for stress response, which provides the theoretical basis for an in-depth study of the reduction system in ahpC-lacking bacteria.

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  • Karthikeyan Ramalingam, Valerie Lee
    Article ID: 2018.05.004
    Published: 2018
    [Advance publication] Released: September 14, 2018

    Acinetobacter baumannii has been well recognized as a problematic human pathogen and several reports has shown the incidence of multidrug and pandrug-resistant A. baumannii strains in infirmary infections. A. baumannii grows only on an air-liquid interface and does not form a contiguous biofilm. Extracellular matrices (ECM) and slanted glass coverslips are (SGC) used as biofilm substrates and biofilms have been investigated by SEM, confocal and crystal violet staining. ECM has shown enhanced biofilm formation under dynamic conditions rather than static conditions. SGC biofilm yield assay has shown higher levels of continuous layers and packed thicker biofilm formation with glass coverslip inserts, up to 1.7 to 3 times higher biofilm formation, than when compared with no glass coverslip inserts. A media immersed ECM study revealed that biofilm grown on extracellular matrixes formed thread-like pili structures, and that these structures had contact with the ECM and also showed excellent cell-to-cell interaction. In summary, A. baumannii showed higher biofilm formation capacities with ECM, while the prominent results were directly related with the biofilm formation capacity of A. baumannii. For the initial step of biofilm formation, adherence is an important factor and, consequently, strains with a comparatively high capability to adhere to extracellular matrices and slanted glass coverslips provide a new method of enhanced biofilm growth for in vitro assays. ECM can be used as a substrate for immersed biofilm formation studies and the SGC method for air-liquid interface exposed biofilm formation studies, and these substrates can provide better biofilm growth and easy handling for in vitro adherence and biofilm assays.

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  • Yan-Jie Zhao, Yuya Sato, Tomohiro Inaba, Tomo Aoyagi, Tomoyuki Hori, H ...
    Article ID: 2018.05.002
    Published: 2018
    [Advance publication] Released: July 31, 2018

    The prokaryotic and eukaryotic microbial communities of activated sludge in a chemical plant wastewater treatment facility, processing relatively oligotrophic wastewater containing aromatic compounds and high-strength bromide ions, were characterized by high-throughput sequencing of rRNA genes based on DNA and RNA extracts. The microbial community structure was distinct from those previously reported from domestic wastewater treatment plants. Several abundant OTUs in the RNA-based prokaryotic community were related to aromatic compound-degrading bacteria, which most likely contributed to the removal of recalcitrant chemicals from the wastewater. Furthermore, both prokaryotic and eukaryotic predators were highly abundant. These might promote stabilization of the microbial food chain and affect biomass in the activated sludge, maintaining the waste-removal function of the microbial community.

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