The Journal of General and Applied Microbiology
Online ISSN : 1349-8037
Print ISSN : 0022-1260
ISSN-L : 0022-1260
早期公開論文
早期公開論文の26件中1~26を表示しています
  • Yuki Kamemoto, Nanaka Funaba, Mayu Kawakami, Katsuhiro Sawasato, Kotok ...
    原稿種別: research-article
    論文ID: 2019.05.001
    発行日: 2019年
    [早期公開] 公開日: 2019/09/10
    ジャーナル フリー 早期公開

    MPIase (membrane protein integrase) is an essential glycolipid that drives protein integration into the inner membrane of E. coli, while glycolipid ECA (enterobacterial common antigen) is a major component at the surface of the outer membrane. Irrespective of the differences in molecular weight, subcellular localization and function in cells, the glycan chains of the two glycolipids are similar, since the repeating unit comprising the glycan chains is the same. A series of biosynthetic genes for ECA, including ones for the corresponding nucleotide sugars, have been identified and extensively characterized. In this study, we found that knockouts as to the respective genes for ECA biosynthesis can grow in the minimum medium with the normal expression level of MPIase, indicating that MPIase can be biosynthesized de novo without the utilization of any compounds generated through ECA biosynthesis. Conversely, ECA was expressed normally upon MPIase depletion. From these results, we conclude that the biosynthetic genes for MPIase and ECA are independent.

  • Hazuki Hasegawa, Tatsuhiro Tsurumaki, Ikki Kobayashi, Sousuke Imamura, ...
    原稿種別: research-article
    論文ID: 2019.05.002
    発行日: 2019年
    [早期公開] 公開日: 2019/09/10
    ジャーナル フリー 早期公開

    Proteins that bind to RNA polymerase (RNAP) sigma factors play important roles in various transcriptional regulations. In this study, we identified a candidate of the principal sigma factor interacting protein in cyanobacteria, named SinA, based on a previous comprehensive protein interaction study (Sato et al., 2007) and analyzed this in the cyanobacterium Synechococcus elongatus PCC 7942. SinA is highly conserved among cyanobacteria and a knock out mutant showed defective growth at a usually permissive high temperature (40°C). Because this observation suggested SinA involvement in heat-inducible transcriptional activation, we examined heat-inducible protein gene hspA expression after temperature upshifts. The second-step induction disappeared after 15 min in the sinA mutant. In vivo pull-down experiments demonstrated the interaction between SinA and the principal sigma factor RpoD1. This SinA-RpoD1 complex was associated with an RNAP core enzyme under growth temperatures, but was dissociated after a temperature upshift. Based on these results, we propose a function of SinA to facilitate the substitution of the principal sigma factor with alternative sigma factors under heat-stressed conditions.

  • Tomoo Ogata, Ryo Ayuzawa, Ryusuke Yamada
    原稿種別: research-article
    論文ID: 2019.05.003
    発行日: 2019年
    [早期公開] 公開日: 2019/09/06
    ジャーナル フリー 早期公開

    Mating is a promising breeding method for industrial yeast. Although sake yeast has a low spore-formation ability, segregants exhibiting a mating type have been isolated from sake yeast K7. Here, we constructed zygotes from a cross between those segregants and a laboratory yeast strain. Because most sake and brewing yeast strains are prototrophs, we developed a PCR-based method to confirm that mating had taken place based on genome sequencing data and differences in nucleotide sequences between the two parental strains. The mated strain, termed S. cerevisiae MITOY123, showed restored spore-formation ability, unlike most sake and brewing yeast strains. By using the mated yeast strain MITOY123, it was possible to carry out tetrad analysis for the trait of the absence of off-flavour due to phenolic products such as 4-vinylguiacol (4-VG) in sake yeast K7. This tetrad analysis indicated that a single genetic region around the gene PAD1 is responsible for the absence of phenolic off-flavour in sake yeast K7. In order to aid the breeding of sake and brewing yeast strains by mating, we also identified a restriction fragment length polymorphism (RFLP) marker for the absence of phenolic off-flavour production in strains derived from sake yeast K7. Collectively, our data show that it is possible to breed new sake and brewing yeast strains by mating and to test for the absence of phenolic off-flavour production in resultant strains easily by RFLP analysis.

  • Sittichoke Ketkaeo, Werasit Sanpamongkolchai, Sumallika Morakul, Shuic ...
    原稿種別: research-article
    論文ID: 2019.04.008
    発行日: 2019年
    [早期公開] 公開日: 2019/08/28
    ジャーナル フリー 早期公開

    Red koji is produced from cultivating rice with Monascus strains that contain various types of fungal secondary metabolites, such as red pigments and monacolin K. Monascus strain also produces citrinin—a mycotoxin. In this study, Monascus purpureus KUPM5 isolated from the Thai fermented food, sufu, was mutagenized to reduce its citrinin production using UV irradiation, NTG treatment, and a combination of UV and NTG. Screening of the mutants using plate bioassay based on the inhibitory effect against Bacillus subtilis enables the selection of 10 mutants. The mutant strains KS301U and KS302U showed an 80% reduction in citrinin production in red koji compared with the wild type (wt), and maintained the ability to produce red pigments similar to the wild type. Activities of enzymes, α-amylase, protease, and lipase, from red koji extract produced by the mutant strain KS302U, were higher than those of the wt, whereas those of the mutant strain KS301U were similar to those of the wt. Consequently, strains KS301U and KS302U were successfully selected as strains suitable for producing red koji and fermented food.

  • Yusuke Suzuki, Atsushi Kouzuma, Kazuya Watanabe
    原稿種別: research-article
    論文ID: 2019.04.007
    発行日: 2019年
    [早期公開] 公開日: 2019/08/23
    ジャーナル フリー 早期公開

    Here, we developed an all-in-one, broad host-range CRISPR/Cas9 vector system widely applicable to genome editing of proteobacteria. Plasmid pBBR1-Cas9 was constructed by cloning the cas9 gene from Streptococcus pyogenes into the broad host-range plasmid pBBR1MCS-2. We evaluated its applicability for frameshift mutagenesis of Shewanella oneidensis MR-1. Significant cell death was observed when MR-1 cells were transformed with a pBBR1-Cas9 derivative that expressed a single-guide RNA targeting the crp gene. However, cell death was partially prevented when a donor DNA fragment containing a modified crp sequence with a frameshift mutation was introduced using the same vector. All transformants (9 colonies) contained the expected frameshift mutation in their chromosomal crp genes. These results indicate that this vector system efficiently introduced CRISPR/Cas9-mediated double-strand DNA breaks and subsequent homology-directed repair. This work provides a simple and powerful genome-editing tool for proteobacteria that can harbor pBBR1-based plasmids.

  • Medhat Ahmed Abu-Tahon, George Saad Isaac
    原稿種別: research-article
    論文ID: 2019.04.006
    発行日: 2019年
    [早期公開] 公開日: 2019/08/20
    ジャーナル フリー 早期公開

    Trichoderma viride AUMC 13021 isolated from Mangrove soil of Ras Mohammed protected area at Sharm El-Sheikh, Egypt, was optimized to enhance chitinase activity under submerged fermentation. The maximum enzyme yield (38.33 U/mg protein) was obtained at 1.4% of colloidal chitin, 96 h of incubation, 35°C, pH 6.5 and 125, rpm and using maltose (1%) and yeast extract (1%) as supplementation of salt basal medium. The enzyme has been purified with an overall yield of 73.1% and 5.48 purification fold, and a specific activity of 210.16 U/mg protein. The molecular mass of the purified chitinase was 62 kDa. Maximal activity of chitinase was recorded at pH 6.5 and 40°C. The highest activity was recorded in the case of colloidal chitin, with an apparent Km value of 6.66 mg/ml and Vmax of 90.8 U/ml. The purified chitinase was activated by Ca+2 and Mn+2 while the activity was inhibited by Hg+2, Zn+2, Cu+2, Co+2, dodecyl sulphate and EDTA. In vivo, the median lethal dose (LD50) was approximately 18.43 mg/kg body weight of Sprague Dawley rats. MTT assay showed that the purified chitinase has a toxic effect to MCF7 with an IC50 value 20 μg/ml, and HCT-116 cell lines with an IC50 value 44 μg/ml. Moreover, the purified enzyme showed significant antifungal activity against Fusarium oxysporum f. sp. lycopersici race 3 the causal agent of tomato wilt.

  • Huilin Yang, Lin Yang, Xiang Li, Hao Li, Zongcai Tu, Xiaolan Wang
    原稿種別: research-article
    論文ID: 2019.04.005
    発行日: 2019年
    [早期公開] 公開日: 2019/08/14
    ジャーナル フリー 早期公開

    A strongly fibrinolytic enzyme was purified from Bacillus amyloliquefaciens Jxnuwx-1, found in Chinese traditional fermented black soya bean (douchi). The molecular mass of the enzyme, estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), was 29 kDa. The optimal pH and temperature for the enzyme were 7.6 and 41°C, respectively. The enzyme was inhibited by phenylmethylsulfonyl fluoride, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, Fe3+, and Fe2+. The highest affinity exhibited by the enzyme was towards N-Succinyl-Ala-Ala-Pro-Phe-pNA. These results indicated that it is a subtilisin-like serine metalloprotease. The enzyme degraded both fibrinogen and fibrin, displaying its highest degrading activity towards the Aα-chains followed by Bβ chains and Cγ chains. The enzyme was also activated by plasminogen, indicating its ability to degrade fibrinogen and fibrin in two ways: (a) by activating plasminogen conversion into plasmin, or (b) by direct hydrolysis. It degraded thrombin, suggesting that it may act as an anticoagulant to prevent thrombosis. Taken together, our results indicate the potential of this enzyme in controlling cardiovascular disease.

  • Thi Hanh Nguyen Vu, Quang Huy Nguyen, Thi My Linh Dinh, Ngoc Tung Quac ...
    原稿種別: research-article
    論文ID: 2019.04.004
    発行日: 2019年
    [早期公開] 公開日: 2019/08/02
    ジャーナル フリー 早期公開

    Endophytic microbes associated with medicinal plants are considered to be potential producers of various bioactive secondary metabolites. The present study investigated the distribution, antimicrobial activity and genetic features of endophytic actinomycetes isolated from the medicinal plant Cinnamomum cassia Presl collected in Hoa Binh province of northern Vietnam. Based on phenotypic characteristics, 111 actinomycetes were isolated from roots, stems and leaves of the host plants by using nine selective media. The isolated actinomycetes were mainly recovered from stems (n = 67, 60.4%), followed by roots (n = 29, 26.1%) and leaves (n = 15, 13.5%). The isolates were accordingly assigned into 5 color categories of aerial mycelium, of which gray is the most dominant (n = 42, 37.8%), followed by white (n = 33; 29.7%), yellow (n = 25; 22,5%), red (n = 8; 7.2%) and green (n = 3; 2.7%). Of the total endophytic actinomycetes tested, 38 strains (occupying 34.2%) showed antimicrobial activity against at least one of nine tested microbes and, among them, 26 actinomycetes (68.4%) revealed anthracycline-like antibiotics production. Analysis of 16S rRNA gene sequences deposited on GenBank (NCBI) of the antibiotic-producing actinomycetes identified 3 distinct genera, including Streptomyces, Microbacterium, and Nocardia, among which Streptomyces genus was the most dominant and represented 25 different species. Further genetic investigation of the antibiotic-producing actinomycetes found that 28 (73.7%) and 11 (28.9%) strains possessed genes encoding polyketide synthase (pks) and nonribosomal peptide synthetase (nrps), respectively. The findings in the present study highlighted endophytic actinomycetes from C. cassia Presl which possessed broad-spectrum bioactivities with the potential for applications in the agricultural and pharmaceutical sectors.

  • Ehab R. El-Helow, Ramy G. Atalla, Wael A. Sabra, Walid A. Lotfy
    原稿種別: research-article
    論文ID: 2019.04.003
    発行日: 2019年
    [早期公開] 公開日: 2019/07/30
    ジャーナル フリー 早期公開

    Pseudomonas aeruginosa is characterized by its capability to produce extracellular virulence proteins and to establish biofilm-based infections that do not respond easily to conventional treatments. However, the physiological conditions that decrease the fitness of such a persistent pathogen would assist the host to defend itself and reduce the infection prevalence. Therefore, developing treatments against P. aeruginosa requires a quantitative understanding of the relationship between bacterial growth kinetics and secretion of alginate and proteins, in addition to the ecological factors that control their synthesis. For this purpose, we examined various environmental factors that affect the specific product yield coefficients (expressed as g product/OD600 (of alginate and extracellular proteins using a mucoid (FRD1) and a non-mucoid (PAO1) clinical isolate of P. aeruginosa, respectively. The results suggested magnesium sulfate, trace elements and hydrogen peroxide as significant variables that positively affect alginate synthesis by the FRD1 cells. However, the production of extracellular proteins by PAO1 was negatively affected by aeration and the concentration of ferrous sulfate. For understanding the kinetics of expressing alginate and extracellular proteins by the cells, a well-controlled 5 L tank bioreactor was used. The results suggested that under the bioreactor controlled conditions, both alginate and extracellular proteins are expressed parallel to biomass increase in the cells of P. aeruginosa.

  • Robin Vivod, Rodrigo Andler, Sylvia Oetermann, Anna-Lena Altenhoff, Ne ...
    原稿種別: research-article
    論文ID: 2019.01.003
    発行日: 2019年
    [早期公開] 公開日: 2019/07/16
    ジャーナル フリー 早期公開

    Nocardia nova SH22a is an actinobacterium capable of degrading the polyisoprenes poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene). Sequencing and annotating the genome of this strain led to the identification of a single gene coding for the key enzyme for the degradation of rubber: the latex clearing protein (Lcp). In this study, we showed that LcpSH22a—contrary to other already characterized rubber cleaving enzymes—is responsible for the initial cleavage of both polyisoprene isomers. For this purpose, lcpSH22a was heterologously expressed in an Escherichia coli strain and purified with a functional His6- or Strep-tag. Applying liquid chromatography electrospray ionization time-of-flight mass spectrometry (LC/ESI-ToF-MS) and a spectrophotometric pyridine hemochrome assay, heme b was identified as a cofactor. Furthermore, heme-associated iron was identified using total reflection X-ray fluorescence (TXRF) analysis and inhibition tests. The enzyme's temperature and pH optima at 30°C and 7, respectively, were determined using an oxygen consumption assay. Cleavage of poly(cis-1,4-isoprene) and poly(trans-1,4-isoprene) by the oxygenase was confirmed via detection of carbonyl functional groups containing cleavage products, using Schiff's reagent and electrospray ionization mass spectrometry (ESI-MS).

  • Yoshio Kimura, Sayaka Kajimoto, Yuuka Yamamoto, Naotaka Tanaka
    原稿種別: research-article
    論文ID: 2019.04.002
    発行日: 2019年
    [早期公開] 公開日: 2019/07/09
    ジャーナル フリー 早期公開

    Myxococcus xanthus Nudix hydrolase 2 (Nud2) hydrolyzed oxidized deoxynucleotides, such as 8-oxo-dGTP, 8-oxo-dGDP, 8-OH-dTP, and 2-OH-dATP, and showed the highest specific activity toward 8-oxo-dGTP. Mn2+ was the most effective co-factor for stimulating oxidized deoxynucleotide hydrolase activity. The Km of Nud2 with 8-oxo-dGTP for Mn2+ was 19-fold lower than that for Mg2+, and was 2-fold lower than that with dGTP for Mn2+. The specificity constant (kcat/Km) for 8-oxo-dGTP was 6-fold higher than that for dGTP. Nud2 contains a similar Nudix motif (84AX590GX7REX2EEXGX). Replacement of Ala84 and/or Gly90 in the Nudix motif of Nud2 by Gly or Glu had negligible effects on 8-oxo-dGTP hydrolase activity, suggesting that a strict Nudix motif sequence is not essential for complete hydrolase activity of Nud2.

  • Nur'Aqilah Farhanah Mohd Mohsi, Atiqqoh Apandi, Megat Johari Megat Moh ...
    原稿種別: research-article
    論文ID: 2019.04.001
    発行日: 2019年
    [早期公開] 公開日: 2019/07/08
    ジャーナル フリー 早期公開

    Prazosin (PRZ), a drug used to treat hypertensive patients, is an emergent contaminant in water systems. PRZ is an alpha-adrenergic receptor blocker used to treat anxiety, and is believed to reach the environment through human excretion, irresponsible disposal of unused medicine, and waste products from manufacturing plants. The purpose of this research was to isolate and characterize potential microbes for PRZ biodegradation and to identify the degradation pathway. After screening, isolated strain STP3 showed a capability for PRZ degradation and was chosen for further analysis. Resting cell assays with PRZ were conducted to identify the intermediate metabolites formed from biodegradation by Fourier transform ion cyclotron resonance mass spectrometer (FT-ICR MS) analysis. Two metabolites degraded from PRZ by STP3 were successfully found, and as these metabolites are derived from the main structure of PRZ, their presence proved PRZ degradation. Draft genome sequencing analysis of STP3 was performed to identify potential enzymes for PRZ biodegradation based on the metabolites found.

  • Junichi Satoh, Shinya Kimata, Shota Nakamoto, Tatsuya Ishii, Eisuke Ta ...
    原稿種別: research-article
    論文ID: 2019.03.001
    発行日: 2019年
    [早期公開] 公開日: 2019/07/05
    ジャーナル フリー 早期公開

    Ferredoxin NADP+ oxidoreductase (Fpr) and oxygen-insensitive NAD(P)H nitroreductase (NfnB) are purified from Escherichia coli JM109 (E. coli JM109) as a predominant free flavin-independent ferric reductase. In the present study, we prepared natural iron storage proteins, E. coli ferritin A (FtnA) and bacterioferritin (Bfr), to show the effective ferrous iron release from these proteins by Fpr and NfnB in the presence of free flavins. Fpr and NfnB showed flavin reductase activity for flavin adenine dinucleotide (FAD), flavin mononucleotide (FMN) and riboflavin, and their ferrous iron release activities were positively associated with the catalytic efficiencies (kcat/Km) for individual flavins. The ferrous iron release activity of E. coli cell-free extracts was affected by flavin reductase activity of the extracts. The Butyl TOYOPEARL column chromatography of the extracts, on the basis of NAD(P)H-dependent flavin reductase activity, resulted in the separation of six active fractions containing Fpr, NfnB, NAD(P)H-quinone oxidoreductase (QOR), flavin reductase (Fre) or alkyl hydroperoxide reductase subunit F (AhpF) as major components. Like Fpr and NfnB, recombinant QOR, Fre, and AhpF showed flavin reductase activity and ferrous iron release activity in the presence of free flavins, indicating an association of flavin reductase activity with ferrous iron releasing activity. Taken together, both free flavin-dependent and free flavin-independent ferric reductases in E. coli require free flavins to mediate an electron transfer from NAD(P)H to ferric iron in the iron storage proteins for the effective ferrous iron release.

  • Zijun Wu, Zhaoyu Kong, Shina Lu, Cheng Huang, Shaoyi Huang, Yinghui He ...
    原稿種別: research-article
    論文ID: 2018.11.004
    発行日: 2019年
    [早期公開] 公開日: 2019/06/27
    ジャーナル フリー 早期公開

    The research purpose was the characterization of indigenous heavy metal-resistant plant growth-promoting bacteria (PGPB) from the farmlands located on the Le'an River basin contaminated by acid mine drainage and their effects on plant growth, nutrient uptake, antioxidant enzyme activities and metal accumulation. The plant growth-promoting (PGP) traits, including 1-aminocyclopropane-1-carboxylic acid deaminase, indoleacetic acid, siderophore, ammonia production and phosphate solubilization, as well as antibiotics, acid/alkali and salt resistance were determined. Ten isolates with relatively high PGP activities were identified to belong to the genera Burkholderia, Paraburkholderia, Cupriavidus, Pseudomonas and Ralstonia. The numerical classification based on bacterial resistant characteristics was mostly consistent with their phylogenetic positions. Burkholderia sp. strain S6-1 and Pseudomonas sp. strain S2-3 possessed both greater PGP activities and resistant characteristics in overall comparison. Compared with non-inoculated plants, strains S6-1 and S2-3 significantly increased the height, dry weight and N uptake of sorghum (Sorghum bicolor L.). The presence of S6-1 significantly increased Pb accumulation and enhanced the translocation of Zn from root to shoot in sorghum. Strain S2-3 helped sorghum to uptake Cu and Zn and improved the remediation effect of sorghum on Cu and Zn. Overall, indigenous PGPB did not show better advantages in improving plant growth than non-indigenous P. putida UW4. Nevertheless, indigenous PGPB can be used as better candidates in heavy metal phytoremediation to minimize the potential risks of introducing invasive microbial species into an agricultural ecosystem.

  • Chuan Wu, Yumeng Chen, Xiaoxue Huang, Shishuai Sun, Jinnan Luo, Zhiwen ...
    原稿種別: research-article
    論文ID: 2019.02.001
    発行日: 2019年
    [早期公開] 公開日: 2019/06/21
    ジャーナル フリー 早期公開

    The filamentous fungus Trichoderma reesei is one of the most important fungi for the production of cellulases and xylanases, which can be used for biofuel production from lignocellulose. We aimed to develop an effective selection marker system for more extensive functional genomic studies in the fungus T. reesei, and to construct better industrial transformants for producing cellulases. Here, we present a novel effective G418 selection marker to use a codon-optimized neomycin phosphotransferase II gene nptII to transform T. reesei. We developed an effective and erasable selection marker, lcNG, and a combined genetic transformation system for gene manipulation in T. reesei using a two-Agrobacterium-mediated transformation method. This transformation strategy combines two steps in the transformation protocol, which saves 15–30-day's time. The system could be a useful tool for the genetic engineering of T. reesei.

  • Mia Fitria Utami, Yoshihiko Matsuda, Ayako Takada, Noritaka Iwai, Taka ...
    原稿種別: research-article
    論文ID: 2019.03.002
    発行日: 2019年
    [早期公開] 公開日: 2019/06/20
    ジャーナル フリー 早期公開

    We previously reported the extracellular production of antibody fragment Fab by Corynebacterium glutamicum. In the course of searching for genes which improve the secretion efficiency of Fab, we coincidentally found that the final growth increased significantly when the NCgl2986 gene encoding an amidase-like protein was overexpressed. This effect was observed when cells were grown on the production medium MMTG, which contains high concentrations of glucose and neutralizing agent CaCO3, but not on MMTG without CaCO3 or Lennox medium. Not only turbidity but also dry cell weight was increased by NCgl2986 overexpression, although the growth rate was not affected. It was recently reported that the Mycobacterium tuberculosis homolog Rv3915 functions as an activator of MurA protein, which catalyzes the initial step of peptidoglycan synthesis. Growth promotion was also observed when the MurA protein was overproduced. His-tagged NCgl2986 protein was purified, but its peptidoglycan hydrolyzing activity could not be detected. These results suggest that NCgl2986 promotes cell growth by activating the peptidoglycan synthetic pathway.

  • Pantira Singkum, Watcharamat Muangkaew, San Suwanmanee, Potjaman Pumee ...
    原稿種別: research-article
    論文ID: 2018.12.002
    発行日: 2019年
    [早期公開] 公開日: 2019/06/18
    ジャーナル フリー 早期公開

    This study examines the ability of the quorum-sensing molecules (QSMs) farnesol and tryptophol to induce programmed cell death of the pathogenic fungus Candida albicans, to alter the expression of apoptosis-related genes, and to reduce the pathogenicity and virulence of C. albicans in Galleria mellonella. Our results showed that both farnesol and tryptophol inhibited C. albicans germ tube formation. In the QSM-treated group, the expression levels of the apoptosis genes increased, whereas the expression level of the anti-apoptosis gene decreased. Further, pretreatment of C. albicans with tryptophol or farnesol prior to G. mellonella larval infection significantly enhanced host survival compared with larvae infected with untreated C. albicans. Thus, farnesol and tryptophol may trigger apoptosis of C. albicans in vitro and reduce the virulence of C. albicans in vivo. Although further study is needed to identify the precise mechanisms underlying the antifungal properties of farnesol and tryptophol, these results suggest that QSMs may be effective agents for controlling fungal infection.

  • Takahiro Yokoi, Mitsuhiro Itaya, Hirotada Mori, Masakazu Kataoka
    原稿種別: research-article
    論文ID: 2018.11.005
    発行日: 2019年
    [早期公開] 公開日: 2019/06/05
    ジャーナル フリー 早期公開

    The Gram-positive bacterium Bacillus subtilis plays important roles in both industrial applications and basic research. However, transformation of competent B. subtilis cells is more difficult to achieve compared with that of Escherichia coli. It has been reported that the conjugative broad host range plasmid RK2 can be transferred to various organisms, including B. subtilis. Nevertheless, the protocol for conjugation from E. coli to B. subtilis has not been properly established. Thus, we optimized interspecies conjugation from E. coli to B. subtilis using the RK2 system. We constructed mobilizable shuttle and integrative vectors pEB1 and pEB2, respectively. pEB1 was used to evaluate the effect of mating media, time, temperature, and genetic background of the recipient and donor strains. We found that conjugation was not significantly affected by the conjugation time or genetic background of the recipient and donor strains. Conjugation on agar was more efficient than that in a liquid medium. A low temperature (16°C and lower) drastically decreased conjugation efficiency. When using the optimized protocol for homologous recombination after conjugation, we could not obtain double crossover mutants, as only single crossover mutants were observed in the initial selection. We then established a two-step homologous recombination method whereby positive colonies were cultivated further, which finally allowed efficient yield of double crossover recombinants. The optimized conjugation method described here allowed facility and efficient gene introduction into B. subtilis from E. coli

  • Medhat Ahmed Abu-Tahon, George Saad Isaac
    原稿種別: research-article
    論文ID: 2019.01.002
    発行日: 2019年
    [早期公開] 公開日: 2019/05/24
    ジャーナル フリー 早期公開

    The aim of this study was to purify L-glutaminase from Aspergillus flavus. The enzyme was purified 12.47-fold from a cell-free extract with a final specific activity of 613.3 U/mg and the yield was 51.11%. The molecular weight of the enzyme, as determined by SDS-PAGE, was found to be 69 kDa. The maximal activity of L-glutaminase was recorded at pH 8 and 40°C. The highest activity was reported towards its natural substrate, L-glutamine, with an apparent Km value of 4.5 mmol and Vmax was 20 Uml–1. The enzyme was activated by Na+ and Co+2, while it was strongly inhibited by iodoacetate, NEM, Zn+2 and Hg+2 at 10 mM. L-glutaminase activity increased gradually with an increase of NaCl concentration up to 15%. In vivo, the median lethal dose (LD50) was approximately 39.4 mg/kg body weight after intraperitoneal injection in Sprague Dawley rats. Also, L-glutaminase showed no observed changes in liver and kidney functions and hematological parameters on rates. Purified A. flavus L-glutaminase had neither an appreciable effect on human platelet aggregation nor hemolytic activity. In addition, MTT assay showed that the purified L-glutaminase has a high toxic effect on Hela and Hep G2 cell lines with an IC50 value 18 and 12 μg/ml, respectively, and a moderate cytotoxic effect on HCT-116 and MCF7 cells, with an IC50 value 44 and 58 μg/ml, respectively.

  • Shuhei Yabe, Chiung-mei Wang, Yu Zheng, Yasuteru Sakai, Keietsu Abe, A ...
    原稿種別: research-article
    論文ID: 2019.01.001
    発行日: 2019年
    [早期公開] 公開日: 2019/05/21
    ジャーナル フリー 早期公開

    Currently, actinomycetes and myxobacteria are the only bacteria believed to form sporangia. Here, we describe a sporangium-forming process identified in Dictyobacter aurantiacus strain S27T belonging to the class Ktedonobacteria in the phylum Chloroflexi. Microscopic observations showed that strain S27T forms a substrate mycelium and subsequently produces globose or subglobose terminal sporangia arising from the vegetative mycelia through short stalk cells. This morphogenetic differentiation is similar to that seen in members of Actinoplanes belonging to the class Actinobacteria. However, unlike in Actinoplanes, motile spores could not be observed. This is the first report of the existence of a bacterium, other than actinomycetes and myxobacteira, with a complex morphogenetic differentiation that forms sporangia and is an important microbiological discovery.

  • Yukiko Shinozaki, Hiroko Kitamoto, Yuka Sameshima-Yamashita, Aya Kinos ...
    原稿種別: research-article
    論文ID: 2018.12.001
    発行日: 2019年
    [早期公開] 公開日: 2019/04/25
    ジャーナル フリー 早期公開

    Siderophores are considered to have a good potential as decontamination agents owing to their metal-chelating abilities. In order to confirm whether siderophores can be used in the recovery of metal ions, a siderophore (or metallophore) exhibiting Co2+-chelating activity was screened to demonstrate its ability to recover Co2+ from an aqueous solution. A siderophore-producing bacterium, Pandoraea sp. HCo-4B, was identified from a screen of Co2+-resistant bacteria grown in an aerobic enrichment culture with a Co2+-supplemented medium. After incubation of the crude extracted siderophore in a Co2+-containing solution, the Co2+-siderophore complex was adsorbed on to a C18 column. The bound Co2+ was eluted from the column by the addition of 10 mM H2SO4. The recovered amount of Co2+ was proportional to the amount of the added siderophore. We observed that the siderophore identified in this study binds to Co2+ at a 1:1 ratio.

  • Takumi Horiike, Osamu Otsuka, Yasuhiro Tanaka, Takeshi Terahara, Chiak ...
    論文ID: 2018.11.003
    発行日: 2019年
    [早期公開] 公開日: 2019/03/29
    ジャーナル フリー 早期公開

    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.

  • Liutengzi Cai, Mishuai Zhang, Tianci Shao, You He, Jingyi Li, Bingjie ...
    論文ID: 2018.11.002
    発行日: 2019年
    [早期公開] 公開日: 2019/03/22
    ジャーナル フリー 早期公開

    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.

  • Naoyuki Tanaka, Tomoyuki Hatano, Soshi Saito, Yukari Wakabayashi, Tets ...
    論文ID: 2018.11.001
    発行日: 2019年
    [早期公開] 公開日: 2019/03/15
    ジャーナル フリー 早期公開

    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.

  • Saori Watahiki, Nobutada Kimura, Atsushi Yamazoe, Takamasa Miura, Yuji ...
    論文ID: 2018.10.003
    発行日: 2019年
    [早期公開] 公開日: 2019/03/08
    ジャーナル フリー 早期公開

    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.

  • Yoichi Noda, Seisuke Arai, Ikuo Wada, Koji Yoda
    論文ID: 2018.10.002
    発行日: 2019年
    [早期公開] 公開日: 2019/03/06
    ジャーナル フリー 早期公開

    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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