The Journal of General and Applied Microbiology
Online ISSN : 1349-8037
Print ISSN : 0022-1260
Advance online publication
Showing 1-26 articles out of 26 articles from Advance online publication
  • 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
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    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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  • Hye Jin Choi, Tae-Yong Jeong, Hyeokjun Yoon, Bo Young Oh, Young Sun Ha ...
    Article ID: 2017.12.007
    Published: 2018
    [Advance publication] Released: July 20, 2018
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    Coastal ecosystems, play critical ecological roles of which tidal flats are a significant component of coastal wetlands, such as habitat and nutrient cycling in aquatic biology. Microbial communities in tidal flats are known to play vital roles of self-purification. And the microbial ecology of the sediment is easily affected by human activities and pollution. In this paper, we applied pyrosequencing technology to investigate microbial communities in three different tidal flats (Ganghwa Island, Ongnyeon land region and Yeongjong Island) on the Incheon, Korea peninsula. A total of 16,906 sequences were obtained. We used these sequences to identify the dominant phyla in the three tidal flats: Proteobacteria, Chloroflexi, Actinobacteria, and Bacteroidetes. The composition of the bacterial community of Ganghwa Island and the Ongnyeon region were more similar to each other than they were to the bacterial community of Yeongjong Island. Simpson's dominance index of Yeongjong Island was higher than that of the other regions, and the Shannon diversity index of this region was the lowest. Previous research of samples in these regions indicated that the three tidal flats had similar geochemical characteristics. However, their bacterial communities were rather distinct. This might be because the analysis of microbial communities and physiochemical analysis have different perspectives. Therefore, the pyrosequencing of a bacterial community with physiochemical analysis is recommended as an effective monitoring tool for the comprehensive management of tidal flats.

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  • Vipavee Cherdvorapong, Hidehisa Fujiki, Wasana Suyotha, Yoichi Takeda, ...
    Article ID: 2018.04.001
    Published: 2018
    [Advance publication] Released: July 17, 2018
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    Extracellular α-1,3-glucanase HF90 (AglST2), with a sodium dodecyl sulfate (SDS)-PAGE-estimated molecular mass of approximately 91 kDa, was homogenously purified from the culture filtrate of Streptomyces thermodiastaticus HF3-3. AglST2 showed a high homology with mycodextranase in an amino acid sequence and demonstrated specificity with an α-1,3-glycosidic linkage of homo α-1,3-glucan. It has been suggested that AglST2 may be a new type of α-1,3-glucanase. The optimum pH and temperature of AglST2 were pH 5.5 and 60°C, respectively. AglST2 action was significantly stimulated in the presence of 5–20% (w/v) NaCl, and 1 mM metal ions Mn2+ and Co2+. On the other hand, it was inhibited by 1 mM of Ag+, Cu2+, Fe2+ and Ni2+. Regarding the stability properties, AglST2 retained more than 80% of its maximum activity over a pH range of 5.0–7.0 at up to 60°C and in the presence of 0–20% (w/v) NaCl. Based on these results, the properties of AglST2 were comparable with those of AglST1, which had been previously purified and characterized from S. thermodiastaticus HF3-3 previously. The N-terminal amino acid sequence of AglST2 showed a good agreement with that of AglST1, suggesting that AglST1 was generated from AglST2 by proteolysis during cultivation. MALDI-TOF mass analysis suggested that AglST1 might be generated from AglST2 by the proteolytic removal of C-terminus polypeptide (approximately 20 kDa). Our investigation thus revealed the properties of AglST2, such as tolerance against high temperature, salts, and surfactants, which have promising industrial applications.

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  • Kaori Ohki, Yu Kanesaki, Noriyuki Suzuki, Maiko Okajima, Tatsuo Kaneko ...
    Article ID: 2018.04.004
    Published: 2018
    [Advance publication] Released: July 12, 2018
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    The clonal strains, phycoerythrin(PE)-rich- and PE-poor strains, of the unicellular, fresh water cyanobacterium Aphanothece sacrum (Suringar) Okada (Suizenji Nori, in Japanese) were isolated from traditional open-air aquafarms in Japan. A. sacrum appeared to be oligotrophic on the basis of its growth characteristics. The optimum temperature for growth was around 20°C. Maximum growth and biomass increase at 20°C was obtained under light intensities between 40 to 80 μmol m–2 s–1 (fluorescent lamps, 12 h light/12 h dark cycles) and between 40 to 120 μmol m–2 s–1 for PE-rich and PE-poor strains, respectively, of A. sacrum . Purified exopolysaccharide (EPS) of A. sacrum has a molecular weight of ca. 104 kDa with five major monosaccharides (glucose, xylose, rhamnose, galactose and mannose; ≥85 mol%). We also deciphered the whole genome sequence of the two strains of A. sacrum. The putative genes involved in the polymerization, chain length control, and export of EPS would contribute to understand the biosynthetic process of their extremely high molecular weight EPS. The putative genes encoding Wzx-Wzy-Wzz- and Wza-Wzb-Wzc were conserved in the A. sacrum strains FPU1 and FPU3. This result suggests that the Wzy-dependent pathway participates in the EPS production of A. sacrum.

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  • Satoshi Endo, Tomoya Maeda, Takahiro Kawame, Noritaka Iwai, Masaaki Wa ...
    Article ID: 2018.05.001
    Published: 2018
    [Advance publication] Released: July 09, 2018
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    Corynebacterium glutamicum is used for the industrial production of various metabolites, including L-glutamic acid and L-lysine. With the aim of understanding the post-transcriptional regulation of amino acid biosynthesis in this bacterium, we investigated the role of RNase E/G in the degradation of mRNAs encoding metabolic enzymes. In this study, we found that the cobalamin-independent methionine synthase MetE was overexpressed in ΔrneG mutant cells grown on various carbon sources. The level of metE mRNA was also approximately 6- to 10-fold higher in the ΔrneG mutant strain than in the wild-type strain. A rifampicin chase experiment showed that the half-life of metE mRNA was approximately 4.2 times longer in the ΔrneG mutant than in the wild-type strain. These results showed that RNase E/G is involved in the degradation of metE mRNA in C. glutamicum.

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  • Gursharan Singh, Sukhpal Singh, Kavleen Kaur, Shailendra Kumar Arya, P ...
    Article ID: 2018.04.002
    Published: 2018
    [Advance publication] Released: June 28, 2018
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    Laccases are unable to oxidize the non-phenolic components of complex lignin polymer due to their less redox potential (E0). Catalytic efficiency of laccases relies on the mediators that potentiates their oxidative strength; for breaking the recalcitrant lignin. Laccase from Bacillus sp. SS4 was evaluated for its compatibility with natural and synthetic mediators. (2 mM). It was found that acetosyringone, vanillin, orcinol and veratraldehyde have no adverse effect on the laccase activity up to 3 h. Syringaldehyde, p-coumaric acid, ferulic acid and hydroquinone reduced the enzyme activity ≥50% after 1.0 h, but laccase activity remained 100 to ~120% in the presence of synthetic mediators HBT (1-Hydroxylbenzotrizole) and ABTS. (2,2'-azino-bis (3-ethylbenzothiazoline-6-sulphonic acid) after 3 h. MgSO4 and MnSO4 (40 mM) increased the enzyme activity 3.5 fold and the enzyme possessed ≥70% activity at a very high concentration. (2 M) of NaCl. The enzyme retained 40–110% activity in the presence of 10% DMSO (dimethylsulfoxide), acetone, methanol and ethyl acetate. On the other hand, CuSO4 (100 μM) induced the laccase production 8.5 fold without increasing the growth of bacterial cells. Laccase from SS4 appropriately decolorized the indigo carmine (50 μM) completely in the presence of acetosyringone (100 μM) within 10 min and 25% decolorization was observed after 4 h without any mediator.

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  • Jannatul Ferdouse, Miyuki Miyagawa, Mikako Hirano, Yuka Kitajima, Shig ...
    Article ID: 2018.04.003
    Published: 2018
    [Advance publication] Released: June 21, 2018
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    At present, the quantitation of the mycelial weight of the industrially important non-pathogenic fungus Aspergillus oryzae, which is used for manufacturing koji, is performed by quantitating N-acetylglucosamine. However, since N-acetylglucosamine is a cell wall component, the extraction procedure is costly and tedious, and its quantitative performance is poor. Here, we report a novel method for the quantitation of A. oryzae mycelial weight. The amount of glycosylceramide significantly correlated with both the mycelial weight of A. oryzae and the amount of N-acetylglucosamine, an established index of the mycelial weight of A. oryzae in koji. This new method is simple and efficient and can be used in the brewing and food industries to determine the mycelial weight of A. oryzae.

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  • Yuki Kobayashi, Kan Tanaka
    Article ID: 2018.01.002
    Published: 2018
    [Advance publication] Released: June 14, 2018
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    We previously showed that nuclear DNA replication (NDR) is regulated by a checkpoint monitoring the occurrence of organelle DNA replication (ODR) in a unicellular red alga Cyanidioschyzon merolae. These analyses depended on the use of chemical CDK inhibitors such as CDK2 inhibitor II and roscovitine, but subsequent analyses yielded conflicting results depending on the experimental conditions. In the present study, we identified significantly short half-lives of the used chemicals in the sulfur acidic cultivation medium, which reconciles the discrepancy among these results.

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  • Tomoko Abe, Kenta Kobayashi, Sho Kawamura, Tatsuya Sakaguchi, Kiwamu S ...
    Article ID: 2018.03.001
    Published: 2018
    [Advance publication] Released: June 12, 2018
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    The adenylation domain of nonribosomal peptide synthetase (NRPS) is responsible for its selective substrate recognition and activation of the substrate (yielding an acyl-O-AMP intermediate) on ATP consumption. DhbF is an NRPS involved in bacillibactin synthesis and consists of multiple domains [adenylation domain, condensation domain, peptidyl carrier protein (PCP) domain, and thioesterase domain]; DhbFA1 and DhbFA2 (here named) are "internal" adenylation domains in the multidomain enzyme DhbF. We firstly succeeded in expressing and purifying the "internal" adenylation domains DhbFA1 and DhbFA2 separately. Furthermore, we initially demonstrated dipeptide synthesis by "internal" adenylation domains. When glycine and L-cysteine were used as substrates of DhbFA1, the formation of N-glycyl-L-cysteine (Gly-Cys) was observed. Furthermore, when L-threonine and L-cysteine were used as substrates of DhbFA2, N-L-threonyl-L-cysteine (Thr-Cys) was formed. These findings showed that both adenylation domains produced dipeptides by forming a carbon-nitrogen bond comprising the carboxyl group of an amino acid and the amino group of L-cysteine, although these adenylation domains are acid-thiol ligase using 4'-phosphopantetheine (bound to the PCP domain) as a substrate. Furthermore, DhbFA1 and DhbFA2 synthesized oligopeptides as well as dipeptides.

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  • Hirofumi Hara, Yus Amira Yusaimi, Siti Norayuni Mohd Zulkeflle, Norio ...
    Article ID: 2018.02.003
    Published: 2018
    [Advance publication] Released: June 06, 2018
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    The emergence of antibiotic resistance among multidrug-resistant (MDR) microbes is of growing concern, and threatens public health globally. A total of 129 Escherichia coli isolates were recovered from lowland aqueous environments near hospitals and medical service centers in the vicinity of Kuala Lumpur, Malaysia. Among the eleven antibacterial agents tested, the isolates were highly resistant to trimethoprim-sulfamethoxazole (83.7%) and nalidixic acid (71.3%) and moderately resistant to ampicillin and chloramphenicol (66.7%), tetracycline (65.1%), fosfomycin (57.4%), cefotaxime (57.4%), and ciprofloxacin (57.4%), while low resistance levels were found with aminoglycosides (kanamycin, 22.5%; gentamicin, 21.7%). The presence of relevant resistance determinants was evaluated, and the genotypic resistance determinants were as follows: sulfonamides (sulI, sulII, and sulIII), trimethoprim (dfrA1 and dfrA5), quinolones (qnrS), β-lactams (ampC and blaCTX-M), chloramphenicol (cmlA1 and cat2), tetracycline (tetA and tetM), fosfomycin (fosA and fosA3), and aminoglycosides (aphA1 and aacC2). Our data suggest that multidrug-resistant E. coli strains are ubiquitous in the aquatic systems of tropical countries and indicate that hospital wastewater may contribute to this phenomenon.

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  • Yan Wang, Tingwei Liu, Shuai Guo, Peng Zhang, Pengyang Sun, Mengqian C ...
    Article ID: 2018.02.002
    Published: 2018
    [Advance publication] Released: May 30, 2018
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    A gene (aga0917) encoding a putative β-agarase was identified from the genome of Pseudoalteromonas fuliginea YTW1-15-1. The nucleotide sequence analysis revealed that aga0917 had significant homology to the agarase genes of the GH16 family. aga0917 encodes a putative protein of 290 amino acids with an estimated molecular mass of 32.5 kDa, including a 21-amino acid signal peptide. A gene fragment encoding only the putative mature form of Aga0917 (269 amino acids) was overexpressed in Escherichia coli BL21 (DE3) pLysS as a 6 × histine-tagged fusion protein (rmAga0917). The Km, Vmax, and kcat for agarose of rmAga0917 were 39.6 mg/mL, 334 (U/mg) of protein, and 178 (1/s), respectively. According to the results of thin-layer chromatography and mass spectrometry analysis, the main end product from agarose with rmAga0917 was neoagarotetraose, in addition to a small amount of neoagarobiose. Notably, the recombinant protein rmAga0917 showed optimum activity at 60°C and retained approximately 100% agarolytic activity after being kept at 40°C for 1 h and 57% residual activity after incubation at 50°C for 1 h. The rmAga0917 exhibited maximum agarase activity at pH 6.0, and retained more than 80% of activity after incubation over a range of pH 4.0–9.0 for 1 h at 4°C.

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  • Hye Young Yoon, Si Young Lee
    Article ID: 2018.02.001
    Published: 2018
    [Advance publication] Released: May 29, 2018
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    Susceptibility testing of bacteria to disinfecting chemical agents isolated from dental unit waterlines (DUWL) is necessary for the development of effective disinfectant products. However, until now, susceptibility tests for chemical agents, which are components of DUWL disinfectant products, have not been conducted on bacteria isolated from DUWL water. The aim of this study was to evaluate and compare the susceptibilities of DUWL isolates in planktonic and biofilm states to cetylpyridinium chloride, as well as to the four chemical agents currently used for DUWL management. A total of 56 isolates, including 12 genera, were identified by 16S rDNA sequencing, and one strain of each genus was selected for susceptibility testing. A total of 12 isolates were used for the susceptibility tests. We determined the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) for the planktonic state and the minimum biofilm inhibitory concentration (MBIC) and minimum biofilm eradication concentration (MBEC) for the biofilm state using microtiter plates. MIC, MBC, MBIC, and MBEC of the 12 isolates for ethanol were the highest, followed by sodium hypochlorite, hydrogen peroxide, and chlorhexidine. Similar to chlorhexidine, the lowest MIC, MBC, MBIC, and MBEC were found in cetylpyridinium chloride. The susceptibilities of the isolates for sodium hypochlorite and ethanol were similar in the planktonic and biofilm states. For hydrogen peroxide and chlorhexidine, the MBIC and MIC were similar, but MBEC was 256 times higher than MBC. The MBIC and MBEC of isolates for cetylpyridinium chloride were 128 and 256 times higher than the MIC and MBC, respectively. As far as we know, this was the first study reporting the susceptibility of DUWL isolates to cetylpyridinium chloride and chemical agents used for disinfecting DUWLs. Cetylpyridinium chloride, for which the DUWL isolates showed the highest susceptibility, could be used for disinfecting DUWLs.

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  • Wangtai Luo, Jing Miao, Zhibin Feng, Ruiyang Lu, Xiaoqiang Sun, Baoshe ...
    Article ID: 2018.01.003
    Published: 2018
    [Advance publication] Released: May 28, 2018
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    In our recent work, we found that pyrrolnitrin, and not phenazines, pyrrolnitrin contributed to the suppression of the mycelia growth of Fusarium graminearum that causes heavy Fusarium head blight (FHB) disease in cereal crops. However, pyrrolnitrin production of Pseudomonas chlororaphis G05 in King's B medium was very low. Although a few regulatory genes mediating the prnABCD (the prn operon, pyrrolnitrin biosynthetic locus) expression have been identified, it is not enough for us to enhance pyrrolnitrin production by systematically constructing a genetically-engineered strain. To obtain new candidate genes involved in regulation of the prn operon expression, we successfully constructed a fusion mutant G05ΔphzΔprn::lacZ, in which most of the coding regions of the prn operon and the phzABCDEFG (the phz operon, phenazine biosynthetic locus) were deleted, and the promoter region plus the first thirty condons of the prnA was in-frame fused with the truncated lacZ gene on its chromosome. The expression of the fused lacZ reporter gene driven by the promoter of the prn operon made it easy for us to detect the level of the prn expression in terms of the color variation of colonies on LB agar plates supplemented with 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-Gal). With this fusion mutant as a recipient strain, mini-Tn5-based random insertional mutagenesis was then conducted. By picking up colonies with color change, it is possible for us to screen and identify new candidate genes involved in regulation of the prn expression. Identification of additional regulatory genes in further work could reasonably be expected to increase pyrrolnitrin production in G05 and to improve its biological control function.

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  • Ryosuke Shigemoto, Takara Matsumoto, Shunsuke Masuo, Naoki Takaya
    Article ID: 2018.01.001
    Published: 2018
    [Advance publication] Released: May 23, 2018
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    Sirtuin is an NAD+-dependent histone deacetylase that is highly conserved among prokaryotes and eukaryotes. Sirtuin deacetylates histones and non-histone proteins, and it is involved in fungal growth and secondary metabolite production. Here, we screened 579 fungal culture extracts that inhibited the histone deacetylase activity of Sirtuin A (SirA), produced by the fungus Aspergillus nidulans. Eight fungal strains containing three Ascomycota, two Basidiomycota and three Deuteromycetes produced SirA inhibitors. We purified the SirA inhibitor from the culture broth of Didymobotryum rigidum JCM 8837, and identified it as 5-methylmellein—a known polyketide. This polyketide and its structurally-related compound, mellein, inhibited SirA activity with IC50 of 120 and 160 μM, respectively. Adding 5-methylmellein to A. nidulans cultures increased secondary metabolite production in the medium. The metabolite profiles were different from those obtained by adding other sirtuin inhibitors nicotinamide and sirtinol to the culture. These results indicated that 5-methylmellein modulates fungal secondary metabolism, and is a potential tool for screening novel compounds derived from fungi.

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  • Md. Abdul Aziz, Rie Ushirokita, Yoshinao Azuma
    Article ID: 2017.12.008
    Published: 2018
    [Advance publication] Released: May 15, 2018
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    Chlamydia pneumoniae is an obligate intracellular pathogen responsible for respiratory diseases, including pneumonia and bronchitis, and is highly involved in chronic diseases, including atherosclerosis, asthma, and Alzheimer's disease. We previously showed that the host apoptotic factor caspase-9 played a crucial role for chlamydial multiplication and host apoptosis inhibition by chlamydial infection. To identify chlamydial genes interacting with human caspase-9, yeast two-hybrid screening was performed and 5 chlamydial genes, including Cpj0838 and pmpG were isolated from the C. pneumoniae genomic library. Pull-down experiments showed that caspase-9 physically bound to the Cpj0838 product and chlamydial cells, which contain PmpG proteins. This study could provide a clue to understanding host-Chlamydia interactions, especially the apoptosis repression by Chlamydia infection.

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  • Hui-Zhong Wang, Min Gou, Yue Yi, Zi-Yuan Xia, Yue-Qin Tang
    Article ID: 2017.12.006
    Published: 2018
    [Advance publication] Released: May 11, 2018
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    Acetate is a significant intermediate of anaerobic fermentation. There are two pathways for converting acetate to CH4 and CO2: acetoclastic methanogenesis by acetoclastic methanogens, and syntrophic acetate oxidation by acetate-oxidizing bacteria (AOB) and hydrogenotrophic methanogens. Detailed investigations of syntrophic acetate-oxidizing bacteria (SAOB) should contribute to the elucidation of the microbial mechanisms of methanogenesis. In this study, we investigated the major phylogenetic groups of acetate-utilizing bacteria (AUB) in a mesophilic methanogenic chemostat fed with acetate as the sole carbon source by using DNA stable isotope probing (SIP) technology. The results indicated that acetoclastic methanogenesis and acetate oxidization/hydrogenotrophic methanogenesis coexisted in the mesophilic chemostat fed with acetate, operated at a dilution rate of 0.1 d–1. OTU Ace13(9-17) (KU869530), Ace13(9-4) (KU667241), and Ace13(9-23) (KU667236), assigned to the phyla Firmicutes and Bacteroidetes, were probably potential SAOB in the chemostat, which needs further investigation. Species in the phyla Proteobacteria, Deferribacteres, Acidobacteria, Spirochaetes and Actinobacteria were probably capable of utilizing acetate for their growth. Methanoculleus was likely to be the preferred hydrogenotrophic methanogen for syntrophy with AOB in the chemostat.

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  • Taejun Chin, Masahiko Ikeuchi
    Article ID: 2017.12.004
    Published: 2018
    [Advance publication] Released: May 08, 2018
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    Sorbitol-6-phosphatase (EC 3.1.3.50) catalyzes sorbitol production from sorbitol-6-phosphate in certain organisms, but has not been identified unequivocally. We screened the activity of the haloacid dehalogenase-like hydrolases (HAD) superfamily and identified four HAD proteins from Escherichia coli as sorbitol-6-phosphatase. Of these proteins, HAD2 (YfbT) exhibited catalytic activity (kcat/Km) that was better than that of the previously reported "preferred" substrate. HAD1 (YniC) and HAD2 exhibited higher sorbitol-6-phosphatase activity than that of HAD12 (YbiV) and HAD13 (YidA). Therefore, genes of HAD may be useful for metabolic engineering of effective sorbitol production.

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  • Shigekazu Yano, Wasana Suyotha, Sumika Zanma, Hiroyuki Konno, Vipavee ...
    Article ID: 2017.12.005
    Published: 2018
    [Advance publication] Released: May 08, 2018
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    α-1,3-Glucanase (Agl-KA) of Bacillus circulans KA-304 consists of an N-terminal discoidin domain (DS1), a carbohydrate binding module family 6 (CBM6), threonine and proline repeats (TP), a second discoidin domain (DS2), an uncharacterized conserved domain (UCD), and a C-terminal catalytic domain. Previously, we reported that DS1, CBM6, and DS2 have α-1,3-glucan-binding activity and contribute to α-1,3-glucan hydrolysis. In this study, UCD deletion mutant (AglΔUCD) was constructed, and its properties were compared with those of Agl-KA. α-1,3-Glucan hydrolyzing, α-1,3-glucan binding, and protoplast-forming activities of AglΔUCD were almost the same as those of Agl-KA. kcat/Km values of AgΔUCD and Agl-KA were 11.4 and 11.1 s–1 mg–1 mL, respectively. AglΔUCD and Agl-KA exhibited similar characteristics, such as optimal pH, pH stability, optimal temperature, and thermostability. These results suggest that UCD is not α-1,3-glucan-binding and flexible linker domain, and that deletion of UCD does not affect the affinity of N-terminal binding domains and the catalytic action of the C-terminal domain. Subsequently, heterologous UCenzyme productivity of AglΔD in Escherichia coli was compared with that of Agl-KA. The productivity of AglΔUCD was about 4-fold larger than that of Agl-KA after an 8-h induction at 30°C. In the case of induction at 20°C, the productivity of AglΔUCD was also larger than that of Agl-KA. These findings indicate that deletion of only UCD enhances the enzyme productivity in E. coli.

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  • Kaori Inoue-Sakamoto, Ehsan Nazifi, Chieri Tsuji, Tomoya Asano, Takumi ...
    Article ID: 2017.12.003
    Published: 2018
    [Advance publication] Released: April 30, 2018
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    The aquatic cyanobacterium Nostoc verrucosum forms macroscopic colonies in streams, and its appearance is superficially similar to that of the terrestrial cyanobacterium Nostoc commune. N. verrucosum is sensitive to desiccation, unlike N. commune, although these Nostoc cyanobacterial species share physiological features, including massive extracellular polysaccharide production and trehalose accumulation capability. In this study, water-soluble sunscreen pigments of mycosporine-like amino acids (MAAs) were characterized in N. verrucosum, and the mysABCD genes responsible for MAA biosynthesis in N. verrucosum and N. commune were compared. N. verrucosum produced porphyra-334 and shinorine, with porphyra-334 accounting for >90% of the total MAAs. Interestingly, porphyra-334 is an atypical cyanobacteial MAA, whereas shinorine is known as a common and dominant MAA in cyanobacteria. Porphyra-334 from N. verrucosum showed little or no radical scavenging activity in vitro, although the glycosylated derivatives of porphyra-334 from N. commune are potent radical scavengers. The presence of the mysABCD gene cluster in N. commune strain KU002 (genotype A) supported its porphyra-334 producing capability via the Nostoc-type mechanism, although the genotype A of N. commune mainly produces the arabinose-bound porphyra-334. The mysABC gene cluster was conserved in N. verrucosum, but the mysD gene was not included in the cluster. These results suggest that the mysABCD gene products are involved in the biosynthesis of porphyra-334 commonly in these Nostoc species, and that the genotype A of N. commune additionally acquired the glycosylation of porphyra-334.

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  • Keitaro Take, Hidehisa Fujiki, Wasana Suyotha, Junji Hayashi, Kazuyosh ...
    Article ID: 2017.12.002
    Published: 2018
    [Advance publication] Released: April 27, 2018
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    Chitinase 1 (Chi1) is an acidic and thermostable hydrolytic enzyme capable of the breakdown of chitin, a resilient biopolymer that is the primary building block of fungi cell walls and marine exoskeletons. In this study, Chi1 was purified from the bacterium Streptomyces thermodiastaticus HF 3-3, and its properties were carefully characterized. The molecular mass of Chi1 was estimated to be approximately 46 kDa and, through sequencing, its N-terminal amino acid sequence was identified as ADSGKVKL. Although the optimal operating temperature and pH for Chi1 were determined to be 65°C and pH 5.5, respectively, the purified enzyme was stable over wide pH (1.5–9) and temperature ranges. Moreover, Chi1 retained 87% of its activity in the presence of 15% NaCl. While Chi1 activity was inhibited by Ag+ and Mn2+, other chemicals tested had no significant effect on its enzymatic activity. The Km and Vmax values of Chi1 for the substrate colloidal chitin were 1.23 ± 0.7 mg/mL and 6.33 ± 1.0 U/mg, respectively. Thin-layer chromatography analysis of the enzymatic reaction end products mainly detected diacetylchitobiose. We also cloned the Chi1 gene and purified the recombinant protein; the properties of the recombinant enzyme were nearly identical to those of the native enzyme. Therefore, Chi1 purified from S. thermodiastaticus HF 3-3 is unique, as it is highly stable under broad range of pH values, temperatures, and chemical exposures. Combined, these properties make this enzyme attractive for use in the industrial bioconversion of chitin.

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  • Vidya Pradeep Kumar, Atul P. Kolte, Arindam Dhali, Chandrashekar Naik, ...
    Article ID: 2017.11.006
    Published: 2018
    [Advance publication] Released: April 25, 2018
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

    Utilization of energy-rich crop residues by ruminants is restricted by the presence of lignin, which is recalcitrant to digestion. Application of lignin degrading enzymes on the lignocellulosic biomass exposes the cellulose for easy digestion by ruminants. Laccases have been found to be considerably effective in improving the digestibility by way of delignification. However, laccase yields from natural hosts are not sufficient for industrial scale applications, which restricts their use. A viable option would be to express the laccase gene in compatible hosts to achieve higher production yields. A codon-optimized synthetic variant of Schizophyllum commune laccase gene was cloned into a pPIC9K vector and expressed in P. pastoris GS115 (his4) under the control of an alcohol oxidase promoter. Colonies were screened for G418 resistance and the methanol utilization phenotype was established. The transformant yielded a laccase activity of 344 U·mL–1 after 5 days of growth at 30°C (0.019 g·mL–1 wet cell weight). The laccase protein produced by the recombinant Pichia clone was detected as two bands with apparent molecular weights of 55 kDa and 70 kDa on SDS-PAGE. Activity staining on native PAGE confirmed the presence of bioactive laccase. Treatment of five common crop residues with recombinant laccase recorded a lignin loss ranging between 1.64% in sorghum stover, to 4.83% in finger millet, with an enhancement in digestibility ranging between 8.71% in maize straw to 24.61% in finger millet straw. Treatment with recombinant laccase was effective in enhancing the digestibility of lignocellulosic biomass for ruminant feeding through delignification. To date, a number of hosts have been adventured to produce laccase in large quantities, but, to our knowledge, there are no reports of the expression of laccase protein from Schizophyllum commune in Pichia pastoris, and also on the treatment of crop residues using recombinant laccase for ruminant feeding.

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  • Jiwei Mao, Quanli Liu, Yuanzi Li, Jiuxia Yang, Xinhao Song, Xiao Liu, ...
    Article ID: 2017.12.001
    Published: 2018
    [Advance publication] Released: April 25, 2018
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

    A biosensor screening assay based on the synthesis of betaxanthin was applied to relatively high throughput screening of the L-tyrosine mutant library. In the assays, fluorescence output showed a linear relationship between extracellular L-tyrosine content and yellow pigment formation. In addition, the yellow pigment accumulation of the L-tyrosine high-yield strain can be easily distinguished with the naked eye compared with the wild-type strain. As a result, numerous mutants that exhibited significantly increased coloration, were screened out after random mutagenesis, and p-coumaric acid production in mutants NK-A3 and NK-B4, were remarkably improved by 4-fold more than that of the wild-type strain. In general, this study provides a novel strategy for screening mutant libraries in the search for highly L-tyrosine-producing strains.

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  • Hung-Yin Pan, Carton W. Chen, Chih-Hung Huang
    Article ID: 2017.11.004
    Published: 2018
    [Advance publication] Released: April 17, 2018
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

    Soil bacteria Streptomyces are the most important producers of secondary metabolites, including most known antibiotics. These bacteria and their close relatives are unique in possessing linear chromosomes, which typically harbor 20 to 30 biosynthetic gene clusters of tens to hundreds of kb in length. Many Streptomyces chromosomes are accompanied by linear plasmids with sizes ranging from several to several hundred kb. The large linear plasmids also often contain biosynthetic gene clusters. We have developed a targeted recombination procedure for arm exchanges between a linear plasmid and a linear chromosome. A chromosomal segment inserted in an artificially constructed plasmid allows homologous recombination between the two replicons at the homology. Depending on the design, the recombination may result in two recombinant replicons or a single recombinant chromosome with the loss of the recombinant plasmid that lacks a replication origin. The efficiency of such targeted recombination ranges from 9 to 83% depending on the locations of the homology (and thus the size of the chromosomal arm exchanged), essentially eliminating the necessity of selection. The targeted recombination is useful for the efficient engineering of the Streptomyces genome for large-scale deletion, addition, and shuffling.

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  • Minoru Sasaki, Yoshitoyo Kodama, Yu Shimoyama, Taichi Ishikawa, Shigen ...
    Article ID: 2017.11.005
    Published: 2018
    [Advance publication] Released: April 17, 2018
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

    Although Streptococcus anginosus constitutes a proportion of the normal flora of the gastrointestinal and genital tracts, and the oral cavity, it has been reported that S. anginosus infection could be closely associated with abscesses at various body sites, infective endocarditis, and upper gastrointestinal cancers. The colonization in an acidic environment due to the aciduricity of S. anginosus could be the etiology of the systemic infection of the bacteria. To elucidate the aciduricity and acid tolerance mechanisms of the microbe, we examined the viability and growth of S. anginosus under acidic conditions. The viabilities of S. anginosus NCTC 10713 and Streptococcus mutans ATCC 25175 at pH 4.0 showed as being markedly higher than those of Streptococcus sanguinis ATCC 10556, Streptococcus gordonii ATCC 10558, and Streptococcus mitis ATCC 49456; however, the viability was partially inhibited by dicyclohexylcarbodiimide, an H+-ATPase inhibitor, suggesting that H+-ATPase could play a role in the viability of S. anginosus under acidic conditions. In addition, S. anginosus NCTC 10713 could grow at pH 5.0 and showed a marked arginine deiminase (ADI) activity, unlike its ΔarcA mutant, deficient in the gene encoding ADI, and other streptococcal species, which indicated that ADI could also be associated with aciduricity. These results suggest that S. anginosus has significant aciduric properties, which can be attributed to these enzyme activities.

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  • Hao Song, Zheng-Lin Yu, Mei-jie Yang, Tao Zhang, Hai-Yan Wang
    Article ID: 2017.11.003
    Published: 2018
    [Advance publication] Released: April 11, 2018
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

    The rapa whelk Rapana venosa is a commercially important gastropod in China, but a major invader worldwide. There is little information on the bacterial community composition in the digestive system of this species, despite the microflora has important roles in digestion, nutrition, disease resistance, and immune response. The present study investigated the bacterial flora community in the esophagus and intestinal tract of wild R. venosa by 16S rRNA gene sequencing. The esophagus and intestine had 1175 and 526 unique operational taxonomic units, respectively, with 616 common to both tissues. The 10 most highly represented microbial genera in the esophagus were Mycoplasma, Bifidobacterium, Escherichia, Shewanella, Vibrio, Lactobacillus, Octadecabacter, Enterococcus, Streptococcus, and Sphingomonas, which accounted for 41.35% of microbes. In the intestine, the most abundant genera were Mycoplasma, Bifidobacterium, Escherichia, Shewanella, Vibrio, Psychrilyobacter, Peptoniphilus, Sporobacterium, Octadecabacter, and Mobiluncusin (representing 62.74% of total microbes). A linear discriminant analysis, coupled with effect size, revealed that 31 taxa were differentially represented in esophagus and intestine bacterial communities. These results demonstrate the diversity of microbiota in the esophagus and intestinal tract of R. venosa and provide a basis for investigations into the physiological and immunological roles of these microorganisms in whelk.

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  • Nobushige Nakazawa, Himiko Yanata, Natsumi Ito, Eri Kaneta, Keitaro Ta ...
    Article ID: 2017.11.002
    Published: 2018
    [Advance publication] Released: March 31, 2018
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

    We analyzed the stress response in a spore clone from Shirakami kodama yeast, Saccharomyces cerevisiae, with an exceptional high tolerance to oxidative stress. The levels of reactive oxygen species (ROS) in this clone were very low, whereas the genes for superoxide dismutase (SOD2) and catalase (CTT1) were highly expressed and those enzymes also had high activities even under non-stress conditions. Both genes are regulated by general stress-responsive transcription factors Msn2 and Msn4, and Yap1, a transcription factor required for oxidative stress tolerance, and the removal of Msn2 or Yap1 caused a significant decrease in CTT1-expression. Under non-stress conditions, Msn2 was ~3.6-fold more abundant in the nucleus of the spore clone compared with a laboratory strain, whereas the nuclear abundance of Yap1 remained unchanged. Thus, a high tolerance to oxidative stress in this spore clone results from a high expression of ROS-degrading enzymes by the abundant accumulation of Msn2 in the nucleus. We found that oxidative stress caused by the presence of furfural did not impair fermentation by this strain, which could make it attractive for ethanol production from lignocellulosic biomass.

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