The Journal of General and Applied Microbiology
Online ISSN : 1349-8037
Print ISSN : 0022-1260
Advance online publication
Showing 1-22 articles out of 22 articles from Advance online publication
  • 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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  • Jing Zheng, Yifan Xia, Qi Liu, Xinyu He, Jiajia Yu, Yongjun Feng
    Article ID: 2018.03.002
    Published: 2018
    [Advance publication] Released: September 06, 2018

    Extracellular DNA (eDNA) is an important polymeric substance that plays essential roles in cell aggregation and nutrient provision for the sessile bacteria. eDNA in bacterial biofilms was extensively studied. Here we found that eDNA also exists in symplasmata, a bacterial cell aggregate, which is different to a biofilm, in the rice enophyte Pantoea agglomerans YS19. We found that exogenous eDNA enhanced the formation and stability of symplasmata significantly, and that, exogenous eDNA also improved the stress resistance and colonization ability of the bacterium on host rice. These results strongly indicate novel roles of the eDNA in Pantoea agglomerans YS19, showing its special relation to the stress-resistance and endophyte-host association of the strain.

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  • Siquan Zeng, Juan Ling, Manzoor Ahmad, Liyun Lin, Yanying Zhang, Cong ...
    Article ID: 2018.02.004
    Published: 2018
    [Advance publication] Released: August 29, 2018

    Fungi are the most suitable cellulase producers attributing to its ability to produce a complete cellulase system. 33 Genus, 175 Species fungi were isolated from Sanya mangrove, Hainan, China. Using congo red cellulose (CMC) medium, five fungi of cellulose-degrading were selected for further study. Molecular biology and morphological identification showed that all of these five fungi belong to Aspergillus fungi. The cellulase produced by these fungi were monitored during liquid state fermentation. The optimum conditions study for enzyme production illustrated that the highest activities appeared at pH 3.0, 35°C after fermentation for 3 days. Beyond that, the enzyme activity of mixed fungi is 11–26% higher than pure. The study demonstrated that mixed culture improved the hydrolysis of fungi cellulase.

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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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  • Vipavee Cherdvorapong, Hidehisa Fujiki, Wasana Suyotha, Yoichi Takeda, ...
    Article ID: 2018.04.001
    Published: 2018
    [Advance publication] Released: July 17, 2018

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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

    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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