The Journal of General and Applied Microbiology
Online ISSN : 1349-8037
Print ISSN : 0022-1260
ISSN-L : 0022-1260
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Displaying 1-23 of 23 articles from this issue
  • Yanxiang Yao, Naren Xi, E Hai, Xiaomin Zhang, Jiayi Guo, Zhi Lin, Wei ...
    Article type: research-article
    Article ID: 2022.05.003
    Published: 2022
    Advance online publication: June 21, 2022
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    Supplementary material

    As a central signaling molecule, c-di-GMP (bis-(3,5)-cyclic diguanosine monophosphate) is becoming the focus for research in bacteria physiology. Pseudomonas aeruginosa PAO1 genome contains highly complicated c-di-GMP metabolizing genes and a number of these proteins have been identified and investigated. Especially, a sophisticated network of these proteins is emerging. In current study, mainly through Bacteria-2-Hybrid assay, we found PA0575 (RmcA), a GGDEF-EAL dual protein, to interact with two other dual proteins of PA4601 (MorA) and PA4959 (FimX). These observations imply the intricacy of c-di-GMP metabolizing protein interactions. Our work thus provides one piece of data to increase the understandings to c-di-GMP signaling.

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  • Tomohiro Inaba, Mami Yamaguchi, Akira Taniguchi, Yuya Sato, Tomo Aoy ...
    Article type: research-article
    Article ID: 2022.04.003
    Published: 2022
    Advance online publication: June 13, 2022
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    Supplementary material

    The decolorization of 11 dyes by granular sludge from an anaerobic expanded granular sludge bed (EGSB) reactor was evaluated. Biological decolorization of Reactive Red 21, 23, and 180, and Reactive Yellow 15, 17, and 23 in model textile wastewater was observed for the first time after a 7-day incubation (over 94% decolorization). According to the sequencing analysis of 16S rRNA gene amplicons from EGSB granular sludge, the operational taxonomic unit related to Paludibacter propionicigenes showed the highest increase in relative abundance ratios in the presence of dyes (7.12 times on average over 11 dyes) compared to those without dyes.

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  • Orn anong Chaiyachet, Ketsara Wongtham, Komsan Sangkasame
    Article type: research-article
    Article ID: 2022.05.002
    Published: 2022
    Advance online publication: June 10, 2022
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    The present study investigated the efficacy of bacterial cellulose production by K. xylinus TISTR 1011 and K. nataicola TISTR 975 using yam bean juice as a nutrient source, and the physicochemical and sensory characteristics of bacterial cellulose were examined. Bacterial cellulose content, production yield, and production rate were significantly higher when K. xylinus TISTR 1011 rather than K. nataicola TISTR 975 was used as the bacterial strain. The analysis of physicochemical characteristics revealed that bacterial cellulose produced by K. xylinus TISTR 1011 using yam bean juice medium had higher scores for CIE L*, a*, and b* values, wet weight, moisture content, firmness, and gel strength than bacterial cellulose produced by K. nataicola TISTR 975. In contrast, sensory evaluation showed that the acceptability scores and preference of all attributes of bacterial cellulose produced by K. nataicola TISTR 975 using yam bean juice medium were higher than those of bacterial cellulose produced by K. xylinus TISTR 1011. The results of this study indicate that yam bean juice from yam bean tubers, an alternative raw material agricultural product, can be used as a nutrient source for producing bacterial cellulose or nata by Komagataeibacter strains.

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  • Kasumi Shimodate, Hiroyuki Honda
    Article type: research-article
    Article ID: 2022.04.002
    Published: 2022
    Advance online publication: June 08, 2022
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    Godo is a traditional fermented soy food made in Aomori prefecture, Japan. It is mainly made of soybeans, rice koji, and salt. Since godo ripens during the long and severe winter in northeast Japan, it is assumed that lactic acid bacteria inhabiting godo have cold tolerance. We aimed to investigate the presence or absence of psychrotrophic lactic acid bacteria in godo. The viable counts of estimated lactic acid bacteria ranged from 106 to 108 cfu/g. In addition, aerobic and anaerobic microorganisms were detected in four godo products though the microbial population differed from sample to sample. Twenty-two bacterial strains were able to be isolated from godo, and all of the isolated strains were Gram-positive and catalase-negative. Some of the isolates grew well at 10°C. The carbohydrate fermentation profile of the selected three strains was determined by API50 CHL analysis. These strains were identified as Leuconostoc mesenteroides, and Latilactobacillus sakei by 16S rRNA gene sequence analysis. Leuconostoc mesenteroides strains HIT231 and HIT252, and Latilactobacillus sakei strain HIT273 could grow at 5°C in MRS broth, but their optimum growth temperature was 20°C-30°C. These results suggest that psychrotrophic lactic acid bacteria presumed to be derived from rice koji are present in godo, which is one of the factors in the low temperature ripening of godo in winter.

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  • Takashi Kuribayashi, Toshiki Sakurai, Akira Hatakeyama, Toshio Joh, Mi ...
    Article type: research-article
    Article ID: 2022.05.001
    Published: 2022
    Advance online publication: June 08, 2022
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    In Saccharomyces cerevisiae, ethyl caprylate is produced by the esterification of caprylic acid, which is synthesized through the action of fatty acid synthase. A recent study reported a yeast mutant with a single nucleotide substitution in the alpha subunit of fatty acid synthase (FAS2) gene (F1279Y; 3836T>A) that produced large amounts of ethyl caprylate. Here, we designed two primer sets (P1/P2 and P3/P4) with mismatches that incorporate restriction sites for the enzymes NdeI and SspI, respectively and developed an easy and rapid polymerase chain reaction-restriction fragment length polymorphism assay to identify yeasts harboring the FAS2-F1279Y mutation associated with high ethyl caprylate productivity.

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  • Zhi Tao, Shiqi Zheng, Shuangyue Liu, Jing Wang, Di Geng, Rufeng Wang
    Article type: research-article
    Article ID: 2021.10.002
    Published: 2022
    Advance online publication: May 31, 2022
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    Supplementary material

    Flavone C-glycosides are not easily degraded because of their strong C-C bond between sugar moieties and aglycones. However, some bacteria such as intestinal species can produce specific enzymes to degrade them. In this study, a bacterial strain P581a, which is capable of deglycosylating flavone C-glycosides, was isolated from human intestinal bacteria and was identified as Enterococcus gallinarum by morphological examination, physiological and biochemical analysis and 16S rRNA gene sequencing. This strain may produce a specific flavonoside glycosidase. The activity of the enzyme in the culture medium containing different quantity of carbon sources was also studied, and it was found that the content of carbon sources is negatively correlated with the deglycosylation efficiency of this strain.

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  • Hidetoshi Inoue, Kumiko Tajima, Cristina Mitsumori, Natsuko Inoue-Kash ...
    Article type: research-article
    Article ID: 2021.11.001
    Published: 2022
    Advance online publication: May 31, 2022
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    Supplementary material

    A genetically modified (GM) strain of the diatom Chaetoceros gracilis expressing the phosphite dehydrogenase gene (ptxD), which is a useful gene both for the biological containment and the avoidance of microbial contamination, was characterized to estimate the risk against the biodiversity by laboratory experiments. GM strain could grow in the medium containing phosphite as a sole source of phosphorus, while its general characteristics such as growth, salt tolerance, heat and dehydration resistance in the normal phosphate-containing medium were equivalent to those of wild type (WT) strain. The increase in potential toxicity of GM strain against plant, crustacean, fish and mammal was also disproved. The dispersal ability of WT strain cultured in an outdoor raceway pond was investigated for 28 days by detecting the psb31 gene in vessels, settled at variable distances (between 5 and 60 m) from the pond. The diatom was detected only in one vessel placed 5 m apart. To estimate the influence on the environment, WT and GM strains were inoculated into freshwater, seawater and soil. The influence on the microbiome in those samples was assessed by 16S rRNA gene amplicon sequencing, in addition to the analysis of the survivability of those strains in the freshwater and the seawater. The results indicated that the effect to the microbiome and the survivability were comparable between WT and GM strains. All results showed that the introduction of the ptxD gene into the diatom had a low risk on biodiversity.

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  • Takuma Suzuki, Kenji Morimoto
    Article type: research-article
    Article ID: 2022.01.004
    Published: 2022
    Advance online publication: May 31, 2022
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    Supplementary material

    Researchers continue to search for efficient processes to reduce the production costs of rare sugars. In this paper, we report a novel D-xylose isomerase from Shinella zoogloeoides NN6 (SzXI) and its application for efficient rare sugar production. Purified SzXI did not show remarkable properties when compared with those of a previously reported D-xylose isomerase. However, NN6 was found to express inducible SzXI and constitutive D-allulose 3-epimerase (SzAE) when cultivated with D-xylose as the sole carbon source. These two enzymes were partially purified and immobilized onto HPA25L, an anion exchange resin. The co-immobilized SzXI and SzAE (i-XA) showed optimal activity at 65°C in sodium phosphate buffer (pH 7.5) and 90°C in sodium phosphate buffer (pH 6.5), respectively. i-XA produced D-ribulose via D-xylulose from D-xylose at a conversion ratio of D-xylose:D-xylulose:D-ribulose of 72:18:10. Furthermore, D-allulose was also produced via D-fructose using D-glucose as the substrate, with a D-allulose yield of 11.2%. This is the first report describing a bacterium expressing D-xylose isomerase and D-allulose 3-epimerase that converts readily available sugars such as D-glucose and D-xylose to rare sugars.

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  • Yuko Yoshimura, Yuri Kobayashi, Takashi Kawaguchi, Shuji Tani
    Article type: research-article
    Article ID: 2021.10.005
    Published: 2022
    Advance online publication: May 23, 2022
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    Supplementary material

    We investigated the effects of deleting major extracellular protease-encoding genes on cellulolytic and xylanolytic enzyme production in Aspergillus aculeatus. We first investigated the effect of prtT deletion, a positive transcription factor for extracellular protease-encoding genes in Aspergillus, on extracellular protease production in A. aculeatus. Genetic analysis indicated that among the major extracellular proteases, pepIIa and pepIIb are controlled by PrtT, but pepI is not. Thus, we generated a mutant with deletion of the two genes prtT and pepIprtTΔpepI) and one with deletion of the three genes pepI, pepIIa, and pepIIbpepIΔIIaΔIIb). Extracellular protease activities decreased in both ΔprtTΔpepI and ΔpepIΔIIaΔIIb to 3% of that in the control strain (MR12). Comparative time-course analyses indicated that endoglucanase activity in ΔprtTΔpepI increased to double that in MR12. Xylanase activities increased in both ΔprtTΔpepI and ΔpepIΔIIaΔIIb to fourfold higher than that in MR12 at maximum. β-Glucosidase activities were increased in ΔprtTΔpepI and ΔpepIΔIIaΔIIb 1.3- and 1.4-fold higher than that in MR12 at maximum, respectively. Residual activities of endoglucanase, xylanase, and β-glucosidase after 7 days of incubation at 37°C in the culture supernatant were 63%, 36%, and 48% of the original in MR12. Residual endoglucanase activities were more than 80% of the original in ΔprtT, ΔprtTΔpepI, and ΔpepIΔIIaΔIIb. Residual xylanase activities were not improved in all test strains. β-Glucosidase remained almost 97% of the original in ΔprtTΔpepI. These findings indicated that the reduction of extracellular proteases effectively improved cellulolytic and xylanolytic enzyme production and stability in A. aculeatus.

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  • Ayan Mahanty, Subhrajyoti Giri, Akas Kar, Shilpi Ghosh
    Article type: research-article
    Article ID: 2022.01.005
    Published: 2022
    Advance online publication: May 23, 2022
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    Rice (Oryza sativa L.) straw is an agricultural byproduct of high yield, and its disposal by burning has detrimental effect on ecosystem. It has potential as source of fermentable sugars for industrial use; however, it requires effective pretreatment to remove lignin. Bacterial enzymes based pretreatment is advantageous due to their extracellular nature, and tolerance to higher temperature, pH and oxygen limitation. We herein report screening of lignocellulose degradation environment of vermicompost for ligninolytic bacteria, and studying role of Micrococcus unnanensis strain B4 in delignification of rice straw. The bacterium was capable to degrade acid soluble and insoluble lignin; and produced lignin degrading laccase and peroxidase having maximum activity at pH 6.5 and 72 h incubation. Both enzymes exhibited alkaline pH stability, and thermal stability with retention of 100 % activity on pre-incubation at 60 oC. The enzymes were used for pretreatment of rice straw using chemicals (acetic acid:hydrogen peroxide) pretreatment as reference. Scanning electron microscopy of pretreated rice straw samples showed alteration in morphology with exposure of cellulosic components. Enzymatically pretreated rice straw on saccharification by a commercial cellulase yielded about 400 mg of reducing sugar per gram, comparable to that released on chemical pretreatment. Hence, pretreatment based on M.unnanensis strain B4 and its ligninolytic enzymes can be an alternative to chemical pretreatment for saccharification of rice straw to fermentable sugars.

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  • Toshio Sakamoto, Yang Wei, Koki Yuasa, Yoshitaka Nishiyama
    Article type: research-article
    Article ID: 2022.01.003
    Published: 2022
    Advance online publication: May 20, 2022
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    Supplementary material

    The terrestrial cyanobacterium Nostoc commune is an anhydrobiotic organism with extreme longevity. Recovery of photosynthesis by rehydration was examined using our laboratory stocks of dry N. commune thalli after long-term storage in a desiccated state. In the samples stored at room temperature for over 8 years, photosynthetic oxygen evolution was barely detectable, whereas oxygen consumption was recovered. There was an exceptional case in which photosynthetic oxygen evolution recovered after 8 years of storage at room temperature. Both photosynthetic oxygen evolution and respiratory oxygen consumption were recovered in dry thalli stored at -20°C for over 15 years. Consistent with the recovery of photosynthetic oxygen evolution, Fv/Fm was detected in the samples stored at -20°C at levels similar to those of freshly collected N. commune colonies. Carotenoids, scytonemin and chlorophyll a appeared to be intact in the dry thalli stored at -20°C, but β-carotene was not detected in the samples stored at room temperature. α-Tocopherol was intact in the samples stored at -20°C but was degraded in the samples stored at room temperature. These results suggest that dry thalli of N. commune are capable of sustaining biological activities for a long time, although they are gradually damaged when stored at room temperature.

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  • Toshikazu Komoda, Yoshitaka Koseki
    Article type: research-article
    Article ID: 2022.03.001
    Published: 2022
    Advance online publication: May 20, 2022
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    In this study, we successfully isolated two compounds, 17T223A (1, C22 H22 O10 ) and 17T223B (2, C22 H20 O9 ), from a culture of Streptomyces sp. 17T223. Spectroscopic analyses revealed that these two compounds belong to the spiroximicin family. The chemical structure of 2 was consistent with that of the established antibiotic spiroximicin, whereas 1 was previously unknown. Furthermore, 1 exhibited moderate radical -scavenging activity, with an ED 50 of 1000 μM, whereas 2 showed no radical -scavenging activity, even at an ED50 of 2000 μM. Significant antimicrobial activity was exhibited by 2 whereas 1 exhibited no antimicrobial activity, suggesting that the epoxide portion of 2 influences its antimicrobial activity.

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  • Yoshihiro Watanabe, Yurika Yoshida, Toshiyuki Tokiwa, Mayuka Higo, Say ...
    Article type: research-article
    Article ID: 2022.03.002
    Published: 2022
    Advance online publication: May 20, 2022
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    Supplementary material

    A new antifungal polyketide, named hakuhybotric acid (1), was isolated from a cultured broth of a mycoparasitic fungus Hypomyces pseudocorticiicola FKI-9008, together with two known analogs, F2928-1 (2) and Cladobotric acid E (3). Their structures were elucidated by MS and NMR analyses. The structure of hakuhybotric acid was the two epoxy groups of F2928-1 converted to olefins. All compounds showed antifungal activity against four different species of Aspergillus spp., the causative agents of aspergillosis. It was suggested that mycoparasitic fungi are a useful source to search antifungal drug lead compounds.

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  • Mitsuhiro Itaya
    Article type: research-article
    Article ID: 2021.12.001
    Published: 2022
    Advance online publication: May 01, 2022
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    Bacillus subtilis Marburg 168 is a unique platform for genome engineering and genome synthesis. Genome scale DNA sequences can be synthesized by repeated integration of small DNA segments in the B. subtilis genome. The small DNA segments are collectively called dominos, and should cover the target genome. The B. subtilis strains which have been designed for use in the domino method are collectively called BGM: Bacillus subtilis Genome for Manipulation. The BGM system has been used to produce various genomes in the B. subtilis genome. The synthesized genomes have been demonstrated to be stably maintained as part of the B. subtilis genome. Instability of the synthesized genomes have been observed in genomes with Guanine plus Cytosine contents much higher or lower than that of BGM. The largest synthesized genome produced using this approach to date is that from Synecchosystis PCC6803, a photosynthetic microbe with a genome size of about 3.5 Mbp.

    The domino method depends on transformation, using the natural competence of B. subtilis. An alternative DNA uptake system, conjugational transfer, has been studied for the past 20 years. A self-transmissible plasmid named pLS20 has been used for the transfer and delivery of large amounts of DNA between B. subtilis. The BGM system is a unique platform for handling very large amounts of DNA from synthesis to dissemination to other cells, and has broad applications in research and practice.

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  • Aya Sato, Misaki Takamatsu, Satona Kobayashi, Michio Ogawa, Yuh Shiwa ...
    Article type: research-article
    Article ID: 2022.01.002
    Published: 2022
    Advance online publication: April 22, 2022
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    Supplementary material

    Among SigA-dependent promoters in Bacillus subtilis, we compared the nucleotide sequences of heat shock responding and non-responding promoters. Chimeric promoter experiments revealed that the heat shock response could be ascribed to the initiation nucleotide (iNTP) of the transcription. Our in vivo reporter assay results indicated that a full response was achieved using GTP, a reduced response was observed using ATP, and no additional expression was observed using UTP or CTP. We then investigated the in vitro transcription assay in more detail. Enhanced transcription that was dependent upon the iNTP was observed when heat treatment was administered during the pre-initiation period. We next analyzed the efficiency of open complex formation using potassium permanganate footprinting, and our results revealed an increase in the ratio of open complex formation at elevated temperatures. Based on this, we suggest that the overall intensification of transcription at high temperatures was derived from the high efficiency of open complex formation together with the high affinity of RNA polymerase (RNAP) for the initiation nucleotide GTP. To determine if this mechanism observed in B. subtilis RNAP is common among bacterial species, we performed similar experiments using Escherichia coli RNAP. Our results indicated that E. coli RNAP also exhibited both temperature- and iNTP-dependent enhancement of transcription. Although the temperature ranges and the ratios of enhancement are somewhat different, the overall heat shock response mechanism mediated by the iNTP of transcription appears to be conserved among bacterial RNAP.

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  • Qian Lin, Jieni Li, Xinru Ling, Xinmei Zhang
    Article type: research-article
    Article ID: 2021.10.001
    Published: 2022
    Advance online publication: April 18, 2022
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    trans-Anethole oxygenase (TAO) is the key enzyme responsible for the oxidation of trans-anethole to p-anisaldehyde. A strain, Paraburkholderia sp. MR185, was isolated from soil in Yulin star anise-planting regions using trans-anethole as a sole carbon source and a gene which encodes a protein with high similarities to a hypothetical protein of Paraburkholderia sp. MM5384-R2 which shows 61.27% identies with TAO from Pseudomonas putida JYR-1 was cloned and sequenced. The gene, tao, was expressed in E. coli cells and its protein product was purified by affinity chromatography through regenerated amorphous cellulose (RAC). SDS-PAGE analysis indicated a clear band of recombinant protein TAO, and its molecular weight, 38.3 kDa, was consistent with the theoretical value. Its enzyme activity of producing p-anisaldehyde from trans-anethole was detected by DNPH (2,4-dinitrophenylhydrazine) chromogenic reaction and HPLC, and the specific activity of TAO reached 3.93 U/mg protein. Immobilized TAO on RAC was used to catalyze the production of p-anisaldehyde from trans-anethole, and the enzyme retained more than 60% of its initial activity after 10 uses. This is the first report on Paraburkholderia TAO.

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  • Yu Shinjyo, Naoya Midorikawa, Takashi Matsumoto, Yuki Sugaya, Yos ...
    Article type: research-article
    Article ID: 2021.09.005
    Published: 2022
    Advance online publication: April 13, 2022
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    Supplementary material

    Recently, the antibacterial effects of essential oils have been investigated in addition to their therapeutic purposes. Owing to their hydrophobic nature, they are thought to perturb the integrity of the bacterial cell membrane, leading to cell death. Against such antibiotic challenges, bacteria develop mechanisms for cell envelope stress responses (CESR). In Bacillus subtilis, a gram-positive sporulating soil bacterium, the extracytoplasmic function (ECF) sigma factor-mediated response system plays a pivotal role in CESR. Among them, σM is strongly involved in response to cell envelope stress, including a shortage of available bactoprenol. Vetiver essential oil, a product of Chrysopogon zizanioides (L.) Roberty root, is also known to possess bactericidal activity. σM was exclusively and strongly induced when the cells were exposed to Vetiver extract, and depletion of multi-ECF sigma factors (ΔsigM, ΔsigW, ΔsigX, and ΔsigV) enhanced sensitivity to it. From this quadruple mutant strain, the suppressor strains, which restored resistance to the bactericidal activity of Vetiver extract, emerged, although attempts to obtain resistant strains from the wild type did not succeed. Whole-genome resequencing of the suppressor strains and genetic analysis revealed inactivation of xseB or pnpA, which code for exodeoxyribonuclease or polynucleotide phosphorylase, respectively. This allowed the quadruple mutant strain to escape from cell death caused by Vetiver extract. Composition analysis suggested that the sesquiterpene, khusimol, might contribute to the bactericidal activity of the Vetiver extract.

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  • Kimihiro Abe, Hiroko Kato, Yuta Hasegawa, Tatsuya Yamamoto, Nobuhik ...
    Article type: research-article
    Article ID: 2021.10.006
    Published: 2022
    Advance online publication: April 13, 2022
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    Supplementary material

    Paenibacillus polymyxa is a spore-forming Gram-positive bacterial species. Both its sporulation process and the spore properties are poorly understood. Here, we investigated sporulation in P. polymyxa ATCC39564. When cultured at 37℃ for 24 h in sporulation medium, more than 80% of the total cells in the culture were spores. Time-lapse imaging revealed that cellular morphological changes during sporulation of P. polymyxa were highly similar to those of B. subtilis. We demonstrated that genetic deletion of spo0A, sigE, sigF, sigG, or sigK, which are highly conserved transcriptional regulators in spore forming bacteria, abolished spore formation. In P. polymyxa, spo0A was required for cell growth in sporulation medium, as well as for the initiation of sporulation. The sigE and sigF mutants formed abnormal multiple asymmetric septa during the early stage of sporulation. The sigG and sigK mutants formed forespores in the sporangium, but they did not become mature. Moreover, fluorescence reporter analysis confirmed compartment-specific gene expression of spoIID and spoVFA in the mother cell and spoIIQ and sspF in the forespore. Transmission electron microscopy imaging revealed that P. polymyxa produces multilayered endospores but lacking a balloon-shaped exosporium. Our results indicate that spore morphogenesis is conserved between P. polymyxa and B. subtilis. However, P. polymyxa genomes lack many homologues encoding spore-coat proteins that are found in B. subtills, suggesting that there are differences in the spore coat composition and surface structure between P. polymyxa and B. subtilis.

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  • Akira Matsumoto, Sung-Jin Kawai, Miwa Yamada
    Article type: research-article
    Article ID: 2021.11.002
    Published: 2022
    Advance online publication: April 13, 2022
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    The marine bacterium Cobetia sp. IU180733JP01 (5-11-6-3) can accumulate poly(3- hydroxybutyrate) [P(3HB)] during cultivation on alginate or waste Laminaria sp. Here, we examined this strain’s ability to utilize various carbon sources for P(3HB) production. When cultured in mineral salt medium containing 1% (w/v) glucose, fructose, glycerol, or gluconic acid, the strain showed better growth and higher P(3HB) production than on alginate, with fructose enabling the highest P(3HB) yield (0.8 ± 0.06 g/L). We also predicted metabolic pathways for P(3HB) synthesis based on draft genome sequence analysis, in which carbon sources are assimilated through Entner–Doudoroff and Embden–Meyerhof pathways, and the resultant acetyl-CoA is converted into P(3HB). Our findings reveal the potential of the 5-11-6-3 strain for application in bioplastic production from not only marine biomass but also other biomass and industrial wastes.

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  • Kyosuke Kita, Sanako Yoshida, Shu Ishikawa, Ken-ichi Yoshida
    Article type: research-article
    Article ID: 2021.11.003
    Published: 2022
    Advance online publication: April 13, 2022
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    Bacteriocins are a large family of peptides synthesized ribosomally by a variety of bacterial species. The genome of one of the thermophilic Gram-positive bacteria, Aeribacillus pallidus PI8, was found to possess an operon comprising five genes possibly involved in the production of a putative bacteriocin that was named pcnABCDE for the production of “pallidocyclicin.” This study investigated the function of the pcn operon experimentally. The heterologous expression of the entire pcn operon from the plasmid was toxic to Escherichia coli but not to Bacillus subtilis. However, when the entire pcn operon was expressed constitutively, even the growth of B. subtilis was impaired, and at least pcnA was implied to serve as the precursor of pallidocyclicin. In addition, a strain of B. subtilis expressing the entire pcn operon from the plasmid showed toxicity to another thermophilic species, Geobacillus kaustophilus, at elevated temperatures, whereas another strain lacking pcnE alone from the pcn operon lost the toxicity, suggesting that pcnE might be involved in the biosynthesis of pallidocyclicin when it is produced in B. subtilis.

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  • Muhammad Imran Firdaus Kamardan, Ezzah Atikah Binti Marsid, Fazrena Na ...
    Article type: research-article
    Article ID: 2021.09.004
    Published: 2022
    Advance online publication: April 08, 2022
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    Supplementary material

    Tropical peatlands account for one of the largest carbon stores in the form of organic matter due to the accumulation of plant litter and waterlogged conditions. Recent anthropogenic disturbances, such as forest fires, agricultural conversion and drainage, in tropical peatlands have caused a vast amount of carbon to be released into the atmosphere, and microbial activities are impacted by these changes. A recent study showed that many phenol- and lignin-degrading bacteria prefer alkaline and neutral pH conditions, while tropical peatland conditions are acidic, possibly changing the mechanisms of the utilization of organic matter from peat soil. The purpose of this study was to isolate and characterize phenolic compound-degrading bacteria from tropical peatlands under acidic conditions due to the lack of information on how the biological processes of microorganisms occur in this unique habitat. Two isolates show the capability to utilize phenolic aldehydes based on building blocks of lignin that are abundant in tropical peatlands, including hydroxyphenyl, guaiacyl and syringyl units. The identification of these isolates by 16S rRNA gene sequence shows that strain S38 is similar to Stenotrophomonas sp., while strain S46 is similar to Burkholderia sp. Further characterization of these isolates shows their ability to degrade 4-hydroxybenzaldehyde and vanillin into phenolic acids within 24 hours of incubation and syringaldehyde within 7 days of incubation. In conclusion, these isolated bacteria show the ability to withstand the acidic environment of tropical peatlands and utilize lignin monomers through unknown metabolic pathways.

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  • Shota Suzuki, Sachie Osada, Daisuke Imamura, Tsutomu Sato
    Article type: research-article
    Article ID: 2021.10.004
    Published: 2022
    Advance online publication: April 06, 2022
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    Supplementary material

    Site-specific recombination (SSR) systems are employed in many genetic mobile elements, including temperate phages, for their integration and excision. Recently, they have also been used as tools for applications in fields ranging from basic to synthetic biology. SPβ is a temperate phage of the Siphoviridae family found in the laboratory standard Bacillus subtilis strain 168. SPβ encodes a serine-type recombinase, SprA, and recombination directionality factor (RDF), SprB. SprA catalyzes recombination between the attachment site of the phage, attP, and that of the host, attB, to integrate phage genome into the attB site of the host genome and generate attL and attR at both ends of the prophage genome. SprB works in conjunction with SprA and switches from attB/attP to attL/R recombination, which leads to excision of the prophage. In the present study, we took advantage of this highly efficient recombination system to develop a site-specific integration and excision plasmid vector, named pSSβ. It was constructed using pUC plasmid and the SSR system components, attP, sprA and sprB of SPβ. pSSβ was integrated into the attB site with a significantly high efficiency, and the resulting pSSβ integrated strain also easily eliminated pSSβ itself from the host genome by the induction of SprB expression with xylose. This report presents two applications using pSSβ that are particularly suitable for gene complementation experiments and for a curing system of SPβ prophage, that may serve as a model system for the removal of prophages in other bacteria.

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  • Manami Kawakami, Satoshi Matsuoka
    Article type: research-article
    Article ID: 2021.08.002
    Published: 2022
    Advance online publication: April 02, 2022
    JOURNAL FREE ACCESS ADVANCE PUBLICATION

    Arabidopsis thaliana monogalactosyldiacylglycerol synthase 1 (AtMGD1) and digalactosyldiacylglycerol synthase 2 (AtDGD2) genes introduced into a Bacillus subtilis chromosome with disrupted galE, which encodes UDP-glucose 4-epi merase, enabled the mutant to produce monogalactosyldiacylglycerol. When galE mutant cells are cultivated in galactose containing medium they show ab normal morphology. This phenotype is correlated with a decrease in the amount of glucolipids. Nucleoids of the ugtP and galE mutants were stained by propidium iodide, which does not permeate intact cell membranes, whereas nucleoids of wild type and of a pssA mutant we examined were not stained. Expression of the AtMGD1 gene in a ugtP galE double mutant restored cell membrane integrity. Expression of galactolipid synthase genes from a multi-copy plasmid, pDGHisN4, allowed higher production of galactolipids. Activation of the extracytoplasmic function sigma factors SigM, SigV, and SigX, in the ugtP mutant was decreased by expression of AtMGD1, and SigX activity was strongly repressed when both AtMGD1 and AtDGD2 genes were expressed in the mutant. We conclude that the number of sugars that bind to diacylglycerol rather than the exact sugar species is important for glycolipid function in B. subtilis.

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