The Journal of General and Applied Microbiology
Online ISSN : 1349-8037
Print ISSN : 0022-1260
ISSN-L : 0022-1260
Advance online publication
Showing 1-27 articles out of 27 articles from Advance online publication
  • Yoshio Inoue, Yukiyo Fukunaga, Hiroshi Katsumata, Shoko Ohji, Akira Ho ...
    Type: research-article
    Article ID: 2019.10.001
    Published: 2020
    [Advance publication] Released: May 26, 2020
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    An aerobic bacterium, designated strain 5N-3 (NBRC 113055), that degrades cis-dichloroethene (cDCE) was isolated from a sea sediment in Japan. Strain 5N-3 was able to degrade a certain amount of cDCE in the presence of pyruvate without the action of inducers. In the presence of inducers, such as phenol and benzene, the strain completely removed cDCE. By the application of 16S ribosomal RNA (16S rRNA) gene sequencing and average nucleotide identity analyses, the strain 5N-3 was identified as Marinobacter salsuginis. On the other hand, identified species of Marinobacter are not known to degrade cDCE at all. A draft genome sequence analysis of the strain 5N-3 suggested that the dmp-homologous operon (operon for phenol degradation) may be contributing to the aerobic degradation of cDCE. This is the first report on an aerobic marine bacterium that has been found to degrade cDCE.

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  • Fan-Ching Hsieh, Lu-Kao Chang, Chih-Hsuan Tsai, Jung-En Kuan, Ke-Feng ...
    Type: research-article
    Article ID: 2019.12.003
    Published: 2020
    [Advance publication] Released: May 19, 2020
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    An Escherichia coli ATP-dependent two-component protease, ClpYQ(HslUV), targets the SulA molecule, an SOS induced protein. ClpY recognizes, unfolds and translocates the substrates into the proteolytic site of ClpQ for degradation. ClpY is divided into three domains N, I and C. The N domain is an ATPase; the C domain allows for oligomerization, while the I domain coordinates substrate binding. In the ClpYQ complex, two layer pore sites, pore I and II, are in the center of its hexameric rings. However, the actual roles of two outer-loop (130~159 aa, L1 and 175~209 aa, L2) of the ClpY-I domain for the degradation of SulA are unclear. In this study, with ATP, the MBP-SulA molecule was bound to ClpY oligomer(s). ClpYΔL1 (ClpY deleted of loop 1) oligomers revealed an excessive SulA-binding activity. With ClpQ, it showed increased proteolytic activity for SulA degradation. Yet, ClpYΔL2 formed fewer oligomers that retained less proteolytic activity, but still had increased SulA-binding activity. In contrast, ClpYΔpore I had a lower SulA-binding activity. ClpYΔ pore I ΔL2 showed the lowest SulA-binding activity. In addition, ClpY (Q198L, Q200L), with a double point mutation in loop 2, formed stable oligomers. It also had a subtle increase in SulA-binding activity, but displayed less proteolytic activity. As a result, loop 2 has an effect on ClpY oligomerization, substrate binding and delivery. Loop 1 has a role as a gate, to prevent excessive substrate binding. Thus, accordingly, ClpY permits the formation of SulA-ClpY(6x), with ATP(s), and this complex then docks through ClpQ(6x) for ultimate proteolytic degradation.

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  • Katsuhiko Okada, Shoko Fujiwara, Mikio Tsuzuki
    Type: research-article
    Article ID: 2020.02.002
    Published: 2020
    [Advance publication] Released: April 24, 2020
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    Photosynthesis is a biological process of energy conversion from solar radiation to useful organic compounds for the photosynthetic organisms themselves. It, thereby, also plays a role of food production for almost all animals on the Earth. The utilization of photosynthesis as an artificial carbon cycle is also attracting a lot of attention regarding its benefits for human life. Hydrogen and biofuels, obtained from photosynthetic microorganisms, such as microalgae and cyanobacteria, will be promising products as energy and material resources. Considering that the efficiency of bioenergy production is insufficient to replace fossil fuels at present, techniques for the industrial utilization of photosynthesis processes need to be developed intensively. Increase in the efficiency of photosynthesis, the yields of target substances, and the growth rates of algae and cyanobacteria must be subjects for efficient industrialization. Here, we overview the whole aspect of the energy production from photosynthesis to biomass production of various photosynthetic microorganisms.

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  • Masakazu Saito, Satoru Watanabe, Kaori Nimura-Matsune, Hirofumi Yoshik ...
    Type: research-article
    Article ID: 2020.02.001
    Published: 2020
    [Advance publication] Released: April 10, 2020
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    Supplementary material

    The CIRCE/HrcA system is highly conserved in cyanobacterial genomes. We have shown that heat-shock induction of the groESL1 operon in the cyanobacterium Synechocystis sp. PCC6803 is negatively regulated by the CIRCE/HrcA system. In Synechococcus elongatus PCC7942, a novel heat shock protein, Orf7.5, is involved in positive regulation of the groESL1 transcription. However, Orf7.5 is not conserved in some cyanobacteria, including Synechocystis sp. PCC6803. The purpose of this study is to evaluate the functional conservation of the CIRCE/HrcA system in S. elongatus PCC7942 and to understand the interplay between the CIRCE/HrcA system and the Orf7.5 regulatory system. We constructed single and double mutants of S. elongatus orf7.5, hrcA and orf7.5/hrcA and heat induction of the groESL1 transcription in these mutants was analyzed. Unexpectedly, derepression of the groESL1 transcription in an hrcA mutant was not observed. In all these mutants, the transcription was greatly suppressed under both normal and heat stress conditions, indicating that both HrcA and Orf7.5 are involved in regulation of the groESL1 transcription in a positive way. Consistent with the decrease in the groESL1 mRNA level, all the single and double mutants showed a great loss of acquired thermotolerance. Heat induction of the orf7.5 promoter activity was totally diminished in the orf7.5 mutant, indicating that Orf7.5 activates its own transcription. Yeast two hybrid analysis showed that the principle sigma factor RpoD1 interacts with Orf7.5. These results indicate that Orf7.5 enhances the transcription of groESL1 and orf7.5 by interacting with RpoD1.

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  • Akira Yasuda, Daichi Inami, Mitsumasa Hanaoka
    Type: research-article
    Article ID: 2020.01.010
    Published: 2020
    [Advance publication] Released: April 07, 2020
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    In cyanobacteria, transcription of a set of genes is specifically induced by high-light-stress conditions. In previous studies, RpaB, a response regulator of the two-component system, was shown to be involved in this regulation in vitro and in vivo. In this study, we examined whether RpaB-dependent transcriptional regulation was extensively observed, not only under high-light-stress conditions but also under various light intensities. Transcription of high-light-dependent genes hliA, nblA and rpoD3 was transiently and drastically induced during a dark-to-light shift in a manner similar to high-light-stress responses. Moreover, expression of these genes was activated under various light-intensity upshift conditions. Phos-tag SDS-PAGE experiments showed that the phosphorylation level of RpaB was decreased along with transcriptional induction of target genes in all of the light environments examined herein. These results suggest that RpaB may be widely involved in transcriptional regulation under dark-to-light and light-intensity upshift conditions and that high-light-responsive genes may be required in various light conditions other than high-light condition. Furthermore, it is hypothesised that RpaB is regulated by redox-dependent signals rather than by high-light-stress-dependent signals.

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  • Haruki Yamamoto, Hiroko Kojima-Ando, Kaori Ohki, Yuichi Fujita
    Type: research-article
    Article ID: 2020.01.009
    Published: 2020
    [Advance publication] Released: April 02, 2020
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    Protochlorophyllide (Pchlide) reduction is the penultimate step of chlorophyll (Chl) biosynthesis, and is catalyzed by two evolutionarily unrelated enzymes: dark-operative Pchlide oxidoreductase (DPOR) and light-dependent Pchlide oxidoreductase (LPOR). Because LPOR is the sole Pchlide reductase in angiosperms, dark-grown seedlings of angiosperms become etiolated. LPOR exists as a ternary complex of Pchlide-NADPH-LPOR to form paracrystalline prolamellar bodies (PLBs) in etioplasts. Because LPOR is distributed ubiquitously across oxygenic phototrophs including cyanobacteria, it would be important to determine whether cyanobacterial LPOR has the ability to form PLBs. We isolated a DPOR-less transformant ΔchlL/LPORox, carrying a plasmid to overexpress cyanobacterial LPOR in the cyanobacterium Leptolyngbya boryana. The transformant did not produce Chl in the dark and became etiolated with an accumulation of Pchlide and LPOR. Novel PLB-like ultrastructures were observed in etiolated cells, which disappeared during the early stage of the light-dependent greening process. However, the rate of Chl production in the greening process of ΔchlL/LPORox was almost the same as that observed in the control cells, which carried an empty vector. An in vitro LPOR assay of extracts of dark-grown ΔchlL/LPORox cells suggested that the PLB-like structures are deficient in NADPH. Low-temperature fluorescence emission spectra of membrane fractions of the etiolated cells indicated the absence of the photoactive form of Pchlide, which was consistent with the inefficiency of the greening process. Cyanobacterial LPOR exhibited an intrinsic ability to form PLB-like ultrastructures in the presence of the co-accumulation of Pchlide; however, the PLB-like structure differed from the authentic PLB regarding NADPH deficiency.

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  • Gen Enomoto, Ayako Kamiya, Yukiko Okuda, Rei Narikawa, Masahiko Ikeuch ...
    Type: research-article
    Article ID: 2020.01.007
    Published: 2020
    [Advance publication] Released: March 30, 2020
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    Supplementary material

    Second messenger molecules are crucial components of environmental signaling systems to integrate multiple inputs and elicit physiological responses. Among various kinds of second messengers, cyclic nucleotides cAMP and cyclic di-GMP (c-di-GMP) play pivotal roles in bacterial environmental responses. However, how these signaling systems are interconnected for a concerted regulation of cellular physiology remains elusive. In a thermophilic cyanobacterium Thermosynechococcus vulcanus strain RKN, incident light color is sensed by cyanobacteriochrome photoreceptors to transduce the light information to the levels of c-di-GMP, which induces cellular aggregation probably via cellulose synthase activation. Herein, we identified that Tlr0485, which is composed of a cGMP-specific phosphodiesterases, adenylate cyclases, and FhlA (GAF) domain and an HD-GYP domain, is a cAMP-activated c-di-GMP phosphodiesterase. We also show biochemical evidence that the two class-III nucleotide cyclases, Cya1 and Cya2, are both adenylate cyclases to produce cAMP in T. vulcanus. The prevalence of cAMP-activated c-di-GMP phosphodiesterase genes in cyanobacterial genomes suggests that the direct crosstalk between cAMP and c-di-GMP signaling systems may be crucial for cyanobacterial environmental responses.

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  • Kazuki Nagata, Katsuaki Oyama, Atsushi Ota, Chihiro Azai, Kazuki Terau ...
    Type: research-article
    Article ID: 2020.01.008
    Published: 2020
    [Advance publication] Released: March 30, 2020
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    Supplementary material

    The cyanobacterial circadian oscillator can be reconstituted by mixing the purified clock proteins KaiA, KaiB, and KaiC with ATP in vitro, leading to a 24-h oscillation of KaiC phosphorylation. The cyanobacterial mutant pr1 carrying valine instead of alanine at position 422 of KaiC (KaiC-A422V) lost the ability to shift the phase of the circadian rhythm. In this study, we analyzed KaiC-A422V to investigate the effect of this single-residue substitution on the in vitro reconstitution of KaiC oscillation. KaiC-A422V exhibited low amplitude oscillations of phosphorylation with a smaller amount of Kai complex than wild-type KaiC (KaiC-WT). Although KaiA can stimulate KaiC phosphorylation, the phosphorylation level of KaiC-A422V is much lower than that of KaiC-WT even at higher KaiA concentrations. It has been suggested that monomer shuffling of KaiC is involved in entraining the in vitro rhythm. To examine whether KaiC-A422V has the capacity for monomer shuffling, we used the difference in the amplitude of the phosphorylation rhythms between KaiC-WT and KaiC-A422V as the indicator of monomer shuffling. When KaiC-A422V and KaiC-WT were mixed, the amplitude of the phosphorylation rhythm changed according to the mixing ratio. This suggests that KaiC-A422V has a reduced ability to shuffle monomers in hexameric KaiC. In addition, the A422V mutation resulted in a change of the stability of the KaiC protein.

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  • Kenjiro Sugiyama, Shinichi Takaichi
    Type: research-article
    Article ID: 2020.01.005
    Published: 2020
    [Advance publication] Released: March 28, 2020
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    Cyanobacteria are oxygenic photoautotrophic prokaryotes containing chlorophylls and carotenoids, and the latter play important roles in light-harvesting, protection of excess light, assembly of pigment-protein complexes, and stabilization of lipid membranes. Cyanobacteria produce many kinds of carotenoids, such as β-carotene, zeaxanthin, echinenone, and myxol glycosides, which have a cyclic structure at one or both end(s). Cyclization of lycopene is a branch point in carotenoid biosynthesis to β-carotene and γ-carotene. Two types of lycopene cyclases, CruA/CruP-type and CrtL-type, are functionally confirmed in only five species, while homologous genes are found in the genomes of most cyanobacteria. This review summarizes the carotenogenesis pathways and the functional enzymes along with genes, focusing particularly on the cyclization of lycopene by distinct types of lycopene cyclases in cyanobacteria.

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  • Naoya Shimada, Yukiko Okuda, Kaisei Maeda, Daisuke Umeno, Shinichi Tak ...
    Type: research-article
    Article ID: 2020.01.003
    Published: 2020
    [Advance publication] Released: March 25, 2020
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    Supplementary material

    Heterologous production of a useful carotenoid astaxanthin was achieved in a cyanobacterium Synechocystis sp. PCC 6803 with the aid of marine bacterial genes. Astaxanthin and its intermediates emerged at high levels, whereas β-carotene and zeaxanthin disappeared in the strain. Total carotenoid accumulation was nearly two fold compared with wild type. The astaxanthin-producing strain was capable of only growing heterotrophically, which was likely due to the absence of β-carotene. Further enhanced accumulation was pursued by gene overexpression for possible rate-limiting steps in the biosynthesis pathway.

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  • Ayumi Kizawa, Takashi Osanai
    Type: research-article
    Article ID: 2020.01.004
    Published: 2020
    [Advance publication] Released: March 13, 2020
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    Supplementary material

    In photosynthetic microorganisms, cell cycle progression depends on day and night cycles; however, how cell division is regulated in response to these environmental changes is poorly understood. RpaA has been implicated in the signal output from both circadian clocks and light/dark conditions in the unicellular spherical-celled cyanobacterium Synechocystis sp. PCC 6803. In the present study, we investigated the involvement of a two-component response regulator RpaA in cell division regulation. Firstly, we examined the effects of rpaA overexpression on cell morphology and the expression levels of cell division genes. We observed an increase in the volume of non-dividing cells and a high proportion of dividing cells in rpaA-overexpressing strains by light microscopy. The expression levels of selected cell division-related genes were higher in the rpaA-overexpressing strain than in the wild type, including minD of the Min system; cdv3 and zipN, which encode two divisome components; and murB, murC, and pbp2, which are involved in peptidoglycan (PG) synthesis. Moreover, in the rpaA-overexpressing strain, the outer membrane and cell wall PG layer were not smooth, and the outer membrane was not clearly visible by transmission electron microscopy. These results demonstrated that rpaA overexpression causes an impaired cell division, which is accompanied by transcriptional activation of cell division genes and morphological changes in the PG layer and outer membrane.

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  • Tomonori Kashimoto, Keita Miyake, Mayuko Sato, Kaisei Maeda, Chikahiro ...
    Type: research-article
    Article ID: 2019.11.008
    Published: 2020
    [Advance publication] Released: March 07, 2020
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    Supplementary material

    The cyanobacterium Acaryochloris marina MBIC 11017 (A. marina 11017) possesses chlorophyll d (Chl. d) peaking at 698 nm as photosystem reaction center pigments, instead of chlorophyll a (Chl. a) peaking at 665 nm. About 95% of the total chlorophylls is Chl. d in A. marina 11017. In addition, A. marina 11017 possesses phycobilisome (PBS) supercomplex to harvest orange light and to transfer the absorbing energy to the photosystems. In this context, A. marina 11017 utilizes both far-red and orange light as the photosynthetic energy source. In the present study, we incubated A. marina 11017 cells under monochromatic orange and far-red light conditions and performed transcriptional and morphological studies by RNA-seq analysis and electron microscopy. Cellular absorption spectra, transcriptomic profiles, and microscopic observations demonstrated that PBS was highly accumulated under an orange light condition relative to a far-red light condition. Notably, transcription of one cpcBA operon encoding the phycobiliprotein of the phycocyanin was up-regulated under the orange light condition, but another operon was constitutively expressed under both conditions, indicating functional diversification of these two operons for light harvesting. Taking the other observations into consideration, we could illustrate the photoacclimation processes of A. marina 11017 in response to orange and far-red light conditions in detail.

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  • Satoru Watanabe, Shunsuke Saito, Yasuhiro Suezaki, Takeshi Seguchi, Ry ...
    Article ID: 2019.11.005
    Published: 2020
    [Advance publication] Released: February 25, 2020
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    In bacterial DNA replication, the initiator protein DnaA binds to the multiple DnaA box sequences located at oriC to facilitate the unwinding of duplex DNA strands. The cyanobacterium Synechococcus elongatus PCC 7942, which contains multiple chromosomal copies per cell, has DnaA box (like sequences around the oriC region, which is located upstream of dnaN. We previously observed the binding of DnaA around the oriC region; however, the DNA-binding specificity of DnaA to DnaA box sequences has not been examined. Here, we analyzed the binding specificity of DnaA protein to the DnaA box in S. elongatus by using bio-layer interferometry (BLI), a method for monitoring intermolecular interactions. We observed that recombinant DnaA protein recognized specifically the DnaA box sequence TTTTCCACA in vitro. In addition, DNA binding activity was significantly increased by R328H mutation of DnaA. This is the first report to characterize DnaA binding to the DnaA box sequence in cyanobacteria.

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  • Tsukasa Saito, Koichiro Awai
    Type: research-article
    Article ID: 2019.11.004
    Published: 2020
    [Advance publication] Released: February 18, 2020
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    Supplementary material

    Heterocysts are the specialized cells for nitrogen fixation in some filamentous cyanobacteria. To protect the oxygen labile nitrogen fixing enzyme, nitrogenase, heterocysts keep their inner environment microoxic by developing layers of barrier on the outside of their outer membranes. Heterocyst specific glycolipids (Hgls) are constituents of the layer of barrier and amphipathic compounds, synthesized from a very long chain fatty alcohol as a hydrophobic tail and a sugar as a polar head. In the model heterocystous cyanobacterium Anabaena sp. PCC 7120, Hgls are made of fatty alcohol with 26 carbons and a glucose, linked by an ether bond in alpha configuration. The fatty alcohol is synthesized via reactions of a polyketide synthase, HglEA. In Anabaena sp. PCC 7120, another polyketide synthase HglE2 shared more than 50% identity in an amino acid sequence with HglEA and is expected to be involved in Hgls synthesis. However, no direct evidence has been reported. Here, we experimentally show that HglEA is the contributor of Hgls synthesis, and that HglE2 is not involved in the development of the heterocyst specific glycolipid layer.

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  • Chihiro Kadooka, Eri Nakamura, Shingo Kubo, Kayu Okutsu, Yumiko Yoshiz ...
    Type: research-article
    Article ID: 2019.09.003
    Published: 2020
    [Advance publication] Released: January 31, 2020
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    Supplementary material

    Naturally occurring fungi have been used in the traditional production of dried bonito, Katsuobushi, in Japan. In this study, we analyzed the fungal population present during Katsuobushi production. Amplicon sequence analysis of ITS1 indicated that Aspergillus spp. are predominant throughout the production process. In addition, culture-dependent analyzes identified three species Aspergillus chevalieri, Aspergillus montevidensis, and Aspergillus sydowii, based on sequencing of benA, caM, and rpb2 genes. A. chevalieri isolates were classified into teleomorphic and anamorphic strains based on morphological analysis. A. chevarieri was the dominant species throughout the production process, whereas A. montevidensis increased and A. sydowii decreased in abundance during Katsuobushi production. Our study will enhance the understanding of fungal species involved in traditional Katsuobushi production.

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  • Kiran Gupta, P. P. Baruah
    Type: research-article
    Article ID: 2019.09.001
    Published: 2020
    [Advance publication] Released: January 24, 2020
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    Cyanobacteria are an important component in the rice field ecosystem and are a well known source of natural biofertilizer. Pesticidal application for the control of pests in rice field soil has led to several environmental problems, and poses a great threat to these beneficial microorganisms. Studies on the impact of pesticides on the diazotrophic growth and survivability of these microorganisms have recently gained much attention. The present paper describes the effects of an iterated use of the insecticide deltamethrin (2.8% EC) on the growth and nitrogen fixation capacity of the filamentous cyanobacterium Calothrix sp. (strain GUEco 1002). This organism has shown a varying degree of sensitivity to the insecticide. For evaluating the deltamethrin toxicity, the test organism was subjected to varying concentrations of deltamethrin i.e. 17.5 ppm, 35 ppm, 70 ppm and 140 ppm based upon LC50 for 20 days. The data obtained in the laboratory revealed that the treatment of the test organism with deltamethrin (17.5–140 ppm) negatively affected its growth, pigments, protein and nitrogen content in a time dose dependent manner. In contrast, carbohydrate content significantly increased with increasing concentrations of deltamethrin, this effect being more prominent at 140 ppm treatment (38%). At this high level (140 ppm), the test organism showed a significant decrease in dry weight biomass (46%), chlorophyll-a (72%), carotenoids (57%), phycocyanin (67%), protein (69%) and nitrogen content (61%) over the control. A little, but insignificant, stimulatory effect on nitrogen content was recorded at 17.5 ppm of the insecticide which however, was the opposite in the case of growth, pigments, carbohydrate and protein content.

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  • Chengchuan Che, Tao Su, Ping Sun, Guizhi Li, Jinfeng Liu, Zengfan Wei, ...
    Type: research-article
    Article ID: 2019.09.002
    Published: 2019
    [Advance publication] Released: December 27, 2019
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    Supplementary material

    Thioredoxins (Trxs) and protein-disulfide isomerases (PDIs) are believed to play a pivotal role in ensuring the proper folding of proteins, facilitating appropriate functioning of proteins, and maintaining intracellular redox homeostasis in bacteria. Two thioredoxins (Trxs) and three thiol-disulfide isomerases (PDIs) have been annotated in Corynebacterium glutamicum. However, nothing is known about their functional diversity in the redox regulation of proteins. Thus, we here analyzed the Trx- and PDI-dependent redox shifts of ribonucleotide reductase (RNR), insulin, 5,5'-dithiobis-(2-nitrobenzoic acid) (DTNB), and several thiol-dependent peroxidases by measuring enzyme activity and thiol status in vitro. We found that the two Trxs and the three PDIs had activities in the cleavage of the disulfidebond, whereas the PDIs had a lower efficiency than the two Trxs. Trx2 could activate thiol-dependent peroxidases with an efficiency comparable with that of Trx1, but the PDIs were inefficient. The redox-active Cys-X-X-Cys motif harbored in both Trxs and PDIs was essential to supply efficiently the donor of reducing equivalents for protein disulfides. In addition, stress-responsive extracytoplasmic function (ECF)-sigma factor H (SigH)-dependent Trxs and PDIs expressions were observed. These results contributed importantly to our overall understanding of reducing functionality of the Trx and PDI systems, and also highlighted the complexity and plasticity of the intracellular redox network.

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  • Ryoji Koike, Yuichi Kato, Shigeki Ehira
    Type: research-article
    Article ID: 2019.10.002
    Published: 2019
    [Advance publication] Released: December 17, 2019
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    Cyanobacteria are a morphologically and physiologically diverse group of bacteria, which contains unicellular and multicellular filamentous strains. Some filamentous cyanobacteria, such as Anabaena sp. strain PCC 7120, form a differentiated cell called a heterocyst. The heterocyst is a specialized cell for nitrogen fixation and is differentiated from a vegetative cell in response to depletion of combined nitrogen in the medium. In Anabaena PCC 7120, it has been demonstrated that hetR, which encodes a transcriptional regulator, is necessary and sufficient for heterocyst differentiation. However, comprehensive genomic analysis of cyanobacteria has shown that hetR is present in non-heterocyst-forming cyanobacteria. Almost all filamentous cyanobacteria have hetR, but unicellular cyanobacteria do not. In this study, we conducted genetic and biochemical analyses of hetR (NIES39_C03480) of the non-heterocyst-forming cyanobacterium Arthrospira platensis NIES-39. HetR of A. platensis was able to complement the hetR mutation in Anabena PCC 7120 and recognized the same DNA sequence as Anabaena HetR. A search of the A. platensis genome revealed the HetR-recognition sequence within the promoter region of NIES39_O04230, which encodes a protein of unknown function. Expression from the NIES39_O04230 promoter could be suppressed by HetR in Anabaena PCC 7120. These data support the conclusion that NIES39_O04230 is regulated by HetR in A. platensis NIES-39.

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  • Satoshi Sasaki, Kurumi Yoshida
    Type: research-article
    Article ID: 2019.07.002
    Published: 2019
    [Advance publication] Released: December 11, 2019
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    Oscillation in bacterial bioluminescence from Photobacterium kishitanii liquid culture was examined regarding reproducibility and bacterial cell activities, i.e., dissolved oxygen (DO) consumption, esterase activity, and product production rate. A frequent increase in DO was suspected to be due to a rapid decrease in luminescence, and a simple model describing not only the monotonous decrease in cell activity, but also the luminescence-DO relationship is proposed.

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  • Eiichiro Kan, Yohei Katsuyama, Jun-ichi Maruyama, Koichi Tamano, Yasuj ...
    Type: research-article
    Article ID: 2019.07.001
    Published: 2019
    [Advance publication] Released: November 27, 2019
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    We recently developed an Aspergillus oryzae strain in which malonyl-coenzyme A (CoA) supply is strengthened by the deletion of snfA and SCAP as an efficient host to produce a plant polyketide, curcumin. Here, we examined the effectiveness of this strain in producing another polyketide, atrochrysone carboxylic acid (ACA), which is synthesized from eight molecules of malonyl-CoA using an iterative type I polyketide synthase, ACA synthase (ACAS), and atrochrysone carboxyl ACP thioesterase (ACTE) in Aspergillus terreus. When ACAS and ACTE were introduced, the A. oryzae ΔsnfAΔSCAP strain produced approximately four times more ACA-related polyketides than did the control strain expressing both genes. This result further demonstrated the availability of the A. oryzae ΔsnfAΔSCAP strain for heterologous polyketide production.

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  • Hirotaka Matsuo, Tomoyasu Hirose, Takayuki Mokudai, Kenichi Nonaka, Yo ...
    Type: research-article
    Article ID: 2019.06.001
    Published: 2019
    [Advance publication] Released: November 15, 2019
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    Supplementary material

    A new chaetochiversin analog, designated chaetochiversin C (1), was discovered from a cultured broth of fungal strain FKI-7792 by physicochemical screening. This strain was identified as a member of genus Neocosmospora based on morphology and DNA barcoding. The partially relative configuration of 1 was determined by 13C-NMR chemical shifts of the acetonide analog of 1. The absolute configuration was determined using an advanced Mosher's method. Compound 1 was assessed for anti-tumor, anti-microbial, and anti-malarial activities, and its ability to scavenge or quench reactive oxygen species (ROS), such as superoxide anion radicals, hydroxy radicals and singlet oxygen (1O2). Compound 1 showed a quenching effect on 1O2.

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  • Tran Vu Ngoc Thi, Duong Duc Hoang Sinh, Le Thi Ha Thanh, Nguyen Duc Hu ...
    Type: research-article
    Article ID: 2019.06.002
    Published: 2019
    [Advance publication] Released: November 12, 2019
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    The present study reports on the cloning, expression and characterization of catechol 1,2-dioxygenase (CAT) of bacterial strains isolated from dioxin-contaminated soils in Vietnam. Two isolated bacterial strains DF2 and DF4 were identified as Burkholderia cepacia based on their 16S rRNA sequences. Their genes coding CAT was amplified with a specific pair of primers. Recombinant CAT (rCAT) was expressed in E. coli M15 cells and its activity was confirmed by the detection of cis,cis-muconic acid, a product from catechol, by high-performance liquid chromatography (HPLC) analysis. The rCAT of DF4 had an optimal pH and temperature of 7 and 30°C, respectively. Metal ions, such as Zn2+ and Mn2+, and surfactants, such as SDS, Tween 20 and Triton X100, strongly inhibited enzyme activity, while K+ slightly increased the activity.

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  • Yuki Kamemoto, Nanaka Funaba, Mayu Kawakami, Katsuhiro Sawasato, Kotok ...
    Type: research-article
    Article ID: 2019.05.001
    Published: 2019
    [Advance publication] Released: September 10, 2019
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

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

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  • Hazuki Hasegawa, Tatsuhiro Tsurumaki, Ikki Kobayashi, Sousuke Imamura, ...
    Type: research-article
    Article ID: 2019.05.002
    Published: 2019
    [Advance publication] Released: September 10, 2019
    JOURNALS FREE ACCESS ADVANCE PUBLICATION
    Supplementary material

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

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  • Tomoo Ogata, Ryo Ayuzawa, Ryusuke Yamada
    Type: research-article
    Article ID: 2019.05.003
    Published: 2019
    [Advance publication] Released: September 06, 2019
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

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

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  • Sittichoke Ketkaeo, Werasit Sanpamongkolchai, Sumallika Morakul, Shuic ...
    Type: research-article
    Article ID: 2019.04.008
    Published: 2019
    [Advance publication] Released: August 28, 2019
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

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

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  • Huilin Yang, Lin Yang, Xiang Li, Hao Li, Zongcai Tu, Xiaolan Wang
    Type: research-article
    Article ID: 2019.04.005
    Published: 2019
    [Advance publication] Released: August 14, 2019
    JOURNALS FREE ACCESS ADVANCE PUBLICATION

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

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