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DOI： 10.1111/jfpp.16367

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Other Link： https://onlinelibrary.wiley.com/doi/full-xml/10.1111/jfpp.16367

Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

The pink-pigmented facultative methylotrophs (PPFMs), a major bacterial group found in the plant phyllosphere, comprise two genera: Methylobacterium and Methylorubrum. They have been separated into three major clades: A, B (Methylorubrum), and C. Within these genera, however, some species lack either pigmentation or methylotrophy, which raises the question of what actually defines the PPFMs. The present study employed a comprehensive comparative genomics approach to reveal the phylogenetic relationship among the PPFMs and to explain the genotypic differences that confer their different phenotypes. We newly sequenced the genomes of 29 relevant-type strains to complete a dataset for almost all validly published species in the genera. Through comparative analysis, we revealed that methylotrophy, nitrate utilization, and anoxygenic photosynthesis are hallmarks differentiating the PPFMs from the other Methylobacteriaceae. The Methylobacterium species in clade A, including the type species Methylobacterium organophilum, were phylogenetically classified into six subclades, each possessing relatively high genomic homology and shared phenotypic characteristics. One of these subclades is phylogenetically close to Methylorubrum species; this finding led us to reunite the two genera into a single genus Methylobacterium. Clade C, meanwhile, is composed of phylogenetically distinct species that share relatively higher percent G+C content and larger genome sizes, including larger numbers of secondary metabolite clusters. Most species of clade C and some of clade A have the glutathione-dependent pathway for formaldehyde oxidation in addition to the H4MPT pathway. Some species cannot utilize methanol due to their lack of MxaF-type methanol dehydrogenase (MDH), but most harbor an XoxF-type MDH that enables growth on methanol in the presence of lanthanum. The genomes of PPFMs encode between two and seven (average 3.7) genes for pyrroloquinoline quinone-dependent alcohol dehydrogenases, and their phylogeny is distinctly correlated with their genomic phylogeny. All PPFMs were capable of synthesizing auxin and did not induce any immune response in rice cells. Other phenotypes including sugar utilization, antibiotic resistance, and antifungal activity correlated with their phylogenetic relationship. This study provides the first inclusive genotypic insight into the phylogeny and phenotypes of PPFMs.

DOI： 10.3389/fmicb.2021.740610

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Authorship：Lead author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER ACADEMIC PRESS INC  

The lanthanide elements (Lns) affect the physiology and growth of certain microorganisms known as "Ln-responsive microorganisms." Among them, in 2011, it was first reported that strains of Methylobacterium exhibited high methanol dehydrogenase (MDH) activity when grown in the presence of Lns; the purified Ln-inducible MDH was identified as XoxF-type MDH, whose catalytic function had previously been unknown. XoxF was the first enzyme to be identified as Ln-dependent, and its function in methylotrophy is more fundamental and important than that of the corresponding Ca2+-dependent MDH MxaFI. XoxF is encoded in the genomes of methylotrophic as well as non-methylotrophic bacteria. Thus, Lns are among the most fascinating and important growth factors for bacteria that potentially utilize methanol. Bacteria that require Lns for methanol growth are called "Ln-dependent methylotrophs." Recent findings indicate that these microorganisms comprise an "Ln-dependent ecosystem" that we have not been able to reconstruct under laboratory conditions without Lns. In this chapter, we summarize methods for (1) screening of Ln-responsive microorganisms, (2) purification of native XoxFs from Ln-dependent methylotrophs, and (3) screening of Ln-dependent methylotrophs from natural environments, while providing a history of the discovery of the Ln-dependent methylotrophs.

DOI： 10.1016/bs.mie.2021.01.031

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY  

Methylotrophs, which can utilize methanol as a sole carbon source, are promising microorganisms to be exploited in a methanol-based bioeconomy, in which a variety of useful compounds are biotechnologically produced from natural gas-derived methanol. Pink-pigmented facultative methylotrophs (PPFMs) are common plant phyllospheric bacteria and are known to enhance seedling growth and total biomass of various plants. However, improvement of crop yield by inoculation of PPFMs at the field level has not been well investigated. We herein describe improvement of crop yield of several rice cultivars by foliar spraying of PPFMs. After selection of PPFM strains and rice cultivars by the in vitro seedling growth test, we further conducted paddy field experiments. The crop yield of the sake-brewing rice Oryza sativa cultivar Hakutsurunishiki was reproducibly improved in a commercial paddy field for over a 5-year period. A one-time foliar spray of PPFM cells (living or killed) or a cell wall polysaccharide fraction, after the heading date, acted in the phyllosphere and effectively improved crop yield. Our results show that the established process with PPFMs is feasible for improvement of food production in the methanol bioeconomy.

DOI： 10.1111/1751-7915.13725

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER GMBH  

Black pepper production in Malaysia was restricted by various diseases. Hazardous chemical products appear to be the best solution to control diseases in black pepper cultivation. However, persistence of chemical residues in peppercorns could affect the quality of exports and consumptions. Application of fertilizers is crucial to sustain pepper growth and high yield. But, continuous use of chemical fertilizers could affect the soil ecosystem and eventually restrict nutrient uptake by pepper roots. Therefore, we propose biological approaches as an alternative solution instead of chemical products to sustain pepper cultivation in Malaysia. In this study, we have isolated a total of seven indigenous rhizobacteria antagonistic to soil-borne Fusarium solani, the causal fungus of slow decline, the most serious debilitating disease of black pepper in Malaysia. The isolated bacteria were identified as Bacillus subtilis, Bacillus siamensis, Brevibacillus gelatini, Pseudomonas geniculata, Pseudomonas beteli, Burkholderia ubonensis and Burkholderia territorii. These bacteria were effective in production of antifungal siderophore with the amount of 53.4 %-73.5 % per 0.5 mL of cell-free supernatants. The bacteria also produced appreciable amount of chitinase with chitinolytic index was ranged from 1.19 to 1.76. The bacteria have shown phosphate solubilizing index within 1.61 to 2.01. They were also efficient in ACC deaminase (0.52 mM-0.62 mM) and ammonia (60.3 mM-75.3 mM) production. The isolated antagonists were efficacious in stimulation of black pepper plant growth and root development through IAA (10.5 μg/mL-42.6 μg/mL) secretion. In conclusion, the isolated rhizobacteria are potent to be developed not only as biocontrol agents to minimize the utilization of hazardous chemicals in black pepper disease management, but also developed as bio-fertilizers to improve black pepper plant growth due to their capabilities in plant growth-promotion.

DOI： 10.1016/j.micres.2020.126549

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

Lanthanide (Ln)-dependent XoxF-type methanol dehydrogenase (MDH) genes can be found in bacteria that are not believed to be methylotrophs, and studies on their methylotrophic pathways and their use of Ln are now emerging. Ln-dependent methanol utilization in Bradyrhizobium sp. strain Ce-3, which belongs to the Bradyrhizobium elkanii superclade (clade II), was investigated in this study. Strain Ce-3 was able to grow in a media containing methanol as a sole carbon source and light Ln (L-Ln, i.e., La3+, Ce3+, Pr3+, and Nd3+), whereas the strain did not show any growth with Ca2+ or the heavy Ln, Sm3+. We found that the uptake of L-Ln is enhanced mainly by methanol and L-Ln species, and the strain incorporates each L-Ln species evenly into the cell. The genome of strain Ce-3 encodes the xox cluster for Ln-dependent methanol dehydrogenase (xoxF) and the enzymes participating in the methanol oxidation pathway (xoxG, fldA, and gfaA) and regulation (xoxR), but the gene encoding formate dehydrogenase (FDH) was not found in the cluster. MDH, formaldehyde dehydrogenase, and FDH activities were induced by methanol/Ln. Moreover, expression of the genes on the xox cluster was upregulated by methanol/Ln. Based on these results, we concluded that strain Ce-3 possesses a complete L-Ln-dependent methanol oxidation pathway, which is dissimilar to plant phyllospheric bacteria, Methylobacterium species, with a transport system for L-Ln species.

DOI： 10.1016/j.jbiosc.2020.07.012

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

A large number of strains in the Rhizobium radiobacter species complex (biovar 1 Agrobacterium) have been known as causative pathogens for crown gall and hairy root diseases. Strains within this complex were also found as endophytes in many plant species with no symptoms. The aim of this study was to reveal the endophyte variation of this complex and how these endophytic strains differ from pathogenic strains. In this study, we devised a simple but effective screening method by exploiting the high resolution power of mass spectrometry. We screened endophyte isolates from young wheat and barley plants, which are resistant to the diseases, and identified seven isolates from wheat as members of the R. radiobacter species complex. Through further analyses, we assigned five strains to the genomovar (genomic group) G1 and two strains to G7 in R. radiobacter Notably, these two genomovar groups harbor many known pathogenic strains. In fact, the two G7 endophyte strains showed pathogenicity on tobacco, as well as the virulence prerequisites, including a 200-kbp Ri plasmid. All five G1 strains possessed a 500-kbp plasmid, which is present in well-known crown gall pathogens. These data strongly suggest that healthy wheat plants are reservoirs for pathogenic strains of R. radiobacter IMPORTANCE Crown gall and hairy root diseases exhibit very wide host-plant ranges that cover gymnosperm and dicot plants. The Rhizobium radiobacter species complex harbors causative agents of the two diseases. Recently, endophyte isolates from many plant species have been assigned to this species complex. We isolated seven endophyte strains belonging to the species complex from wheat plants and revealed their genomovar affiliations and plasmid profile. The significance of this study is the finding of the genomovar correlation between the endophytes and the known pathogens, the presence of a virulence ability in two of the seven endophyte strains, and the high ratio of the pathogenic strains in the endophyte strains. This study therefore provides convincing evidence that could unravel the mechanism that maintains pathogenic agents of this species and sporadically delivers them to susceptible plants.

DOI： 10.1128/AEM.00671-20

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

Lanthanides (Ln) are an essential cofactor for XoxF-type methanol dehydrogenases (MDHs) in Gram-negative methylotrophs. The Ln3+ dependency of XoxF has expanded knowledge and raised new questions in methylotrophy, including the differences in characteristics of XoxF-type MDHs, their regulation, and the methylotrophic metabolism including formaldehyde oxidation. In this study, we genetically identified one set of Ln3+- and Ca2+-dependent MDHs (XoxF1 and MxaFI), that are involved in methylotrophy, and an ExaF-type Ln3+-dependent ethanol dehydrogenase, among six MDH-like genes in Methylobacterium aquaticum strain 22A. We also identified the causative mutations in MxbD, a sensor kinase necessary for mxaF expression and xoxF1 repression, for suppressive phenotypes in xoxF1 mutants defective in methanol growth even in the absence of Ln3+. Furthermore, we examined the phenotypes of a series of formaldehyde oxidation-pathway mutants (fae1, fae2, mch in the tetrahydromethanopterin (H4MPT) pathway and hgd in the glutathione-dependent formaldehyde dehydrogenase (GSH) pathway). We found that MxaF produces formaldehyde to a toxic level in the absence of the formaldehyde oxidation pathways and that either XoxF1 or ExaF can oxidize formaldehyde to alleviate formaldehyde toxicity in vivo. Furthermore, the GSH pathway has a supportive role for the net formaldehyde oxidation in addition to the H4MPT pathway that has primary importance. Studies on methylotrophy in Methylobacterium species have a long history, and this study provides further insights into genetic and physiological diversity and the differences in methylotrophy within the plant-colonizing methylotrophs.

DOI： 10.3390/microorganisms8060822

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

© 2020 Elsevier Inc. XoxF-type methanol dehydrogenase was recently found to be lanthanide-dependent, while its counterpart MxaF is Ca2+-dependent. The lanthanide (Ln) series consists of 15 different elements, all of which exist in nature, although at different relative abundances. XoxF from Methylorubrum extorquens strain AM1 has been shown to be induced by four light Ln species (La3+ to Nd3+). The preference of XoxFs for certain co-existing Ln species and the catalytic activity and stability of XoxF metallated with different Ln species have not been well investigated. In this study, we found that (i) strain AM1 cells preferentially utilize La3+ rather than Nd3+ for growth, (ii) XoxF purified from cells grown with a La3+ and Nd3+ mixture contained a larger proportion of La3+, and (iii) La3+-metallated XoxF has higher activity and thermal stability than Nd3+-metallated XoxF, although (iv) both enzymes showed unchanged surface charges. Thermal shift assay in particular revealed that metallation affects the temperature for subunit denaturation but not for subunit dissociation. We concluded that, although La3+ and Nd3+ have similar distributions in nature, XoxF could chose La3+ preferentially, likely because of its higher Lewis acidity, which is important for the catalytic activity of the enzyme.

DOI： 10.1016/j.enzmictec.2020.109518

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOLOGY SOC  

A new lanthanide (Ln3+)-dependent methanol-utilizing bacterial strain, La3113T, was isolated from rice field soil and its taxonomic position was investigated using polyphasic approaches. The strain was aerobic, Gram-stain-negative, strongly motile, catalase-positive and cytochrome oxidase-positive. It could neither catalyse the hydrolysis of urea nor reduce nitrate to nitrite. Growth was observed within a temperature range of 10-40 °C and a pH range of 6-8, with optimum growth at 28 °C and pH 7. Methylamine was utilized as the single source of energy, carbon and nitrogen, and it was oxidized by methylamine dehydrogenase. C16 : 1  ω7c, C16 : 1  ω6c and C16 : 0 were the dominant cellular fatty acids. Its draft genome (2.67 Mbp and 44.9 mol% G+C content) encodes genes including three Ln3+-dependent methanol dehydrogenase (XoxF-type MDH) genes, those for formaldehyde assimilation (ribulose monophosphate pathway), formate dehydrogenases and methylamine dehydrogenases, but not Ca2+-dependent MDH (MxaFI-MDH), which characterizes the species as a Ln3+-dependent methylotroph. The 16S rRNA gene sequence showed that strain La3113T belongs to the genus Methylotenera and is closely related to Methylotenera mobilis JLW8T (98.29 % identity). The digital DNA-DNA hybridization (dDDH) values (less than 30 %) and average nucleotide identity (ANI) values (less than 85 %) between genomes of strain La3113T and related type strains were lower than the thresholds for species delineation (70 % for dDDH and 95-96 % for ANI). On the basis of these polyphasic approaches, we propose a novel Methylotenera species, Methylotenera oryzisoli sp. nov. (type strain La3113T=NBRC 111954T=DSM 103219T).

DOI： 10.1099/ijsem.0.004098

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI  

Geobacillus sp. JF8 is a thermophilic biphenyl and naphthalene degrader. To identify the naphthalene degradation genes, cis-naphthalene dihydrodiol dehydrogenase was purified from naphthalene-grown cells, and its N-terminal amino acid sequence was determined. Using a DNA probe encoding the N-terminal region of the dehydrogenase, a 10-kb DNA fragment was isolated. Upstream of nahB, a gene for dehydrogenase, there were two open reading frames which were designated as nahAc and nahAd, respectively. The products of nahAc and nahAd were predicted to be alpha and beta subunit of ring-hydroxylating dioxygenases, respectively. Phylogenetic analysis of amino acid sequences of NahB indicated that it did not belong to the cis-dihydrodiol dehydrogenase group that includes those of classical naphthalene degradation pathways. Downstream of nahB, four open reading frames were found, and their products were predicted as meta-cleavage product hydrolase, monooxygenase, dehydrogenase, and gentisate 1,2-dioxygenase, respectively. A reverse transcriptase-PCR analysis showed that transcription of nahAcAd was induced by naphthalene. These findings indicate that we successfully identified genes involved in the upper pathway of naphthalene degradation from a thermophilic bacterium.

DOI： 10.3390/microorganisms8010044

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE INC  

© 2019 Elsevier Inc. The legume symbiotic nitrogen-fixing bacterium, B. diazoefficiens strain USDA110, utilizes methanol for growth in the presence of light lanthanides, such as La3+, Ce3+, Pr3+ or Nd3+, and its cells possess significant methanol dehydrogenase (MDH) activity. We purified MDH to homogeneity from B. diazoefficiens strain USDA110 grown in a methanol/Ce3+ medium; the protein was identified as XoxF5-type MDH (blr6213). The purified XoxF contained 0.58 cerium atoms per enzyme subunit. Moreover, the in-solution structure of XoxF was analyzed by small angle X-ray scattering (SAXS) analysis; the radius of gyration (Rg) and maximum particle dimension (Dmax) of XoxF were calculated to be 32.3 and 96.8 Å, respectively, suggesting that XoxF adopts a dimer structure in solution. These results show that B. diazoefficiens strain USDA110 has XoxF, a lanthanides-dependent MDH, which has methanol oxidation activity and is induced by methanol/lanthanaides, and that lanthanide is one of the important factors in methanol utilization by the strain.

DOI： 10.1016/j.enzmictec.2019.109371

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Authorship：Last author,　Corresponding author   Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Motile bacteria often exhibit chemotaxis toward favorable compounds. However, the diversity of bacteria that are attracted to a given substance is largely unknown. This study aimed to reveal the diversity of bacteria with natural chemotaxis towards methanol. We tried to enrich environmental chemotactic bacteria using a glass capillary that is half-filled with methanol solidified with agarose as a trap ("chemotaxis fishing"). The pilot experiment using methanol-chemotactic Methylobacterium aquaticum strain 22A enriched the cells by 46-fold. The method was then applied to bacterial suspensions from paddy water and plants. Depending on the isolation sources and the methods of motility induction, methylotrophic bacteria were enriched 1.2-330-fold. The fished isolates belong to 32 species in 18 genera, mainly containing Acinetobacter, Methylobacterium and Pseudomonas species. Our chemotaxis fishing unveiled a part of diversity of the bacteria with natural chemotaxis towards methanol.

DOI： 10.1080/09168451.2019.1637715

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Plants use many natural products to counter pests and diseases in nature. In rice, direct defense mechanisms include broad range of secondary metabolites, such as phenolamides (PA), diterpene phytoalexins, and flavonoid sakuranetin. Recently, accumulation of PAs in rice was shown to be under control of microbial symbionts in honeydew (HD), digestive waste from the rice brown planthopper (Nilaparvata lugens; BPH), but whether HD microbiota can also promote diterpene phytoalexins, momilactone A (MoA) and MoB, has not been reported. Here, we demonstrate that crude HD, but not a filtered one, induces MoA and MoB in rice, suggesting the involvement of BPH-HD endosymbionts. Consequently, microbial strains previously isolated from HD could promote MoA and MoB levels in wounded rice leaves, suggesting that rice indeed responds to BPH by cumulative chemical defense that involves both PA and diterpene phytoalexin pathways.

DOI： 10.1080/15592324.2019.1655335

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：OXFORD UNIV PRESS  

Feeding of sucking insects, such as the rice brown planthopper (Nilaparvata lugens; BPH), causes only limited mechanical damage on plants that is otherwise essential for injury-triggered defense responses against herbivores. In pursuit of complementary BPH elicitors perceived by plants, we examined the potential effects of BPH honeydew secretions on the BPH monocot host, rice (Oryza sativa). We found that BPH honeydew strongly elicits direct and putative indirect defenses in rice, namely accumulation of phytoalexins in the leaves, and release of volatile organic compounds from the leaves that serve to attract natural enemies of herbivores, respectively. We then examined the elicitor active components in the honeydew and found that bacteria in the secretions are responsible for the activation of plant defense. Corroborating the importance of honeydew-associated microbiota for induced plant resistance, BPHs partially devoid of their microbiota via prolonged antibiotics ingestion induced significantly less defense in rice relative to antibiotic-free insects applied to similar groups of plants. Our data suggest that rice plants may additionally perceive herbivores via their honeydew-associated microbes, allowing them to discriminate between incompatible herbivores-that do not produce honeydew-and those that are compatible and therefore dangerous.

DOI： 10.1093/jxb/erz041

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Other Link： http://orcid.org/0000-0001-9840-8845

Language：English   Publishing type：Research paper (scientific journal)   Publisher：Springer Netherlands  

The presence of 1-aminocyclopropane-1-carboxylate (ACC) deaminase determines the ability of bacteria to increase the resistance of plants to various types of stress. The genes of ACC deaminase (acdS) and the closely related enzyme d-cysteine desulfhydrase (dcyD) were searched in type strains of various representatives of the genus Methylobacterium. Using PCR screening and in silico searching in the available complete genome sequences of type strains, the genes were found in 28 of 48 species of the genus. Phylogenetic analysis of amino acid sequences of proteins revealed two large groups of sequences of the AcdS protein and one of the DcyD protein. The distribution of these groups correlates well with the phylogenetic tree based on the sequences of the 16S rRNA genes, which apparently indicates a different evolutionary adaptation to association with plants in the representatives of these groups. For the first time for aerobic methylotrophs it was demonstrated that the gene dcyD encodes d-cysteine desulfhydrase by cloning and recombinant protein characterization.

DOI： 10.1007/s10482-018-1061-5

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Blackwell Publishing Ltd  

Recently, it has been found that two types of methanol dehydrogenases (MDHs) exist in Gram-negative bacterial methylotrophs, calcium-dependent MxaFI-MDH and lanthanide-dependent XoxF-MDH and the latter is more widespread in bacterial genomes. We aimed to isolate and characterize lanthanide-dependent methylotrophs. The growth of strain La2-4T on methanol, which was isolated from rice rhizosphere soil, was strictly lanthanide dependent. Its 16S rRNA gene sequence showed only 93.4% identity to that of Methylophilus luteus MimT, and the name Novimethylophilus kurashikiensis gen. nov. sp. nov. is proposed. Its draft genome (ca. 3.69 Mbp, G + C content 56.1 mol%) encodes 3579 putative CDSs and 84 tRNAs. The genome harbors five xoxFs but no mxaFI. XoxF4 was the major MDH in the cells grown on methanol and methylamine, evidenced by protein identification and quantitative PCR analysis. Methylamine dehydrogenase gene was absent in the La2-4T genome, while genes for the glutamate-mediated methylamine utilization pathway were detected. The genome also harbors those for the tetrahydromethanopterin and ribulose monophosphate pathways. Additionally, as known species, isolates of Burkholderia ambifaria, Cupriavidus necator and Dyadobacter endophyticus exhibited lanthanide dependent growth on methanol. Thus, lanthanide can be used as an essential growth factor for methylotrophic bacteria that do not harbor MxaFI-MDH.

DOI： 10.1111/1462-2920.14062

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：American Society for Microbiology  

Methylobacterium species are representative of methylotrophic bacteria. Their genomes usually encode two types of methanol dehydrogenases (MDHs): MxaF and XoxF. The former is a Ca2+-dependent enzyme, and the latter was recently determined to be a lanthanide-dependent enzyme that is necessary for the expression of mxaF. This finding revealed the unexpected and important roles of lanthanides in bacterial methylotrophy. In this study, we performed transcriptome sequencing (RNA-seq) analysis using M. aquaticum strain 22A grown in the presence of different lanthanides. Expression of mxaF and xoxF1 genes showed a clear inverse correlation in response to La3+. We observed downregulation of formaldehyde oxidation pathways, high formaldehyde dehydrogenase activity, and low accumulation of formaldehyde in the reaction with cells grown in the presence of La33+
this might be due to the direct oxidation of methanol to formate by XoxF1. Lanthanides induced the transcription of AT-rich genes, the function of most of which was unknown, and genes possibly related to cellular survival, as well as other MDH homologues. These results revealed not only the metabolic response toward altered primary methanol oxidation, but also the possible targets to be investigated further in order to better understand methylotrophy in the presence of lanthanides.

DOI： 10.1128/mSphere.00462-17

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

Ergothioneine (EGT) is a sulfur-containing, anti-oxidative amino acid derived from histidine. EGT is synthesized in bacteria and fungi but not in animals and plants, and is now recognized as important for human health. Its cost-effective fermentative production has not been elucidated due to the lack of information for productive microorganisms. In this study, we doubled the gene copy for EGT synthesis and deleted the histidine ammonia-lyase gene in a potent EGT-producing methylotrophic bacterium Methylobacterium aquaticum strain 22A, and optimized its culture conditions, resulting in increased EGT production of 7.0 mg EGT/g dry cell weight and 100 μg EGT/5 mL/7 days. In addition, through screening we found EGT-producing eukaryotic strains of Aureobasidium pullulans and Rhodotorula mucilaginosa, which can produce 1.0 and 3.2 mg EGT/g dry cell weight, 70 and 120 μg EGT/5 mL/7 days, respectively. This study proposes practical uses of potent EGT-producing recombinant Methylobacterium species and non-recombinant yeast and fungal strains.

DOI： 10.1016/j.jbiosc.2018.05.021

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Elsevier B.V.  

Oharaeibacter diazotrophicus strain SM30T, isolated from rice rhizosphere, is an aerobic, facultative lanthanide (Ln3+)-utilizing methylotroph and diazotroph that belongs to the Methylocystaceae family. In this research, the complete genome sequence of strain SM30T was determined, and its methylotrophy modules were characterized. The genome consists of one chromosome and two plasmids, comprising a total of 5,004,097 bp, and the GC content was 71.6 mol%. A total of 4497 CDSs, 67 tRNA, and 9 rRNA were encoded. Typical alpha-proteobacterial methylotrophy genes were found: pyrroloquinoline quinone (PQQ)-dependent methanol dehydrogenase (MDH) (mxaF and xoxF1-4), methylotrophy regulatory proteins (mxbDM and mxcQE), PQQ synthesis, H4F pathway, H4MPT pathway, formate oxidation, serine cycle, and ethylmalonyl-CoA pathway. SDS-PAGE and subsequent LC–MS analysis, and qPCR analysis revealed that MxaF and XoxF1 were the dominant MDH in the absence or presence of lanthanum (La3+), respectively. The growth of MDH gene-deletion mutants on alcohols and qPCR results indicated that mxaF and xoxF1 are also involved in ethanol and propanol oxidation, xoxF2 participates in methanol oxidation in the presence of La3+, while xoxF3 was associated with methanol and ethanol oxidation in the absence of La3+, implying that XoxF3 is a calcium (Ca2+)-binding XoxF. Four Ln3+ such as La3+, cerium (Ce3+), praseodymium (Pr3+), and neodymium (Nd3+) served as cofactors for XoxF1 by supporting ΔmxaF growth on methanol. Some heavier lanthanides inhibited growth of SM30 on methanol. This study contributes to the understanding of the function of various XoxF-type MDHs and their roles in methylotrophs.

DOI： 10.1016/j.jbiosc.2018.05.023

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOLOGY SOC  

A novel facultatively methanol-utilizing bacterial strain, SM30(T), was isolated from rice rhizosphere. Strain SM30(T) was Gram-stain-negative, aerobic, motile, short rods, and grew optimally at pH 7 and at 28 degrees C. It could tolerate 0 to 2%(w/v) NaCl. Based on 16S rRNA gene sequence comparisons, strain SM30(T) was most closely related to Pleomorphomonas oryzae DSM 16300(T), with a low similarity of 94.17 %. One of the lanthanide metals, lanthanum, could enhance its growth slightly on methanol. Phylogenetic trees, based on the mxaF, xoxF and cpn60 genes of SM30(T) showed its distinct phylogenetic position with respect to species with validly published names. Polymerase chain reaction (PCR) amplification of the nifH and growth on nitrogen-free medium indicated that strain SM30(T) is a diazotroph. The major cellular fatty acids were summed feature 8 (containing 18:1 omega 7c and 18:1 omega 6c) and cyclo 19:0 omega 8c. The major quinone was ubiquinone 10. The DNA G+C content was 74.6 mol%. Based on the genotypic and phenotypic characteristics, strain SM30(T) represents a novel genus and species, for which the name Oharaeibacter diazotrophicus gen. nov., sp. nov. is proposed with the type strain SM30(T) (= NBRC 111955(T) = DSM 102969(T)).

DOI： 10.1099/ijsem.0.001660

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：NATURE PUBLISHING GROUP  

Viruses typically encode the capsid that encases their genome, while satellite viruses do not encode a replicase and depend on a helper virus for their replication(1). Here, we report interplay between two RNA viruses, yado-nushi virus 1 (YnV1) and yado-kari virus 1 (YkV1), in a phytopathogenic fungus, Rosellinia necatrix(2). YkV1 has a close phylogenetic affinity to positive-sense, single-stranded (+)ssRNA viruses such as animal caliciviruses(3), while YnV1 has an undivided double-stranded (ds) RNA genome with a resemblance to fungal toti-viruses(4). Virion transfection and infectious full-length cDNA transformation has shown that YkV1 depends on YnV1 for viability, although it probably encodes functional RNA-dependent RNA polymerase (RdRp). Immunological and molecular analyses have revealed trans-encapsidation of not only YkV1 RNA but also RdRp by the capsid protein of the other virus (YnV1), and enhancement of YnV1 accumulation by YkV1. This study demonstrates interplay in which the capsidless (+) ssRNA virus (YkV1), hijacks the capsid protein of the dsRNA virus (YnV1), and replicates as if it were a dsRNA virus.

DOI： 10.1038/NMICROBIOL.2015.1

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.53.744

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：FRONTIERS MEDIA SA  

Metabolomic analysis revealed that Methylobacterium cells accumulate a large amount of ergothioneine (EGT), which is a sulfur containing, non-proteinogenic, antioxidative amino acid derived from histidine. EGT biosynthesis and its role in methylotrophy and physiology for plant surface-symbiotic Methylobacterium species were investigated in this study. Almost all Methylobacterium type strains can synthesize FGT. We selected one of the most productive strains (M. aquaticum strain 22A isolated from a moss), and investigated the feasibility of fermentative EGT production through optimization of the culture condition. Methanol as a carbon source served as the best substrate for production. The productivity reached up to 1000 mu g/100 ml culture (1200 mu g/g wet weight cells, 6.3 mg/g dry weight) in 38 days. Next, we identified the genes (egtBD) responsible for EGT synthesis, and generated a deletion mutant defective in EGT production. Compared to the wild type, the mutant showed better growth on methanol and on the plant surface as well as severe susceptibility to heat treatment and irradiation of ultraviolet (UV) and sunlight. These results suggested that EGT is not involved in methylotrophy, but is involved in their phyllospheric lifestyle fitness of the genus in natural conditions.

DOI： 10.3389/fmicb.2015.01185

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：Prince of Songkla University  

Enterocin CE5-1 produced by Enterococcus faecium CE5-1 isolated from the chicken gastrointestinal tract was active in the wide range of pH 2-10 and temperature 30-100°C and sensitive to proteolytic enzymes and α-amylase. It remained active after storage at -20°C for 2 months. Moreover, enterocin CE5-1 showed antibacterial activity against lactobacilli, bacilli, listeria, staphylococci and enterococci, especially antibiotic-resistant enterococci. In vitro study of enterocin CE5-1 decreased the population of Ent. faecalis VanB from 6.03 to 4.03 log CFU/ml. The lethal mode of action of enterocin CE5-1 appeared to be pore and filament formation in the cell wall. PCR sequencing analysis revealed the presence of two open reading frames (ORFs), containing enterocin CE5-1 (entCE5-1) and enterocin immunity (entI) gene. Therefore, enterocin CE5-1 from Ent. faecium CE5-1 could possibly be used as an antimicrobial agent to control foodborne pathogen, spoilage bacteria and antibiotic-resistant enterococci in foods, feeds and the environments.

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCIENCE BV  

Despite the importance of bryophyte-associated microorganisms in various ecological aspects including their crucial roles in the soil-enrichment of organic mass and N-2 fixation, nonetheless, little is known about the microbial diversity of the bryophyte phyllospheres (epi-/endophytes). To get insights into bacterial community structures and their dynamics on the bryophyte habitats in different ecosystems and their potential biological roles, we utilized the 16S rRNA gene PCR-DGGE and subsequent phylogenetic analyses to investigate the bacterial community of eight bryophyte species collected from three distinct ecosystems from western Japan. Forty-two bacterial species belonging to gamma-proteobacteria and Firmicutes with 71.4% and 28.6%, respectively, were identified among 90 DGGE gel band population. These DGGE-bands were assigned to 13 different genera with obvious predomination the genus Clostridium with 21.4% from the total bacterial community. These analyses provide new insights into bryophyte-associated bacteria and their relations to the ecosystems. (C) 2014 Production and hosting by Elsevier B.V. on behalf of King Saud University.

DOI： 10.1016/j.sjbs.2014.07.009

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Methylobacterium species colonize plant surfaces and utilize methanol emitted from plants. Methylobacterium aquaticum strain 22A was isolated from a hydroponic culture of a moss, Racomitrium japonicum, and is a potent plant growth promoter. The complete genome sequencing of the strain confirmed the presence of genes related to plant growth promotion and methylotrophy.

DOI： 10.1128/genomeA.00266-15

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：PUBLIC LIBRARY SCIENCE  

Methylobacterium species frequently inhabit plant surfaces and are able to utilize the methanol emitted from plants as carbon and energy sources. As some of the Methylobacterium species are known to promote plant growth, significant attention has been paid to the mechanism of growth promotion and the specificity of plant-microbe interactions. By screening our Methylobacterium isolate collection for the high growth promotion effect in vitro, we selected some candidates for field and pot growth tests for rice and barley, respectively. We found that inoculation resulted in better ripening of rice seeds, and increased the size of barley grains but not the total yield. In addition, using whole-cell matrix-assister laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, we identified and classified Methylobacterium isolates from Methylobacterium-inoculated rice plants. The inoculated species could not be recovered from the rice plants, and in some cases, the Methylobacterium community structure was affected by the inoculation, but not with predomination of the inoculated species. The isolates from non-inoculated barley of various cultivars grown in the same field fell into just two species. These results suggest that there is a strong selection pressure at the species level of Methylobacterium residing on a given plant species, and that selection of appropriate species that can persist on the plant is important to achieve growth promotion.

DOI： 10.1371/journal.pone.0129509

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Despite tremendous advances in understanding the microbial degradation of acrylamide, reports on the nature of the two-dimensional protein patterns for acrylamide-degrading bacteria are not yet available. This work, focusing on the acrylamide-inducible proteins, studied the response of Enterobacter aerogenes, a novel acrylamide-degrading bacterium, to acrylamide. Proteome analysis was applied using 2D-polyacrylamide gel electrophoresis and matrixassisted laser desorption/ionisation-time of flight mass spectrometry to identify proteins differentially expressed from E. aerogenes grown on acrylamide. Six protein homologues with amidohydrolase, urease accessory protein, quaternary ammonium compound resistance proteins, dipeptide transport protein, Omp36 osmoporin and large conductance mechanosensitive channel proteins (MscL) are seemingly involved in acrylamide stress response and its degradation. Five proteins identified as GroEL-like chaperonin, ArsR-transcriptional regulator, Ts- and Tu-elongation factor and trigger factor and four proteins (phosphoglycerate kinase, ATP synthase β-subunit, malate dehydrogenase and succinyl-CoA synthetase α-subunit) responsive for the adaption of E. aerogenes in the presence of acrylamide.

DOI： 10.12982/cmujns.2014.0016

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS LTD  

Pink-pigmented facultative methylotrophs (PPFMs) are major inhabitants of the phyllosphere. In a preceding study, we found that perilla plants harbor a dominant population of PPFMs on their leaves and seeds, and that the closest relative of PPFMs (Methylbacterium sp. strain OR01 as representative strain) isolated from red perilla seeds was M. fujisawaense DSM5686(T). In the present study, the specific interaction between red perilla and Methylobacterium species was investigated. All the PPFMs isolated from red perilla seeds harvested in the Ohara area of Kyoto, Japan in 2009, 2010, and 2011 and the PPFMs isolated from red perilla leaves planted at four geographically different places in Japan had 16S rRNA sequences identical to that of strain OR01. Direct transmission of PPFMs from seeds to leaves and the competitiveness of strain OR01 were confirmed. This report is the first step toward understanding the species-level specificity of the interaction between perilla plants and Methylobacterium species.

DOI： 10.1271/bbb.130207

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

A new multichannel microdevice flow system with stainless steel flow chamber was used for architecture visualization, development monitoring and structural quantification of GFP-labeled Pseudomonas aeruginosa PAO1 live biofilms. Direct in situ investigations using confocal laser scanning microscopy (CLSM) at 72 h revealed structural pattern differences as a result of nutrient concentration gradients. When grown in LB medium, round, dispersed cellular aggregates were formed whereas in 1/3-diluted LB medium, biofilms were mostly flat and compact. However, COMSTAT analyses showed no considerable differences in biomass and thickness between the two LB concentrations. Characterization of time-dependent development of biofilms grown in 1/3-diluted LB medium showed full maturation of colonies by 120 h reaching maximum biomass at 17.1 mu m(3)/mu m(2) and average thickness at 44.4 mu m. Consequent thinning and formation of openings through interior in colonies occurred by 168 h. These results suggest that the new system tested allowed a fast and thick biofilm development on the surface of the stainless steel flow chamber. These findings may provide better estimates of biofilm activity and systematic evaluation of the effects of different parameters on biofilm morphology and development in industrial and biomedical systems. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2012.09.018

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：AMER SOC MICROBIOLOGY  

Treatment of Pseudomonas aeruginosa PAO1 flow biofilms with a D-amino acid mixture caused significant reductions in cell biomass by 75% and cell viability by 71%. No biofilm disassembly occurred, and matrix production increased by 30%, thereby providing a thick protective cover for remaining viable or persister cells.

DOI： 10.1128/AEM.02911-12

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MICROBIOLOGY SOC  

Pink-pigmented, facultatively methylotrophic bacteria, strains 87eT and 99bT, were isolated from the bryophytes Haplocladium microphyllum and Brachythecium plumosum, respectively. The cells of both strains were Gram-reaction-negative, motile, non-spore-forming rods. On the basis of 16S rRNA gene sequence similarity, strains 87eT and 99bT were found to be related to Methylobacterium organophilum ATCC 27886T (97.1 % and 97.7%, respectively). Strains 87eT and 99bT showed highest 16S rRNA gene similarity to Methylobacterium gnaphalii 23eT (98.3 and 99.0%, respectively). The phylogenetic similarities to all other species of the genus Methylobacterium with validly published names were less than 97%. Major cellular fatty acids of both strains were C18:1ω7c and C18:0. The results of DNA-DNA hybridization, phylogenetic analyses based on 16S rRNA and cpn60 gene sequences, fatty acid profiles, whole-cell matrix- assisted, laser-desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) analysis, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strains 87eT and 99bT from their phylogenetically closest relatives. We propose that strains 87eT and 99bT represent novel species within the genus Methylobacterium, for which the names Methylobacterium haplocladii sp. nov. (type strain 87eT=DSM 24195T=NBRC 107714T) and Methylobacterium brachythecii sp. nov. (type strain 99bT=DSM 24105T=NBRC 107710T) are proposed. © 2013 IUMS.

DOI： 10.1099/ijs.0.048215-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER INTERNATIONAL PUBLISHING AG  

Rice (Oryza sativa L.) is the most important staple food crop in many developing countries, and is ranked third in Kenya after maize and wheat. Continuous cropping without replenishing soil nutrients is a major problem in Kenya resulting to declining soil fertility. The use of chemical fertilizers to avert the problem of low soil fertility is currently limited due to rising costs and environmental concerns. Many soil micro-organisms are able to solubilize the unavailable phosphorus, increase uptake of nitrogen and also synthesize growth promoting hormones including auxin. The aim of this study was to isolate and characterize phyllosphere, rhizoplane and rhizosphere micro-organisms from Kenyan rice with growth promoting habits. In this study whole plant rice samples were collected from different rice growing regions of Kenya. 76.2%, over 80% and 38.5% of the bacterial isolates were positive for phosphate solubilization, nitrogenase activity and IAA production whereas 17.5% and 5% of the fungal isolates were positive for phosphate solubilization and IAA production respectively. Hence these micro-organisms have potential for utilization as bio-fertilizers in rice production. © 2013 Mwajita et al.
licensee Springer.

DOI： 10.1186/2193-1801-2-606

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SPRINGER  

Biosurfactant-producing bacteria were isolated from mangrove sediment samples collected in the southern part of Thailand by an enrichment-culture technique in which lubricating oil was the sole carbon source. A total of 1,600 colonies were obtained, which were screened for biosurfactant production using the qualitative drop-collapsing test in a mineral salts medium containing 1% of different carbon sources (commercial sugar, glucose, molasses, and used lubricating oil). Ninety-five isolates were positive for biosurfactant production based on the results of this test, among which 20 could reduce the surface tension of the 48-h culture supernatant. The phylogenetic position of these 20 isolates was evaluated by 16S rRNA gene sequence analysis. The production of biosurfactants was determined for strains representative of eight different bacterial genera. Leucobacter komagatae 183, one of the newly isolated strains showing biosurfactant production, produced extracellular biosurfactants which reduced the surface tension of the culture supernatant from 72.0 to 32.0 m/Nm. Eighteen strains released extracellular emulsifiers able to stabilize the emulsion formed. Among these, the strains L. komagatae 183 and Ochrobactrum anthropi 11/6 exhibited emulsification activities comparable to those of synthetic surfactants. Overall, the new biosurfactant-producing strains isolated in this study display promising features for the future development and use in economically efficient industrial-scale biotechnological processes.

DOI： 10.1007/s13213-012-0424-9

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

A pink-pigmented, facultatively methylotrophic bacterium, strain 23e(T), was isolated from the leaves of Gnaphalium spicatum (cudweed). The cells of strain 23e(T) were Gram-reaction negative, motile and non-spore-forming rods. On the basis of 16S rRNA gene sequence similarities, strain 23e(T) was related to Methylobacterium organophilum ATCC 27886(T) (97.1 %) and Methylobacterium marchantiae JT1(T) (97 %), and the phylogenetic similarities to all other Methylobacterium species with validly published names were less than 97%. Major cellular fatty acids were C(18:1 omega)7c, C-16:00 and C-18:0. The results of DNA DNA hybridization, phylogenetic analyses based on 16S rRNA and cpn60 gene sequences, fatty acid profiles, whole-cell matrix-assisted laser desorption/ionization time of flight/MS analysis, physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 23e(T) from the phylogenetically closest relatives. We propose that strain 23e(T) represents a novel species within the genus Methylobacterium, for which the name Methylobacterium gnaphalii sp. nov. is proposed. The type strain is 23e(T) (=DSM 24027(T)=NBRC 107716(T)).

DOI： 10.1099/ijs.0.037713-0

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：TAYLOR & FRANCIS INC  

Worldwide contamination by acrylamide, a neurotoxicant and carcinogen in animals, is becoming a significant problem. We isolated three novel acrylamide-degrading bacteria from domestic wastewater in Chonburi, Thailand. Using biochemical characteristics and 16S rRNA gene sequencing, the strains were identified as Klebsiella pneumoniae, Kluyvera georgiana and Enterococcus faecalis. K. georgiana strain No. 2 was selected for further characterization due to its degradation potential of high concentrations of acrylamide at the mesophilic temperatures. The strain grew well in the presence of acrylamide at concentrations to 0.5 % (w/v), pH 5.0 to 7.0 and 37 degrees C. Degradation of acrylamide to acrylic acid began after 30 min of cultivation as a biomass-dependent manner. Mass balance analysis revealed 92.3 % conversion of acrylamide to acrylic acid and two lower polarity compounds. Strain No. 2 degraded many aliphatic amides but not iodoacetamide and thioacetamide. High degradation level (>80 %) was found with propionamide, cyanoacetamide and acetamide. Moderate degradation was obtained in the order of formamide > butyramide > lactamide > urea while sodium azide provided 34% degradation. These findings render this novel bacterium attractive for biodegradation of acrylamide and other aliphatic amides in the environment.

DOI： 10.1080/10934529.2012.680312

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：ELSEVIER SCI LTD  

Lactic acid bacteria (LAB) were isolated from traditional Thai fermented fish, Plasom at various fermentation periods. It was found that 138 isolates exhibited a clear zone and growth on MRS agar supplemented with CaCO3; however, only 133 isolates were identified as LAB. Only 14 strains showed excellent inhibition zone diameters on agar when Salmonella sp. was used as an indicator for preliminary detection of antagonistic activity. Staphylococcus aureus was used for secondary screening for antagonistic activity of these 14 strains. It was found that only 7 strains exhibited good inhibition zone diameters on agar, and all of them could inhibit E. coli as the third indicator. The strains which exhibited widest zones of inhibition against Escherichia coli, S. aureus and Salmonella sp. were LPS04, LPS17, and LD219 and LPS18 respectively. Tested pathogenic strains were inhibited by 4 selected LAB. The strain which showed the best lactic acid production and pH reduction ability was LD219. Using 16s rDNA sequence analysis, LD219 was identified as Streptococcus salivarius, LPS04. LPS17 and LPS18 were identified as Enterococcus faecalis. Plasom was produced by using a mixed culture (LD219, LPS04, LPS17 and LPS18) as a starter culture compared with spontaneous and back-slopping processes. Significant differences (p < 0.05) in pH, its titratable acidity as lactic acid and number of total viable counts (TVC), LAB and Enterobacteriaceae were found in these Plasom at 0 and 8 days. However, no significant difference (p > 0.05) was observed in terms of colour, smell, taste, sour, texture and overall acceptance of Plasm produced by non-starter cultured, back-slopping and starter cultured processes. (C) 2010 Elsevier Ltd. All rights reserved.

DOI： 10.1016/j.foodcont.2010.09.010

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Quantitative analysis of the target microorganism in microbial communities is important for the assessment of bacterial activity in environment. Here we present a method of a combination of fluorescent in situ hybridization (FISH) method and live/dead staining which allows in situ identification and analysis of physiological status of specific bacteria. (C) 2010, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2010.07.004

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Language：Japanese   Publisher：Japan Society for Bioscience, Biotechnology, and Agrochemistry  

DOI： 10.1271/kagakutoseibutsu.48.598

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Other Link： https://jlc.jst.go.jp/DN/JALC/00356205982?from=CiNii

Language：English   Publishing type：Research paper (scientific journal)   Publisher：WILEY-V C H VERLAG GMBH  

Transcription factor Stb5p, previously known as one of the multidrug resistance gene regulators in Saccharomyces cerevisiae, was shown here to play an essential role in acetaldehyde tolerance. A mutant strain, Delta stb5 exhibited increased acetaldehyde sensitivity, and failed to induce genes such as GND1, TKL1 and TAL1 involved in the pentose phosphate pathway (PPP) upon acetaldehyde stress. Using this strain it was revealed that Stb5p acts as a repressor for PGI1 encoding glucose-6-phosphate isomerase under acetaldehyde stress. In reverse, overexpression of Stb5p reinforced acetaldehyde tolerance to the yeast. Furthermore, various deletion mutants of the genes involved in glycolysis showed increased acetaldehyde tolerance compared to the wild-type strain. From these results, it was suggested that Stb5p participates in acetaldehyde tolerance by regulating expression of the PPP genes and PGI1, and that down-regulation of glycolytic pathway may lead to vitalization of PPP and to increased acetaldehyde tolerance.

DOI： 10.1002/jobm.200900391

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：MDPI AG  

A highly specific, high throughput-amenable bacterial biosensor for chemically induced cellular oxidation was developed using constitutively expressed redox-sensitive green fluorescent protein roGFP2 in E. coli (E. coli-roGFP2). Disulfide formation between two key cysteine residues of roGFP2 was assessed using a double-wavelength ratiometric approach. This study demonstrates that only a few minutes were required to detect oxidation using E. coli-roGFP2, in contrast to conventional bacterial oxidative stress sensors. Cellular oxidation induced by hydrogen peroxide, menadione, sodium selenite, zinc pyrithione, triphenyltin and naphthalene became detectable after 10 seconds and reached the maxima between 80 to 210 seconds, contrary to Cd(2+), Cu(2+), Pb(2+), Zn(2+) and sodium arsenite, which induced the oxidation maximum immediately. The lowest observable effect concentrations (in ppm) were determined as 1.0 x 10(-7) (arsenite), 1.0 x 10(-4) (naphthalene), 1.0 x 10(-4) (Cu(2+)), 3.8 x 10(-4) (H(2)O(2)), 1.0 x 10(-3) (Cd(2+)), 1.0 x 10(-3) (Zn(2+)), 1.0 x 10(-2) (menadione), 1.0 (triphenyltin), 1.56 (zinc pyrithione), 3.1 (selenite) and 6.3 (Pb(2+)), respectively. Heavy metal-induced oxidation showed unclear response patterns, whereas concentration-dependent sigmoid curves were observed for other compounds. In vivo GSH content and in vitro roGFP2 oxidation assays together with E. coli-roGFP2 results suggest that roGFP2 is sensitive to redox potential change and thiol modification induced by environmental stressors. Based on redox-sensitive technology, E. coli-roGFP2 provides a fast comprehensive detection system for toxicants that induce cellular oxidation.

DOI： 10.3390/s100706290

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC BIOSCIENCE BIOENGINEERING JAPAN  

A new method for isolating targeted live bacterial cells was established with the use of cell sorting by flow cytometry (FCM) based on the fluorescence of the intermediate metabolite of biphenyl degradation. During biphenyl degradation, a PCB degrader, Comamonas testosteroni 02, produces a meta-cleavage intermediate metabolite, 2-hydroxy-6-oxo-6-phenylhexa-2, 4-dienoic acid (HOPDA), which emits green fluorescence. HOPDA was produced from 2,3-dihydroxy biphenyl as a substrate, but it was not appropriate for labeling cells because it was released from the cells into the medium. When we used 4-n-butylbiphenyl and 4-n-heptylbiphenyl, we found that the cells produced and accumulated 2,3-dihydroxy intermediate metabolites. By the addition of synthesized 2,3-dihydroxy-4'-butylbiphenyl (2,3-DHBBP), we were able to label the cells with strong green fluorescence, suggesting the persistence of fluorescent intermediate metabolite in the cells by the introduction of the alkyl tail. 2,3-DHBBP was then used to label strain TK102 and the cells were sorted with FCM. The sorting efficiency of FCM was defined as the percentage of colony numbers per sorting events. Strain TK102 cells were successfully enriched by 4.1-fold from the mixture with environmental indigenous bacteria with a sorting efficiency of 7.3%. The method we present here serves as a basic technique for the specific and direct isolation of live bacterial cells which contain dioxygenases active on dihydroxylated aromatic compounds. (C) 2009, The Society for Biotechnology. japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.11.023

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Cell-free extracts from a PEG 4000-utilizing bacterium, Sphingopyxis macrogoltabida strain 103, grown on glucose and PEG 4000 medium were separated into cytoplasmic, membrane-bound and signal protein fractions. Each fraction was analyzed by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE). A total of 19 differentially-expressed proteins by PEG were in-gel trypsin digested and the digestion mixtures were analyzed by MALDI-TOF mass spectrometry to determine the molecular masses of the resulting tryptic peptides. Ten proteins in the cytoplasmic fraction showed homology to fatty acyl CoA synthetase, IEA two-component response regulator, permease, LacI-transcription regulator, galactinol synthase, coenzyme PQQ synthesis protein, transcription regulator, translation-initiation factor like protein, CheY-like two-component response regulator and hypothetical protein. Three proteins in the membrane-bound fraction were identified as LysR-transcription regulator, GutR-transcription regulator and two-component response regulator. Six proteins in the signal protein fraction were polyphosphate kinase, ATP sulfurylase, amino acid permease, sigma-54 dependent transcription regulator, fatty-acid-CoA ligase and LysR-transcription regulator. These proteins are expected to be relevant to PEG metabolism by S. macrogoltabida strain 103.

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We hypothesize the following items based on 41 autopsy cases with or without head injuries especially on subependymal injuries at the anterior horns of the lateral cerebral ventricles. The subependymal injuries at the regions (1) are not seen in fatal cases that did not have intracranial injuries, (2) have nothing to do with technical procedures for removal of the whole brain at autopsy, (3) are frequently associated with axonal injuries, (4) are presumed to be formed by rotating head injuries in a sagittal direction. © 2009 Elsevier Ireland Ltd. All rights reserved.

DOI： 10.1016/j.legalmed.2009.02.061

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Language：English   Publishing type：Research paper (scientific journal)   Publisher：SOC GENERAL MICROBIOLOGY  

Rhodotorula glutinis IFO1125 was found to acquire increased aluminium (Al) resistance from 50 mu M to more than 5 mM by repetitive culturing with stepwise increases in Al concentration at pH 4.0. To investigate the mechanism underlying this novel phenomenon, wild-type and Al- resistant cells were compared. Neither cell type accumulated the free form of Al (Al3+) added to the medium. Transmission electron microscopic analyses revealed a greater number of mitochondria in resistant cells. The formation of small mitochondria with simplified cristae structures was observed in the wild-type strain grown in the presence of Al and in resistant cells grown in the absence of Al. Addition of Al to cells resulted in high mitochondrial membrane potential and concomitant generation of reactive oxygen species (ROS). Exposure to Al also resulted in elevated levels of oxidized proteins and oxidized lipids. Addition of the antioxidants a-tocopherol and ascorbic acid alleviated the Al toxicity, suggesting that ROS generation is the main cause of Al toxicity. Differential display analysis indicated upregulation of mitochondrial genes in the resistant cells. Resistant cells were found to have 2.5- to 3-fold more mitochondrial DNA (mtDNA) than the wild-type strain. Analysis of tricarboxylic acid cycle and respiratory-chain enzyme activities in wild-type and resistant cells revealed significantly reduced cytochrome c oxidase activity and resultant high ROS production in the latter cells. Taken together, these data suggest that the adaptive increased resistance to Al stress in resistant cells resulted from an increased number of mitochondria and increased mtDNA content, as a compensatory response to reduced respiratory activity caused by a deficiency in complex IV function.

DOI： 10.1099/mic.0.2007/016048-0

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Polypropylene glycol (PPG)-assimilating Sphingobium sp. strain PW-1 was grown on 0.5% PPG 700. PPG and its metabolites were analyzed by MALDI-TOF mass spectrometry. An oxidized form of a terminal alcohol group appeared with each molecular species as a metabolite. Cell-free extract showed PPG dehydrogenase activity. In this way, the oxidative metabolic pathway for PPG was confirmed.

DOI： 10.1271/bbb.70749

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Sphingopyxis terrae, and Sphingopyxis macrogoltabida strains 103 and 203, can degrade polyethylene glycols (PEGs). They differ in the following respects: (i) different substrate specificities (chain length) of assimilable PEG, (ii) PEG-inducible or constitutive PEG-degradative proteins, and (iii) symbiotic or axenic degradation of PEG. S. terrae was able to incorporate PEG 6000, but strain 103 could not incorporate more than PEG 4000, suggesting that the difference in assimilable PEG chain length depends on the ability to take up substrate. PEG-degradative genes (pegB, C, D, A, E and R) from these strains were cloned. Their primary structures shared a high homology of more than 99%. The peg genes encode a TonB-dependent receptor (pegB), a PEG-aldehyde dehydrogenase (pegC), a permease (pegD), a PEG dehydrogenase (pegA) and an acyl-CoA ligase (pegE), and in the opposite orientation, an AraC-type transcription regulator (pegR). The peg operon was flanked by two different sets of transposases. These three strains contained large plasmids and the operon was located in one of the large plasmids in S. terrae. The peg genes could be detected in other PEG-degrading sphingomonads. These results suggest that the peg genes have evolved in a plasmid-mediated manner. An insertion of a transposon gene (pegF) between pegD and pegA in strain 203 was found, which caused the constitutive expression of pegA in this strain.

DOI： 10.1099/mic.0.2006/000992-0

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A gene for periplasmic poly(vinyl alcohol) (PVA) dehydrogenase (PVADH) was cloned, based on the N-terminal amino acid sequence of the purified PVADH from Sphingomonas sp. 113P3 and the sequence of the gene for PVADH (pvaA, GenBank accession no. AB 190288). The recombinant PVADH tagged with hexahistidine was expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme had the same characteristics as the purified enzyme from Sphingomonas sp. strain 113P. In addition to PVA, the recombinant PVADH could oxidize glycols such as polypropylene glycols and 1,3-butane/cyclohexanediol and 2,4-pentanediol, but neither primary nor secondary alcohols. The amino acid sequence of the recombinant PVADH showed similarity with those of PVADH from Pseudomonas sp. strain VM15C, putative PVADHs from Azoarcus sp. EbN1, and Xanthomonas species (54-25% identity), and the quinohaemoprotein alcohol dehydrogenases (OH-ADHs) from Comamonas testosteroni, Ralstonia eutropha and Pseudomonas putida (25-29% identity). PVADHs from strains 113P3 and VM15C have a conserved superbarrel domain (SD), probable PQQ-binding amino acids in the SID and a haem-binding domain (HBD) (they should be designated QH-PVADHs), but the positions of the amino acid sequences for the HBD and SID are the reverse of those of OH-ADHs. A protein structure of QH-PVADHs is proposed. Results of dot-blot hybridization and RT-PCR indicated that the three genes encoding oxidized PVA hydrolase, PVADH and cytochrome c are expressed constitutively and form an operon.

DOI： 10.1099/mic.0.28848-0

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Polyethylene glycol dehydrogenase (PEGDH) from Sphingopyxis terrae (formerly Sphingomonas terrae) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from Aspergillus niger and Penicillium amagasakiense. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q(10) was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.

DOI： 10.1128/AEM.02174-05

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Because of the large-scale production and widespread use of poly(ethylene glycol) (PEG) and polyvinyl alcohol) (PVA), these products eventually find their way into the natural environment. Their microbial metabolic pathways have been well studied. Our laboratory has cloned the relevant genes involved in the degradation of PEG and PVA from Sphingomonas sp. strains 103 and 113P3, respectively. A 13.3 kb DNA fragment containing the PEG dehydrogenase gene was cloned and sequenced. This report presents data about the operon related to PEG degradation and the regulation of this operon. Similar gene structures were found in other PEGutilizing sphingomonads. In addition, the PVA degradation operon, which includes genes for PVA dehydrogenase and oxidized PVA hydrolase, was also investigated. These genes were located in tandem with little intergenic space between them, suggesting that both genes are expressed constitutively and polycistronically. © 2006 American Chemical Society.

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Oxidized polyvinyl alcohol hydrolase (OPH) and polyvinyl alcohol dehydrogenase were found to be constitutively present in the periplasm of Sphingromonas sp. strain 113P3 (formerly Pseudomonas sp. 113P3). The OPH was purified to homogeneity with a yield of 40% and a 5-9-fold increase in specific activity. The enzyme was a homodimer consisting of 35 kDa subunits. Its activity was inhibited by PMSF, Hg2+ and Zn2+ . The enzyme hydrolysed oxidized polyvinyl alcohol (oxidized PVA) and p-nitrophenyl acetate (PNPA), but did not hydrolyse any of the mono- or diketones tested. K-m and V-max, values for oxidized PVA and PNPA were 0-2 and 0(.)3 mM, and 0(.)1 and 3(.)4 mu mol min(-1) mg(-1), respectively. The gene for OPH was cloned and sequenced. Sequencing analysis revealed that the open reading frame consisted of 1095 bp, corresponding to a protein of 364 amino acids residues, encoding a signal peptide and a mature protein of 34 and 330 amino acids residues, respectively. The presence of a serine-hydrolase motif (a lipase box; Gly-X-Ser-X-Gly) strongly suggested that the enzyme belongs to the serine-hydrolase family. The protein exhibited homology with OPH of the Pseudomonas sp. strain VM15C (63% identity) and the polyhydroxybutyrate depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti (29-32% identity). The oph gene was expressed in Escherichia coli under the control of the lac promoter. The recombinant protein had the same molecular mass and N-terminal amino acid sequence as the purified OPH from strain 113P3.

DOI： 10.1099/mic.0.27655-0

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NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C2 to C14 and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes. © 1989, American Association for Cancer Research. All rights reserved.

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Methylobacterium species are methylotrophic bacteria that widely inhabit plant surfaces. In addition to studies on methylotrophs as model organisms, research has also been conducted on their mechanism of plant growth promotion as well as the species-species specificity of plant-microbe interaction. We employed whole-cell matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (WC-MS) analysis, which enables the rapid and accurate identification of bacteria at the species level, to identify Methylobacterium isolates collected from the rice seeds of different cultivars harvested in Japan, Thailand, and Kenya. Rice seeds obtained from diverse geographical locations showed different communities of Methylobacterium species. We found that M. fujisawaense, M. aquaticum, M. platani, and M. radiotolerans are the most frequently isolated species, but none were isolated as common species from 18 seed samples due to the highly biased communities in some samples. These findings will contribute to the development of formulations containing selected species that promote rice growth, though it may be necessary to customize the formulations depending on the cultivars and farm conditions. (C) 2016, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2016.09.001

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Algal bloom is typically caused by aberrant propagation of a single species, resulting in its predomination in the local population. While environmental factors including temperature and eutrophication are linked to bloom, the precise mechanism of its formation process is still obscure. Here, we isolated a bacterial strain that promotes growth of Heterosigma akashiwo, a Raphidophyceae that causes harmful algal blooms. Based on 16S rRNA gene sequence, the strain was identified as Altererythrobacter ishigakiensis, a member of the class Alphaproteobacteria. When added to culture, this strain facilitated growth of H. akashiwo and increased its cell culture yield significantly. Importantly, this strain did not affect the growth of other raphidophytes, Chattonella ovate and C. antiqua, indicating that it promotes growth of H. akashiwo in a species-specific manner. We also found that, in co-culture, H. akashiwo suppressed the growth of C. ovate. When A. ishigakiensis was added to the mixed culture, H. akashiwo growth was facilitated while C ovate propagation was markedly suppressed, indicating that the presence of the bacterium enhances the dominance of H. akashiwo over C ovate. This is the first example of selective growth promotion of H. akashiwo by a marine bacterium, and may exemplify importance of symbiotic bacterium on algal bloom forming process in general. (C) 2016 Elsevier B.V. All rights reserved.

DOI： 10.1016/j.hal.2016.11.009

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Methylobacterium species colonize plant surfaces and utilize methanol emitted from plants. Methylobacterium aquaticum strain 22A was isolated from a hydroponic culture of a moss, Racomitrium japonicum, and is a potent plant growth promoter. The complete genome sequencing of the strain confirmed the presence of genes related to plant growth promotion and methylotrophy.

DOI： 10.1128/genomeA.00266-15

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Language：English   Publisher：American Society for Microbiology  

Methylobacterium species colonize plant surfaces and utilize methanol emitted from plants. Methylobacterium aquaticum strain 22A was isolated from a hydroponic culture of a moss, Racomitrium japonicum, and is a potent plant growth promoter. The complete genome sequencing of the strain confirmed the presence of genes related to plant growth promotion and methylotrophy.

DOI： 10.1128/genomeA.00266-15

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Language：English   Publisher：FRONTIERS MEDIA SA  

Metabolomic analysis revealed that Methylobacterium cells accumulate a large amount of ergothioneine (EGT), which is a sulfur containing, non-proteinogenic, antioxidative amino acid derived from histidine. EGT biosynthesis and its role in methylotrophy and physiology for plant surface-symbiotic Methylobacterium species were investigated in this study. Almost all Methylobacterium type strains can synthesize FGT. We selected one of the most productive strains (M. aquaticum strain 22A isolated from a moss), and investigated the feasibility of fermentative EGT production through optimization of the culture condition. Methanol as a carbon source served as the best substrate for production. The productivity reached up to 1000 mu g/100 ml culture (1200 mu g/g wet weight cells, 6.3 mg/g dry weight) in 38 days. Next, we identified the genes (egtBD) responsible for EGT synthesis, and generated a deletion mutant defective in EGT production. Compared to the wild type, the mutant showed better growth on methanol and on the plant surface as well as severe susceptibility to heat treatment and irradiation of ultraviolet (UV) and sunlight. These results suggested that EGT is not involved in methylotrophy, but is involved in their phyllospheric lifestyle fitness of the genus in natural conditions.

DOI： 10.3389/fmicb.2015.01185

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Other Link： http://search.jamas.or.jp/link/ui/2016231746

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Other Link： http://search.jamas.or.jp/link/ui/2016231754

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A new multichannel microdevice flow system with stainless steel flow chamber was used for architecture visualization, development monitoring and structural quantification of GFP-labeled Pseudomonas aeruginosa PAO1 live biofilms. Direct in situ investigations using confocal laser scanning microscopy (CLSM) at 72 h revealed structural pattern differences as a result of nutrient concentration gradients. When grown in LB medium, round, dispersed cellular aggregates were formed whereas in 1/3-diluted LB medium, biofilms were mostly flat and compact. However, COMSTAT analyses showed no considerable differences in biomass and thickness between the two LB concentrations. Characterization of time-dependent development of biofilms grown in 1/3-diluted LB medium showed full maturation of colonies by 120 h reaching maximum biomass at 17.1 mu m(3)/mu m(2) and average thickness at 44.4 mu m. Consequent thinning and formation of openings through interior in colonies occurred by 168 h. These results suggest that the new system tested allowed a fast and thick biofilm development on the surface of the stainless steel flow chamber. These findings may provide better estimates of biofilm activity and systematic evaluation of the effects of different parameters on biofilm morphology and development in industrial and biomedical systems. (C) 2012, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2012.09.018

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Treatment of Pseudomonas aeruginosa PAO1 flow biofilms with a D-amino acid mixture caused significant reductions in cell biomass by 75% and cell viability by 71%. No biofilm disassembly occurred, and matrix production increased by 30%, thereby providing a thick protective cover for remaining viable or persister cells.

DOI： 10.1128/AEM.02911-12

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In the methylotrophic bacterium Methylobacterium extorquens strain AM1, MxaF, a Ca2+-dependent methanol dehydrogenase (MDH), is the main enzyme catalyzing methanol oxidation during growth on methanol. The genome of strain AM1 contains another MDH gene homologue, xoxF1, whose function in methanol metabolism has remained unclear. In this work, we show that XoxF1 also functions as an MDH and is La3+-dependent. Despite the absence of Ca2+ in the medium strain AM1 was able to grow on methanol in the presence of La3+. Addition of La3+ increased MDH activity but the addition had no effect on mxaF or xoxF1 expression level. We purified MDH from strain AM1 grown on methanol in the presence of La3+, and its N-terminal amino acid sequence corresponded to that of XoxF1. The enzyme contained La3+ as a cofactor. The Delta mxaF mutant strain could not grow on methanol in the presence of Ca2+, but was able to grow after supplementation with La3+. Taken together, these results show that XoxF1 participates in methanol metabolism as a La3+-dependent MDH in strain AM1.

DOI： 10.1371/journal.pone.0050480

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Language：English   Publisher：ACADEMIC JOURNALS  

This study was conducted with the aim of isolating and identifying endophytic bacteria associated with bananas in Kenya and assessing their functional potentiality as biological fertilizers. Banana material was collected from two different banana cultivars in five different geographical regions and bacteria were isolated using five different isolation media. Whole-cell matrix-assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF/MS) analysis was used for microorganism profiling. Protein from the living cells were extracted using the ethanol/formic acid extraction procedure and intact molecular weights of the ionized proteins directly measured and the pattern of the protein molecular weights used as fingerprints. Forty three isolates were selected for partial 16S rRNA gene sequencing. Isolates were characterized on the basis of their in-vitro plant growth-promoting activities that included abilities to fix free nitrogen, solubilize phosphates and produce siderophores. The isolates were identified as Serratia, Pseudomonas, Rahnella, Enterobacter, Raoultella, Yokenella, Bacillus, Klebsiella, Yersinia and Ewingella species. Siderophore production activity was detected with all the Pseudomonas isolates as determined on blue Chrome Azurol S (CAS) agar plates. Twenty seven isolates were observed to solubilize phosphates, with Rahnella isolates showing the highest potential as determined on NBRIP growth medium. All the isolates grew on solid nitrogen-source free medium, suggesting their ability to fix nitrogen. In conclusion, endophytic bacteria of bananas in Kenya were isolated and identified, and Rahnella and Pseudomonas isolates proposed as potential microbial biofertilizers for sustainable banana production in Kenya.

DOI： 10.5897/AJMR12.1170

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Methylobacterium species are ubiquitous alpha-proteobacteria that reside in the phyllosphere and are fed by methanol that is emitted from plants. In this study, we applied whole-cell matrix-assisted laser desorption/ionization time-of-flight mass spectrometry analysis (WC-MS) to evaluate the diversity of Methylobacterium species collected from a variety of plants. The WC-MS spectrum was reproducible through two weeks of cultivation on different media. WC-MS spectrum peaks of M. extorquens strain AM1 cells were attributed to ribosomal proteins, but those were not were also found. We developed a simple method for rapid identification based on spectra similarity. Using all available type strains of Methylobacterium species, the method provided a certain threshold similarity value for species-level discrimination, although the genus contains some type strains that could not be easily discriminated solely by 16S rRNA gene sequence similarity. Next, we evaluated the WC-MS data of approximately 200 methylotrophs isolated from various plants with MALDI Biotyper software (Bruker Daltonics). Isolates representing each cluster were further identified by 16S rRNA gene sequencing. In most cases, the identification by WC-MS matched that by sequencing, and isolates with unique spectra represented possible novel species. The strains belonging to M. extorquens, M. adhaesivum, M. marchantiae, M. komagatae, M. brachiatum, M. radiotolerans, and novel lineages close to M. adhaesivum, many of which were isolated from bryophytes, were found to be the most frequent phyllospheric colonizers. The WC-MS technique provides emerging high-throughputness in the identification of known/novel species of bacteria, enabling the selection of novel species in a library and identification without 16S rRNA gene sequencing.

DOI： 10.1371/journal.pone.0040784

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A pink-pigmented, facultatively methylotrophic bacterium, strain 35a(T), was isolated from the leaves of Oxalis comiculata. Cells of strain 35a(T) were Gram-reaction-negative, motile, non-spore-forming rods. The highest 16S rRNA gene pairwise sequence similarities for strain 35a(T) were found with the strains of Methylobacterium iners 5317S-33(T) (96.7 %), 'Methylobacterium soil YIM 48816 (96.6%) and Methylobacterium jeotgali S2R03-9(T) (96.3 %). 16S rRNA gene sequence similarities with the type strains of all other recognized species of the genus Methylobacterium were below 96%. Major cellular fatty acids were C-18:1 omega 7c, C-18:0 and C-16:0, The results of DNA-DNA hybridization experiments, analysis of cpn60 gene sequences, fatty acid profiles, whole-cell MALDI-TOF/MS spectral pattern analysis, and physiological and biochemical tests allowed genotypic and phenotypic differentiation of strain 35a(T) from its nearest phylogenetic neighbours. Strain 35a(T) is therefore considered to represent a novel species within the genus Methylobacterium, for which the name Methylobacterium oxalidis sp. nov. is proposed. The type strain is 35a(T) (=DSM 24028(T) = NBRC 107715(T)).

DOI： 10.1099/ijs.0.033019-0

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Here we report the biological and molecular attributes of a novel dsRNA virus isolated from Rosellinia necatrix, a filamentous phytopathogenic fungus. The virus, termed Rosellinia necatrix quadrivirus 1 (RnQV1), forms rigid spherical particles approximately 45 nm in diameter in infected mycelia. The particles contain 4 dsRNA segments, dsRNA1 to dsRNA4, with a size range of 4.9 to 3.7 kbp, each possessing a single large ORF. A comparison of the virus-infected and -cured isogenic fungal strains suggested that RnQV1 infection has no appreciable phenotypic effects. Phylogenetic analysis using the dsRNA3-encoded RdRp sequence revealed that RnQV1 is more distantly related to quadripartite chrysoviruses than to monopartite totiviruses, and is placed in a distinct group from other mycoviruses. No significant sequence similarities were evident between known proteins and RnQV1 structural proteins shown to be encoded by dsRNA2 or dsRNA4. These suggest that RnQV1 is a novel latent virus, belonging to a new family. (C) 2012 Elsevier Inc. All rights reserved.

DOI： 10.1016/j.virol.2012.01.013

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Bryophytes, or mosses, are considered the most maintenance-free materials for roof greening. Racomitrium species are most often used due to their high tolerance to desiccation. Because they grow slowly, a technology for forcing their growth is desired. We succeeded in the efficient production of R. japonicum in liquid culture. The structure of the microbial community is crucial to stabilize the culture. A culture-independent technique revealed that the cultures contain methylotrophic bacteria. Using yeast cells that fluoresce in the presence of methanol, methanol emission from the moss was confirmed, suggesting that it is an important carbon and energy source for the bacteria. We isolated Methylobacterium species from the liquid culture and studied their characteristics. The isolates were able to strongly promote the growth of some mosses including R. japonicum and seed plants, but the plant-microbe combination was important, since growth promotion was not uniform across species. One of the isolates, strain 22A, was cultivated with R. japonicum in liquid culture and in a field experiment, resulting in strong growth promotion. Mutualistic symbiosis can thus be utilized for industrial moss production.

DOI： 10.1371/journal.pone.0033800

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Language：English   Publisher：CZECH ACADEMY AGRICULTURAL SCIENCES  

Bacteriocin-producing lactic acid bacteria (LAB) were isolated and screened from the gastrointestinal tract (GIT) of Thai indigenous chickens. The bacteriocinogenic activities and the primary probiotic properties were determined. The bacteriocins produced by 14 strains of selected LAB displayed inhibitory activity against indicator strains after the supernatants were neutralized with NaOH in the following species: Lactobacillus sakei subsp. sakei JCM1157, Enterococcus faecalis VanB, Bacillus sp., and Listeria monocytogenes. The antagonistic activity of selected LAB was inactivated or decreased after being treated with proteolytic enzymes (alpha-chymotrypsin and trypsin). CR5-1 strain exhibited the highest level of activity (5120 AU/ml) in the stationary phase against L. sakei subsp. sakei JCM1157 in MRS broth at 37 degrees C. The nine isolates of selected LAB were investigated for primary probiotic properties. The survival of the nine isolates was found to decrease approximately by 3 log CFU/ml after passing through the gastrointestinal conditions. All isolates exhibited protein digestion on agar plates but no isolates showed the ability to digest starch and lipid. Most of them showed high susceptibilities to some antibiotics (penicillin G, tetracycline and erythromycin). Thirteen LAB strains producing bacteriocin with strongly inhibitory activity were identified as Lactobacillus salivarius and only one strain was identified by 16S rDNA sequence analysis as Lactobacillus agilis.

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Five isolates, designated TA2, TA4, TA25(T), KOx(T) and NS15(T) were isolated in previous studies by enrichment in mineral medium with potassium oxalate as the sole carbon source and were characterized using a polyphasic approach. The isolates were Gram-reaction-negative, aerobic, non-spore-forming rods. Phylogenetic analyses based on 16S rRNA and DNA gyrase B subunit (gyrB) gene sequences confirmed that the isolates belonged to the genus Pandoraea and were most closely related to Pandoraea sputorum and Pandoraea pnomenusa (97.2-99.70/0 16S rRNA gene sequence similarity). The isolates could be differentiated from their closest relatives on the basis of several phenotypic characteristics. The major cellular fatty acid profiles of the isolates comprised C(16:0), C(18:1)omega 7c, C(17:0) cyclo and summed feature 3 (C(16:1)omega 7c and/or iso-C(15:0) 2-OH). On the basis of DNA DNA hybridization studies and phylogenetic analyses, the isolates represent three novel species within the genus Pandoraea, for which the names Pandoraea oxalativorans sp. nov. (TA25(T) =NBRC 106091(T) =CCM 7677(T) =DSM 23570(T)), Pandoraea faecigallinarum sp. nov. (KOx(T) =NBRC 106092(T) =CCM 2766(T) =DSM 23572(T)) and Pandoraea vervacti sp. nov. (NS15(T) =NBRC 106088(T) =CCM 7667(T) =DSM 235711) are proposed.

DOI： 10.1099/ijs.0.026138-0

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The use of Racomitrium japonicum, a drought resistant bryophyte used for roof-greening, is gradually increasing. However, its utilization is hampered by slow growth rate. Here we isolated culturable bacteria from hydroponic cultivation samples to identify isolates that could promote moss growth. Most of the isolates belonged to Pseudomonas, Rhodococcus, and Duganella species. The isolates were biochemically characterized according to their type of interaction with plants, i.e., production of auxin, siderophores, or hydrogen cyanate, growth in the absence of an added nitrogen source, calcium phosphate solubilization, utilization of sugars, polymers, or aliphatic compounds, and antifungal activity. The isolates were applied to sterile protonemata and non-sterile adult gametophytes of R japonicum to evaluate their effect on plant growth. Furthermore, we isolated fungi that inhibited moss growth. Our results suggest that the microbial community structure in hydroponic cultures is important to stabilize moss production and the isolates that promote moss growth have potential to be utilized as biofertilizers for moss production. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2011.03.012

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Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant-and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single-and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species.

DOI： 10.1371/journal.ppat.1002146

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Non-retroviral RNA virus sequences (NRVSs) have been found in the chromosomes of vertebrates and fungi, but not plants. Here we report similarly endogenized NRVSs derived from plus-, negative-, and double-stranded RNA viruses in plant chromosomes. These sequences were found by searching public genomic sequence databases, and, importantly, most NRVSs were subsequently detected by direct molecular analyses of plant DNAs. The most widespread NRVSs were related to the coat protein (CP) genes of the family Partitiviridae which have bisegmented dsRNA genomes, and included plant-and fungus-infecting members. The CP of a novel fungal virus (Rosellinia necatrix partitivirus 2, RnPV2) had the greatest sequence similarity to Arabidopsis thaliana ILR2, which is thought to regulate the activities of the phytohormone auxin, indole-3-acetic acid (IAA). Furthermore, partitivirus CP-like sequences much more closely related to plant partitiviruses than to RnPV2 were identified in a wide range of plant species. In addition, the nucleocapsid protein genes of cytorhabdoviruses and varicosaviruses were found in species of over 9 plant families, including Brassicaceae and Solanaceae. A replicase-like sequence of a betaflexivirus was identified in the cucumber genome. The pattern of occurrence of NRVSs and the phylogenetic analyses of NRVSs and related viruses indicate that multiple independent integrations into many plant lineages may have occurred. For example, one of the NRVSs was retained in Ar. thaliana but not in Ar. lyrata or other related Camelina species, whereas another NRVS displayed the reverse pattern. Our study has shown that single-and double-stranded RNA viral sequences are widespread in plant genomes, and shows the potential of genome integrated NRVSs to contribute to resolve unclear phylogenetic relationships of plant species.

DOI： 10.1371/journal.ppat.1002146

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The use of Racomitrium japonicum, a drought resistant bryophyte used for roof-greening, is gradually increasing. However, its utilization is hampered by slow growth rate. Here we isolated culturable bacteria from hydroponic cultivation samples to identify isolates that could promote moss growth. Most of the isolates belonged to Pseudomonas, Rhodococcus, and Duganella species. The isolates were biochemically characterized according to their type of interaction with plants, i.e., production of auxin, siderophores, or hydrogen cyanate, growth in the absence of an added nitrogen source, calcium phosphate solubilization, utilization of sugars, polymers, or aliphatic compounds, and antifungal activity. The isolates were applied to sterile protonemata and non-sterile adult gametophytes of R japonicum to evaluate their effect on plant growth. Furthermore, we isolated fungi that inhibited moss growth. Our results suggest that the microbial community structure in hydroponic cultures is important to stabilize moss production and the isolates that promote moss growth have potential to be utilized as biofertilizers for moss production. (C) 2011, The Society for Biotechnology, Japan. All rights reserved.

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A cryptic plasmid, pSM103mini, was found in polyethylene-glycol degrading bacterium Sphingopyxis macrogoltabida, strain 103. The plasmid was 4,328-bp long and its GC content was 57.5%. It contained four open reading frames, but only two of them showed significant similarity to reported proteins. ORF3 and ORF4 were assumed to encode resolvase and replication protein (RepA) respectively. Downstream of ORF4 we found complex repeat sequences. These together with 0121;3 and 4 were necessary and sufficient for plasmid maintenance in strain 103, as analyzed by constructing deletion plasmids. The pHSG398-fused plasmid (pHSG-SM103mini) functioned as a shuttle vector between strain 103 and Escherichia coli. The plasmid constructed was maintained in strain 103 and its close relative, S. macrogoltabida strain 203, but not efficiently in PEG-degrading S. terrae.

DOI： 10.1271/bbb.100650

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A cryptic plasmid, pSM103mini, was found in polyethylene-glycol degrading bacterium Sphingopyxis macrogoltabida, strain 103. The plasmid was 4,328-bp long and its GC content was 57.5%. It contained four open reading frames, but only two of them showed significant similarity to reported proteins. ORF3 and ORF4 were assumed to encode resolvase and replication protein (RepA) respectively. Downstream of ORF4 we found complex repeat sequences. These together with 0121;3 and 4 were necessary and sufficient for plasmid maintenance in strain 103, as analyzed by constructing deletion plasmids. The pHSG398-fused plasmid (pHSG-SM103mini) functioned as a shuttle vector between strain 103 and Escherichia coli. The plasmid constructed was maintained in strain 103 and its close relative, S. macrogoltabida strain 203, but not efficiently in PEG-degrading S. terrae.

DOI： 10.1271/bbb.100650

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Rhodotorula glutinis IFO1125 acquired increased aluminum (Al) resistance from 50 mu M to more than 5 mM by repetitive culturing with stepwise increases in Al concentration at pH 4.0. In our previous study we performed differential display to find that three genes (RgFET3, RgGET3, and RgCMK) encoding proteins homologous to Saccharomyces cerevisiae FET3p, GET3p, and CMK1p and CMK2p, respectively, were up-regulated in the Al-resistant cells. In this study we cloned these genes and found they were indeed up-regulated in Al-resistant strains. The cloned genes were introduced into S. cerevisiae and corresponding mutants to test their relevance to Al resistance. The introduction of RgFET3 and RgGET3 conferred Al resistance to the host, but that of RgCMK did not. Green fluorescent protein (GFP)-tagged RgFet3p was localized at the cell periphery in the host. GFP-tagged RgGet3p formed more punctate bodies in the host under Al stress than in the absence of Al. Different growth responses to Fe (III), Cu (II), Ca ions, and cyclosporin A in the wild type and resistant cells of R. glutinis suggested the involvement and possible links of the three genes in Al resistance. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.

DOI： 10.1016/j.jbiosc.2009.10.015

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DOI： 10.1016/j.jbiosc.2009.08.257

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This study was conducted in order to evaluate the probiotic properties of lactic acid bacteria (LAB) isolated from intestinal tract of broilers and Thai indigenous chickens. The major properties, including the gastric juice and bile salts tolerance, starch, protein and lipid digesting capabilities, and the inhibition on certain pathogenic bacteria were investigated. Three-hundred and twenty-two and 226 LAB strains were isolated from ten broilers and eight Thai indigenous chickens, respectively. The gastrointestinal transit tolerance of these 548 isolates was determined by exposing washed cell suspension at 41A degrees C to simulated gastric juice (pH 2.5) containing pepsin (3 mg ml(-1)), and to simulated small intestinal juice (pH 8.0) in the presence of pancreatin (1 mg ml(-1)) and 7% fresh chicken bile, mimicking the gastrointestinal environment. The survival of 20 isolates was found after passing through the gastrointestinal conditions. The survival rates of six strains; KT3L20, KT2CR5, KT10L22, KT5S19, KT4S13 and PM1L12 from the sequential study were 43.68, 37.56, 33.84, 32.89, 31.37 and 27.19%, respectively. Twelve isolates exhibited protein digestion on agar plate but no isolates showed the ability to digest starch and lipid. All 20 LAB showed the antimicrobial activity against Salmonella sp., Staphylococcus aureus and Escherichia coli except one strain which did not show the inhibitory activity toward E. coli. Accordingly, five isolates of selected LAB (KT2L24, KT3L20, KT4S13, KT3CE27 and KT8S16) can be classified as the best probiotics and were identified as Enterococcus faecalis, Enterococcus durans, Enterococcus faecium, Pediococcus pentosaceus, and Enterococcus faecium, respectively. The survival rate of microencapsulation of E. durans KT3L20 under simulated small intestine juice after sequential of simulated gastric juice was also investigated. An extrusion technique exhibited a higher survival rate than emulsion technique and free cell, respectively.

DOI： 10.1007/s11274-009-0020-8

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Language：Japanese   Publisher：日本生物工学会  

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Other Link： https://projects.repo.nii.ac.jp/?action=repository_uri&item_id=303491

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Sphingopyxis terrae and the Sphingopyxis macrogoltabida strains 103 and 203 are able to degrade polyethylene glycol (PEG). They possess the peg operon, which is responsible for the conversion of PEG to PEG-carboxylate-coenzyme A (CoA). The upstream (3.0 kb) and downstream (6.5 kb) regions of the operon in strain 103 were cloned and sequenced. The structure was well conserved between S. macrogoltabida strain 203 and S. terrae, except that two sets of transposases are absent in strain 203. The downstream region contains the genes for PEG-carboxylate dehydrogenase (PCDH), glutathione S-transferase (GST), tautomerase, and a hypothetical protein. The genes for pcdh and gst were transcribed constitutively and monocistronically, indicating that their transcription is independent of the operon regulation. PCDH and GST were expressed in Escherichia coli and characterized biochemically. PCDH is a homotetramer of 64-kDa subunits and contains one molecule of flavin adenine dinucleotide per subunit. The enzyme dehydrogenates PEG-carboxylate to yield glyoxylate, suggesting that the enzyme is the third enzyme involved in PEG degradation. GST is a homodimer of 28-kDa subunits. GST activity was noncompetitively inhibited by acyl-CoA and PEG-carboxylate-CoA, suggesting the interaction of GST with them. The proposed role for GST is to buffer the toxicity of PEG-carboxylate-CoA.

DOI： 10.1007/s00253-008-1635-7

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Rhodotorula glutinis IFO1125 was found to acquire increased aluminium (Al) resistance from 50 mu M to more than 5 mM by repetitive culturing with stepwise increases in Al concentration at pH 4.0. To investigate the mechanism underlying this novel phenomenon, wild-type and Al- resistant cells were compared. Neither cell type accumulated the free form of Al (Al3+) added to the medium. Transmission electron microscopic analyses revealed a greater number of mitochondria in resistant cells. The formation of small mitochondria with simplified cristae structures was observed in the wild-type strain grown in the presence of Al and in resistant cells grown in the absence of Al. Addition of Al to cells resulted in high mitochondrial membrane potential and concomitant generation of reactive oxygen species (ROS). Exposure to Al also resulted in elevated levels of oxidized proteins and oxidized lipids. Addition of the antioxidants a-tocopherol and ascorbic acid alleviated the Al toxicity, suggesting that ROS generation is the main cause of Al toxicity. Differential display analysis indicated upregulation of mitochondrial genes in the resistant cells. Resistant cells were found to have 2.5- to 3-fold more mitochondrial DNA (mtDNA) than the wild-type strain. Analysis of tricarboxylic acid cycle and respiratory-chain enzyme activities in wild-type and resistant cells revealed significantly reduced cytochrome c oxidase activity and resultant high ROS production in the latter cells. Taken together, these data suggest that the adaptive increased resistance to Al stress in resistant cells resulted from an increased number of mitochondria and increased mtDNA content, as a compensatory response to reduced respiratory activity caused by a deficiency in complex IV function.

DOI： 10.1099/mic.0.2007/016048-0

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Polypropylene glycol (PPG)-assimilating Sphingobium sp. strain PW-1 was grown on 0.5% PPG 700. PPG and its metabolites were analyzed by MALDI-TOF mass spectrometry. An oxidized form of a terminal alcohol group appeared with each molecular species as a metabolite. Cell-free extract showed PPG dehydrogenase activity. In this way, the oxidative metabolic pathway for PPG was confirmed.

DOI： 10.1271/bbb.70749

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Sphingopyxis macrogoltabida strain 103 possesses polyethylene-glycol (PEG)-inducible pegBCDAE operon encoding the genes relevant to PEG degradation. PEG is converted to PEG-carboxylate by PegA (PEG dehydrogenase) and PegC (PEG-aldehyde dehydrogenase). In this study, the recombinant PegE (homologous to acyl-CoA synthetases) was characterized. PegE was an acyl-CoA synthetase active for PEG-carboxylate and fatty acids. Judging from the nature of this kind of protein (located on the cytoplasmic membrane as a translocator), PegE might be responsible for the translocation of PEG-carboxylate from the periplasm into the cytoplasm or for the detoxification of strong acidity of the substrate.

DOI： 10.1007/s00203-007-0320-z

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The upstream and downstream regions of the tentative pva operon including genes encoding oxidized polyvinyl alcohol (PVA) hydrolase (oph), PVA dehydrogenase (pvaA) and cytochrome c (cytC) from Sphingopyxis sp. strain 113P3 were sequenced. The resultant 7.9 kb sequence contained orf1 in the upstream region and orf2 and orf3 in the downstream region. Reverse transcription-polymerase chain reaction (PCR) analyses revealed that the intergenic regions between orf1 and oph or between cytC and orf2 were expressed neither in PVA medium nor glucose medium, indicating that the pva operon consists of three genes. A transcription start site was determined by 5'-rapid amplification of cDNA ends to be 428 bp upstream of the start codon of the oph. The stop codon of cytC was followed by a sequence of inverted repeats that could function as a factor-independent transcription terminator. Strain 113P3 had one megaplasmid including the pva operon. Northern blot hybridization for the three genes revealed that mRNA size was approximately 3 to 4 kb and expression was elevated in PVA medium compared to glucose medium.

DOI： 10.1007/s00253-008-1348-y

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Sphingopyxis terrae, and Sphingopyxis macrogoltabida strains 103 and 203, can degrade polyethylene glycols (PEGs). They differ in the following respects: (i) different substrate specificities (chain length) of assimilable PEG, (ii) PEG-inducible or constitutive PEG-degradative proteins, and (iii) symbiotic or axenic degradation of PEG. S. terrae was able to incorporate PEG 6000, but strain 103 could not incorporate more than PEG 4000, suggesting that the difference in assimilable PEG chain length depends on the ability to take up substrate. PEG-degradative genes (pegB, C, D, A, E and R) from these strains were cloned. Their primary structures shared a high homology of more than 99%. The peg genes encode a TonB-dependent receptor (pegB), a PEG-aldehyde dehydrogenase (pegC), a permease (pegD), a PEG dehydrogenase (pegA) and an acyl-CoA ligase (pegE), and in the opposite orientation, an AraC-type transcription regulator (pegR). The peg operon was flanked by two different sets of transposases. These three strains contained large plasmids and the operon was located in one of the large plasmids in S. terrae. The peg genes could be detected in other PEG-degrading sphingomonads. These results suggest that the peg genes have evolved in a plasmid-mediated manner. An insertion of a transposon gene (pegF) between pegD and pegA in strain 203 was found, which caused the constitutive expression of pegA in this strain.

DOI： 10.1099/mic.0.2006/000992-0

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The ethoxy chains of short ethoxy chain nonylphenol (NPEOav2.0, containing average 2.0 ethoxy units) were dehydrogenated by cell-free extracts from Ensifer sp. strain AS08 grown on a basal medium supplemented with NPEOav2.0. The reaction was coupled with the reduction in 3-(4,5-dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide and phenazine methosulfate. The enzyme ( NPEOav2.0 dehydrogenase; NPEO-DH) was purified to homogeneity with a yield of 20% and a 56-fold increase in specific activity. The molecular mass of the native enzyme was 120 kDa, consisting of two identical monomer units ( 60 kDa). The gene encoding NPEO-DH was cloned, which consisted of 1,659 bp, corresponding to a protein of 553 amino acid residues. The deduced amino acid sequence agreed with the N-terminal amino acid sequence of the purified NPEO-DH. The presence of a flavin adenine dinucleotide (FAD)-binding motif and glucose-methanol-choline (GMC) oxidoreductase signature motifs strongly suggested that the enzyme belongs to the GMC oxidoreductase family. The protein exhibited homology (40-45% identity) with several polyethylene glycol dehydrogenases (PEG-DHs) of this family, but the identity was lower than those ( approximately 58%) among known PEG-DHs. The substrate- binding domain was more hydrophobic compared with those of glucose oxidase and PEG-DHs. The recombinant protein had the same molecular mass as the purified NPEO-DH and dehydrogenated PEG400-2000, NPEOav2.0 and its components, and NPEOav10, but only slight or no activity was found using diethylene glycol, triethylene glycol, and PEG200.

DOI： 10.1007/s00253-006-0620-2

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The genes for polyethylene glycol (PEG) catabolism (pegB, C, D, A and E) in Sphingopyxis macrogoltabida strain 103 were shown to form a PEG-inducible operon. The pegR gene, encoding an AraC-type regulator in the downstream area of the operon, is transcribed in the reverse direction. The transcription start sites of the operon were mapped, and three putative a sigma(70)-type promoter sites were identified in the pegB, pegA and pegR promoters. A promoter activity assay showed that the pegB promoter was induced by PEG and oligomeric ethylene glycols, whereas the pegA and pegR promoters were induced by PEG. Deletion analysis of the pegB promoter indicated that the region containing the activator-binding motif of an AraC/XyIS-type regulator was required for transcription of the pegBCDAE operon. Gel retardation assays demonstrated the specific binding of PegR to the pegB promoter. Transcriptional fusion studies of pegR with pegA and pegB promoters suggested that PegR regulates the expression of the pegBCDAE operon positively through its binding to the pegB promoter, but PegR does not bind to the pegA promoter. Two specific binding proteins for the pegA promoter were purified and identified as a GaIR-type regulator and an H2A histone fragment (histone-like protein, HU). The binding motif of a GaIR/Lacl-type regulator was found in the pegA and pegR promoters. These results suggested the dual regulation of the pegBCDAE operon through the pegB promoter by an AraC-type regulator, PegR (PEG-independent), and through the pegA and pegR promoters by a GaIR/Lacl-type regulator together with HU (PEG-dependent).

DOI： 10.1099/mic.0.29127-0

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Ensifer sp. strain AS08 and Pseudomonas sp. strain AS90 degrading short ethoxy (EO) chain-nonylphenol (NP) [NPEOav2.0 containing NP mono- similar to tetraethoxylates (NP1EO similar to NP4EO); average 2.0 EO units] were isolated by enrichment cultures. Both strains grew on NP but not on octyl- and nonylphenol polyethoxylates (NPEOs) (average 10 EO units). Growth and degradation of NPEOav2.0 was increased with increased concentrations of yeast extract (0.02-0.5%) in a culture medium. Culture supernatants of both strains grown on NPEOav2.0 were analyzed by high-performance liquid chromatography, showing degradation of NP4EO-NP1EO. The metabolites from nonylphenol diethoxylate (NP2EO) by resting cells of both strains were identified by gas chromatography-mass spectrometry as nonylphenoxyethoxyacetic acid, NP1EO, nonylphenoxyacetic acid (NP1EC), and NP, while those from NP1EO were identified as NP1EC and NP. Cell-free extracts from strain AS08 grown on NPEOav2.0 dehydrogenated NPEOs, NPEOav2.0, NP2EO, NP1EO, and PEG 400, but the extracts were inactive toward di- similar to tetraethylene glycol. Aldehydes were formed in the reaction mixture of each substrate with cell-free extracts. From these results, the aerobic metabolic pathway for short EO chain-NP is proposed: A terminal alcohol group of the EO chain is oxidized to a carboxylic acid via an aldehyde, and then one EO unit is removed. This process is repeated until NP is produced.

DOI： 10.1007/s00253-005-0288-z

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A gene for periplasmic poly(vinyl alcohol) (PVA) dehydrogenase (PVADH) was cloned, based on the N-terminal amino acid sequence of the purified PVADH from Sphingomonas sp. 113P3 and the sequence of the gene for PVADH (pvaA, GenBank accession no. AB 190288). The recombinant PVADH tagged with hexahistidine was expressed in Escherichia coli and purified to homogeneity. The recombinant enzyme had the same characteristics as the purified enzyme from Sphingomonas sp. strain 113P. In addition to PVA, the recombinant PVADH could oxidize glycols such as polypropylene glycols and 1,3-butane/cyclohexanediol and 2,4-pentanediol, but neither primary nor secondary alcohols. The amino acid sequence of the recombinant PVADH showed similarity with those of PVADH from Pseudomonas sp. strain VM15C, putative PVADHs from Azoarcus sp. EbN1, and Xanthomonas species (54-25% identity), and the quinohaemoprotein alcohol dehydrogenases (OH-ADHs) from Comamonas testosteroni, Ralstonia eutropha and Pseudomonas putida (25-29% identity). PVADHs from strains 113P3 and VM15C have a conserved superbarrel domain (SD), probable PQQ-binding amino acids in the SID and a haem-binding domain (HBD) (they should be designated QH-PVADHs), but the positions of the amino acid sequences for the HBD and SID are the reverse of those of OH-ADHs. A protein structure of QH-PVADHs is proposed. Results of dot-blot hybridization and RT-PCR indicated that the three genes encoding oxidized PVA hydrolase, PVADH and cytochrome c are expressed constitutively and form an operon.

DOI： 10.1099/mic.0.28848-0

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Polyethylene glycol dehydrogenase (PEGDH) from Sphingopyxis terrae (formerly Sphingomonas terrae) is composed of 535 amino acid residues and one flavin adenine dinucleotide per monomer protein in a homodimeric structure. Its amino acid sequence shows 28.5 to 30.5% identity with glucose oxidases from Aspergillus niger and Penicillium amagasakiense. The ADP-binding site and the signature 1 and 2 consensus sequences of glucose-methanol-choline oxidoreductases are present in PEGDH. Based on three-dimensional molecular modeling and kinetic characterization of wild-type PEGDH and mutant PEGDHs constructed by site-directed mutagenesis, residues potentially involved in catalysis and substrate binding were found in the vicinity of the flavin ring. The catalytically important active sites were assigned to His-467 and Asn-511. One disulfide bridge between Cys-379 and Cys-382 existed in PEGDH and seemed to play roles in both substrate binding and electron mediation. The Cys-297 mutant showed decreased activity, suggesting the residue's importance in both substrate binding and electron mediation, as well as Cys-379 and Cys-382. PEGDH also contains a motif of a ubiquinone-binding site, and coenzyme Q(10) was utilized as an electron acceptor. Thus, we propose several important amino acid residues involved in the electron transfer pathway from the substrate to ubiquinone.

DOI： 10.1128/AEM.02174-05

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A gene (pegC) encoding aldehyde dehydrogenase (ALDH) was located 3.4 kb upstream of a gene encoding polyethylene glycol (PEG) dehydrogenase (pegA) in Sphingomonas macrogoltabidus strain 103. ALDH was expressed in Escherichia coli and purified on a Ni-nitrilotriacetic acid agarose column. The recombinant enzyme was a homotetramer consisting of four 46.1-kDa subunits. The alignment of the putative amino acid sequence of the cloned enzyme showed high similarity with a group of NAD(P)-dependent ALDHs (identity 36-52%); NAD-binding domains (Rossmann fold and four glycine residues) and catalytic residues (Glu225 and Cys259) were well conserved. The cofactor, which was extracted from the purified enzyme, was tightly bound to the enzyme and identified as NADP. The enzyme contained 0.94 mol NADP per subunit. The enzyme was activated by Ca2+, but by no other metals; no metal (Zn, Fe, Mg, or Mn) was detected in the purified recombinant enzyme. Activity was inhibited by p-chloromercuric benzoate, and heavy metals such as Hg, Cu, Pb and Cd, indicating that a cysteine residue is involved in the activity. Enzyme activity was independent of N,N-dimethyl-p-nitrosoaniline as an electron acceptor. Trans-4-(N,N-dimethylamino)-cinnamaldehyde was not oxidized as a substrate, but the compound worked as an inhibitor for the enzyme, as did pyrazole. The enzyme acted on n-aldehydes C-2-C-14) and PEG-aldehydes. Thus the enzyme was concluded to be a novel Ca2+-activating nicotinoprotein (NADP-containing) PEG-aldehyde dehydrogenase involved in the degradation of PEG in S. macrogoltabidus strain 103.

DOI： 10.1007/s00253-005-1936-z

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High performance liquid chromatography( HPLC) on a normal phase silica-gel column with isocratic elution method was established to determine nonylphenol ( NP ) and short ethoxy chain nonylphenols ( NPnEO n = 1 similar to 3 ). NP and NPnEO were separated on a Cosmosil packed column 5SL- II (250 mm x 4. 6 mm i. d. 5 mu m) using ethyl acetate-ethanol as mobile phase at a flow rate of 1. 0 mL/min and detected at 281 run. Within 10 minutes all of the target compounds were eluted and separated. The limit of detection for NP and NPnEO ( n = 1 similar to 3 ) were 0. 1 mg/L and 0. 5 mg/L ( n = 1 similar to 3) respectively. The relative standard deviations of NP, NPEO, NP2EO and NP3EO were 1. 4%, 1. 6%, 2. 5% and 1. 4%, respectively.

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Oxidized polyvinyl alcohol hydrolase (OPH) and polyvinyl alcohol dehydrogenase were found to be constitutively present in the periplasm of Sphingomonas sp. strain 113P3 (formerly Pseudomonas sp. 113P3). The OPH was purified to homogeneity with a yield of 40 % and a 5.9-fold increase in specific activity. The enzyme was a homodimer consisting of 35 kDa subunits. Its activity was inhibited by PMSF, Hg(2+) and Zn(2+). The enzyme hydrolysed oxidized polyvinyl alcohol (oxidized PVA) and p-nitrophenyl acetate (PNPA), but did not hydrolyse any of the mono- or diketones tested. K(m) and V(max) values for oxidized PVA and PNPA were 0.2 and 0.3 mM, and 0.1 and 3.4 micromol min(-1) mg(-1), respectively. The gene for OPH was cloned and sequenced. Sequencing analysis revealed that the open reading frame consisted of 1095 bp, corresponding to a protein of 364 amino acids residues, encoding a signal peptide and a mature protein of 34 and 330 amino acids residues, respectively. The presence of a serine-hydrolase motif (a lipase box; Gly-X-Ser-X-Gly) strongly suggested that the enzyme belongs to the serine-hydrolase family. The protein exhibited homology with OPH of the Pseudomonas sp. strain VM15C (63 % identity) and the polyhydroxybutyrate depolymerases from Mesorhizobium loti, Rhizobium sp. and Sinorhizobium meliloti (29-32 % identity). The oph gene was expressed in Escherichia coli under the control of the lac promoter. The recombinant protein had the same molecular mass and N-terminal amino acid sequence as the purified OPH from strain 113P3.

DOI： 10.1099/mic.0.27655-0

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Pseudonocardia sp. strain K1 is the only Gram-positive bacterium among the bacteria aerobically metabolizing polyethylene glycol (PEG). Generally, PEG is metabolized by an oxidative pathway in which a terminal alcohol group of PEG is oxidized to aldehyde and to carboxylic acid and then an ether bond is oxidatively cleaved. As the cell-free extract of Pseudonocardia sp. strain K1 has PEG dehydrogenase, PEG aldehyde dehydrogenase and diglycolic acid (DGA) dehydrogenase (DGADH) activities, all of which are constitutively formed, the strain has a metabolic pathway similar to that so far known. We purified an ether bond-splitting enzyme as DGADH. The molecular mass of the enzyme was estimated to be 55 kDa; and it consisted of two identical subunits. The enzyme oxidatively cleaved both an ether bond of PEG 3000 dicarboxylic acid and DGA. The N-terminal amino acid sequence of the purified enzyme had high homology with various superoxide dismutases and the enzyme had also superoxide dismutase activity. The atomic absorption spectrum showed that approximately one atom of Fe was included in each subunit of the enzyme. DGADH activity increased in the cells grown in a PEG medium supplemented with FeCl3. Thus, we concluded that the enzyme purified from Pseudonocardia sp. strain K1 is a new ether bond-splitting enzyme.

DOI： 10.1007/s00253-004-1709-0

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A gene encoding an ether-bond-cleaving enzyme, diglycolic acid dehydrogenase (DGADH) from polyethylene glycol-utilizing Pseudonocardia sp. strain K1, was cloned and expressed in Escherichia coli. The deduced amino acid sequence had a high homolog with superoxide dismutases (SODs) from various bacteria. The recombinant protein showed the same activities as those of DGADH front Pseudonocardia sp. strain K1, namely. SOD activity and ether-bond-cleaving activity.

DOI： 10.1263/jbb.98.313

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When aluminum (Al) was added to a culture, growth of Rhodotorula glutinis IFO1125 was temporarily arrested, showing longer lag phases, depending on the Al concentrations (50-300 muM) added, but the growth rates were not affected at all. Resistant strains obtained by one round of plate treatment containing Al reverted the resistance level to the wild-type level when cultivated without Al. Repeated Al treatments, however, induced heritable and stable Al resistance, the level of which was increased up to 4,000 muM by stepwise increments in Al concentrations. Thus, the heritable Al resistance adaptively acquired was due neither to adaptation nor to mutation, but to a mechanism which has yet to be studied. Heritable Al resistance seemed to release the Al inhibition of magnesium uptake.

DOI： 10.1007/s00253-003-1546-6

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The enzymological and genetic aspects of microbial metabolism of hydrocarbons have been extensively revealed. Such molecular information is useful for understanding the bioremediation of oil spill environments and production of hydrocarbon-specific fine chemicals.

DOI： 10.1016/S1369-5274(03)00053-5

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Two genes encoding acyl-CoA dehydrogenases, acdA and acdB, arranged in tandem, were found in the chromosomal DNA of Acinetobacter sp. strain M-1. AcdA was purified from the parental strain and AcdB was purified from an Escherichia coli strain expressing the cloned gene. The substrate specificities of the two enzymes suggest that AcdA is a medium-chain acyl-CoA dehydrogenase and that AcdB is a long-chain acyl-CoA dehydrogenase. Characterization of n-alkane metabolism in Acinetobacter sp. strain M-1 has revealed parallel pathways as well as enzymes with overlapping specificities in a single pathway. The two acyl-CoA dehydrogenases described here provide another example of the physiological complexity underlying n-alkane utilization.

DOI： 10.1263/jbb.94.326

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Acinetobacter sp. strain M-1 accumulated a large amount of wax esters from an n-alkane under nitrogen-limiting conditions. Under the optimized conditions with n-hexadecane as the substrate, the amount of hexadecyl hexadecanoate in the cells reached 0.17 g/g of cells (dry weight). Electron microscopic analysis revealed that multilayered disk-shaped intracellular inclusions were formed concomitant with wax ester formation. The contribution of acyl-CoA reductase to wax ester synthesis was evaluated by gene disruption analysis.

DOI： 10.1128/AEM.68.3.1192-1195.2002

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Language：English   Publisher：AMER SOC MICROBIOLOGY  

In the long-chain n-alkane degrader Acinetobacter sp. strain M-1, two alkane hydroxylase complexes are switched by controlling the expression of two n-alkane hydroxylase-encoding genes in response to the chain length of n-alkanes, while rubredoxin and rubredoxin ruductase are encoded by a single gene and expressed constitutively.

DOI： 10.1128/JB.183.5.1819-1823.2001

Web of Science

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Language：English   Publisher：AMER SOC MICROBIOLOGY  

NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp, strain M-l was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C-2 to C-14 and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes.

DOI： 10.1128/AEM.66.12.5231-5235.2000

Web of Science

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Language：Japanese  

J-GLOBAL

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Language：English   Publisher：AMER SOC MICROBIOLOGY  

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp, strain M-1 cells grown on n-hexadecane as a sole carbon source, The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, cor:responding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases, The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis, rAld1 showed enzyme activity toward n-alkanals (C-4 to C-14), with a preference, for longer carbon chains within the tested range; the highest activity was obtained with tetradecanal, The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1 Delta) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1 Delta strain became much lower than that in the wild-type strain. These and other results imply that a soluble long chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp, strain M-1.

DOI： 10.1128/AEM.66.8.3481-3486.2000

Web of Science

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Language：Japanese  

CiNii Article

CiNii Books

J-GLOBAL

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Language：Japanese  

J-GLOBAL

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Language：English  

NADPH-dependent alkylaldehyde reducing enzyme, which was greatly induced by n-hexadecane, from Acinetobacter sp. strain M-1 was purified and characterized. The purified enzyme had molecular masses of 40 kDa as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and 160 kDa as determined by gel filtration chromatography. The enzyme, which was shown to be highly thermostable, was most active toward n-heptanal and could use n-alkylaldehydes ranging from C2 to C14 and several substituted benzaldehydes, including the industrially important compounds cinnamyl aldehyde and anisaldehyde, as substrates. The alrA gene coding for this enzyme was cloned, and its nucleotide sequence was determined. The deduced amino acid sequence encoded by the alrA gene exhibited homology to the amino acid sequences of zinc-containing alcohol dehydrogenases from various sources. The gene could be highly expressed in Escherichia coli, and the product was purified to homogeneity by simpler procedures from the recombinant than from the original host. Our results show that this enzyme can be used for industrial bioconversion of useful alcohols and aldehydes.

DOI： 10.1128/AEM.66.12.5231-5235.2000

Scopus

PubMed

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Language：English  

A long-chain aldehyde dehydrogenase, Ald1, was found in a soluble fraction of Acinetobacter sp. strain M-1 cells grown on n-hexadecane as a sole carbon source. The gene (ald1) was cloned from the chromosomal DNA of the bacterium. The open reading frame of ald1 was 1,512 bp long, corresponding to a protein of 503 amino acid residues (molecular mass, 55,496 Da), and the deduced amino acid sequence showed high similarity to those of various aldehyde dehydrogenases. The ald1 gene was stably expressed in Escherichia coli, and the gene product (recombinant Ald1 [rAld1]) was purified to apparent homogeneity by gel electrophoresis. rAld1 showed enzyme activity toward n-alkanals (C4 to C14), with a preference for longer carbon chains within the tested range
the highest activity was obtained with tetradecanal. The ald1 gene was disrupted by homologous recombination on the Acinetobacter genome. Although the ald1 disruptant (ald1Δ) strain still had the ability to grow on n-hexadecane to some extent, its aldehyde dehydrogenase activity toward n-tetradecanal was reduced to half the level of the wild-type strain. Under nitrogen-limiting conditions, the accumulation of intracellular wax esters in the ald1Δ strain became much lower than that in the wild-type strain. These and other results imply that a soluble long-chain aldehyde dehydrogenase indeed plays important roles both in growth on n-alkane and in wax ester formation in Acinetobacter sp. strain M-1.

DOI： 10.1128/AEM.66.8.3481-3486.2000

Scopus

PubMed

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Language：Japanese  

CiNii Article

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Language：Japanese  

CiNii Article

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Language：Japanese  

CiNii Article

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Language：Japanese  

CiNii Article

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Language：Japanese   Publisher：社団法人日本農芸化学会  

CiNii Article

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Language：English   Publisher：Society of Fermentation and Bioengineering, Japan  

n-Alkane oxidation in a long chain n-alkane utilizer, Acinetobacter sp. M- l, was investigated. In Acinetobacter, n-alkanes have been postulated to be converted to the acid via a non-conventional oxidation pathway: n-alkane → n-alkyl hydroperoxide → aldehyde → acid (Finnerty, W.R.: Lipids of Acinetobacter. In Proceedings of the World Conference on Biotechnology for the Fats and Oils Industry, 184-188, 1988). However, there is little biochemical information on the enzymes involved in the postulated pathway, particularly the enzyme catalyzing the first step. In this study, we purified an n-alkane-oxidizing enzyme to apparent homogeneity by SDS-PAGE. The enzyme was a flavoprotein, and required molecular oxygen and Cu2+ for its activity, but did not require a reduced coenzyme such as NAD(P)H. A hydroperoxide was detected as a product of the enzyme reaction. We assume that the n-alkane-oxidizing enzyme is a dioxygenase. In addition, as a fatty alcohol does not appear to be an intermediate, fatty alcohol dehydrogenase is assumed not to participate in the n-alkane oxidation. The validity of the postulated pathway is supported by the following observations: (i) n-alkane monooxygenase activity was not detected, (ii) fatty alcohol dehydrogenase activities were low, and (iii) NAD(P)H-dependent long chain fatty aldehyde dehydrogenase activities were strongly induced in n-alkane-grown cells. NAD(P)H-dependent fatty aldehyde reductase activity was also found in n- alkane-grown cells, which may contribute to the formation of waxes that are cell reserve substances in n-alkane-utilizing Acinetobacter.

DOI： 10.1016/0922-338X(96)80578-2

Scopus

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Event date： 2022.9.14

Language：English   Presentation type：Oral presentation (general)  

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Venue：岡山理科大学  

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Venue：Okayama International Center  

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Venue：Okayama International Center  

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Venue：関西大学  

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Venue：名古屋 名城大学  

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Venue：名古屋 名城大学  

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Venue：名古屋 名城大学  

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