Lot-to-lot variation in the microbiota during the brewing process of kimoto-type Japanese rice wine.

Kimoto-type Japanese rice wine (sake) has a wide variety of flavors, as the predominant microbes, including lactic acid bacteria (LAB) and nitrate-reducing bacteria, that spontaneously proliferate in the fermentation starter vary depending on the brewery. In this study, we traced the microbiota in four lots of starters manufactured in a newly established brewery and evaluated the lot-to-lot variation and characteristics of the microbiota in the brewery. The results of a 16S ribosomal RNA amplicon analysis showed that the starters brewed in the second brewing year had a more diverse microbiota than those in the first brewing year. Among the LAB predominated at the middle production stage, lactococci, including Leuconostoc spp., were detected in all the lots, while lactobacilli predominated for the first time in the second year. These results suggest that repeated brewing increased microbial diversity and altered the microbial transition pattern in the kimoto-style fermentation starters. Phylogenetic analyses for the LAB isolates from each starter identified Leuconostoc suionicum, Leuconostoc citreum, and Leuconostoc mesenteroides as predominant lactococci as well as a unique lactobacillus in place of Latilactobacillus sakei. We also found that a rice koji-derived Staphylococcus gallinarum with nitrate-reducing activity was generally predominant during the early production stage, suggesting that there was a case in which staphylococci played a role in nitrite production in the starters. These findings are expected to contribute to the understanding of the diversity of microbiota in kimoto-type sake brewing and enable control of the microbiota for consistent sake quality.

Bradyrhizobium ottawaense efficiently reduces nitrous oxide through high nosZ gene expression.

N2O is an important greenhouse gas influencing global warming, and agricultural land is the predominant (anthropogenic) source of N2O emissions. Here, we report the high N2O-reducing activity of Bradyrhizobium ottawaense, suggesting the potential for efficiently mitigating N2O emission from agricultural lands. Among the 15 B. ottawaense isolates examined, the N2O-reducing activities of most (13) strains were approximately five-fold higher than that of Bradyrhizobium diazoefficiens USDA110T under anaerobic conditions. This robust N2O-reducing activity of B. ottawaense was confirmed by N2O reductase (NosZ) protein levels and by mitigation of N2O emitted by nodule decomposition in laboratory system. While the NosZ of B. ottawaense and B. diazoefficiens showed high homology, nosZ gene expression in B. ottawaense was over 150-fold higher than that in B. diazoefficiens USDA110T, suggesting the high N2O-reducing activity of B. ottawaense is achieved by high nos expression. Furthermore, we examined the nos operon transcription start sites and found that, unlike B. diazoefficiens, B. ottawaense has two transcription start sites under N2O-respiring conditions, which may contribute to the high nosZ expression. Our study indicates the potential of B. ottawaense for effective N2O reduction and unique regulation of nos gene expression towards the high performance of N2O mitigation in the soil.

Evolution of rhizobial symbiosis islands through insertion sequence-mediated deletion and duplication.

Symbiosis between organisms influences their evolution via adaptive changes in genome architectures. Immunity of soybean carrying the Rj2 allele is triggered by NopP (type III secretion system [T3SS]-dependent effector), encoded by symbiosis island A (SymA) in B. diazoefficiens USDA122. This immunity was overcome by many mutants with large SymA deletions that encompassed T3SS (rhc) and N2 fixation (nif) genes and were bounded by insertion sequence (IS) copies in direct orientation, indicating homologous recombination between ISs. Similar deletion events were observed in B. diazoefficiens USDA110 and B. japonicum J5. When we cultured a USDA122 strain with a marker gene sacB inserted into the rhc gene cluster, most sucrose-resistant mutants had deletions in nif/rhc gene clusters, similar to the mutants above. Some deletion mutants were unique to the sacB system and showed lower competitive nodulation capability, indicating that IS-mediated deletions occurred during free-living growth and the host plants selected the mutants. Among 63 natural bradyrhizobial isolates, 2 possessed long duplications (261-357 kb) harboring nif/rhc gene clusters between IS copies in direct orientation via homologous recombination. Therefore, the structures of symbiosis islands are in a state of flux via IS-mediated duplications and deletions during rhizobial saprophytic growth, and host plants select mutualistic variants from the resultant pools of rhizobial populations. Our results demonstrate that homologous recombination between direct IS copies provides a natural mechanism generating deletions and duplications on symbiosis islands.

Levels of Periplasmic Nitrate Reductase during Denitrification are Lower in Bradyrhizobium japonicum than in Bradyrhizobium diazoefficiens.

Soybean plants host endosymbiotic dinitrogen (N2)-fixing bacteria from the genus Bradyrhizobium. Under oxygen-limiting conditions, Bradyrhizobium diazoefficiens and Bradyrhizobium japonicum perform denitrification by sequentially reducing nitrate (NO3-) to nitrous oxide (N2O) or N2. The anaerobic reduction of NO3- to N2O was previously shown to be lower in B. japonicum than in B. diazoefficiens due to impaired periplasmic nitrate reductase (Nap) activity in B. japonicum. We herein demonstrated that impaired Nap activity in B. japonicum was due to low Nap protein levels, which may be related to a decline in the production of FixP and FixO proteins by the cbb3-type oxidase.

Diversity of Bradyrhizobium in Non-Leguminous Sorghum Plants: B. ottawaense Isolates Unique in Genes for N2O Reductase and Lack of the Type VI Secretion System.

Diverse members of Bradyrhizobium diazoefficiens, B. japonicum, and B. ottawaense were isolated from the roots of field-grown sorghum plants in Fukushima, and classified into "Rhizobia" with nodulated soybeans, "Free-living diazotrophs", and "Non-diazotrophs" by nitrogen fixation and nodulation assays. Genome analyses revealed that B. ottawaense members possessed genes for N2O reduction, but lacked those for the Type VI secretion system (T6SS). T6SS is a new bacterial weapon against microbial competitors. Since T6SS-possessing B. diazoefficiens and B. japonicum have mainly been isolated from soybean nodules in Japan, T6SS-lacking B. ottawaense members may be a cryptic lineage of soybean bradyrhizobia in Japan.

An Alkane Sulfonate Monooxygenase Is Required for Symbiotic Nitrogen Fixation by Bradyrhizobium diazoefficiens (syn. Bradyrhizobium japonicum) USDA110T.

Sulfur (S)-containing molecules play an important role in symbiotic nitrogen fixation and are critical components of nitrogenase and other iron-S proteins. S deficiency inhibits symbiotic nitrogen fixation by rhizobia. However, despite its importance, little is known about the sources of S that rhizobia utilize during symbiosis. We previously showed that Bradyrhizobium diazoefficiens USDA110T can assimilate both inorganic and organic S and that genes involved in organic S utilization are expressed during symbiosis. Here, we show that a B. diazoefficiens USDA110T mutant with a sulfonate monooxygenase (ssuD) insertion is defective in nitrogen fixation. Microscopy analyses revealed that the ΔssuD mutant was defective in root hair infection and that ΔssuD mutant bacteroids showed degradation compared to the wild-type strain. Moreover, the ΔssuD mutant was significantly more sensitive to hydrogen peroxide-mediated oxidative stress than the wild-type strain. Taken together, these results show that the ability of rhizobia to utilize organic S plays an important role in symbiotic nitrogen fixation. Since nodules have been reported to be an important source of reduced S used during symbiosis and nitrogen fixation, further research will be needed to determine the mechanisms involved in the regulation of S assimilation by rhizobia.IMPORTANCE Rhizobia form symbiotic associations with legumes that lead to the formation of nitrogen-fixing nodules. Sulfur-containing molecules play a crucial role in nitrogen fixation; thus, the rhizobia inside nodules require large amounts of sulfur. Rhizobia can assimilate both inorganic (sulfate) and organic (sulfonates) sources of sulfur. However, very little is known about rhizobial sulfur metabolism during symbiosis. In this report, we show that sulfonate utilization by Bradyrhizobium diazoefficiens is important for symbiotic nitrogen fixation in both soybean and cowpea. The symbiotic defect is probably due to increased sensitivity to oxidative stress from sulfur deficiency in the mutant strain defective for sulfonate utilization. The results of this study can be extended to other rhizobium-legume symbioses, as sulfonate utilization genes are widespread in these bacteria.

Symbiotic incompatibility between soybean and Bradyrhizobium arises from one amino acid determinant in soybean Rj2 protein.

Cultivated soybean (Glycine max) carrying the Rj2 allele restricts nodulation with specific Bradyrhizobium strains via host immunity, mediated by rhizobial type III secretory protein NopP and the host resistance protein Rj2. Here we found that the single isoleucine residue I490 in Rj2 is required for induction of symbiotic incompatibility. Furthermore, we investigated the geographical distribution of the Rj2-genotype soybean in a large set of germplasm by single nucleotide polymorphism (SNP) genotyping using a SNP marker for I490. By allelic comparison of 79 accessions in the Japanese soybean mini-core collection, we suggest substitution of a single amino acid residue (R490 to I490) in Rj2 induces symbiotic incompatibility with Bradyrhizobium diazoefficiens USDA 122. The importance of I490 was verified by complementation of rj2-soybean by the dominant allele encoding the Rj2 protein containing I490 residue. The Rj2 allele was also found in Glycine soja, the wild progenitor of G. max, and their single amino acid polymorphisms were associated with the Rj2-nodulation phenotype. By SNP genotyping against 1583 soybean accessions, we detected the Rj2-genotype in 5.4% of G. max and 7.7% of G. soja accessions. Distribution of the Rj2-genotype soybean plants was relatively concentrated in the temperate Asian region. These results provide important information about the mechanism of host genotype-specific symbiotic incompatibility mediated by host immunity and suggest that the Rj2 gene has been maintained by environmental conditions during the process of soybean domestication.

Breast Cancer Metastasis in the Skin with Hyperkeratotic Pigmentation Caused by Melanocyte Colonization.

Pigmented breast cancer in the skin caused by nonneoplastic melanocytes of epidermal origin is a rare condition of metastasis from breast cancer, but the pathogenesis of this phenomenon is almost unknown. In this report, we describe a case of breast cancer metastasis in the skin with prominent hyperkeratotic pigmentation caused by nonneoplastic melanocyte colonization. Immunohistochemical staining revealed that the metastatic tumor cells produced IL-23, which is reported not only to induce IL-17 but also to inhibit cell apoptosis in breast cancer cells, which affects tumor progression. In addition to IL-23, substantial numbers of IL-17-producing cells were detected at the peritumoral area, suggesting that IL-17 might induce not only melanogenesis but also keratinocyte proliferation and tumorigenesis. Our report suggests possible mechanisms of hyperkeratotic pigmentation of breast cancer metastasis in the skin.

Variation in bradyrhizobial NopP effector determines symbiotic incompatibility with Rj2-soybeans via effector-triggered immunity.

Genotype-specific incompatibility in legume-rhizobium symbiosis has been suggested to be controlled by effector-triggered immunity underlying pathogenic host-bacteria interactions. However, the rhizobial determinant interacting with the host resistance protein (e.g., Rj2) and the molecular mechanism of symbiotic incompatibility remain unclear. Using natural mutants of Bradyrhizobium diazoefficiens USDA 122, we identified a type III-secretory protein NopP as the determinant of symbiotic incompatibility with Rj2-soybean. The analysis of nopP mutations and variants in a culture collection reveal that three amino acid residues (R60, R67, and H173) in NopP are required for Rj2-mediated incompatibility. Complementation of rj2-soybean by the Rj2 allele confers the incompatibility induced by USDA 122-type NopP. In response to incompatible strains, Rj2-soybean plants activate defense marker gene PR-2 and suppress infection thread number at 2 days after inoculation. These results suggest that Rj2-soybeans monitor the specific variants of NopP and reject bradyrhizobial infection via effector-triggered immunity mediated by Rj2 protein.

Effect of Exogenous General Plant Growth Regulators on the Growth of the Duckweed Lemna minor.

Gibberellic acid (GA3), indole-3-acetic acid (IAA), salicylic acid (SA), abscidic acid (ABA), jasmonic acid (JA) 1-amino cyclopropane-1-carboxylic acid (ACC) and aminoethoxyvinylglycine (AVG) are popular growth regulators of plants. However, the effects of their exogenous addition on the biomass production of aquatic plants, including Lemnoideae plants, "duckweeds," are largely unknown. In this study, the growth of Lemna minor was tested for 10 d in Hoagland medium containing each compound at different concentrations of 0-50 µM. GA3, IAA, and SA were found to have no apparent positive effect on the growth at all concentrations tested. Conversely, ACC and JA moderately and AVG and ABA severely inhibited the growth of L. minor. Among the tested compounds, ascorbic acid had an apparent growth-promoting effect.

Complete Genome Sequence of Bradyrhizobium diazoefficiens USDA 122, a Nitrogen-Fixing Soybean Symbiont.

We report the complete genome sequence of Bradyrhizobium diazoefficiens USDA 122, a nitrogen-fixing soybean symbiont. The genome consists of a 9.1 Mb circular chromosome, and 8,551 coding sequences (CDSs) were predicted on the genome. The sequence will provide insight into the evolution of rhizobial genome, and the symbiotic compatibility with host plants.

Metagenomic Analysis Revealed Methylamine and Ureide Utilization of Soybean-Associated Methylobacterium.

Methylobacterium inhabits the phyllosphere of a large number of plants. We herein report the results of comparative metagenome analyses on methylobacterial communities of soybean plants grown in an experimental field in Tohoku University (Kashimadai, Miyagi, Japan). Methylobacterium was identified as the most dominant genus (33%) among bacteria inhabiting soybean stems. We classified plant-derived Methylobacterium species into Groups I, II, and III based on 16S rRNA gene sequences, and found that Group I members (phylogenetically close to M. extorquens) were dominant in soybean-associated Methylobacterium. By comparing 29 genomes, we found that all Group I members possessed a complete set of genes for the N-methylglutamate pathway for methylamine utilization, and genes for urea degradation (urea carboxylase, urea amidolyase, and conventional urease). Only Group I members and soybean methylobacterial isolates grew in a culture supplemented with methylamine as the sole carbon source. They utilized urea or allantoin (a urea-related compound in legumes) as the sole nitrogen source; however, group III also utilized these compounds. The utilization of allantoin may be crucial in soybean-bacterial interactions because allantoin is a transported form of fixed nitrogen in legume plants. Soybean-derived Group I strain AMS5 colonized the model legume Lotus japonicus well. A comparison among the 29 genomes of plant-derived and other strains suggested that several candidate genes are involved in plant colonization such as csgG (curli fimbriae). Genes for the N-methylglutamate pathway and curli fimbriae were more abundant in soybean microbiomes than in rice microbiomes in the field. Based on these results, we discuss the lifestyle of Methylobacterium in the legume phyllosphere.

Complete Genome Sequence of Methylobacterium sp. Strain AMS5, an Isolate from a Soybean Stem.

Nonrhizobial Methylobacterium spp. inhabit the phyllosphere of a wide variety of plants. We report here the complete genome sequence of Methylobacterium sp. AMS5, which was isolated from a soybean stem. The information is useful for understanding the molecular mechanisms of the interaction between nonrhizobial Methylobacterium spp. and plants.

Bacterial clade with the ribosomal RNA operon on a small plasmid rather than the chromosome.

rRNA is essential for life because of its functional importance in protein synthesis. The rRNA (rrn) operon encoding 16S, 23S, and 5S rRNAs is located on the "main" chromosome in all bacteria documented to date and is frequently used as a marker of chromosomes. Here, our genome analysis of a plant-associated alphaproteobacterium, Aureimonas sp. AU20, indicates that this strain has its sole rrn operon on a small (9.4 kb), high-copy-number replicon. We designated this unusual replicon carrying the rrn operon on the background of an rrn-lacking chromosome (RLC) as the rrn-plasmid. Four of 12 strains close to AU20 also had this RLC/rrn-plasmid organization. Phylogenetic analysis showed that those strains having the RLC/rrn-plasmid organization represented one clade within the genus Aureimonas. Our finding introduces a previously unaddressed viewpoint into studies of genetics, genomics, and evolution in microbiology and biology in general.

Characterization of a Functional Role of the Bradyrhizobium japonicum Isocitrate Lyase in Desiccation Tolerance.

Bradyrhizobium japonicum is a nitrogen-fixing symbiont of soybean. In previous studies, transcriptomic profiling of B. japonicum USDA110, grown under various environmental conditions, revealed the highly induced gene aceA, encoding isocitrate lyase (ICL). The ICL catalyzes the conversion of isocitrate to succinate and glyoxylate in the glyoxylate bypass of the TCA cycle. Here, we evaluated the functional role of B. japonicum ICL under desiccation-induced stress conditions. We purified AceA (molecular mass = 65 kDa) from B. japonicum USDA110, using a His-tag and Ni-NTA column approach, and confirmed its ICL enzyme activity. The aceA mutant showed higher sensitivity to desiccation stress (27% relative humidity (RH)), compared to the wild type. ICL activity of the wild type strain increased approximately 2.5-fold upon exposure to 27% RH for 24 h. The aceA mutant also showed an increased susceptibility to salt stress. Gene expression analysis of aceA using qRT-PCR revealed a 148-fold induction by desiccation, while other genes involved in the glyoxylate pathway were not differentially expressed in this condition. Transcriptome analyses revealed that stress-related genes, such as chaperones, were upregulated in the wild-type under desiccating conditions, even though fold induction was not dramatic (ca. 1.5-2.5-fold).

Survival and Competitiveness of Bradyrhizobium japonicum Strains 20 Years after Introduction into Field Locations in Poland.

It was previously demonstrated that there are no indigenous strains of Bradyrhizobium japonicum forming nitrogen-fixing root nodule symbioses with soybean plants in arable field soils in Poland. However, bacteria currently classified within this species are present (together with Bradyrhizobium canariense) as indigenous populations of strains specific for nodulation of legumes in the Genisteae tribe. These rhizobia, infecting legumes such as lupins, are well established in Polish soils. The studies described here were based on soybean nodulation field experiments, established at the Poznan University of Life Sciences Experiment Station in Gorzyn, Poland, and initiated in the spring of 1994. Long-term research was then conducted in order to study the relation between B. japonicum USDA 110 and USDA 123, introduced together into the same location, where no soybean rhizobia were earlier detected, and nodulation and competitive success were followed over time. Here we report the extra-long-term saprophytic survival of B. japonicum strains nodulating soybeans that were introduced as inoculants 20 years earlier and where soybeans were not grown for the next 17 years. The strains remained viable and symbiotically competent, and molecular and immunochemical methods showed that the strains were undistinguishable from the original inoculum strains USDA 110 and USDA 123. We also show that the strains had balanced numbers and their mobility in soil was low. To our knowledge, this is the first report showing the extra-long-term persistence of soybean-nodulating strains introduced into Polish soils and the first analyzing the long-term competitive relations of USDA 110 and USDA 123 after the two strains, neither of which was native, were introduced into the environment almost 2 decades ago.

Symbiosis island shuffling with abundant insertion sequences in the genomes of extra-slow-growing strains of soybean bradyrhizobia.

Extra-slow-growing bradyrhizobia from root nodules of field-grown soybeans harbor abundant insertion sequences (ISs) and are termed highly reiterated sequence-possessing (HRS) strains. We analyzed the genome organization of HRS strains with the focus on IS distribution and symbiosis island structure. Using pulsed-field gel electrophoresis, we consistently detected several plasmids (0.07 to 0.4 Mb) in the HRS strains (NK5, NK6, USDA135, 2281, USDA123, and T2), whereas no plasmids were detected in the non-HRS strain USDA110. The chromosomes of the six HRS strains (9.7 to 10.7 Mb) were larger than that of USDA110 (9.1 Mb). Using MiSeq sequences of 6 HRS and 17 non-HRS strains mapped to the USDA110 genome, we found that the copy numbers of ISRj1, ISRj2, ISFK1, IS1632, ISB27, ISBj8, and IS1631 were markedly higher in HRS strains. Whole-genome sequencing showed that the HRS strain NK6 had four small plasmids (136 to 212 kb) and a large chromosome (9,780 kb). Strong colinearity was found between 7.4-Mb core regions of the NK6 and USDA110 chromosomes. USDA110 symbiosis islands corresponded mainly to five small regions (S1 to S5) within two variable regions, V1 (0.8 Mb) and V2 (1.6 Mb), of the NK6 chromosome. The USDA110 nif gene cluster (nifDKENXSBZHQW-fixBCX) was split into two regions, S2 and S3, where ISRj1-mediated rearrangement occurred between nifS and nifB. ISs were also scattered in NK6 core regions, and ISRj1 insertion often disrupted some genes important for survival and environmental responses. These results suggest that HRS strains of soybean bradyrhizobia were subjected to IS-mediated symbiosis island shuffling and core genome degradation.

Draft Genome Sequence of Acinetobacter calcoaceticus Strain P23, a Plant Growth-Promoting Bacterium of Duckweed.

Acinetobacter calcoaceticus strain P23 is a plant growth-promoting bacterium, which was isolated from the surface of duckweed. We report here the draft genome sequence of strain P23. The genome data will serve as a valuable reference for understanding the molecular mechanism of plant growth promotion in aquatic plants.

Designing display primaries with currently available light sources for UHDTV wide-gamut system colorimetry.

The wide-gamut system colorimetry has been standardized for ultra-high definition television (UHDTV). The chromaticities of the primaries are designed to lie on the spectral locus to cover major standard system colorimetries and real object colors. Although monochromatic light sources are required for a display to perfectly fulfill the system colorimetry, highly saturated emission colors using recent quantum dot technology may effectively achieve the wide gamut. This paper presents simulation results on the chromaticities of highly saturated non-monochromatic light sources and gamut coverage of real object colors to be considered in designing wide-gamut displays with color filters for the UHDTV.