Microbial composition and metabolic profiles during machine-controlled intra-factory fermentation of cocoa beans harvested in semitropical area of Japan.

Cocoa bean fermentation is typically performed in a spontaneous manner on farms in tropical countries or areas and involves several microbial groups. Metabolism by microbes markedly affects the quality of cocoa beans fermented and the chocolate produced thereof. The present study characterized the microbiota and their metabolic profiles in temperature- and humidity-controlled intra-factory cocoa fermentation in a semitropical area of Japan. Although environmental factors were uniform, the microbiota of cocoa beans subjected to intra-factory fermentation was not stable between tests, particularly in terms of the cell count levels and species observed. Fermentation was sometimes delayed, and fermenting microbes were present at very low levels after 24 hr of fermentation. Due to the unstable microbiota, the profiles of water-soluble compounds differed between tests, indicating the unstable qualities of the fermented cocoa beans. These results suggest the necessity of starter cultures not only in on-farm fermentation but also in machine-controlled intra-factory cocoa fermentation.

Chromosome-level genome assemblies of Cutaneotrichosporon spp. (Trichosporonales, Basidiomycota) reveal imbalanced evolution between nucleotide sequences and chromosome synteny.

BACKGROUND: Since DNA information was first used in taxonomy, barcode sequences such as the internal transcribed spacer (ITS) region have greatly aided fungal identification; however, a barcode sequence alone is often insufficient. Thus, multi-gene- or whole-genome-based methods were developed. We previously isolated Basidiomycota yeasts classified in the Trichosporonales. Some strains were described as Cutaneotrichosporon cavernicola and C. spelunceum, whereas strain HIS471 remained unidentified. We analysed the genomes of these strains to elucidate their taxonomic relationship and genetic diversity. RESULTS: The long-read-based assembly resulted in chromosome-level draft genomes consisting of seven chromosomes and one mitochondrial genome. The genome of strain HIS471 has more than ten chromosome inversions or translocations compared to the type strain of C. cavernicola despite sharing identical ITS barcode sequences and displaying an average nucleotide identity (ANI) above 93%. Also, the chromosome synteny between C. cavernicola and the related species, C. spelunceum, showed significant rearrangements, whereas the ITS sequence identity exceeds 98.6% and the ANI is approximately 82%. Our results indicate that the relative evolutionary rates of barcode sequences, whole-genome nucleotide sequences, and chromosome synteny in Cutaneotrichosporon significantly differ from those in the model yeast Saccharomyces. CONCLUSIONS: Our results revealed that the relative evolutionary rates of nucleotide sequences and chromosome synteny are different among fungal clades, likely because different clades have diverse mutation/repair rates and distinct selection pressures on their genomic sequences and syntenic structures. Because diverse syntenic structures can be a barrier to meiotic recombination and may lead to speciation, the non-linear relationships between nucleotide and synteny diversification indicate that sequence-level distances at the barcode or whole-genome level are not sufficient for delineating species boundaries.

yaaJ, the tRNA-Specific Adenosine Deaminase, Is Dispensable in Bacillus subtilis.

Post-transcriptional modifications of tRNA are crucial for their core function. The inosine (I; 6-deaminated adenosine) at the first position in the anticodon of tRNAArg(ICG) modulates the decoding capability and is generally considered essential for reading CGU, CGC, and CGA codons in eubacteria. We report here that the Bacillus subtilis yaaJ gene encodes tRNA-specific adenosine deaminase and is non-essential for viability. A ß-galactosidase reporter assay revealed that the translational activity of CGN codons was not impaired in the yaaJ-deletion mutant. Furthermore, tRNAArg(CCG) responsible for decoding the CGG codon was dispensable, even in the presence or absence of yaaJ. These results strongly suggest that tRNAArg with either the anticodon ICG or ACG has an intrinsic ability to recognize all four CGN codons, providing a fundamental concept of non-canonical wobbling mediated by adenosine and inosine nucleotides in the anticodon. This is the first example of the four-way wobbling by inosine nucleotide in bacterial cells. On the other hand, the absence of inosine modification induced +1 frameshifting, especially at the CGA codon. Additionally, the yaaJ deletion affected growth and competency. Therefore, the inosine modification is beneficial for translational fidelity and proper growth-phase control, and that is why yaaJ has been actually conserved in B. subtilis.

Evaluation of rRNA depletion methods for capturing the RNA virome from environmental surfaces.

OBJECTIVE: Metatranscriptomic analysis of RNA viromes on built-environment surfaces is hampered by low RNA yields and high abundance of rRNA. Therefore, we evaluated the quality of libraries, efficiency of rRNA depletion, and viral detection sensitivity using a mock community and a melamine-coated table surface RNA with levels below those required (< 5 ng) with a library preparation kit (NEBNext Ultra II Directional RNA Library Prep Kit). RESULTS: Good-quality RNA libraries were obtained from 0.1 ng of mock community and table surface RNA by changing the adapter concentration and number of PCR cycles. Differences in the target species of the rRNA depletion method affected the community composition and sensitivity of virus detection. The percentage of viral occupancy in two replicates was 0.259 and 0.290% in both human and bacterial rRNA-depleted samples, a 3.4 and 3.8-fold increase compared with that for only bacterial rRNA-depleted samples. Comparison of SARS-CoV-2 spiked-in human rRNA and bacterial rRNA-depleted samples suggested that more SARS-CoV-2 reads were detected in bacterial rRNA-depleted samples. We demonstrated that metatranscriptome analysis of RNA viromes is possible from RNA isolated from an indoor surface (representing a built-environment surface) using a standard library preparation kit.

Hybrid Genome Assembly of Short and Long Reads in Galaxy.

Galaxy is a web browser-based data analysis platform that is widely used in biology. Public Galaxy instances allow the analysis of data and interpretation of results without requiring software installation. NanoGalaxy is a public Galaxy instance with tools and workflows for nanopore data analysis. This chapter describes the steps involved in performing genome assembly using short and long reads in NanoGalaxy.

TFIID dependency of steady-state mRNA transcription altered epigenetically by simultaneous functional loss of Taf1 and Spt3 is Hsp104-dependent.

In Saccharomyces cerevisiae, class II gene promoters have been divided into two subclasses, TFIID- and SAGA-dominated promoters or TFIID-dependent and coactivator-redundant promoters, depending on the experimental methods used to measure mRNA levels. A prior study demonstrated that Spt3, a TBP-delivering subunit of SAGA, functionally regulates the PGK1 promoter via two mechanisms: by stimulating TATA box-dependent transcriptional activity and conferring Taf1/TFIID independence. However, only the former could be restored by plasmid-borne SPT3. In the present study, we sought to determine why ectopically expressed SPT3 is unable to restore Taf1/TFIID independence to the PGK1 promoter, identifying that this function was dependent on the construction protocol for the SPT3 taf1 strain. Specifically, simultaneous functional loss of Spt3 and Taf1 during strain construction was a prerequisite to render the PGK1 promoter Taf1/TFIID-dependent in this strain. Intriguingly, genetic approaches revealed that an as-yet unidentified trans-acting factor reprogrammed the transcriptional mode of the PGK1 promoter from the Taf1/TFIID-independent state to the Taf1/TFIID-dependent state. This factor was generated in the haploid SPT3 taf1 strain in an Hsp104-dependent manner and inherited meiotically in a non-Mendelian fashion. Furthermore, RNA-seq analyses demonstrated that this factor likely affects the transcription mode of not only the PGK1 promoter, but also of many other class II gene promoters. Collectively, these findings suggest that a prion or biomolecular condensate is generated in a Hsp104-dependent manner upon simultaneous functional loss of TFIID and SAGA, and could alter the roles of these transcription complexes on a wide variety of class II gene promoters without altering their primary sequences. Therefore, these findings could provide the first evidence that TFIID dependence of class II gene transcription can be altered epigenetically, at least in Saccharomyces cerevisiae.

Replacement of water yam (Dioscorea alata L.) indigenous root endophytes and rhizosphere bacterial communities via inoculation with a synthetic bacterial community of dominant nitrogen-fixing bacteria.

Biofertilizers containing high-density plant growth-promoting bacteria are gaining interest as a sustainable solution to environmental problems caused by eutrophication. However, owing to the limitations of current investigative techniques, the selected microorganisms are not always preferred by the host plant, preventing recruitment into the native microbiota or failing to induce plant growth-promoting effects. To address this, five nitrogen-fixing bacteria previously isolated from water yam (Dioscorea alata L.) plants and showing dominant abundance of 1% or more in the water yam microbiota were selected for analysis of their plant growth-promoting activities when used as a synthetic bacterial inoculant. Water yam cv. A-19 plants were inoculated twice at 10 and 12 weeks after planting under greenhouse conditions. Bacterial communities in root, rhizosphere, and bulk soil samples were characterized using high-throughput 16S rRNA amplicon sequencing. Compared with non-inoculated plants, all bacterial communities were significantly altered by inoculation, mainly at the genus level. The inoculation effects were apparently found in the root communities at 16 weeks after planting, with all inoculated genera showing dominance (in the top 35 genera) compared with the control samples. However, no significant differences in any of the growth parameters or nitrogen contents were observed between treatments. At 20 weeks after planting, the dominance of Stenotrophomonas in the inoculated roots decreased, indicating a decline in the inoculation effects. Interestingly, only the Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium clade was dominant (>1% relative abundance) across all samples, suggesting that bacteria related to this clade are essential core bacteria for water yam growth. This is the first report on addition of a synthetic nitrogen-fixing bacterial community in water yam plants showing that native bacterial communities can be replaced by a synthetic bacterial community, with declining in the effects of Stenotrophomonas on the modified communities several weeks after inoculation.

Urban Microbiomes in Narita, Chiba, Japan: Shotgun Metagenome Sequences of a Train Station.

Here, we performed shotgun metagenome sequencing of swab samples collected on floors at a train station in Narita City, Chiba, Japan. The taxonomic analysis revealed that Actinobacteria and Proteobacteria were the dominant phyla. The data will contribute to insight into the microbiome community on the surfaces of urban built environments.

Weizmannia acidilactici sp. nov., a lactic acid producing bacterium isolated from soils.

The strains designed PP-18T, JC-4 and JC-7 isolated from soils, were Gram-stain-positive rods, facultative anaerobe, endospore-forming bacteria. The strains produced l-lactic acid from glucose. They showed positive for catalase but negative for oxidase, nitrate reduction and arginine hydrolysis. Strains P-18T, JC-4 and JC-7 were closely related to Weizmannia coagulans LMG 6326T (97.27-97.64%) and W. acidiproducens KCTC 13078T (96.46-96.74%) based on 16S rRNA gene sequence similarity, respectively. They contained meso-diaminopimelic acid in cell wall peptidoglycan and had seven isoprene units (MK-7) as the predominant menaquinone. The major cellular fatty acids of strain PP-18T were iso-C15:0, anteiso-C17:0, iso-C16:0 and anteiso-C15:0. The ANIb and ANIm values among the genomes of strains PP-18T, JC-4 and JC-7 are above 99.4% while their ANIb and ANIm values among them and W. coagulans LMG 6326T and W. acidiproducens KCTC 13078T were ranged from 76.61 to 79.59%. These 3 strains showed the digital DNA-DNA hybridization (dDDH) values of 20.7-23.6% when compared with W. coagulans LMG 6326T and W. acidiproducens DSM 23148T. The DNA G + C contents of strains PP-18T, JC-4 and JC-7 were 45.82%, 45.86% and 45.86%, respectively. The major polar lipids were diphosphatidylglycerol, phosphatidylglycerol and phosphoglycolipids. The results of phenotypic and chemotaxonomic characteristics and whole-genome analysis indicated that the strains PP-18T, JC-4 and JC-7 should be represented as a novel species within the genus Weizmannia for which the name Weizmannia acidilactici sp. nov. is proposed. The type strain is PP-18T (=KCTC 33974T = NBRC 113028T = TISTR 2515T).

Unbiased prediction of off-target sites in genome-edited rice using SITE-Seq analysis on a web-based platform.

Genome-editing using the CRISPR-Cas9 system has the potential to substantially accelerate crop breeding. Since off-target editing is one of problems, a reliable method for comprehensively detecting off-target sites is needed. A number of in silico methods based on homology to on-target sequence have been developed, however the prediction without false negative is still under discussion. In this study, we performed a SITE-Seq analysis to predict potential off-target sites. SITE-Seq analysis is a comprehensive method that can detect double-strand breaks in vitro. Furthermore, we developed a systematic method using SITE-Seq in combination with web-based Galaxy system (Galaxy for Cut Site Detection), which can perform reproducible analyses without command line operations. We conducted a SITE-Seq analysis of a rice genome targeted by OsFH15 gRNA-Cas9 as a model, and found 41 candidate off-target sites in the annotated regions. Detailed amplicon-sequencing revealed mutations at one off-target site in actual genome-edited rice. Since this off-target site has an uncommon protospacer adjacent motif, it is difficult to predict using in silico methods alone. Therefore, we propose a novel off-target assessment scheme for genome-edited crops that combines the prediction of off-target candidates by SITE-Seq and in silico programs and the validation of off-target sites by amplicon-sequencing.

Clarification of relationship between single-nucleotide polymorphism panels of Shiga toxin-producing Escherichia coli O157:H7/H- strains.

Eighty strains of enterohemorrhagic Escherichia coli O157:H7/H- were analyzed by three single-nucleotide polymorphism (SNP) panels using whole-genome sequencing data. The partial concordance of SNP types among the different SNP panels was observed on minimum spanning trees reconstructed with SNP data. As for lineage I/II strains, some of the clade 7 strains belonged to one unique SNP type as determined by three panels, suggesting that clade 7 should be divided into at least two genotypes, namely, the unique type and the rest. In addition, clade 8 contained two unique genotypes, which was consistent with the previous prediction. Similarly, for lineage II, clade 12 should be divided into three genotype strains. In contrast, many strains of several clades belonging to lineage I were clustered into the same node on each minimum spanning tree upon testing with the three SNP panels. Previous studies reported that lineage I diverged more recently than lineages I/II and II. Such low diversity in lineage I in this study may have arisen because this lineage has not accumulated SNPs because of its relatively recent divergence. Based on the concordance observed in this study, some of the previously published O157 genotype distribution data were successfully interpreted to clarify the clade distribution, which was well supported by previous literature.

Bacterial Community of Water Yam (Dioscorea alata L.) cv. A-19.

The bacterial community of water yam (Dioscorea alata L.) cv. A-19 is vital because it may promote plant growth without the need for fertilization. However, the influence of fertilization practices on the composition and proportion of the bacterial community of water yam cv. A-19 has not yet been extensively examined. Therefore, we herein investigated the diversity and composition of the bacterial community of water yam cv. A-19 cultivated with and without chemical fertilization using amplicon community profiling based on 16S rRNA gene sequences. No significant difference was detected in the growth of plants cultivated with or without chemical fertilization. Alpha diversity indices were significantly dependent on each compartment, and a decrease was observed in indices from the belowground (rhizosphere and root) to aboveground compartments (stem and leaf). The bacterial composition of each compartment was clustered into three groups: bulk soil, rhizosphere and root, and stem and leaf. Chemical fertilization did not significantly influence the diversity or composition of the water yam cv. A-19 bacterial community. It remained robust in plants cultivated with chemical fertilization. The amplicon community profiling of bacterial communities also revealed the dominance of two bacterial clades, the Allorhizobium-Neorhizobium-Pararhizobium-Rhizobium clade and Burkholderia-Caballeronia-Paraburkholderia clade, with and without chemical fertilization. This is the first study to characterize the bacterial community of water yam cv. A-19 cultivated with and without chemical fertilization.

Novel heat shock response mechanism mediated by the initiation nucleotide of transcription.

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

Orthologs of Saccharomyces cerevisiae SFH2, genes encoding Sec14 family proteins, implicated in utilization of n-alkanes and filamentous growth in response to n-alkanes in Yarrowia lipolytica.

The dimorphic yeast Yarrowia lipolytica has an ability to assimilate n-alkanes as carbon and energy sources. In this study, the roles of orthologs of Saccharomyces cerevisiae SEC14 family gene SFH2, which we named SFH21, SFH22, SFH23 and SFH24, of Y. lipolytica were investigated. The transcript levels of SFH21, SFH22 and SFH23, determined by RNA-seq analysis, qRT-PCR analysis and northern blot analysis, were found to increase in the presence of n-alkanes. The deletion mutant of SFH21, but not that of SFH22, SFH23 or SFH24, showed defects in growth in the media containing n-alkanes and in filamentous growth on the solid media containing n-alkanes. Additional deletions of SFH22 and SFH23 significantly exaggerated the defect in filamentous growth of the deletion mutant of SFH21, and expression of SFH22 or SFH24 using the SFH21 promoter partially suppressed the growth defect of the deletion mutant of SFH21 on n-alkanes. These results suggest that SFH2 orthologs are involved in the utilization of n-alkanes and filamentous growth in response to n-alkanes in Y. lipolytica.

Draft Genome Sequence of the Polychlorinated Biphenyl Degrader Comamonas testosteroni Strain YAZ2, Isolated from a Natural Landscape in the Tohoku Region of Japan.

We report a draft genome sequence of Comamonas testosteroni strain YAZ2, a polychlorinated biphenyl (PCB) degrader that was isolated from a PCB-unpolluted environment. The assembled genome contains a single 5.4-Mb chromosome and an 87-kb plasmid. The bph gene cluster, which is involved in PCB degradation, was found on the chromosome.

Identification and structural characterisation of a catecholate-type siderophore produced by Stenotrophomonas maltophilia K279a.

Siderophores are produced by several bacteria that utilise iron in various environments. Elucidating the structure of a specific siderophore may have valuable applications in drug development. Stenotrophomonas maltophilia, a Gram-negative bacterium that inhabits a wide range of environments and can cause pneumonia, produces siderophores. However, the structure was unknown, and therefore, in this study, we aimed to elucidate it. We purified siderophores from cultures of S. maltophilia K279a using preparative reversed-phase HPLC. The structure was analysed through LC-MS and 1H and 13C NMR. The results demonstrated that S. maltophilia K279a produces 2,3-dihydroxybenzoylserine (DHBS), a monomer unit of enterobactin. We suggested the uptake of Iron(III) by the DHBS complex. DHBS production by S. maltophilia K279a could be attributed to an incomplete enterobactin pathway. Drugs targeting DHBS synthesis could prevent S. maltophilia infection.

Draft genome sequencing of Sporolactobacillus terrae SBT-1, an efficient bacterium to ferment concentrated sugar to D-lactic acid.

Recently, the industrial-scale development of microbial D-lactic acid production has been discussed. In this study, the efficiency of the new isolate Sporolactobacillus terrae SBT-1 for producing D-lactic acid under challenge conditions was investigated. The isolate SBT-1 exhibited superior activity in fermenting a very high glucose or sucrose concentration to D-lactic acid compared to the other S. terrae isolates previously reported in the literature; therefore, SBT-1 could overcome the limitations of effective lactic acid production. In batch cultivation using 360 g/L glucose, SBT-1 produced 290.30 g/L D-lactate with a sufficiently high glucose conversion yield of 86%, volumetric productivity of 3.02 g/L h, and optical purity of 96.80% enantiomer excess. SBT-1 could also effectively utilize 440 g/L sucrose as a sole carbon source to produce 276.50 g/L lactic acid with a conversion yield of 90%, a production rate of 2.88 g/L h, and an optical purity of 98%. D-Lactic acid fermentation by two other related producers, S. inulinus NRIC1133T and S. terrae NRIC0357T, was compared with fermentation by isolate SBT-1. The experimental data revealed that SBT-1 possessed the ability to ferment relatively high glucose or sucrose concentrations to D-lactic acid without obvious catabolite repression and byproduct formation compared to the two reference strains. In draft genome sequencing of S. terrae SBT-1, the results provided here can promote further study to overcome the current limitations for the industrial-scale production of D-lactic acid.

Niche-specific adaptation of Lactobacillus helveticus strains isolated from malt whisky and dairy fermentations.

Lactobacillus helveticus is a well characterized lactobacillus for dairy fermentations that is also found in malt whisky fermentations. The two environments contain considerable differences related to microbial growth, including the presence of different growth inhibitors and nutrients. The present study characterized L. helveticus strains originating from dairy fermentations (called milk strains hereafter) and malt whisky fermentations (called whisky strains hereafter) by in vitro phenotypic tests and comparative genomics. The whisky strains can tolerate ethanol more than the milk strains, whereas the milk strains can tolerate lysozyme and lactoferrin more than the whisky strains. Several plant-origin carbohydrates, including cellobiose, maltose, sucrose, fructooligosaccharide and salicin, were generally metabolized only by the whisky strains, whereas milk-derived carbohydrates, i.e. lactose and galactose, were metabolized only by the milk strains. Milk fermentation properties also distinguished the two groups. The general genomic characteristics, including genomic size, number of coding sequences and average nucleotide identity values, differentiated the two groups. The observed differences in carbohydrate metabolic properties between the two groups correlated with the presence of intact specific enzymes in glycoside hydrolase (GH) families GH1, GH4, GH13, GH32 and GH65. Several GHs in the milk strains were inactive due to the presence of stop codon(s) in genes encoding the GHs, and the inactivation patterns of the genes encoding specific enzymes assigned to GH1 in the milk strains suggested a possible diversification manner of L. helveticus strains. The present study has demonstrated how L. helveticus strains have adapted to their habitats.