Draft genome sequence and morphological data of Planifilum fimeticola PLACP1, a thermophilic chloramphenicol-resistant bacterium isolated from thermophilic sludge.

Planifilum is a thermophilic aerobic Actinomyces often found in compost that is suggested to play a primary role in the degradation of organic matter and is a potential antibiotic-resistance gene (ARG)-hosting bacterium during the composting process. Planifilum fimeticola PLACP1 was isolated from thermophilic sludge on a Columbia plate supplemented with chloramphenicol. PLACP1 was Gram-stain-positive with cells longer than 20 µm that branched and intertwined with each other. A draft genome sequence of P. fimeticola PLACP1 was generated using the Illumina NovaSeq system and deposited in the National Center for Biotechnology Information database under the BioProject accession numbers PRJDB17484 and SAMD00736731. The genome sequence comprised 3,395,140 bp, with 57.97 % GC content and 3,368 genes, including 3,267 protein-coding, 6 rRNA, and 56 tRNA genes. Based on the Comprehensive Antibiotic Resistance Database, 237 predicted gene products were related to ARGs, including 44 macrolide antibiotic-related genes (19 %) as the largest group. This dataset will be beneficial for the morphological identification, comparative genomic analyses, and ARG research in the genus Planifilum.

CRISPRi knockdown of the cyabrB1 gene induces the divergently transcribed icfG and sll1783 operons related to carbon metabolism in the cyanobacterium Synechocystis sp. PCC 6803.

Most cyanobacterial genomes possess more than two copies of genes encoding cyAbrBs (cyanobacterial AbrB-like proteins) having an AbrB-like DNA-binding domain at their C-terminal region. Accumulating data suggest that a wide variety of metabolic and physiologic processes are regulated by cyAbrBs. In this study, we investigated the function of the essential gene cyabrB1 (sll0359) in Synechocystis sp. PCC 6803 by using CRISPR interference technology. The conditional knockdown of cyabrB1 caused increases of cyAbrB2 transcript and protein levels. However, the effect of cyabrB1 knockdown on global gene expression profile was quite limited compared to the previously reported profound effect of knockout of cyabrB2. Among 24 up-regulated genes, 16 genes were members of the divergently transcribed icfG and sll1783 operons related to carbon metabolism. The results of this and previous studies indicate the different contributions of two cyAbrBs to transcriptional regulation of genes related to carbon, hydrogen and nitrogen metabolism. Possession of a pair of cyAbrBs has been highly conserved during the course of evolution of the cyanobacterial phylum, suggesting physiological significance of transcriptional regulation attained by their interaction.

Implication of amino acid metabolism and cell surface integrity for the thermotolerance mechanism in the thermally adapted acetic acid bacterium Acetobacter pasteurianus TH-3.

IMPORTANCE: Acetobacter pasteurianus, an industrial vinegar-producing strain, is suffered by fermentation stress such as fermentation heat and/or high concentrations of acetic acid. By an experimental evolution approach, we have obtained a stress-tolerant strain, exhibiting significantly increased growth and acetic acid fermentation ability at higher temperatures. In this study, we report that only the three gene mutations of ones accumulated during the adaptation process, ansP, dctD, and glnD, were sufficient to reproduce the increased thermotolerance of A. pasteurianus. These mutations resulted in cell envelope modification, including increased phospholipid and lipopolysaccharide synthesis, increased respiratory activity, and cell size reduction. The phenotypic changes may cooperatively work to make the adapted cell thermotolerant by enhancing cell surface integrity, nutrient or oxygen availability, and energy generation.

Relationship between microorganisms and volatile components in each fermentation process in the kusaya gravy that plays an important role in the manufacturing of kusaya, a traditional Japanese fermented fish product.

The relationship between the microbiota and volatile components of kusaya gravy involved in the manufacturing of kusaya, a traditional Japanese fermented fish product, in the Izu Islands (Niijima and Hachijojima) and the fermentation processes are not clear. In this study, we aimed to investigate the relationship between the microbiota and volatile compounds involved in the manufacturing and management of kusaya gravy. 16S ribosomal RNA (rRNA) gene-based amplicon sequencing revealed that the microbiota in kusaya gravy was significantly different between the two islands, and the microbiota hardly changed during each fermentation process. Gas chromatography-mass spectrometry analysis also revealed that the volatile components were strongly related to the microbiota in kusaya gravy, with Hachijojima samples containing sulfur-containing compounds and Niijima samples containing short-chain fatty acids. Therefore, our findings suggest that kusaya gravy is a characteristic fermented gravy with a stable microbiota, and the fermented pickling gravy is fermented by microorganisms.

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.

GENETIC VARIATION OF LEPTOTROMBIDIUM (ACARI: TROMBICULIDAE) MITES CARRYING ORIENTIA TSUTSUGAMUSHI, THE BACTERIAL PATHOGEN CAUSING SCRUB TYPHUS.

Leptotrombidium (Acari: Trombiculidae) mites are carriers of Orientia tsutsugamushi, the bacterial pathogen causing scrub typhus in humans. Classification of Leptotrombidium is vital because limited mite species carry O. tsutsugamushi. Generally, Leptotrombidium at the larval stage (approximately 0.2 mm in size) are used for morphological identification. However, morphological identification is often challenging because it requires considerable skills and taxonomic expertise. In this study, we found that the full-length sequences of the mitochondrial cytochrome c oxidase subunit 1 gene varied among the significant Leptotrombidium. On the basis of these, we modified the canonical deoxyribonucleic acid (DNA) barcoding method for animals by redesigning the primer set to be suitable for Leptotrombidium. Polymerase chain reaction with the redesigned primer set drastically increased the detection sensitivity, especially against Leptotrombidium scutellare (approximately 17% increase), one of the significant mites carrying O. tsutsugamushi. Phylogenetic analysis showed that the samples morphologically classified as L. scutellare and Leptotrombidium pallidum were further split into 3 and 2 distinct subclusters respectively. The mean genetic distance (p-distance) between L. scutellare and L. pallidum was 0.2147, whereas the mean distances within each species were 0.052 and 0.044, respectively. Within L. scutellare, the mean genetic distances between the 3 subclusters were 0.1626-0.1732, whereas the distances within each subcluster were 0.003-0.017. Within L. pallidum, the mean genetic distance between the 2 subclusters was 0.1029, whereas the distances within each subcluster were 0.010-0.013. The DNA barcoding uncovered a broad genetic diversity of Leptotrombidium, especially of L. scutellare and L. pallidum, the notable species carrying O. tsutsugamushi. We conclude that the DNA barcoding using our primers enables precise and detailed classification of Leptotrombidium and implies the existence of a subgenotype in Leptotrombidium that had not been found by morphological identification.

Glucose controls manganese homeostasis through transcription factors regulating known and newly identified manganese transporter genes in Bacillus subtilis.

Mn2+ is an essential nutrient whose concentration is tightly controlled in bacteria. In Bacillus subtilis, the Mn2+-activated transcription factor MntR controls Mn2+ transporter genes. However, factors regulating intracellular Mn2+ concentration are incompletely understood. Here, we found that glucose addition induces an increase in intracellular Mn2+ concentration. We determined this upshift was mediated by glucose induction of the major Mn2+ importer gene mntH by the transcription factor AhrC, which is known to be involved in arginine metabolism and to be indirectly induced by glucose. In addition, we identified novel AhrC-regulated genes encoding the Mn2+ importer YcsG and the ABC-type exporter YknUV. We found the expression of these genes was also regulated by glucose and contributes to the glucose induction of Mn2+ concentrations. ycsG expression is regulated by MntR as well. Furthermore, we analyzed the interaction of AhrC and MntR with the promoter driving ycsG expression and examined the Mn2+-dependent induction of this promoter to identify the transcription factors responsible for the Mn2+ induction. RNA-Seq revealed that disruption of ahrC and mntR affected the expression of 502 and 478 genes, respectively (false discovery rate, <0.001, log2[fold change] ≥ |2|. The AhrC- and/or MntR-dependent expression of twenty promoters was confirmed by LacZ analysis, and AhrC or MntR binding to some of these promoters was observed via EMSA. The finding that glucose promotes an increase in intracellular Mn2+ levels without changes in extracellular Mn2+ concentrations is reasonable for the bacterium, as intracellular Mn2+ is required for enzymes and pathways mediating glucose metabolism.

Symbiosis of Carpenter Bees with Uncharacterized Lactic Acid Bacteria Showing NAD Auxotrophy.

Eusocial bees (such as honey bees and bumble bees) harbor core gut microbiomes that are transmitted through social interaction between nestmates. Carpenter bees are not eusocial; however, recent microbiome analyses found that Xylocopa species harbor distinctive core gut microbiomes. In this study, we analyzed the gut microbiomes of three Xylocopa species in Japan between 2016 and 2021 by V1 to V2 region-based 16S rDNA amplicon sequencing, and 14 candidate novel species were detected based on the full-length 16S rRNA gene sequences. All Xylocopa species harbor core gut microbiomes consisting of primarily lactic acid bacteria (LAB) that were phylogenetically distant from known species. Although they were difficult to cultivate, two LAB species from two different Xylocopa species were isolated by supplementing bacterial culture supernatants. Both genomes exhibited an average LAB genome size with a large set of genes for carbohydrate utilization but lacked genes to synthesize an essential coenzyme NAD, which is unique among known insect symbionts. Our findings of phylogenetically distinct core LAB of NAD auxotrophy reflected the evolution of Xylocopa-restricted bacteria retention and maintenance through vertical transmission of microbes during solitary life. We propose five candidate novel species belonging to the families Lactobacillaceae and Bifidobacteriaceae, including a novel genus, and their potential functions in carbohydrate utilization. IMPORTANCE Recent investigations found unique microbiomes in carpenter bees, but the description of individual microbes, including isolation and genomics, remains largely unknown. Here, we found that the Japanese Xylocopa species also harbor core gut microbiomes. Although most of them were difficult to isolate a pure colony, we successfully isolated several strains. We performed whole-genome sequencing of the isolated candidate novel species and found that the two Lactobacillaceae strains belonging to the Xylocopa-specific novel LAB clade lack the genes for synthesizing NAD, a coenzyme central to metabolism in all living organisms. Here, we propose a novel genus for the two LAB species based on very low 16S rRNA gene sequence similarities and genotypic characters.

Identification of Capsular Polysaccharide Synthesis Loci Determining Bacteriophage Susceptibility in Tetragenococcus halophilus.

Bacteriophages infecting Tetragenococcus halophilus, a halophilic lactic acid bacterium, have been a major industrial concern due to their detrimental effects on the quality of food products. Previously characterized tetragenococcal phages displayed narrow host ranges, but there is little information on these mechanisms. Here, we revealed the host's determinant factors for phage susceptibility using two virulent phages, phiYA5_2 and phiYG2_4, that infect T. halophilus YA5 and YG2, respectively. Phage-resistant derivatives were obtained from these host strains, and mutations were found at the capsular polysaccharide (CPS) synthesis (cps) loci. Quantification analysis verified that capsular polysaccharide production by the cps derivatives from YG2 was impaired. Transmission electron microscopy observation confirmed the presence of filamentous structures outside the cell walls of YG2 and their absence in the cps derivatives of YG2. Phage adsorption assays revealed that phiYG2_4 adsorbed to YG2 but not its cps derivatives, which suggests that the capsular polysaccharide of YG2 is the specific receptor for phiYG2_4. Interestingly, phiYA5_2 adsorbed and infected cps derivatives of YG2, although neither adsorption to nor infection of the parental strain YG2 by phiYA5_2 was observed. The plaque-surrounding halos formed by phiYA5_2 implied the presence of the virion-associated depolymerase that degrades the capsular polysaccharide of YA5. These results indicated that the capsular polysaccharide is a physical barrier rather than a binding receptor for phiYA5_2 and that phiYA5_2 specifically overcomes the capsular polysaccharide of YA5. Thus, it is suggested that tetragenococcal phages utilize CPSs as binding receptors and/or degrade CPSs to approach host cells. IMPORTANCE T. halophilus is a halophilic lactic acid bacterium that contributes to the fermentation processes for various salted foods. Bacteriophage infections of T. halophilus have been a major industrial problem causing fermentation failures. Here, we identified the cps loci in T. halophilus as genetic determinants of phage susceptibility. The structural diversity of the capsular polysaccharide is responsible for the narrow host ranges of tetragenococcal phages. The information provided here could facilitate future studies on tetragenococcal phages and the development of efficient methods to prevent bacteriophage infections.

Membrane Potential-requiring Succinate Dehydrogenase Constitutes the Key to Propionate Oxidation and Is Unique to Syntrophic Propionate-oxidizing Bacteria.

Propionate oxidation in Pelotomaculum thermopropionicum is performed under a thermodynamic limit. The most energetically unfavorable reaction in the propionate oxidation pathway is succinate oxidation. Based on previous genomic and transcriptomic ana-lyses, succinate oxidation in P. thermopropionicum under propionate-oxidizing conditions is conducted by the membrane-bound forms of two succinate dehydrogenases (SDHs). We herein examined the activity of SDH, the mechanisms underlying the succinate oxidation reaction in P. thermopropionicum, and the importance of the protein sequences of related genes. SDH activity was highly localized to the membrane fraction. An ana-lysis of the soluble fraction revealed that fumarate reductase received electrons from NADH, suggesting the involvement of membrane-bound SDH in propionate oxidation. We utilized an uncoupler and inhibitors of adenosine triphosphate (ATP) synthase and membrane-bound SDH to investigate whether the membrane potential of P. thermopropionicum supports propionate oxidation alongside hydrogen production. These chemicals inhibited hydrogen production, indicating that membrane-bound SDH requires a membrane potential for succinate oxidation, and this membrane potential is maintained by ATP synthase. In addition, the phylogenetic distribution of the flavin adenine dinucleotide-binding subunit and conserved amino acid sequences of the cytochrome b subunit of SDHs in propionate-oxidizing bacteria suggests that membrane-bound SDHs possess specific conserved amino acid residues that are strongly associated with efficient succinate oxidation in syntrophic propionate-oxidizing bacteria.

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.

Stepwise metabolic engineering of Corynebacterium glutamicum for the production of phenylalanine.

Corynebacterium glutamicum was metabolically engineered to produce phenylalanine, a valuable aromatic amino acid that can be used as a raw material in the food and pharmaceutical industries. First, a starting phenylalanine-producer was constructed by overexpressing tryptophan-sensitive 3-deoxy-D-arabino-heptulosonate-7-phosphate synthase and phenylalanine- and tyrosine-insensitive bifunctional enzyme chorismate mutase prephenate dehydratase from Escherichia coli, followed by the inactivation of enzymes responsible for the formation of dihydroxyacetone and the consumption of shikimate pathway-related compounds. Second, redirection of the carbon flow from tyrosine to phenylalanine was attempted by deleting of the tyrA gene encoding prephenate dehydrogenase, which catalyzes the committed step for tyrosine biosynthesis from prephenate. However, suppressor mutants were generated, and two mutants were isolated and examined for phenylalanine production and genome sequencing. The suppressor mutant harboring an amino acid exchange (L180R) on RNase J, which was experimentally proven to lead to a loss of function of the enzyme, showed significantly enhanced production of phenylalanine. Finally, modifications of phosphoenolpyruvate-pyruvate metabolism were investigated, revealing that the inactivation of either phosphoenolpyruvate carboxylase or pyruvate carboxylase, which are enzymes of the anaplerotic pathway, is an effective means for improving phenylalanine production. The resultant strain, harboring a phosphoenolpyruvate carboxylase deficiency, synthesized 50.7 mM phenylalanine from 444 mM glucose. These results not only provided new insights into the practical mutations in constructing a phenylalanine-producing C. glutamicum but also demonstrated the creation of a potential strain for the biosynthesis of phenylalanine-derived compounds represented by plant secondary metabolites.

De novo genome assembly and analysis of Zalaria sp. Him3, a novel fructooligosaccharides producing yeast.

BACKGROUND: Zalaria sp. Him3 was reported as a novel fructooligosaccharides (FOS) producing yeast. However, Zalaria spp. have not been widely known and have been erroneously classified as a different black yeast, Aureobasidium pullulans. In this study, de novo genome assembly and analysis of Zalaria sp. Him3 was demonstrated to confirm the existence of a potential enzyme that facilitates FOS production and to compare with the genome of A. pullulans. RESULTS: The genome of Zalaria sp. Him3 was analyzed; the total read bases and total number of reads were 6.38 Gbp and 42,452,134 reads, respectively. The assembled genome sequence was calculated to be 22.38 Mbp, with 207 contigs, N50 of 885,387, L50 of 10, GC content of 53.8%, and 7,496 genes. g2419, g3120, and g3700 among the predicted genes were annotated as cellulase, xylanase, and ß-fructofuranosidase (FFase), respectively. When the read sequences were mapped to A. pullulans EXF-150 genome as a reference, a small amount of reads (3.89%) corresponded to the reference genome. Phylogenetic tree analysis, which was based on the conserved sequence set consisting of 2,362 orthologs in the genome, indicated genetic differences between Zalaria sp. Him3 and Aureobasidium spp. CONCLUSION: The differences between Zalaria and Aureobasidium spp. were evident at the genome level. g3700 identified in the Zalaria sp. Him3 likely does not encode a highly transfructosyl FFase because the motif sequences were unlike those in other FFases involved in FOS production. Therefore, strain Him3 may produce another FFase. Furthermore, several genes with promising functions were identified and might elicit further interest in Zalaria yeast.

Causality Verification for the Correlation between the Presence of Nonstarter Bacteria and Flavor Characteristics in Soft-Type Ripened Cheeses.

Flavor characteristics of ripened cheese are established by various bacteria, such as lactic acid bacteria, Actinobacteria, and Proteobacteria, which spontaneously develop during the cheese-manufacturing process. We previously revealed the relationship between bacterial microbiota and flavor components in soft-type ripened cheeses by using a multiomics approach that combined metagenomics and metabolomics; however, we could not establish a causal relationship. This study aimed to substantiate the causal nature of the correlations revealed by the multiomics approach by using cheese-ripening tests with single isolate inoculation. The bacterial diversity and composition in surface mold-ripened cheeses from Japan and France varied, depending on the differences between the milks (pasteurized or raw), cheese positions (core or rind), and manufacturers. Although the volatile compounds did not clearly reflect the distinctive characteristics of the cheese samples, nonstarter lactic acid bacteria, Actinobacteria, and Proteobacteria positively correlated with ketones and sulfur compounds, as evidenced by a Spearman's correlation analysis. Cheese-ripening tests conducted after inoculation with single bacterial strains belonging to the above-mentioned taxa confirmed that these bacteria formed volatile compounds, in agreement with the correlations observed. In particular, various flavor compounds, such as acids, esters, ketones, and sulfur compounds, were detected in cheese inoculated with Pseudoalteromonas sp. TS-4-4 strain. These findings provide important insights into the role of nonstarter bacteria in the development of cheese flavor and into the effectiveness of the multiomics approach in screening for bacteria that can improve the quality of cheese products. IMPORTANCE Our previous study revealed that the existence of various bacteria, such as lactic acid bacteria, Actinobacteria, and Proteobacteria, clearly correlated with the abundance of flavor components, such as volatile compounds, in soft-type ripened cheeses via a multiomics approach that used 16S rRNA gene amplicon sequencing and headspace gas chromatography-mass spectrometry. However, this approach only showed correlations derived from statistical analyses rather than causal relationships. Therefore, in the present study, we performed cheese-ripening tests using nonstarter bacteria to substantiate the correlations revealed by the multiomics approach in soft-type ripened cheese. Our results suggest the capability of nonstarter bacteria, such as Proteobacteria, to impart flavor to cheese and the effectiveness of the multiomics approach in screening for microbial isolates that can improve the quality of cheese. Overall, our research provides new insights into the importance of bacteria in cheese production.

Development of a stable semi-continuous lipid production system of an oleaginous Chlamydomonas sp. mutant using multi-omics profiling.

BACKGROUND: Microalgal lipid production has attracted global attention in next-generation biofuel research. Nitrogen starvation, which drastically suppresses cell growth, is a common and strong trigger for lipid accumulation in microalgae. We previously developed a mutant Chlamydomonas sp. KAC1801, which can accumulate lipids irrespective of the presence or absence of nitrates. This study aimed to develop a feasible strategy for stable and continuous lipid production through semi-continuous culture of KAC1801. RESULTS: KAC1801 continuously accumulated > 20% lipid throughout the subculture (five generations) when inoculated with a dry cell weight of 0.8-0.9 g L-1 and cultured in a medium containing 18.7 mM nitrate, whereas the parent strain KOR1 accumulated only 9% lipid. Under these conditions, KAC1801 continuously produced biomass and consumed nitrates. Lipid productivity of 116.9 mg L-1 day-1 was achieved by semi-continuous cultivation of KAC1801, which was 2.3-fold higher than that of KOR1 (50.5 mg L-1 day-1). Metabolome and transcriptome analyses revealed a depression in photosynthesis and activation of nitrogen assimilation in KAC1801, which are the typical phenotypes of microalgae under nitrogen starvation. CONCLUSIONS: By optimizing nitrate supply and cell density, a one-step cultivation system for Chlamydomonas sp. KAC1801 under nitrate-replete conditions was successfully developed. KAC1801 achieved a lipid productivity comparable to previously reported levels under nitrogen-limiting conditions. In the culture system of this study, metabolome and transcriptome analyses revealed a nitrogen starvation-like response in KAC1801.

Periplasmic dehydroshikimate dehydratase combined with quinate oxidation in Gluconobacter oxydans for protocatechuate production.

Protocatechuate (3,4-dihydroxybenzoate) has antioxidant properties and is a raw material for the production of muconic acid, which is a key compound in the synthesis of polymers such as nylon and polyethylene terephthalate. Gluconobacter oxydans strain NBRC3244 has a periplasmic system for oxidation of quinate to produce 3-dehydroquinate. Previously, a periplasmic 3-dehydroshikimate production system was constructed by heterologously expressing Gluconacetobacter diazotrophicus dehydroquinate dehydratase in the periplasm of G. oxydans strain NBRC3244. 3-Dehydroshikimate is converted to protocatechuate by dehydration. In this study, we constructed a G. oxydans strain that expresses the Acinetobacter baylyi quiC gene, which encodes a dehydroshikimate dehydratase of which the subcellular localization is likely the periplasm. We attempted to produce protocatechuate by co-cultivation of two recombinant G. oxydans strains-one expressing the periplasmically targeted dehydroquinate dehydratase and the other expressing A. baylyi dehydroshikimate dehydratase. The co-cultivation system produced protocatechuate from quinate in a nearly quantitative manner.

Mutations in degP and spoT Genes Mediate Response to Fermentation Stress in Thermally Adapted Strains of Acetic Acid Bacterium Komagataeibacter medellinensis NBRC 3288.

An acetic acid bacterium, Komagataeibacter medellinensis NBRC 3288, was adapted to higher growth temperatures through an experimental evolution approach in acetic acid fermentation conditions, in which the cells grew under high concentrations of ethanol and acetic acid. The thermally adapted strains were shown to exhibit significantly increased growth and fermentation ability, compared to the wild strain, at higher temperatures. Although the wild cells were largely elongated and exhibited a rough cell surface, the adapted strains repressed the elongation and exhibited a smaller cell size and a smoother cell surface than the wild strain. Among the adapted strains, the ITO-1 strain isolated during the initial rounds of adaptation was shown to have three indel mutations in the genes gyrB, degP, and spoT. Among these, two dispensable genes, degP and spoT, were further examined in this study. Rough cell surface morphology related to degP mutation suggested that membrane vesicle-like structures were increased on the cell surface of the wild-type strain but repressed in the ITO-1 strain under high-temperature acetic acid fermentation conditions. The ΔdegP strain could not grow at higher temperatures and accumulated a large amount of membrane vesicles in the culture supernatant when grown even at 30°C, suggesting that the degP mutation is involved in cell surface stability. As the spoT gene of ITO-1 lost a 3'-end of 424 bp, which includes one (Act-4) of the possible two regulatory domains (TGS and Act-4), two spoT mutant strains were created: one (ΔTGSAct) with a drug cassette in between the 5'-half catalytic domain and 3'-half regulatory domains of the gene, and the other (ΔAct-4) in between TGS and Act-4 domains of the regulatory domain. These spoT mutants exhibited different growth responses; ΔTGSAct grew better in both the fermentation and non-fermentation conditions, whereas ΔAct-4 did only under fermentation conditions, such as ITO-1 at higher temperatures. We suggest that cell elongation and/or cell size are largely related to these spoT mutations, which may be involved in fermentation stress and thermotolerance.

Ribitol-Containing Wall Teichoic Acid of Tetragenococcus halophilus Is Targeted by Bacteriophage phiWJ7 as a Binding Receptor.

Tetragenococcus halophilus, a halophilic lactic acid bacterium, is used in the fermentation process of soy sauce manufacturing. For many years, bacteriophage infections of T. halophilus have been a major industrial problem that causes fermentation failure. However, studies focusing on the mechanisms of tetragenococcal host-phage interactions are not sufficient. In this study, we generated two phage-insensitive derivatives from the parental strain T. halophilus WJ7, which is susceptible to the virulent phage phiWJ7. Whole-genome sequencing of the derivatives revealed that insertion sequences were transposed into a gene encoding poly(ribitol phosphate) polymerase (TarL) in both derivatives. TarL is responsible for the biosynthesis of ribitol-containing wall teichoic acid, and WJ7 was confirmed to contain ribitol in extracted wall teichoic acid, but the derivative was not. Cell walls of WJ7 irreversibly adsorbed phiWJ7, but those of the phage-insensitive derivatives did not. Additionally, 25 phiWJ7-insensitive derivatives were obtained, and they showed mutations not only in tarL but also in tarI and tarJ, which are responsible for the synthesis of CDP-ribitol. These results indicate that phiWJ7 targets the ribitol-containing wall teichoic acid of host cells as a binding receptor. IMPORTANCE Information about the mechanisms of host-phage interactions is required for the development of efficient strategies against bacteriophage infections. Here, we identified the ribitol-containing wall teichoic acid as a host receptor indispensable for bacteriophage infection. The complete genome sequence of tetragenococcal phage phiWJ7 belonging to the family Rountreeviridae is also provided here. This study could become the foundation for a better understanding of host-phage interactions of tetragenococci.