Mutualistic Interactions between Dinoflagellates and Pigmented Bacteria Mitigate Environmental Stress.

Scleractinian corals form symbiotic relationships with a variety of microorganisms, including endosymbiotic dinoflagellates of the family Symbiodiniaceae, and with bacteria, which are collectively termed coral holobionts. Interactions between hosts and their symbionts are critical to the physiological status of corals. Coral-microorganism interactions have been studied extensively, but dinoflagellate-bacterial interactions remain largely unexplored. Here, we developed a microbiome manipulation method employing KAS-antibiotic treatment (kanamycin, ampicillin, and streptomycin) to favor pigmented bacteria residing on cultured Cladocopium and Durusdinium, major endosymbionts of corals, and isolated several carotenoid-producing bacteria from cell surfaces of the microalgae. Following KAS-antibiotic treatment of Cladocopium sp. strain NIES-4077, pigmented bacteria increased 8-fold based on colony-forming assays from the parental strain, and 100% of bacterial sequences retrieved through 16S rRNA amplicon sequencing were affiliated with the genus Maribacter. Microbiome manipulation enabled host microalgae to maintain higher maximum quantum yield of photosystem II (variable fluorescence divided by maximum fluorescence [Fv/Fm]) under light-stress conditions, compared to the parental strain. Furthermore, by combining culture-dependent and -independent techniques, we demonstrated that species of the family Symbiodiniaceae and pigmented bacteria form strong interactions. Dinoflagellates protected bacteria from antibiotics, while pigmented bacteria protected microalgal cells from light stress via carotenoid production. Here, we describe for the first time a symbiotic relationship in which dinoflagellates and bacteria mutually reduce environmental stress. Investigations of microalgal-bacterial interactions further document bacterial contributions to coral holobionts and may facilitate development of novel techniques for microbiome-mediated coral reef conservation. IMPORTANCE Coral reefs cover less than 0.1% of the ocean floor, but about 25% of all marine species depend on coral reefs at some point in their life cycles. However, rising ocean temperatures associated with global climate change are a serious threat to coral reefs, causing dysfunction of the photosynthetic apparatus of endosymbiotic microalgae of corals, and overproducing reactive oxygen species harmful to corals. We manipulated the microbiome using an antibiotic treatment to favor pigmented bacteria, enabling their symbiotic microalgal partners to maintain higher photosynthetic function under insolation stress. Furthermore, we investigated mechanisms underlying microalgal-bacterial interactions, describing for the first time a symbiotic relationship in which the two symbionts mutually reduce environmental stress. Our findings extend current insights about microalgal-bacterial interactions, enabling better understanding of bacterial contributions to coral holobionts under stressful conditions and offering hope of reducing the adverse impacts of global warming on coral reefs.

Peptide barcoding for one-pot evaluation of sequence-function relationships of nanobodies.

Optimisation of protein binders relies on laborious screening processes. Investigation of sequence-function relationships of protein binders is particularly slow, since mutants are purified and evaluated individually. Here we developed peptide barcoding, a high-throughput approach for accurate investigation of sequence-function relationships of hundreds of protein binders at once. Our approach is based on combining the generation of a mutagenised nanobody library fused with unique peptide barcodes, the formation of nanobody-antigen complexes at different ratios, their fine fractionation by size-exclusion chromatography and quantification of peptide barcodes by targeted proteomics. Applying peptide barcoding to an anti-GFP nanobody as a model, we successfully identified residues important for the binding affinity of anti-GFP nanobody at once. Peptide barcoding discriminated subtle changes in KD at the order of nM to sub-nM. Therefore, peptide barcoding is a powerful tool for engineering protein binders, enabling reliable one-pot evaluation of sequence-function relationships.

Porphyromonas gingivalis induces entero-hepatic metabolic derangements with alteration of gut microbiota in a type 2 diabetes mouse model.

Periodontal infection induces systemic inflammation; therefore, aggravating diabetes. Orally administered periodontal pathogens may directly alter the gut microbiota. We orally treated obese db/db diabetes mice using Porphyromonas gingivalis (Pg). We screened for Pg-specific peptides in the intestinal fecal specimens and examined whether Pg localization influenced the intestinal microbiota profile, in turn altering the levels of the gut metabolites. We evaluated whether the deterioration in fasting hyperglycemia was related to the changes in the intrahepatic glucose metabolism, using proteome and metabolome analyses. Oral Pg treatment aggravated both fasting and postprandial hyperglycemia (P < 0.05), with a significant (P < 0.01) increase in dental alveolar bone resorption. Pg-specific peptides were identified in fecal specimens following oral Pg treatment. The intestinal Pg profoundly altered the gut microbiome profiles at the phylum, family, and genus levels; Prevotella exhibited the largest increase in abundance. In addition, Pg-treatment significantly altered intestinal metabolite levels. Fasting hyperglycemia was associated with the increase in the levels of gluconeogenesis-related enzymes and metabolites without changes in the expression of proinflammatory cytokines and insulin resistance. Oral Pg administration induced gut microbiota changes, leading to entero-hepatic metabolic derangements, thus aggravating hyperglycemia in an obese type 2 diabetes mouse model.

Produced ß-hydroxybutyrate after ß-hydroxy-ß-methylbutyrate (HMB) administration may contribute HMB function in mice.

ß-Hydroxy-ß-methylbutyrate (HMB) is an intermediate in the metabolism of the branched-chain amino acid leucine. HMB has several demonstrated effects on skeletal muscle function, some of which are contradictory. In addition, the effect of exogenous HMB intake on the levels of intermediate metabolites is not known. Therefore, we investigated changes in HMB metabolites after oral HMB administration in mice. First, ICR mice were treated with either distilled water or HMB (0.215 g/10 mL/kg). Sampling was performed at 0, 1, 6, 12, and 24 h after administration. Next, ICR mice were given distilled water or HMB (0.215 g/10 mL/kg/d) for 10 d. Mice given HMB shown a significant increase in liver ß-methylcrotonyl-CoA and increased ß-hydroxybutyrate in plasma and the gastrocnemius muscle 1 h after HMB administration. Mice administered HMB for 10 d showed significantly decreased food intake and body weight; however, the relative weight of the gastrocnemius muscle was significantly increased. These results may be attributed to an increase in ß-hydroxybutyrate resulting from exogenous HMB, since ß-hydroxybutyrate inhibits food intake and suppresses skeletal muscle catabolism. In conclusion, ß-hydroxybutyrate, a metabolite of HMB, was found to play an important role in the function of HMB.

A three-component monooxygenase from Rhodococcus wratislaviensis may expand industrial applications of bacterial enzymes.

The high-valent iron-oxo species formed in the non-heme diiron enzymes have high oxidative reactivity and catalyze difficult chemical reactions. Although the hydroxylation of inert methyl groups is an industrially promising reaction, utilizing non-heme diiron enzymes as such a biocatalyst has been difficult. Here we show a three-component monooxygenase system for the selective terminal hydroxylation of α-aminoisobutyric acid (Aib) into α-methyl-D-serine. It consists of the hydroxylase component, AibH1H2, and the electron transfer component. Aib hydroxylation is the initial step of Aib catabolism in Rhodococcus wratislaviensis C31-06, which has been fully elucidated through a proteome analysis. The crystal structure analysis revealed that AibH1H2 forms a heterotetramer of two amidohydrolase superfamily proteins, of which AibHm2 is a non-heme diiron protein and functions as a catalytic subunit. The Aib monooxygenase was demonstrated to be a promising biocatalyst that is suitable for bioprocesses in which the inert C-H bond in methyl groups need to be activated.

Selected reaction monitoring for the quantification of Escherichia coli ribosomal proteins.

Ribosomes are the sophisticated machinery that is responsible for protein synthesis in a cell. Recently, quantitative mass spectrometry (qMS) have been successfully applied for understanding the dynamics of protein complexes. Here, we developed a highly specific and reproducible method to quantify all ribosomal proteins (r-proteins) by combining selected reaction monitoring (SRM) and isotope labeling. We optimized the SRM methods using purified ribosomes and Escherichia coli lysates and verified this approach as detecting 41 of the 54 r-proteins separately synthesized in E. coli S30 extracts. The SRM methods will enable us to utilize qMS as a highly specific analytical tool in the research of E. coli ribosomes, and this methodology have potential to accelerate the understanding of ribosome biogenesis, function, and the development of engineered ribosomes with additional functions.

Neuronal subclass-selective proteomic analysis in Caenorhabditis elegans.

Neurons are categorised into many subclasses, and each subclass displays different morphology, expression patterns, connectivity and function. Changes in protein synthesis are critical for neuronal function. Therefore, analysing protein expression patterns in individual neuronal subclass will elucidate molecular mechanisms for memory and other functions. In this study, we used neuronal subclass-selective proteomic analysis with cell-selective bio-orthogonal non-canonical amino acid tagging. We selected Caenorhabditis elegans as a model organism because it shows diverse neuronal functions and simple neural circuitry. We performed proteomic analysis of all neurons or AFD subclass neurons that regulate thermotaxis in C. elegans. Mutant phenylalanyl tRNA synthetase (MuPheRS) was selectively expressed in all neurons or AFD subclass neurons, and azido-phenylalanine was incorporated into proteins in cells of interest. Azide-labelled proteins were enriched and proteomic analysis was performed. We identified 4,412 and 1,834 proteins from strains producing MuPheRS in all neurons and AFD subclass neurons, respectively. F23B2.10 (RING-type domain-containing protein) was identified only in neuronal cell-enriched proteomic analysis. We expressed GFP under the control of the 5' regulatory region of F23B2.10 and found GFP expression in neurons. We expect that more single-neuron specific proteomic data will clarify how protein composition and abundance affect characteristics of neuronal subclasses.

Dramatic dietary shift maintains sequestered toxins in chemically defended snakes.

Unlike other snakes, most species of Rhabdophis possess glands in their dorsal skin, sometimes limited to the neck, known as nucho-dorsal and nuchal glands, respectively. Those glands contain powerful cardiotonic steroids known as bufadienolides, which can be deployed as a defense against predators. Bufadienolides otherwise occur only in toads (Bufonidae) and some fireflies (Lampyrinae), which are known or believed to synthesize the toxins. The ancestral diet of Rhabdophis consists of anuran amphibians, and we have shown previously that the bufadienolide toxins of frog-eating species are sequestered from toads consumed as prey. However, one derived clade, the Rhabdophis nuchalis Group, has shifted its primary diet from frogs to earthworms. Here we confirm that the worm-eating snakes possess bufadienolides in their nucho-dorsal glands, although the worms themselves lack such toxins. In addition, we show that the bufadienolides of R. nuchalis Group species are obtained primarily from fireflies. Although few snakes feed on insects, we document through feeding experiments, chemosensory preference tests, and gut contents that lampyrine firefly larvae are regularly consumed by these snakes. Furthermore, members of the R. nuchalis Group contain compounds that resemble the distinctive bufadienolides of fireflies, but not those of toads, in stereochemistry, glycosylation, acetylation, and molecular weight. Thus, the evolutionary shift in primary prey among members of the R. nuchalis Group has been accompanied by a dramatic shift in the source of the species' sequestered defensive toxins.

A Zeaxanthin-Producing Bacterium Isolated from the Algal Phycosphere Protects Coral Endosymbionts from Environmental Stress.

Reef-building corals form a complex consortium with photosynthetic algae in the family Symbiodiniaceae and bacteria, collectively termed the coral holobiont. These bacteria are hypothesized to be involved in the stress resistance of the coral holobiont, but their functional roles remain largely elusive. Here, we show that cultured Symbiodiniaceae algae isolated from the reef-building coral Galaxea fascicularis are associated with novel bacteria affiliated with the family Flavobacteriaceae Antibiotic treatment eliminated the bacteria from cultured Symbiodiniaceae, resulting in a decreased maximum quantum yield of PSII (variable fluorescence divided by maximum fluorescence [Fv/Fm]) and an increased production of reactive oxygen species (ROS) under thermal and light stresses. We then isolated this bacterial strain, named GF1. GF1 inoculation in the antibiotic-treated Symbiodiniaceae cultures restored the Fv/Fm and reduced the ROS production. Furthermore, we found that GF1 produces the carotenoid zeaxanthin, which possesses potent antioxidant activity. Zeaxanthin supplementation to cultured Symbiodiniaceae ameliorated the Fv/Fm and ROS production, suggesting that GF1 mitigates thermal and light stresses in cultured Symbiodiniaceae via zeaxanthin production. These findings could advance our understanding of the roles of bacteria in Symbiodiniaceae and the coral holobiont, thereby contributing to the development of novel approaches toward coral protection through the use of symbiotic bacteria and their metabolites.IMPORTANCE Occupying less than 1% of the seas, coral reefs are estimated to harbor ∼25% of all marine species. However, the destruction of coral reefs has intensified in the face of global climate changes, such as rising seawater temperatures, which induce the overproduction of reactive oxygen species harmful to corals. Although reef-building corals form complex consortia with bacteria and photosynthetic endosymbiotic algae of the family Symbiodiniaceae, the functional roles of coral-associated bacteria remain largely elusive. By manipulating the Symbiodiniaceae bacterial community, we demonstrated that a bacterium that produces an antioxidant carotenoid could mitigate thermal and light stresses in cultured Symbiodiniaceae isolated from a reef-building coral. Therefore, this study illuminates the unexplored roles of coral-associated bacteria under stressful conditions.

YAP1 mediates survival of ALK-rearranged lung cancer cells treated with alectinib via pro-apoptotic protein regulation.

Despite the promising clinical efficacy of the second-generation anaplastic lymphoma kinase (ALK) inhibitor alectinib in patients with ALK-rearranged lung cancer, some tumor cells survive and eventually relapse, which may be an obstacle to achieving a cure. Limited information is currently available on the mechanisms underlying the initial survival of tumor cells against alectinib. Using patient-derived cell line models, we herein demonstrate that cancer cells survive a treatment with alectinib by activating Yes-associated protein 1 (YAP1), which mediates the expression of the anti-apoptosis factors Mcl-1 and Bcl-xL, and combinatorial inhibition against both YAP1 and ALK provides a longer tumor remission in ALK-rearranged xenografts when compared with alectinib monotherapy. These results suggest that the inhibition of YAP1 is a candidate for combinatorial therapy with ALK inhibitors to achieve complete remission in patients with ALK-rearranged lung cancer.

Domain swapping of complementarity-determining region in nanobodies produced by Pichia pastoris.

Easy preparation of chimeric nanobodies with various scaffolds is important for customizing abilities of nanobodies toward practical utilization. To accomplish high-throughput production of various nanobodies, utilization of microbes is an attractive option. In the present study, various chimeric nanobodies were prepared using the methylotrophic yeast Pichia pastoris. We designed chimeric nanobodies with complementarity-determining regions (CDRs) against green fluorescent protein (GFP) or cluster of differentiation 4 (CD4) based on the scaffold of GFP-nanobody. FLAG-tagged chimeric nanobodies were prepared by one-step cloning and produced using P. pastoris. Secreted chimeric nanobodies were purified from the culture media of P. pastoris transformants. Relative binding abilities of purified chimeric nanobodies to GFP and CD4 was tested using a BIACORE T-200. P. pastoris successfully produced a high yield of FLAG-tagged chimeric nanobodies. FLAG-tagged GFP- and CD4-nanobodies were shown to specifically bind to GFP and CD4, respectively. Chimeric nanobodies, in which the CDR2 or 3 of GFP-nanobody was replaced with CDRs of CD4-nanobody, acquired the ability to bind to CD4 without binding to GFP. These results demonstrate successful production of functional chimeric nanobodies using P. pastoris. These results also suggest that swapping of CDRs, especially CDRs 2 or 3, potentially enables a novel method of creating nanobodies.

High-throughput identification of peptide agonists against GPCRs by co-culture of mammalian reporter cells and peptide-secreting yeast cells using droplet microfluidics.

Since G-protein coupled receptors (GPCRs) are linked to various diseases, screening of functional ligands against GPCRs is vital for drug discovery. In the present study, we developed a high-throughput functional cell-based assay by combining human culture cells producing a GPCR, yeast cells secreting randomized peptide ligands, and a droplet microfluidic device. We constructed a reporter human cell line that emits fluorescence in response to the activation of human glucagon-like peptide-1 receptor (hGLP1R). We then constructed a yeast library secreting an agonist of hGLP1R or randomized peptide ligands. We demonstrated that high-throughput identification of functional ligands against hGLP1R could be performed by co-culturing the reporter cells and the yeast cells in droplets. We identified functional ligands, one of which had higher activity than that of an original sequence. The result suggests that our system could facilitate the discovery of functional peptide ligands of GPCRs.

Temporal proteome dynamics of Clostridium cellulovorans cultured with major plant cell wall polysaccharides.

BACKGROUND: Clostridium cellulovorans is a mesophilic, cellulosome-producing bacterium containing 57 genomic cellulosomal enzyme-encoding genes. In addition to cellulosomal proteins, C. cellulovorans also secretes non-cellulosomal proteins to degrade plant cell wall polysaccharides. Unlike other cellulosome-producing Clostridium species, C. cellulovorans can metabolize all major plant cell wall polysaccharides (cellulose, hemicelluloses, and pectins). In this study, we performed a temporal proteome analysis of C. cellulovorans to reveal strategies underlying plant cell wall polysaccharide degradation. RESULTS: We cultured C. cellulovorans with five different carbon sources (glucose, cellulose, xylan, galactomannan, and pectin) and performed proteome analysis on cellular and secreted proteins. In total, we identified 1895 cellular proteins and 875 secreted proteins. The identified unique carbohydrate-degrading enzymes corresponding to each carbon source were annotated to have specific activity against each carbon source. However, we identified pectate lyase as a unique enzyme in C. cellulovorans cultivated on xylan, which was not previously associated with xylan degradation. We performed k-means clustering analysis for elucidation of temporal changes of the cellular and secreted proteins in each carbon sources. We found that cellular proteins in most of the k-means clusters are involved in carbohydrate metabolism, amino acid metabolism, translation, or membrane transport. When xylan and pectin were used as the carbon sources, the most increasing k-means cluster contained proteins involved in the metabolism of cofactors and vitamins. In case of secreted proteins of C. cellulovorans cultured either on cellulose or xylan, galactomannan, and pectin, the clusters with the most increasing trend contained either 25 cellulosomal proteins and five non-cellulosomal proteins or 8-19 cellulosomal proteins and 9-16 non-cellulosomal proteins, respectively. These differences might reflect mechanisms for degrading cellulose of other carbon source. Co-abundance analysis of the secreted proteins revealed that proteases and protease inhibitors accumulated coordinately. This observation implies that the secreted protease inhibitors and proteases protect carbohydrate-degrading enzymes from an attack from the plant. CONCLUSION: In this study, we clarified, for the first time, the temporal proteome dynamics of cellular and secreted proteins in C. cellulovorans. This data will be valuable in understanding strategies employed by C. cellulovorans for degrading major plant cell wall polysaccharides.

Evaluation of meter-long monolithic columns for selected reaction monitoring mass spectrometry.

Proteome is extremely complex as many proteins with a large dynamic range are involved. Nano-liquid chromatography/mass spectrometry-based proteomics has made it possible to separate and identify thousands of proteins in one shot. Although the number of identified proteins in proteomics has significantly improved, it is necessary to increase detection sensitivity to clearly identify low-abundant proteins. In this study, we developed meter-long monolithic columns with a small inner diameter and applied them to selected reaction monitoring-based proteomics for improving proteomic detection sensitivity. Bovine serum albumin tryptic digests were analyzed with optimized selected reaction monitoring methods, and separation efficiency and detection sensitivity in each monolithic column were evaluated. As a result, peak capacity increased by about 1.8-fold and peak area of peptide levels increased by about 2.3-fold. Although flow rate was reduced during analysis with columns of a smaller inner diameter, the peak area reproducibility was maintained. These data displayed the value of meter-long monolithic columns with small inner diameter for selected reaction monitoring-based proteomics.

Peptide barcoding for establishment of new types of genotype-phenotype linkages.

Measuring binding properties of binders (e.g., antibodies) is essential for developing useful experimental reagents, diagnostics, and pharmaceuticals. Display technologies can evaluate a large number of binders in a high-throughput manner, but the immobilization effect and the avidity effect prohibit the precise evaluation of binding properties. In this paper, we propose a novel methodology, peptide barcoding, to quantitatively measure the binding properties of multiple binders without immobilization. In the experimental scheme, unique peptide barcodes are fused with each binder, and they represent genotype information. These peptide barcodes are designed to have high detectability for mass spectrometry, leading to low identification bias and a high identification rate. A mixture of different peptide-barcoded nanobodies is reacted with antigen-coated magnetic beads in one pot. Peptide barcodes of functional nanobodies are cleaved on beads by a specific protease, and identified by selected reaction monitoring using triple quadrupole mass spectrometry. To demonstrate proof-of-principle for peptide barcoding, we generated peptide-barcoded anti-CD4 nanobody and anti-GFP nanobody, and determined whether we could simultaneously quantify their binding activities. We showed that peptide barcoding did not affect the properties of the nanobodies, and succeeded in measuring the binding activities of these nanobodies in one shot. The results demonstrate the advantages of peptide barcoding, new types of genotype-phenotype linkages.

Pentenediol-Type Compounds Specifically Bind to Voltage-Dependent Anion Channel 1 in Saccharomyces cerevisiae Mitochondria.

Voltage-dependent anion channel 1 (VDAC1) situated in the outer mitochondrial membrane regulates the transfer of various metabolites and is a key player in mitochondria-mediated apoptosis. Although many small chemicals that modulate the functions of VDAC1 have been reported to date, most, if not all, of them cannot be regarded as specific reagents due to their interactions with other transporters or enzymes. By screening our chemical libraries using isolated Saccharomyces cerevisiae mitochondria, we found pentenediol (PTD)-type compounds (e.g., PTD-023) as new specific inhibitors of VDAC1. PTD-023 inhibited overall ADP-uptake/ATP-release reactions in isolated mitochondria at a single digit µM level. To identify the binding position of PTDs in VDAC1 by visualizing PTD-bound peptides, we conducted ligand-directed tosyl (LDT) chemistry using the synthetic LDT reagent t-PTD-023 derived from the parent PTD-023 in combination with mutagenesis experiments. t-PTD-023 made a covalent bond predominantly and subsidiarily with nucleophilic Cys210 and Cys130, respectively, indicating that PTDs bind to the region interactive with both residues. Site-directed mutations of hydrogen bond-acceptable Asp139 and Glu152 to Ala, which were selected as potential interactive partners of the critical pentenediol moiety based on the presumed binding model of PTDs in VDAC1, resulted in a decrease in susceptibility against PTD-023. This result strongly suggests that PTDs bind to VDAC1 through a specific hydrogen bond with the two residues. The present study is the first to demonstrate the binding position of specific inhibitors of VDAC1 at the amino acid level.

Alectinib Resistance in ALK-Rearranged Lung Cancer by Dual Salvage Signaling in a Clinically Paired Resistance Model.

The mechanisms responsible for the development of resistance to alectinib, a second-generation anaplastic lymphoma kinase (ALK) inhibitor, are still unclear, and few cell lines are currently available for investigating ALK-rearranged lung cancer. To identify the mechanisms underlying acquired resistance to alectinib, two patient-derived cell lines were established from an alectinib-naïve ALK-rearranged lung cancer and then after development of alectinib resistance. The properties acquired during treatments were detected by comparisons of the two cell lines, and then functional analyses were performed. Coactivation of c-Src and MET was identified after the development of alectinib resistance. Combinatorial therapy against Src and MET significantly restored alectinib sensitivity in vitro (17.2-fold). Increased apoptosis, reduction of tumor volume, and inhibition of MAPK and PI3K/AKT signaling molecules for proliferation and survival were observed when the three kinases (Src, MET, and ALK) were inhibited. A patient-derived xenograft from the alectinib-resistant cells indicated that combination therapy with a saracatinib and crizotinib significantly decreased tumor size in vivo. To confirm the generality, a conventional alectinib-resistant cell line model (H2228-AR1S) was established from NCI-H2228 cells (EML4-ALK variant 3a/b). In H2228-AR1S, combination inhibition of Src and MET also restored alectinib sensitivity. These data reveal that dual salvage signaling from MET and Src is a potential therapeutic target in alectinib-resistant patients. IMPLICATIONS: This study demonstrates the feasibility to elucidate personalized drug-resistance mechanisms from individual patient samples.

Ruegeria sp. Strains Isolated from the Reef-Building Coral Galaxea fascicularis Inhibit Growth of the Temperature-Dependent Pathogen Vibrio coralliilyticus.

The coral microbiome has attracted increased attention because of its potential roles in host protection against deadly diseases. However, little is known about the role of coral-associated bacteria against the temperature-dependent opportunistic pathogen Vibrio coralliilyticus. In this study, we tested whether bacteria associated with the reef-building coral Galaxea fascicularis could inhibit the growth of V. coralliilyticus. Twenty-nine cultivable bacteria were successfully isolated from a healthy colony of G. fascicularis kept in an aquarium. Among the bacterial isolates, three Ruegeria sp. strains inhibited the growth of V. coralliilyticus P1 as a reference strain and Vibrio sp. isolated in this study. Ruegeria sp. strains were also detected from other G. fascicularis colonies in the aquarium and in previous field studies by 16S rRNA amplicon sequencing, suggesting that Ruegeria sp. strains are common among G. fascicularis colonies. These results illuminate the potential role of Ruegeria sp. in protecting corals against pathogenic Vibrio species.

Comparative Proteomic Analysis of Lithospermum erythrorhizon Reveals Regulation of a Variety of Metabolic Enzymes Leading to Comprehensive Understanding of the Shikonin Biosynthetic Pathway.

Plants produce a large variety of specialized (secondary) metabolites having a wide range of hydrophobicity. Shikonin, a red naphthoquinone pigment, is a highly hydrophobic metabolite produced in the roots of Lithospermum erythrorhizon, a medicinal plant in the family Boraginaceae. The shikonin molecule is formed by the coupling of p-hydroxybenzoic acid and geranyl diphosphate, catalyzed by a membrane-bound geranyltransferase LePGT at the endoplasmic reticulum, followed by cyclization of the geranyl chain and oxidations; the latter half of this biosynthetic pathway, however, has not yet been clarified. To shed light on these steps, a proteome analysis was conducted. Shikonin production in vitro was specifically regulated by illumination and by the difference in media used to culture cells and hairy roots. In intact plants, however, shikonin is produced exclusively in the root bark of L. erythrorhizon. These features were utilized for comparative transcriptome and proteome analyses. As the genome sequence is not known for this medicinal plant, sequences from de novo RNA-seq data with 95,861 contigs were used as reference for proteome analysis. Because shikonin biosynthesis requires copper ions and is sensitive to blue light, this methodology identified strong candidates for enzymes involved in shikonin biosynthesis, such as polyphenol oxidase, cannabidiolic acid synthase-like and neomenthol dehydrogenase-like proteins. Because acetylshikonin is the main end product of shikonin derivatives, an O-acetyltransferase was also identified. This enzyme may be responsible for end product formation in these plant species. Taken together, these findings suggest a putative pathway for shikonin biosynthesis.