“BIOLOGY OF SYMBIOSIS”
WORKSHOP
August 21st and 22nd, 2017 (Monday and Tuesday)
Lecture Hall (Koryuto Hall), Main Office Building, RIKEN Yokohama Campus
1Meeting of “Biology of Symbiosis” projectAugust 21st and 22nd, 2017 (Monday and Tuesday)
Lecture Hall (Koryuto Hall), Main Office Building, RIKEN Yokohama Campus

Program August 21st
10:30-10:40 Opening remarks by Ken SHIRASU
10:40-11:00 Ken SHIRASU (CSRS, Plant Immunity Research Group), Chair“Rhizosphere interactions regulate secondary metabolite production of root-associatedmicrobes”
11:00-11:20 Paul SCHULZE-LEFERT (Max Planck Institute, Cologne)“Rhizobial root microbiota membership predisposed convergent evolution of nitrogen-fixing
symbiosis with legumes”
11:20-11:40 Makoto HAYASHI (CSRS, Plant Symbiosis Research Team)“Underground symbiosis between plants and microbes: its significance and molecular
evolution”
11:40-11:55 Miki FUJITA (CSRS, Gene Discovery Research Group)“Development of “RIPPS”, an automated system for evaluating plant environmental stressresponse”
12:00-12:55 Lunch
13:00-13:20 Masahira HATTORI (IMS, Laboratory for Microbiome)“Long-read metagenomics of human gut microbiomes”
13:20-13:40 Hiroshi OHNO (IMS, Laboratory for Intestinal Ecosystem), Chair“Characteristics of M cells and follicle-associated epithelium”
13:40-14:00 Masayuki AMAGAI (IMS, Skin homeostasis)“Stratum corneum as niche for skin microbiome”
14:00-14:20 Kenya HONDA (IMS, Gut Homeostasis)“Modulation of the immune system by the gut microbiota”
14:20-14:40 Break
14:40-15:00 Sidonia FAGARASAN (IMS, Laboratory for Mucosal Immunity), Chair“Host-gut microbiota mutualism by IgA-glycan”
15:00-15:20 Hidewaki NAKAGAWA (IMS, Laboratory for Genome Sequencing Analysis)“Cancer genome sequencing for pathogen and symbiosis in human cancers”215:20-15:40 Toru TAKUMI (BSI, Laboratory for Mental Biology)“The gut-brain axis in autism”
15:40-16:00 Atsushi IRIKI (BSI, Laboratory for Symbolic Cognitive Development)“Primate model of “Mind-Body Interaction” through the mechanisms of gut-brain axis via
microbiome.”
16:00-16:20 Break
16:20-16:40 Hiroyuki OSADA (CSRS, Chemical Biology Research Group), Chair“Activation of biosynthesis of tenuazonic acid in a plant pathogenic fungus”
16:40-17:00 Katsuyuki SHIROGUCHI (QBiC, Lab for Integrative Omics)“Toward "The third generation" bacterial microbiota analysis”
17:00-17:15 Hiroyoshi AOKI (Center for Advanced Photonics, Ultrahigh Precision Optics Technology
Team)“Agarose Gel Microcapsule: Pico-liter-scale DNA Amplification Microvessel for Single Cell
Genomics.”
17:15-17:30 Akiko MINODA (CLST, Epigenome Technology Exploration Unit)“Studying the effect of different microbiota at the cell type and subpopulations landscape levelby single cell RNA-seq”
17:30-17:45 Arnaud GERMOND (QBiC, Laboratory for Comprehensive Bioimaging)“Raman spectroscopy as a tool for cell-line discrimination and gene expression monitoring”
18:00- Get-together party with beverage3Program August 22nd
09:30-09:40 Opening remarks by Moriya OHKUMA
09:40-10:00 Chikara FURUSAWA (QBiC, Laboratory for Multiscale Biosystem Dynamics), Chair“Analysis of phenotypic evolution and symbiosis using microbial laboratory evolution”
10:00-10:20 Shunji TAKAHASHI (CSRS, Natural Product Biosynthesis Unit)“Unveiling Chemical Signal and Biosynthetic Mechanism of Reveromycin”
10:20-10:40 Ryo NAKABAYASHI (CSRS, Metabolomics Research Group)“Metabolome analysis in the parasitism between Solanum lycopersicum and Cuscuta
campestris”
10:40-11:00 Shigehiro KURAKU (CLST, Phyloinformatics Unit)“Genome evolution and ecosystem of marine vertebrates”
11:00-11:15 Break
11:15-11:35 Ryuhei NAKAMURA (CSRS, Biofunctional Catalyst Research Team), Chair“Energy Flow Organized by Benthic Ecosystems”
11:35-11:55 Jun KIKUCHI (CSRS, Environmental Metabolic Analysis Research Team)“Marine and fishery product studies assisted by metabolic profiling with data science”
11:55-12:10 Shigeharu MORIYA (CSRS, Biomass research platform team)“RNA-seq approaching to unlock the ecological functions of natural microbiome system”
12:15-13:05 PI Lunch
13:10-13:30 Yuichi HONGO/Hirokazu KUWAHARA (Tokyo Technology Institute, BRC Microbe
Division)“Functional genomics of multiple endo- and ectosymbionts of termite gut protists”
13:30-13:45 Akiko FUJIWARA (CSRS, Chemical Genomics Research Group)“Elucidation of molecular mechanisms for multiple endosymbiotic systems and development ofnovel control technologies by the inhibition of endosymbiotic machinery in Bemisia tabaci, theagricultural pest”
13:45-14:00 Seok-Won KIM (IMS, Laboratory for Integrated Bioinformatics)“Analysis of the onset mechanism of obesity and type 2 diabetes correlated with gut
microbiota using data mining”
14:00-14:20 Moriya OHKUMA (BRC, Japan Collection of Microorganisms), Chair“Single-cell genome analysis of termite gut symbionts”
14:20-14:30 Closing remarks by Hiroshi OHNO
4

List of Speakers on August 21st
Ken SHIRASU (CSRS, Plant Immunity Research Group)“Rhizosphere interactions regulate secondary metabolite production ofroot-associated microbes”

Many plant roots-associated actinomycetes are known antagonists toward plant pathogens, often by
producing various types of antibiotics. To investigate whether plants influence the root-associatedactinomycetes for antibiotic production, we assayed the antibiotic activities of the strains isolated from
Arabidopsis, the model plant. We show that the isolates altered their antibiotic activities in the presence of
the Arabidopsis root extracts. In particular, Streptosporangium sp. AEG048, produced the red pigment when
cultured with the root extracts but not with shoot extracts. Scanning electron microscopy (SEM) revealed
that the hyphae of AEG048 extensively colonized the root surface, especially around the root hairs. To
identify the secondary metabolite gene cluster(s) responsible for the red pigment production and its
regulation in AEG048, we sequenced its genome using PacBio Single Molecule Real-Time (SMRT)
sequencing. Based on antiSMASH analysis and phylogenetic analysis of the chain length factor of type II
polyketide synthases (PKSs), we identified at least one type-II PKS that is predicted to synthesize the red
pigment. The Arabidopsis-AEG048 pair and the genomic information on AEG048 provide a novel model
system for studying of plant-actinomycete interactions. In addition, using bacterial culture collections fromMax Plank Institute, their anti-Ralstonia activities were also altered by root extract, suggesting that plants
control their associating microbes to produce anti-pathogenic compounds for self-protection.

Paul SCHULZE-LEFERT (Max Planck Institute, Cologne)"Rhizobial root microbiota membership predisposed convergent evolutionof nitrogen-fixing symbiosis with legumes"

Rhizobia are a paraphyletic group of soil-borne bacteria defined by their ability to induce nodule
organogenesis in legume roots and fix atmospheric nitrogen for plant growth. In non-leguminous plants,
species within the Rhizobiales order define a core lineage of the plant microbiota, suggesting alternative
forms of interactions with plant hosts. We compared more than 1,300 whole-genome sequences of5

Rhizobiales isolates, including microbiota members from non-legumes, and show that the set of genes
required for nodulation and nitrogen fixation in legume symbiosis was acquired multiple independent times
within each Rhizobiales sublineage. The majority of root-associated rhizobia colonize and promote root
growth in the crucifer Arabidopsis without nitrogen fixation, indicating these are rhizobial traits of anancestral root association. Thus, the capacity for nodulation and nitrogen fixation in legumes was likely
acquired from a predisposed root association in multiple subsequent events, constituting an example
of convergent evolution.

Makoto HAYASHI (CSRS, Plant Symbiosis Research Team)“Underground symbiosis between plants and microbes: its significance andmolecular evolution”

Land plants typically associate with soil-born microbes in order to obtain minerals that are essential for plant
growth. There are two major types of root symbioses; one is association with arbuscular mycorrhizal fungi,
the other is that with nitrogen-fixing bacteria. The latter symbiosis is limited to certain plant species, so
called Nitrogen Fixing Clade (NFC). Since NFC is monophyletic, it is hypothesized that the most recent
common ancestor had acquired the ability to evolve the mechanism involved in this association.
We are interested in how plants establish this intimate relationship, especially in development of root
nodules that offer a milieu for nitrogen fixation. So far, molecular genetic analyses tell us the outline of plantcomponents, from bacterial recognition to onset of nodule organogenesis: 3 membrane-bound receptor-likekinases, 5 nuclear membrane-associated proteins, and 5 nuclear proteins. Although most of them are also
required for arbuscular mycorrhiza, a transcription factor called NIN is specific to nodulation. Since theectopic expression of Nin confers nodule-like structures to roots, we believe NIN is the central player for
nodule organogenesis. We hypothesize that the recruitment of NIN was the key for evolution of nodulation.
Currently we are investigating how Nin expression is regulated, and how NIN regulates downstream gene
expression.
_______________________________________________________________________________________

6

Miki FUJITA (CSRS, Gene Discovery Research Group)“Development of “RIPPS”, an automated system for evaluating plant
environmental stress response”

Plant health and fitness are greatly impacted by the rhizospheric microbiomes, as their interactions are
co-adapted and co-evolved across time and space. Despite considerable evidence in the previous reports that
beneficial or detrimental rhizospheric microbiomes can alter in plant growth, morphology, and mineralcontents, the mechanism in which the reciprocal interactions affect plant health and fitness is largely
unknown. To understand the effect of rhizospheric microbes on plant growth, development, and stressresponses, phenotyping systems with strict regulation of environmental conditions and precise quantificationof plant growth are expected to be useful. Recently we have developed such automatic phenotyping systemthat enables us to evaluate plant growth responses to a wide spectrum of environmental conditions. The
system named RIPPS (RIKEN Plant Phenotyping System) can control individual soil moisture incontinuously rotating 120 pots by a combination of automatic weighing and watering systems. RIPPS also
can monitor each rosette size and expansion rate every two hours. We are also developing imaging
techniques using various cameras to analyze plant water status. In this seminar, we will demonstrate theutility of the RIPPS in evaluating drought or salinity tolerance and water use efficiency.
_______________________________________________________________________________________
Masahira HATTORI (IMS, Laboratory for Microbiome)“Long-read metagenomics of human gut microbiomes”

Our team is working on metagenomics of human and other microbiomes to elucidate their ecological andbiological features related to host’s physiologies via microbe-host interaction.
Metagenomics using large datasets of short-read (~300 bp) sequencing is the most popular approach toevaluate the human gut microbiome. However, short-read sequencing has several limitations inreconstruction of the whole microbial genomes including mobile genetic elements (MGEs) such asplasmids/phages and epigenetic base modifications. We applied single-molecule real-time (SMRT)
7

sequencing by PacBio to metagenomics of human gut microbiomes. We successfully obtained datasets withthe average read-length of >9 kb by sequencing DNA templates prepared by the enzymatic lysis method,which was much longer than 2.7 kb previously published. The computational analysis identified manynear-complete microbial genomes and complete circular sequences of putative MGEs, which included
hitherto unknown species and MGEs. The comparison between the reconstructed and known genomes
revealed large differences mostly due to indels of large genomic islands. We also found that some of the
MGEs were highly prevalent among human gut microbiomes from different nations. Furthermore, the
SMRT sequencing identified methylation motifs, many of which were novel. Thus, this study provides novel
views of the human gut microbiome structure.
_______________________________________________________________________________________
Hiroshi OHNO (IMS, Laboratory for Intestinal Ecosystem)“Characteristics of M cells and follicle-associated epithelium”

Huge numbers of commensal bacteria inhabit our gut.

However, we do not host this gut commensal

microbiota unconditionally; rather we try to contain them by secreting mucus, antimicrobial molecules, andIgA.

To evoke intestinal immune responses to produce IgA against commensal microbes, thegut-associated lymphoid tissue (GALT) needs antigenic microbial supply across the intestinal epithelial
barrier. This microbial delivery across the epithelium is done by M cells, a subset of intestinal epithelial cells
specialized for microbial uptake and delivery to the GALT.

M cells exist scattered in the area of epithelium

covering the GALT lymphoid follicles, called follicle-associated epithelium (FAE).

We have identified thetranscription factor Spi-B as a master regulator of M-cell differentiation, and bacterial uptake receptors on M
cells, including GP2 and prion protein.

Recently, we have also found that IL-22BP, an endogenous

inhibitor of the cytokine IL-22 signaling, confers the characteristic of FAE that enables intestinal microbes
easy access to the epithelial layer and hence aids the their uptake by M cells.

8Masayuki AMAGAI (IMS, Skin homeostasis)“Stratum corneum as niche for skin microbiome”

Enhanced cutaneous sensitization by skin barrier dysfunction has been extensively investigated as an initial
important step for the onset of allergic disorders. Staphylococcus aureus-dominant dysbiosis in the skinplays an important pathogenic role for development of atopic dermatitis (AD). To understand how thedysbiosis is induced on the surface of stratum corneum (SC), we have been characterizing the normal natureof SC as niche for skin microbiome.
SC consists of stacked dead corneocytes which are generated from terminally-differentiated keratinocytes instratum granulosum (SG). Visualization of SC by time-of-flight secondary-ion-mass-spectrometry(TOF-SIMS) demonstrated that SC has at least three layers of distinct functional properties; upper
sponge-like layer which allows passive influx and efflux of ions, middle hydration layer, and lower barrier
layer which is abrogated by filaggrin deficiency.
The pH of SC is considered to be acidic in general while its precise pH distribution and dynamic changes indifferent environments within SC layers remain to be clarified. Transgenic mice with a ratiometric pH
biosensor with pH sensitive fluorescent protein, VenusH148G, and pH insensitive protein, mCherry, weregenerated as a tool to visualize in vivo pH in SC and dissect a role of pH in SC homeostasis. Confocal
microscopic analysis of living pH imaging demonstrated that SC has at least two distinct middle-acidic andupper-neutral layers with cross section view, rather than gradual pH changes across the layers. Furthermore,
the upper-neutral layers showed mosaic patterns of uneven pH distribution by differentiated units with en
face view. These findings indicated the dynamic and complex nature of SC in terms of pH regulation.
Uncovering the physiological mechanisms of SC homeostasis will lead us to develop a novel body surface
tactics to conquer the dysbiosis of the skin.
_______________________________________________________________________________________

9

Kenya HONDA (IMS, Gut Homeostasis)“Modulation of the immune system by the gut microbiota”

The mammalian alimentary tract harbors hundreds of species of commensal microbes that critically
influence a multitude of host physiological functions, including the immune system. Manipulation of thegut microbiota holds great promise for the treatment of inflammatory and allergic diseases. However, to datethere are only a handful of examples of single species or defined communities of bacteria that can be used toactivate and polarize distinct subsets of immune cells. By combining anaerobic culturing and gnotobiotic
technique, together with the latest high-throughput sequencing technique, we have built a method
to understand the role of individual components of commensal microbes, particularly their immunologicalattributes.

Indeed, we

have

succeededinisolation

of

human

and

mouse

gut-associatedcommensal bacterial strains that specifically affect the development and function of Th17 cells, Treg cells,
and Th1 cells.
In this study, we have identified and isolatedg-expressing CD8+ T cells in the intestine. IFNg+ CD8+ T cells
were abundantly present in the intestine kept under specific-pathogen free conditions, whereas few
IFNg+ CD8+ T cells were found in germ-free (GF) mice. To investigate whether the human gut microbiota
was able to induce IFNg+ CD8+ T cells, GF mice were gavaged with stool samples collected fromsix healthy volunteers. IFNg+ CD8+ T cells were variably induced, and we selected a mouse that exhibitedthe highest frequency of IFNg+ CD8+ T cells. Cecal contents were collected from the selected mouse were
inoculated into another GF mice, and treated with antibiotics in the drinking water in order toconcentrate responsible microbes for IFNg+ CD8+ T cell induction. Ampicillin (Amp) treatment enhanced
induction of colonic IFNg+ CD8+ T cells. Cecal contents were collected from Amp-treated mice andcultured in vitro. We succeeded in isolation of 26 strains, and selected 11 strains, which were correlated withthe frequency of IFNg+ CD8+ T cells. The mixture of 11 strains sufficiently induced IFNg+ CD8+ T cells
when colonized in GF mice. Our findings can be potentially applied to treatment/prevention ofviral infection and cancer.
_______________________________________________________________________________________

10!

Sidonia FAGARASAN (IMS, Laboratory for Mucosal Immunity)“Host-gut microbiota mutualism by IgA-glycan”

Immunoglobulin A (IgA) is the most abundant antibody isotype and a major regulator of symbiotic bacteria
in the gut. We previously found that IgAs induced upon stimulation by symbiotic bacteria are coating
bacteria but such coating contributes to retention rather than elimination of the bacteria. However, themechanistic details of how IgAs add symbiosis remained largely unknown.

Increasing number ofexperimental data indicate that the generation of gut IgAs is regulated by complex mechanisms. For
example, while the majority of IgA are generated by reactions that are T cell-dependent, other IgAs can be
induced in a T cell-independent manner. In addition, IgAs can be produced under normal and inflammatory
conditions, yet whether they have different functional properties is not yet clear.

We hypothesize that

post-translational modification of secretory IgA might be regulated, at least in part, by glycan moiety of theantibody and such modifications may impact the symbiotic role of IgA. We are currently analyzing theimpact of IgA-glycan on the function and composition of the gut microbiota. Here, we will discuss ourrecent findings about the role of IgA-glycosylation that hopefully will contribute to understanding of
host-microbiota mutualism mediated by secretory IgAs in the gut.
_______________________________________________________________________________________
Hidewaki NAKAGAWA (IMS, Laboratory for Genome Sequencing Analysis)“Cancer genome sequencing for pathogen and symbiosis in human cancers”

Many types of cancers are asscoiated with virus and bactria infection. Hepatitis virus (HBV and HCV)
infection is strongly associated with liver cancer, HPV with cervical and head&neck cancer, and H.pylori
and EBV with gastric cancer. Some of virus infection can induce carcinogenesis through genomic alterationsin the host.
We here examined whole genome seauencing (WGS) and RNA-seq data of 300 human liver cancers (NatGenet 48:500-509, 2016), and found some pathogen and symbiosis in human liver (cancer) tissues in11

addition to HBV and HCV. To detect HBV virus and its integration, we aligned unmapped reads to 79
different HBV genome sequences, and inferred genotypes of the infected HBV in each sample. 74 samples
had ≥ 10 reads mapped to the HBV genomes. Of the 74 samples, nine patients were HBV negative by theirserological tests, suggesting their “occult” HBV infection. Our analysis identified 223 HBV integration
events into human genome. Furthermore, we performed ultra-deep sequencing targeting HBV in humanliver tissues and human-hepatocyte chimeric mice with HBV infection, and characterized 1,684 HBV
integration sites. To explore any potential of other pathogens and symbiosis, we performed de novo
assembly of unmapped reads of RNA-seq data from 244 liver cancer and non-cancer liver samples. In
addition to sequence contigs derived from HBV, HCV, and several bacteria, we detected some long contigs
aligned to adeno-associated virus (AAV) in two liver cancer samples, which were validated to be integrated
into human genome.
To make profiles of pathogen and symbiosis in human, we are now analyzing pathogens and symbiosis in
human tissues (liver, digest organs, and prostate) by sequencing DNAs/RNAs from these human tissues.

Toru TAKUMI (BSI, Laboratory for Mental Biology)“The gut-brain axis in autism”

Autism spectrum disorder (ASD) is a childhood neuropsychiatric disorder and one of the most famous
neurodevelopmental disorders. Besides major symptoms such as impaired social communication andrepetitive behavior, gut and sleep problems are frequent comorbidity. The genetic contribution of the diseasecauses in ASD is higher than that in other psychiatric diseases, however, the genetic mechanism is not
simple but complex as many other multi-factorial diseases and environmental causes are not excluded. On
the other hand, the gut microbiota is essential in human health and its disturbance may be involved invarious diseases. The gut is also known as a second brain and the gut-brain axis may also be involved indiffer disorders including ASD. We are interested in the gut-brain axis in ASD and studying how this axis isregulated and contributes to the pathogenesis by using different mouse models of ASD.
_______________________________________________________________________________________

12

Atsushi IRIKI (BSI, Laboratory for Symbolic Cognitive Development)“Primate model of “Mind-Body Interaction” through the mechanisms ofgut-brain axis via microbiome.”

States of human’s conscious mind has impacts on bodily states, such as social stress-induced weakening of
immune reactions and alteration in metabolic regulatory organ, but no animal models reflecting more highly
developed brain function for such mind-body interactions exists to the date. Marmoset Wasting Syndrome(MWS, lethal dysfunction of immune system suspected) is a common problem in captivity among colonies
across the world, with unknown mechanisms and thus no treatment established. Social and mental stress inthe captivity has been highly suspected, as no evidence of this symptom observed in the wild habitat.
Occasionally, our preliminary fecal microbiota transplantation (FMT) have in part attenuated the more sever
form of symptoms. Therefore, MWS could be an ideal primate model for studying human mind-bodyinteractions, with most likely implication of understanding microbiome mechanisms.
_______________________________________________________________________________________
Hiroyuki OSADA (CSRS, Chemical Biology Research Group)“Activation of biosynthesis of tenuazonic acid in a plant pathogenic fungus”

Plant pathogenic fungi produce a wide variety of small molecules which contain unique and complex
structures. However, most biosynthetic gene clusters are not working in the laboratory condition. Moreover,
the relationship between pathogenicity and the small molecules remains unclear. To activate the silent geneclusters in fungi, successful approaches such as epigenetic regulation, promoter exchange, and heterologous
expression have been reported in these days.
Tenuazonic acid (TeA) is a well-known mycotoxin produced by various plant pathogenic fungi, but its
biosynthetic gene has been unknown to date. Thus, we undertook this study to know the biosynthetic
mechanism of TeA in Pyricularia oryzae (Rice blast fungus). P. oryzae did not produce TeA under thelaboratory condition. However, the production of TeA was induced by the disruption of OSM1 gene which is
involved in the response to environmental signals. Interestingly, the addition of 1% DMSO also induced TeA
production in P. oryzae.

TeA is synthesized from isoleucine and acetoacetyl-coenzyme A by TeA
13

synthetase 1 (TAS1). TAS1 is a unique non-ribosomal peptide synthetase and polyketide synthase(NRPS-PKS) hybrid enzyme.
_______________________________________________________________________________________
Katsuyuki SHIROGUCHI (QBiC, Lab for Integrative Omics)“Toward "The third generation" bacterial microbiota analysis”

We are developing a novel high-throughput cell-number quantification method which counts the number ofbacteria at single-cell level based on single-base identification in 16S rRNA sequences.

Using this method,

we try to understand the cell-cell network, and to identify "master" bacteria which may affect thecell-number distribution in a bacteria community.
distribution using, e.g.,

Eventually, we would like to control the cell-number

the "master" bacteria.

_______________________________________________________________________________________
Hiroyoshi AOKI (Center for Advanced Photonics, Ultrahigh Precision Optics
Technology Team)“Agarose Gel Microcapsule: Pico-liter-scale DNA Amplification Microvessel forSingle Cell Genomics.”

Whole genome amplification (WGA) revealed a world of diverse and complex yet-uncultured
microorganisms (YUM) in symbiotic microbiomes. Single cell genomics of YUM provided us physiological
functions of the individual YUM. However, the single cell WGA had some technical problems: DNA
contamination and amplification bias in next generation sequencing (NGS). To overcome the technicalissues, we developed agarose gel microcapsule (AGM) for the single cell genomics, which was consisted ofan alginate-sol core and an agarose-gel shell. In a closed microtube, the AGM was easily prepared bygelating emulsion of the alginate gel–the agarose sol with cooling, and then the alginate-gel core was solatedwith EDTA. The agarose gel shell prevented the external DNA contamination whereas it allowed to diffuse
low molecular WGA reagents. In the AGM, the alginate core embedded single cell and worked as aseparable pL-scale reaction microvessel for WGA. Since the amplification bias was suppressed in quite low
reaction volume, the alginate core would be effective for prevention of the amplification bias.
14Thus, the AGM provided the pL-scale DNA amplification microvessel, which was able to isolated single cellfor NGS. The AGM will accelerate the microbiome network analysis by single YUM genomics.
_______________________________________________________________________________________
Akiko MINODA (CLST, Epigenome Technology Exploration Unit)“Studying the effect of different microbiota at the cell type and subpopulations
landscape level by single cell RNA-seq”

Different environments give different signals to the host that may result in epigenetic changes. Thesechanges in turn may result in different distribution of cell types or subpopulations. In this project, we areinterested in determining how the host plant is affected in the presence of different microbiota, for example
at the roots. To address this question, we will utilize single cell transcriptome technology, which is a
powerful tool in identifying cell types and subpopulations in tissues that are constituted from multiple cell
types. I will present our ongoing efforts on single cell analyses with mouse tissues, where we have been able
to identify novel subpopluations.
Lastly, we hope to carry out and integrate additional omics analyses such as ATAC-seq (open chromatin
regions) both at the single cell and bulk levels to further understand the effect of different microbiomes atthe chromatin level.
_______________________________________________________________________________________
Arnaud GERMOND (QBiC, Laboratory for Comprehensive Bioimaging)“Raman spectroscopy as a tool for cell-line discrimination and gene
expression monitoring”

Raman spectroscopy is a laser microscopy technique that characterizes thevibration of molecule, allowing to monitor the chemical fingerprint of a given biological entity in a fast,
non-labelling and non-destructive manner. Single a Raman spectrum has been proved to be a faithful
signature of the metabolic state of living cells (1-2), since it holds information about the major cellularcompounds (protein, RNA, DNA, cytochrome, aromatic compounds, pigments, …). Multivariate statisticalanalyses of Raman spectra allow to perform cell-line discrimination and classification with very low error
15

rates. In RIKEN, I built an automated Raman spectroscopy platform to discriminate cell lines (strains) of
human cells and microorganisms with a single-cell resolution. Previous studies showed the possibility tomonitor the effects of antibiotic drugs in term of exposure and concentration. No study, however, has tried to
identify strains according to their acquired resistance or to the mechanism of antibiotic resistance (ribosomal
RNA, cell-wall,…). My recent work demonstrates the ability of Raman spectroscopy to identify the
population of E. coli strains with acquired antibiotic resistance, and also to classify strains by the molecularmechanism of their respective antibiotic resistance. Three individual experiments were performed to show
the results are reproducible. Finally, in my most recent work, I built a predictive model to monitor the gene
expression based on the sole Raman spectral information. Major biological functions of E. coli populations
were successfully predicted. We envision that our approach will find a broad range of applications in
non-invasive biological and biomedical research, ranged from diagnosis to monitoring of gene expression at
single-cell level.
References
1. Visualizing the appearance and disappearance of the attractor of differentiation using Raman
spectral imaging. Taro Ichimura, Liang-da Chiu, Katsumasa Fujita, Hiroaki Machiyama, Satoshi
Kawata, Tomonobu M. Watanabe, Hideaki Fujita. Scientific Reports, doi:10.1038/srep11358 (Jun.
2015)
2. Visualizing Cell State Transition Using Raman Spectroscopy. Taro Ichimura, Liang-da Chiu,
Katsumasa Fujita, Satoshi Kawata, Tomonobu M. Watanabe, Toshio Yanagida, Hideaki Fujita. PLoS
One, 9(1), e84478 (Jan. 2014)_______________________________________________________________________________________

16

List of Speakers on August 22nd
Chikara FURUSAWA (QBiC, Laboratory for Multiscale Biosystem Dynamics)“Analysis of phenotypic evolution and symbiosis using microbial laboratory
evolution”

In the process of symbiotic formation, organisms change their phenotype adaptively each otherin a flexible and robust manner. Here, to understand the nature of robust adaptive evolution
and possible changes of phenotype-genotype mappings, we studied laboratory evolution of
microorganisms under various conditions. In the first project, we analyzed evolutionary
dynamics of E. coli under 96 different stress conditions including addition of acids, metals,
surfactants, antibiotics, and so on, and the changes of phenotypes and genotypes were
quantified. The results of these comprehensive analyses suggested that the expression changes
were restricted to a low-dimensional dynamics, while diverse genomic changes can contributeto similar phenotypic changes. In the second project, to understand the contribution of
expansion of genotype space to evolutionary dynamics, we performed laboratory evolution ofcells obtained by protoplast fusion. We obtained actively growing protoplast cells, called L-form
cells, from various microorganism species. Then, cells after protoplast fusion were cultured
under various stress conditions, to obtain stably fused cells. The preliminary results showedthat after laboratory evolution of fused cells, a mixture of ancestor phenotypes, i.e., acquisitionof resistances to two different stresses was observed, suggesting that intra-cellular symbiosis
contributed to the evolution. We expect that the analyses of evolutionary constraints andevolvability of microorganisms will contribute to capturing potentiality of symbiotic formation
among microorganisms.

17

Shunji TAKAHASHI (CSRS, Natural Product Biosynthesis Unit)“Unveiling Chemical Signal and Biosynthetic Mechanism of Reveromycin”

RM-A is a polyketide natural product isolated from Streptomyces. Microorganisms utilize a variety ofchemical signals. Streptomyces, a soil-dwelling gram-positive bacteria, utilize autoregulators to produce avariety of specialized metabolites (SMs). Chemical signals derived from extra-species/environmental stimuli
also induce SM production in Streptomyces species. Improved production of bioactive SMs has been along-standing goal. SM production by Actinomycetes has been explored by modifying the components of theculture medium. By testing various culture media, we found that reveromycin (RM) production by
Streptomyces reveromyceticus SN-593 was enhanced by adding tomato juice to the culture medium. Thisobservation led us to speculate that naturally existing extracellular chemicals can activate secondary
metabolism. Based on the screening a natural product library, we found that β-carboline chemical signallingenhanced RM production by activating the LuxR family transcriptional regulator in the RM gene cluster.
We also analysed biosynthetic mechanism of RM. The biosynthesis includes 2-alkylmalonyl-CoA formation,
polyketide truncation, spiroacetal formation, hydroxylation of spiroacetal structure, and subsequentsuccinylation. Among these, we focused on the mechanism of succinylation of tertiary alcohol in the last
step of biosynthesis. By chemical synthesis approach, the succinylation required high pressure (1.5 Gpa).
However, S. reveromyceticus SN-593 performs the reaction under normal culture condition. Therefore, it isof interest to understand the mechanism. We found that three enzyme (RevK, RevL, and RevM) wererequired to catalyse succinylation in the presence of ATP, fumaric acid, NADH, and RM-T1. To understandthe mode of reaction we performed crystal structure analysis.
_______________________________________________________________________________________
Ryo NAKABAYASHI (CSRS, Metabolomics Research Group)“Metabolome analysis in the parasitism between Solanum lycopersicum and Cuscuta campestris”
It is suggested that plants exert specialized metabolites (previously called secondary metabolites) to defend
against parasitic plants, but little is known so far. Here, metabolome analysis using liquid
chromatography-mass spectrometry was performed to understand specialized metabolites in tomato(Solanum lycopersicum) induced by Cuscuta campestris. The comparative analysis of the metabolome in
Cuscuta, tomatoes, and safflower showed that tomato-specific metabolites induced by Cuscuta.
Computational metabolome annotation found that dehydrophenolamides including kukoamine A are highly18induced by Cuscuta as well as phenolics and glycoalkaloids. The accumulation of the dehydrophenolamides
decreased in jai1 mutant, which lacks the jasmonic acid (JA) signaling, revealed that the biosynthesis of thedehydrophenolamides are under the JA signaling. A discriminate analysis between tomatoes that killed
Cucuta and ones which healthfully grew Cuscuta showed that the metabolites are associated with thedefense to Cuscuta.
_______________________________________________________________________________________
Shigehiro KURAKU (CLST, Phyloinformatics Unit)“Genome evolution and ecosystem of marine vertebrates”

Phyloinformatics Unit in RIKEN Kobe was initially launched as a DNA sequencing core facility of Center
for Developmental Biology (CDB) and later in 2014 fused into Center for Life Science Technologies(CLST). Its current activities range from original technical development for next-generation sequencing andgenome informatics to technical support for sequencing data production for whole RIKEN. Based on
experience of participating multiple international consortia for sequencing and analyzing vertebrate genomes,
it now autonomously conducts de novo genome sequencing and analysis of several early vertebrates
including elasmobranch sharks whose genome sizes exceed those of many other vertebrates. In particular,
our ongoing analyses focus on evolution of protein-coding gene repertoire responsible for embryonicdevelopment, endocrine control of homeostasis, and physiology. In the near future, the scope of the unit will
include monitoring ecosystems of marine organisms based on environmental DNA analysis andhost-parasite interaction in collaboration with local aquaria. This presentation will cover some of the unit’s
past achievements, overview of ongoing projects, and the near future perspectives.
_______________________________________________________________________________________

19

Ryuhei NAKAMURA (CSRS, Biofunctional Catalyst Research Team)“Energy Flow Organized by Benthic Ecosystems”

As represented by three key energy metabolisms in biology (i.e. photosynthesis, respiration, andfermentation), life can be regarded as the material organized by energy flow. Like individual organisms,
highly complicated ecosystems stand on the sophisticated mechanisms of energy flow generated by the
intricate chain of various metabolic interaction between bacteria and animals. If the modest biological andphysical disturbance to healthy ecosystems occurs, it may return to original status, as opposed to becoming
very different ecosystems. Such an ability is called energy homeostasis and is a key feature for ecosystem torecover original energy flow state from excess amount of energy input by human activity. Therefore,
understanding the mechanisms how ecosystems maintain energy flow is of fundament importance to keepecosystems healthy and enable sustainable harvest and production from the ecosystems.
To deepen our understanding of energy homeostasis, we have applied the knowledge and techniquedeveloped in the field of electromicrobiology to coastal benthic ecosystems. In this presentation, I will
introduce a novel electrochemical system to monitor, control, and optimize microbial metabolisms in thereconstituted benthic ecosystem and discuss how benthos and microbes cooperatively maintain energy
homeostasis.
_______________________________________________________________________________________Jun KIKUCHI (CSRS, Environmental Metabolic Analysis Research Team)“Marine and fishery product studies assisted by metabolic profiling with data
science”

Data-driven approaches were applied to investigate the temporal and spatial changes of natural fishes andenvironmental waters in Japan(1-6). To do this, NMR-based metabolic profiling, ICP-OES-based ionomic
profiling, as well as NGS-based microbial profiling were employed to obtain the “big-data” from natural
ecosystems (7,8). I will also introduce the first achievement of collaborative research with Fishery Research
Agency, for the trans-omics approach to characterize fish nutritional biorhythm in Leopard coral groupers
(9).

20

References
1) Yoshida et al. (2014) Sci. Rep. 4, 7005
2) Asakura et al. (2014) PeerJ 2, e556 (2014).
3) Misawa et al. (2016) Anal. Chem. 88, 6130-6134.
4) Asakura et al. (submitted).
5) Wei et al. (submitted).
6) Uchimiya et al. (submitted).
7) Kikuchi et al. (invited) Prog. NMR Spect.
8) Kikuchi & Yamada (invited) Analyst
9) Mekuchi et al. (in press) Sci. Rep.
_______________________________________________________________________________________
Shigeharu MORIYA (CSRS, Biomass research platform team)“RNA-seq approaching to unlock the ecological functions of natural
microbiome system”

The current expansion of next-generation high-throughput sequencing has promoted various applications forthe biological sciences, one of which is microbial ecology analysis. Because most natural microbialcommunity members are uncultivable, such a meta-genomic and/or meta-transcriptomic analysis is one of
the few ways to access information regarding the community structures of environmental microbialcommunities and the genetic activities in these communities.
Here, we developed a quantitative analysis pipeline for both microbial community structure and gene
expression. Mapping-based analysis is designed with ordinary transcriptome analysis tools, a public small
subunit RNA gene database and self-assembled contigs as a structural gene reference database. This new
pipeline, All RNA Information sequencing (ARIseq), can be applied to any environmental samples.
We performed this analysis on samples from an acidic mine drainage biofilm ecosystem and confirmed
estimates of its quantity by quantitative PCR. In addition to identifying an iron oxidation bacterial-based
community that has been reported previously, the results of this analysis suggest that target ecosystems
include a specific diatom community that may have an important role in this environment.
Also, we will report about progress and future plan of research about termite symbiotic system by using ofmeta- or single cell- transcriptome analysis.
_______________________________________________________________________________________

21

Yuichi HONGO/Hirokazu KUWAHARA (Tokyo Technology Institute, BRC
Microbe Division)“Functional genomics of multiple endo- and ectosymbionts of termite gut protists”

Termites harbor symbiotic protists and prokaryotes in their hindgut, which are unique to termites. Certainprokaryotic species are specifically associated with the protist cells: this multilayer symbiosis is a prominent
feature of the termite gut ecosystem. We have been investigating the functions of such unculturable endo- or
ectosymbiotic prokaryotes of the cellulolytic gut protists by analyzing their genome sequences. We will
present the newest results from the genome analysis of cytoplasmic, nucleoplasmic, and surface-embedded
bacterial symbionts of the Trichonympha protists in the termite gut.
_______________________________________________________________________________________
Akiko FUJIWARA (CSRS, Chemical Genomics Research Group)“Elucidation of molecular mechanisms for multiple endosymbiotic systems anddevelopment of novel control technologies by the inhibition of endosymbiotic
machinery in Bemisia tabaci, the agricultural pest”

Bemisia tabaci is a pest insect on agricultural crops worldwide. They are highly resistant to major
insecticides. Therefore, novel pest control methods that are effective and environmental-friendly are needed.
B. tabaci harbors endosymbiotic bacteria. The symbionts are critical to survival and reproduction of host,because they supply host with essential nutrients. The aim of this study is to elucidate the molecularmechanisms and develop the novel control technologies by targeting endosymbiotic machinery. In ourprevious studies, several candidate genes related to the symbiotic systems were discovered. We’ve analyzed
for localization and function of symbiotic factors in preparatory for screening for inhibitors of symbiotic
mechanism by chemical biology high throughput methods. In this workshop, a summary of present research
is presented.
_______________________________________________________________________________________

22

Seok-Won KIM (IMS, Laboratory for Integrated Bioinformatics)“Analysis of the onset mechanism of obesity and type 2 diabetes
correlated with gut microbiota using data mining”

Recently, many studies have reported that disorders in the gastrointestinal tract are not only
associated with intestinal diseases such as colitis or colon cancer but also connected to obesity
or metabolic diseases like type 2 diabetes and arteriosclerosis, and connected to systemic
diseases such as allergic rhinitis and autoimmune diseases. Therefore, it is considered that
understanding and proper controlling intestinal environment changes related to obesity or adisease such as type 2 diabetes can lead to a better prevention, diagnosis, and development oftherapy. We collect clinical information such as lifestyle, gut microbiota, metabolic functions,and genetic polymorphisms from patients' medical checkup, and data from animal
experiments using disease model mice. These data are integrated and analyzed transversely toreveal the correlation between obesity or type 2 diabetes and shape of gut microbiota. From
understanding the relations between onset mechanism of lifestyle diseases and gut microbiotachange, our goal is to establish methods to prevent the diseases. This initial study allows us todevelop the platform for analysis and to perform a proof of concept with model cases of type 2
diabetes. Later, it is expected to apply the same workflow to other diseases related to an
imbalance of gut microbiota.
_______________________________________________________________________________________Moriya OHKUMA (BRC, Japan Collection of Microorganisms)“Single-cell genome analysis of termite gut symbionts”

Functional roles of individual microbial symbionts and their interactions among members of symbiotic
microbial communities are important for understanding the symbioses. Unfortunately, most symbionts are
formidably difficult to culture and usually comprise a complex community, which resist standard
microbiological and molecular biological studies. We are investigating termite-gut microbial symbionts
23particularly through single-cell genome analyses. There are several species-specific associations between gut
cellulolytic protists and bacteria belonging to phyla Spirochaetes, Bacteroidetes, and others. The
associations involve both intracellular endosymbiosis and cell-surface attached ectosymbioses. The
associated bacteria comprise a majority of the gut community and play roles in the host nutrition. We also
investigate the gut microbial community of a termite species that typically lack gut cellulolytic protists andthus bacteria are responsible for lignocellulose digestion. In addition to studies on mechanisms for efficient
digestion and metabolisms of the symbionts, comparative studies of various symbionts are also beneficial tounderstand their coevolution.
_______________________________________________________________________________________

24