Microbiology and Biochemistry of Secondary Metabolites Unit

Assistant Professor Dr. Holger Jenke-Kodama

Abstract

This research unit works on bacterial model organisms, toxin-producing dinoflagellates and microbial communities associated to marine invertebrates. The common theme behind all projects is to gain a better understanding of the biological functions of secondary metabolites. Marine toxins belonging to the palytoxin family, i. e. palytxoin itself and its numerous structural congeners, have been a focus area of our research since 2012. Apart from the marine environment, which is for a research unit being located in Okinawa a natural choice of subject, we have also become interested in the microbial aspects of soil biology. In fiscal year 2013 (April 2013 - March 2014), we continued various projects, most of them being now in the stage of mansucript preparation.  We continued our collaborative projects with the University of the Ryukyus to become better integrated in the Okinawan research community. The staff of the unit changed considerably due to a relatively high fluctuation at the end of FY 2012 and the beginning of FY 2013. We presented some research results at international conferences.

1. Staff

• Dr. Holger Jenke-Kodama, Assistant Professor
• Dr. Tomoko Yamazaki, Group Leader/Lab Manager (from January 2013)
• Dr. Hiroshi Izumi, Researcher (from March 2013)
• Dr. Sung-Yin Yang, Researcher (from April 2013)
• Yosuke Taira, MSc, Technician
• Akiko Sato, MSc, Technician (from March 2013)
• Kevin Wakeman, BSc, Technician (from April 2013)
• Dr. Satoe Aratake, Research Assistant (from April 2013)
• Ryoko Uchida, Research Administrator

2. Collaborations

• Theme: Botryococcene biosynthesis in the green microalga Botryococcus braunii
  • Type of collaboration: Joint research
  • Researchers:
    • Dr. Shigeru Okada, Associate Professor, Graduate School of Agriculture and Life Sciences, The University of Tokyo
• Theme: Microbial communities in Palythoa tuberculosa and palytoxin production
  • Type of collaboration: Joint research
  • Researchers:
    • Dr. James D. Reimer, Associate Professor, Department of Chemistry, Biology and Marine Science, University of the Ryukyus
• Theme: Effects of the Kaichu-Doro causeway on biodiversity
  • Type of collaboration: Joint research
  • Researchers:
    • Dr. James D. Reimer, Associate Professor, Department of Chemistry, Biology and Marine Science, University of the Ryukyus

 

3. Activities and Findings

3.1 Palytoxin and related compounds

Palytoxin (PTX) was first isolated from the marine invertebrate Palythoa toxica in 1971. Since then PTX has been captivating both chemists and biologists because of its complex structure, extraordinary toxicity and unusual molecular mechanisms. Compared to other secondary metabolites PTX has a huge molecular mass of 2680 and has the second longest contiguous carbon chain ever reported for a natural product. Altough its discovery was reported more than 40 years ago, it has remained unclear what organism produces the compound. Several pieces of evidence hint to bacteria being the producers, but over the last ten years scientist have found more and more evidence for the hypothesis that dinoflagellates, a type of mostly marine alage, might be th real producers. Nowadays there is a whole family of similar structures known, which is called the palytoxin type of compounds. It includes the original PTX and numerous structurally very similar natural products, so-called congeners, for instance 42-hydroxy-palytoxin (42-OH-PTX), ostreocin and various ovatoxins.

After completion of our survey in Kabira Bay (Ishigaki island, Okinawa, Japan), as described in the annual report of FY2012, we established a new sampling site for Palythoa tuberculosa, the dominant Palythoa species found in the Okinawa area. This new sampling site is in Okinawa-hontou, i. e. the main island of Okinawa prefecture, where OIST is located. The new sampling site has provided much better sampling conditions, shorter ways to the laboratory and thus a clear improvement in possibilities for analysis and number of samples that can be processed at the same time. In addition, we started to sample Palythoa tuberculosa colonies also at different locations around the main island of Okinawa for a comparative analysis.

Having sampled and chemically analysed more than one hundred Palythoa colonies, we made two main observations. Firstly, the average PTX content showed considerable differences between sampling sites. Especially interesting is that there seems to be a geographical pattern in the distribution of PTX, which could mean that the PTX producer has higher population densities under certain environmental conditions or, alternatively, that the production rate of PTX depends strongly on certain environmental factor. Secondly, in all samples that have been analysed so far, we found only the original PTX and its structural congener 42-OH-PTX but never even traces of the other very similar compounds mentioned above. This means that we have to take into account the possibility that certain compounds of the PTX structural family are produced by bacteria, wheras other members of that family are produced by dinoflagellates. The next step in this research activity will be to analyse composition of dinoflagellate populations found in close proximity to Palythoa colonies in order to identify possible producers and try to correlate their densities with the PTX contents of the colonies. This will be done by classical microscopic techniques, amplicon high-throughput sequencing and quantitative PCR targeting the known toxic dinoflagellate species.

This project is conducted in close collaboration with Dr. James D. Reimer (Associate Professor, The University of the Ryukyus).

 

3.2 Microbial communities of the marine invertebrate Palythoa tuberculosa

Related to the project described in section 3.1, samples colonies of the marine invertebrate Palythoa tuberculosa are also analysed with regard to the microbial communities associated with their gastric cavaties. Palythoa have a simple body plan with polyps that have tentacles and one mouth opening that leads to a single central cavity, the so-called gastric cavity. Since one of hour hypotheses is that the organism producing PTX enters from outside or lives for longer periods of times inside the animals, the first entry point to be considered is the gastric cavity. The concept is to investigate the microbial community structure inside the cavity by means of high-throughput amplicon sequencing, which targets not only bacteria but also eukaryotes, fungi and archae. The community composition data obtained can then be correlated to the PTX contents found in the colonies.

One major result is that PTX seems to have a considerable effect on the microbial community composition, which in several cases can be called dramatic. At the moment we do not know any mechanisms behind this effect. It could be a direct activity of PTX or a secondary effect, for example a kind of stress reaction of the animal. Further studies will help clarify the underlying mechanism.

 

 

3.2 Biochemodiversity studies in soil environments

Compared to aquatic environments, soil and sediment are highly complex environments for microbial organisms. The main reasons are that population densities are in average much higher and microorganisms have to live in very close contact to each other. Due to these phenomena the genomes of bacteria living in soil are typically larger than their aquatic counperparts. Furthermore, soil bacteria are well known producers of secondary metabolites, many of which have been used in the development of medicaments, for example antibiotics, antifungal substances and cytotoxic compounds for cancer treatment. One group of soil bacteria that has been used for many decades as a rich source of pharmaceutically intersting compounds are the Streptomycetes.

Last fiscal year we reported the completion of the first phase of the Kaichu-Doro project (see annual reports for FY2012 and FY2011). In a follow-up study we have been using the sediment material gained by drilling corse at several sampling points along the Kaichu-Doro for a more detailed molecular analysis. The aim of this project is to correlate the structure of microbial communities (including bacteria, archaea, fungi and microbial eukaryotes) with the biochemodiversity. The term biochemodiversity describes the diversity of compounds in a certain area, which is produced by living organsims as primary or secondary metabolites. We have initiated the development of a new analysis method based on extraction procedures and screening the extracts by means of liquid chromatography coupled with mass spectrometry (LC-MS). This method aims at automatic identification of characteristic peaks and pek groups in the mass spectrogramm. This information can then be used for a statistical analysis of identified patterns, which finally results in a kind of "fingerprint" for each sample. Such fingerprints will be useful for correlation analysis with microbial community structures. At the moment, the development of those analysis tools is ongoing. We expect first results for FY 2014.

 

4. Publications

4.1 Journals

Uchida, H., Y. Taira, T. Yasumoto (2013) Structural elucidation of palytoxin analogs produced by the dinoflagellate Ostreopsis ovata IK2 strain by complementray use of positive and negative ion liquid chromatography/quadrupole time-of-flight mass spectrometry. Rap Comm Mass Spec 27: 1999-2008.

Sarai, C., A. Yamaguchi, H. Kawami, K. Matsuoka (2013) Two new species formally attributed to Protoperidinium oblongum (Aurivillius) Park et Dodge (Peridiniales, Dinophyceae) in coastal areas of Japan. Palaeobot Palynol 192: 103-118.

Nishimura, T.. S. Sato, W. Tawong, H. Sakanari, K. Uehara, M. M. Rahman Shah, S. Suda, T. Yasumoto, Y. Taira, H. Yamaguchi and M. Adachi (2013). Genetic diversity and distribution of the ciguatera-causing dinoflagellate Gambierdiscus spp. (dinophyceae) in coastal areas of Japan. PLoS One 8(4): e60882.

Mertens, K. N., A. Yamaguchi, Y. Takano, V. Pospelova, M. J. Head, T. Radi, A.J. Pienkowski, A. de Vernal, H. Kawami, K. Matsuoka (2013) A new heterotrophic dinoflagellate from the North-eastern Pacific, Protoperidinium fukuyoi: Cyst-theca relationship, phylogeny, distribution and ecology. J Eukaryot Microbiol 60: 545-563.

Izumi, H., E. Sagulenko, R. I. Webb, J. A. Fuerst (2013) Isolation and diversity of planctomycetes from the sponge Niphates sp., seawater and sediment of Moreton Bay, Australia. Antonie van Leeuwenhoek 104: 533-546.

 

4.2 Books and Other One-Time Publications

Nothing to report.

4.3 Oral and Poster Presentations

Z. Kulberg, N. Satoh, H. Jenke-Kodama. Transcriptome analysis of an Okinawan sponge. Gordon Research Conference on Marine Molecular Ecology, 11th-16th August 2013, Hong Kong, People's Republic of China. [Poster presentation]

H. Jenke-Kodama, M. Tamura, Y. Taira, J. D. Reimer. Analysis of palytoxin production in Palythoa tuberculosa by an integrated metagenomics and transcriptomics approach, Ocean Science Meeting 2014, 23rd-28th February 2014, Honolulu, Hawaii, USA. [Oral presentation]

T. Yamazaki, Y. Taira, J. D. Reimer, H. Jenke-Kodama. Seasonility of palytoxin distribution patterns and its correlation with microbial communities, Ocean Science Meeting 2014, 23rd-28th February 2014, Honolulu, Hawaii, USA. [Poster presentation]

 

5. Intellectual Property Rights and Other Specific Achievements

Nothing to report.