Mobilomics of toxin production in cyanobacteria

mobilomics

Trichomes of the bloom-forming cyanobacteria Planktothrix rubescens and Planktothrix agardhii

Logo FWFProjektleitung/project management: Doz. Dr. Rainer Kurmayer
Research Institute for Limnology, Mondsee
of the University of Innsbruck
Dauer/project period: from March 2012 to Feb 2016
Projektfinanzierung/project funded by:
Austrian Science Fund (FWF), P24070


Summary

Harmful algal blooms (HABs) formed by freshwater cyanobacteria have been repeatedly associated with the toxification of humans and livestock. Up to date only one genome from toxin-producing cyanobacteria has been completely elucidated. Efforts to form one contiguous sequence representing the whole genome have been hampered by the high amount of repetitive elements such as mobile transposable elements (TEs). Previous research has revealed that the genetic basis of secondary metabolite production is frequently influenced by TEs, however, only the quantitative comparison of complete genomes can reveal the influence of TEs on the interrelation between secondary metabolite production and ongoing speciation processes.

We have totally sequenced the genome of the bloom-forming and toxic cyanobacterium Planktothrix agardhii, which will serve as the reference genome. In this project we will sequence eight additional clonal isolates (strains) of Planktothrix spp. representing eight different phylogenetic lineages comprising 140 strains which have been characterized genetically by genotyping (using Multi Locus Sequence Typing) as well as for secondary metabolite production by chemical analytical techniques. The strains have been collected from 40 waterbodies located in 17 countries on three different continents (Europe, North America, Africa) and evolved both in the presence and in the absence of toxins.


Kurzfassung

Massenaufkommen von toxischen Algen in Gewässern haben in der Vergangenheit zu Vergiftungen von Tieren und Menschen geführt. Durch die Entschlüsselung des Erbguts dieser toxischen Blütenbildner versucht man, wichtige ökologische Anpassungen in Erfahrung zu bringen, um durch spezielle Maßnahmen diese Algen an ihrer Ausbreitung zu hindern. Die Entschlüsselung der Genome dieser Organismen wird durch sich wiederholende DNA (repetitive DNA) Sequenzen behindert, die von sogenannten mobilen Elementen (ME) stammen, die definitionsgemäß ihre Position im Genom spontan verändern. Der Anteil dieser ME am gesamten Genom ist beträchtlich (1-11%). Es wird vermutet, dass diese ME zur physiologischen Plastizität eines bestimmten Organismus durch Restrukturierung des Genoms wesentlich beitragen, jedoch können lediglich in ihrer Gesamtheit assemblierte Genome bezüglich dieses Einflusses analysiert werden.

In den letzten Jahren ist es uns gelungen, das Genom des blütenbildenden und toxischen Cyanobakteriums Planktothrix agardhii zu assemblieren und die vorhandenen ME systematisch zu erfassen. Mithilfe dieses Referenzgenoms sollen in diesem Projekt acht weitere Genome von Isolaten derselben Art jedoch unterschiedlicher ökologischer Einnischung sequenziert und assembliert werden. Diese Vertreter wurden aus einem Stammbaum mit insgesamt 140 Isolaten ausgewählt und repräsentieren je eine über ein bis drei Kontinente (Eurasien, Nordamerika, Afrika) verbreitete Verwandschaftslinie, die entweder toxische oder harmlose Genotypen enthält.


Objectives

During the project we aim to find out

  1. the TE induced variation and innovation in secondary metabolite production in specific Planktothrix lineages;

  2. secondary metabolite synthesis genes which have not been affected by TE inactivation and which are therefore of potential selective importance;

  3. the activity and specificity of frequent TEs through in vitro experiments;

  4. the relationship between the frequency of active TEs and the secondary metabolite synthesis recombination potential in populations in the wild.


Methods

While the total genome assembly will reveal the full potential of present TEs, the comparative analysis of the 140 strains will show, which TEs and which TE induced mutations are specific to individual lineages. The activity and the specificity of TEs will be probed in vivo and in vitro. Since the TE sequences are known, the TE insertion sites can be elucidated by sequencing the flanking regions directly from field populations. This cultivation independent approach would elucidate TE induced mutations in toxin production at an early stage.

mobilomicsmobilomics

In lakes, recombination processes as initiated by transposable elements will be investigated during algal bloom formation.


Innovative aspects

This project is the first to study speciation within a well defined HAB genus distributed over a wide geographical scale in relation to the occurrence of TEs in cyanobacteria. Since this project will reveal the entire set of secondary metabolite gene clusters of different lineages it will go far beyond monocausal gene vs. environment studies and will further elucidate the relationship between toxigenicity and other ecophysiological characters. Thus it will be elucidated which toxin-producing ecotypes are most likely to profit from the predicted scenario of a global increase in HAB occurrence.


Cooperation partners

  • Dr. Judith Blom
    University of Zürich, Institute of Plant Biology, Limnological Station, Kilchberg, Switzerland

  • Prof. Dr. Karl Gademann
    University of Basel, Department of Chemistry, Basel, Switzerland

  • Dr. Alexander Goesmann
    University of Bielefeld, Centrum für Biotechnologie – CeBiTec, Bielefeld, Germany

  • Dr. Dan Kramer
    CyanoBiotech GmbH, Berlin, Germany


Project News

October 2016

Presentation of project results at the 10th International Conference of Toxic cyanobacteria in Wuhan, China, 23-27 October:

Entfellner, E, Frei, M, Christiansen, G, Deng, L, Blom, J, Kurmayer, R. (2016) Evolution of anabaenopeptin results in phylogenetic fixation of a meta peptide synthesis gene cluster in cyanobacteria. 27 October (Presentation).

and

Invited lecture at Nanjing Institute of Geography and Limnology, Chinese Academy of Sciences, Nanjiing, China: Kurmayer, R. (28.10.2016) The genetic basis of cyanoHABs worldwide, recent insights.

 

August 2016

Rainer Kurmayer became Associate Editor for the very successful Open Access Journal “Frontiers in Microbiology, Section Aquatic Microbiology” http://journal.frontiersin.org/journal/microbiology/section/aquatic-microbiology .

 

June 2016

Mutagenization workshop on the toxigenic cyanobacterium Planktothrix,  13 – 17 June 2016 with ten participants (Czech Republic: 1, France: 4, Greece: 2, Hungaria: 1, Poland: 2). The transformation technique that is used for experimental Planktothrix mutagenesis has been disseminated by a so-called “mutagenisation workshop” which has been organized already in July 2011 but now repeated since it has attracted scientists and PhD students from Europe and overseas.

Mutagenization course Mondsee 2016 

group picture of the Mutagenization course in Mondsee 2016, photo: ILIM

February 2016

New Publication in BMC Microbiology:

Chen, Q, Christiansen, G, Deng, L, Kurmayer, R (in press) Emergence of nontoxic mutants as revealed by single filament analysis in bloom-forming cyanobacteria of the genus Planktothrix. BMC Microbiology

Popular scientific contribution:

Rainer Kurmayer : Rolle von toxischen Peptiden für die Bildung von Algenblüten. bioskop, 1. Februar 2016.

http://www.austrianbiologist.at/bioskop/2016/02/rolle-von-toxischen-peptiden-fuer-die-bildung-von-algenblueten/

January 2016

New Publication in Harmful Algae:

Kurmayer, R., Deng, L., Entfellner, E. (in press) Role of toxic and bioactive secondary metabolites in colonization and bloom formation by filamentous cyanobacteria Planktothrix. Harmful Algae

Start of Doc-fellowship funded by the Austrian Academy of Sciences (ÖAW):

Elisabeth Entfellner: Toward an Understanding of Secondary Metabolic Diversity Among Algal Bloom-forming Cyanobacteria (Jan 2016 - Jan 2019), Link: http://www.uibk.ac.at/limno/personnel/elisabeth-holzweber/

November 2015

Seminar talk at the EAWAG aquatic research, in Dübendorf (Switzerland), 12. November 2015

Kurmayer, R. Evolutionary Ecology of harmful cyanobacteria.

August 2015

Conference presentation at the 15th International Symposium on Phototrophic Prokaryotes, Tübingen, 06.08.2015, http://www.ispp15.org/program/program-august-6-2015/

Kurmayer, R., Holzweber, E.; Frei, M.; Christiansen, C.: Microevolution of bioactive peptide gene clusters in the bloom-forming cyanobacterium Planktothrix spp.

April, 2015

Global Solutions to Regional Problems, 13-14 April 2015

Collecting Global Expertise to Address the Problem of Harmful Algal Blooms Workshop at Bowling Green State University. http://www.bgsu.edu/research/lake-erie-and-watershed-studies.html

Invited talk R. Kurmayer: CHAB occurrences worldwide – recent insights.

Jan 2015

Pavla Urbankova from Charles university in Prague starts her Erasmus+ traineeship fellowship on Cyanobacteria single filament genetics (Jan-Apr 2015). Single filaments of cyanobacteria Planktothrix will be analyzed for the composition of associated heterotrophic bacteria. Good luck with the work!

Oct 2014

New Publication in ISME Journal:

Kurmayer R, Blom JF, Deng L, Pernthaler J (2014). Integrating phylogeny, geographic niche partitioning and secondary metabolite synthesis in bloom-forming Planktothrix. ISME J. 9: 909-921, doi:10.1038/ismej.2014.189.

http://www.nature.com/ismej/journal/vaop/ncurrent/full/ismej2014189a.html

Aug 2014

New publication in Harmful Algae:

Kohler E, Grundler V, Häussinger D, Kurmayer R, Gademann K, Pernthaler J et al (2014). The toxicity and enzyme activity of a chlorine and sulfate containing aeruginosin isolated from a non-microcystin-producing Planktothrix strain. Harmful Algae 39: 154-160. doi:10.1016/j.hal.2014.07.003

http://www.sciencedirect.com/science/article/pii/S1568988314001292

June 2014

Nadine Münzberg and Eva Szekeres (HBLA Ursprung-Elixhausen, Salzburg) absolve a 6-weeks practical course analyzing single filaments of the cyanobacterium Planktothrix and associated bacteria isolated from Lake Mondsee.

May 2014

New publication in Applied and Environmental Microbiology: Christiansen G, Goesmann A, Kurmayer R (2014). Elucidation of insertion elements encoded on plasmids and in vitro construction of shuttle vectors from the toxic cyanobacterium Planktothrix. Appl Environ Microbiol. doi: 10.1128/AEM.01188-14

http://aem.asm.org/content/early/2014/06/03/AEM.01188-14.abstract

Feb 2014

New Publication in Natural Products and Bioprospecting: Niedermeyer TJ, Schmieder P, Kurmayer R (2014). Isolation of Microcystins from the Cyanobacterium Planktothrix rubescens Strain No80. Nat Prod Bioprospect: 1-9.

http://link.springer.com/article/10.1007%2Fs13659-013-0001-3

Jan 2014

Veronika Ostermaier defended her PhD thesis at the Univ. of Vienna: “Environmental regulation of microcystin genotype abundance and the resulting net production of the toxic heptapeptide microcystin among bloom-forming cyanobacteria”, 166 pp. Thank you for your great job and best wishes!

Elisabeth Holzweber starts her PhD thesis “Analysis of Genetic Recombination Phenomena in Cyanotoxin Gene Clusters as Induced by Mobile Transposable Elements” at the University of Innsbruck. Welcome on board!

Nov 2013

New publication in PLOS One: Ostermaier V, Christiansen G, Schanz F, Kurmayer R (2013). Genetic Variability of Microcystin Biosynthesis Genes in Planktothrix as Elucidated from Samples Preserved by Heat Desiccation during Three Decades. PLoS ONE 8: e80177.

http://www.plosone.org/article/info%3Adoi%2F10.1371%2Fjournal.pone.0080177

Sep 2013

PhD candidate Qin Chen (Northwest A&F University, Yangling, Shaanxi Province) successfully finished her research stay (23 months) after characterizing ongoing mutations within the microcystin synthesis gene cluster from 800 individually isolated filaments of the cyanobacterium Planktothrix spp. (Publication in prep.)

Aug 2013

Mark Frei submitted his MSc thesis “Verbreitung und Rekombination von Genen zur Synthese von Toxinen und Peptiden in Cyanobakterien” at the University of Vienna, 120pp.

June 2013

Satellite Event - Cyanobacteria and Cyanotoxins in Aquatic Environments, 25 June 2013. Organized in collaboration with the American Chemical Society and CYANOCOST Action ES 1105. http://www.icce2013.org/satellite_cyan.html

Invited talk R. Kurmayer: “Molecular ecology and evolution of toxin-producing cyanobacteria in drinking and recreational water.”

April 2013

New publication in Applied Environmental Microbiology:  Rohrlack, T.*, Christiansen, G., Kurmayer. R. (2013) Putative Antiparasite Defensive System Involving Ribosomal and Nonribosomal Oligopeptides in Cyanobacteria of the Genus Planktothrix. http://aem.asm.org/content/79/8/2642.long

*Corresponding author

December 2012

New publication in BMC Biology: Ostermaier, V., Schanz, F., Köster, O., Kurmayer, R.* (2012) Stability of toxin gene proportion in red-pigmented populations of the cyanobacterium Planktothrix during 29 years of re-oligotrophication of Lake Zürich. (http://www.biomedcentral.com/1741-7007/10/100/abstract )

*Corresponding author

August 2012
Katharina Moosbrugger (FH Joanneum) started her MTA position in the project. Welcome on board!

May 2012
We are currently announcing a MTA/BMA position for 24 months

Kick off meeting of the COST Action ES1105 entitled “Cyanobacterial blooms and toxins in water resources: Occurrence, impacts and management” involving 23 European partners. http://www.cost.eu/domains_actions/essem/Actions/ES1105

March 2012
Cand. MSc. Mark Frei (University of Vienna) started his diploma thesis entitled
"Genetic variation and recombination processes affecting the synthesis of bioactive peptides in the cyanobacterium Planktothrix", Welcome on board!


Contact and Links

Dr. Rainer Kurmayer
Project leader
rainer.kurmayer@uibk.ac.at

Dr. Sabine Wanzenböck
Public Relations
sabine.wanzenboeck@uibk.ac.at

Research Institute for Limnology, Mondsee
of the University of Innsbruck
Herzog-Odilostrasse 101
5310 Mondsee
AUSTRIA