Comparative genome analysis in social amoebas

Lead Research Organisation: University of Dundee
Department Name: College of Life Sciences

Abstract

Biologists try to understand how complex multicellular organisms have evolved from simple single-celled ancestors. We know in theory how this happened: spontaneous mutations in the genes of earlier organisms caused small changes in the developmental program of their off-spring. This sometimes resulted in an improved adult that more successfully reproduced, and therefore gradually replaced the earlier form. However, to really understand this process and prove that it actually occurred, we have to trace back which genes were mutated and how this mutation changed gene function and consequently the developmental program. Because it is difficult to obtain such detailed information for complex organisms like ourselves, we investigate this problem in the social amoebas. Social amoebas feed as single cells on bacteria in forest soil. However, when starving, they come together and form a fruiting structure, in which a proportion of cells is preserved as spores. The other cells are sacrificed to form a stalk that aids in spore dispersal. This life style depends on mutual collaboration and specialization of cells. In the course of evolution the social amoebae have progressed from basal species that form structures with 10-100 cells and only two cell-types, to advanced species that form structures with over 100.000 cells and up to five cell types. One advanced species, Dictyostelium discoideum, is used widely as a model system to understand how cells move, feed and propagate and how they communicate with each other to achieve multicellularity. The D.discoideum genome has been completely sequenced, which means that we have a complete inventory of all the genes that control these processes. D.discoideum uses cyclic AMP (cAMP) as the major signal molecule for cell-cell communication. It acts as a chemoattractant to bring starving cells together, and then continues to guide cells to build a fruiting body. cAMP also induces the differentiation of the spores and regulates the process of spore germination. In previous BBSRC-funded research we constructed a family tree of the social amoebas, which shows that they are subdivided into four major groups. D.discoideum belongs to the most evolved group 4. From species in all four groups, we obtained fragments of the genes that are necessary for cAMP signalling by gene amplification. This suggests that many roles of cAMP are conserved. However, between groups, we observed changes in the stage of development at which these genes are active. One such as change gave rise to the use of cAMP as chemoattractant in the group 4 species. Gene amplification can only be used for very deeply conserved genes and only provides information on small regions of DNA. For many reasons it would be much better to compare species evolution at the level of the entire genome. With this project we therefore propose to sequence the genome of Polysphondylium pallidum to completion. This work will be performed in collaboration with a German team, who already obtained funding for draft sequencing of the P.pallidum genome. P.pallidum is particularly suitable for evolutionary studies because it occupies a basal position in the family tree and it is one of the few Dictyostelids that is readily accessible for gene manipulation. The complete P.pallidum genome sequence will give us the complete inventory and sequences of all cAMP signalling genes, and very importantly, will also tell us which genes are missing. By identifying gene losses and gains, and by comparing genes that are conserved between D.discoideum and P.pallidum, we can detect the genetic changes that occurred in the course of evolution. The completed P.pallidum genome will also be of great benefit for the Dictyostelium and broader research community. For instance, it can be used to identify conserved regions in proteins with important roles, that are thus far not well characterized.

Technical Summary

Social amoebae display conditional multicellularity with a broad range of forms. One species, D.discoideum, is widely used to investigate a range of cell- and developmental questions. Its genome is completely sequenced by an international consortium. We use the social amoebas to study the origin of phenotypic diversity across species and started with the construction of their molecular phylogeny. This tree shows subdivision into four major groups: D.discoideum is placed in the most derived group 4, while the root of the tree lies between groups 1 and 2. Cyclic AMP is a major regulator of D.discoideum development, and we used a PCR-based approach to identify cAMP signalling genes in species that span the phylogeny. This work showed that the use of extracellular cAMP as chemoattractant in group 4 species is derived from a role in coordinating fruiting body formation in basal species. Proximal-to-distal addition of novel promoters appeared to provide existing genes with novel roles in Dictyostelid evolution. The PCR approach has many obvious limitations, which would be overcome if representative genome sequences were available. The Dictyostelium Sequencing Consortium aim to sequence at least one genome from each of the 4 groups. The US team started draft sequencing of another group 4 species. The German team obtained funding for draft sequencing of the D.fasciculatum (group 1) and P.pallidum (group 2). The latter species is important because of its basal position and genetic tractability. The draft sequences provide a preliminary gene inventory. However, our research requires complete information about taxon specific gene gains- and losses, as well as complete promoter sequences. I therefore request funding for sequencing the P.pallidum genome to completion. The information will be used to study the evolution of developmental signalling and to test the hypothesis that elaboration of promoters is a major mechanism for generation of phenotypic novelty.
 
Description We are interested in changes at the genome level that caused the evolution of multicellular complexity. This project was part of a larger initiative for comparative genome analysis in the Dictyostelid social amoeba, organisms which still live at the boundary of uni- and multicellularity
The goal of the research project was to sequence and assemble the genome of one Dictyostelid, Polysphondylium
pallidum, to completion.
We sequenced this genome to 15-fold coverage together with our collaborator Gernot Gloeckner in Jena, Germany and
finished the assembly to a very high standard (only 52 gaps left). We electronically assigned gene models and performed
extensive manual gene model annotation and bioinformatic analysis of conservation of genes and gene families. The
genome sequence has been deposited in the public domain (Genbank). We also contributed to the sequencing of the Dictyostelum fasciculatum genome in Dr Gloeckner laboratory, by providing cultures, genomic DNA, cultures and a developmental time course of messenger RNA for RNA sequencing and we performed extensive annotation and bioinformatics on the D.fasciculatum genome
In addition we have also completely sequenced and assembled the genome of Dictyostelium lacteum. Gene annotation and bioinformatic analysis of this genome is still in
progress
Together with the previously completed genome of the model species Dictyostelium discoideum, we have now obtained
complete genome sequences from a representative species from each of the four major groups of Dictyostelia.
Exploitation Route Comparative genomics is crucial to understanding how genes evolved and assumed novel roles. The sequence genomes are a crucial resources for evolutionary biologist who aim to understand the evolution of multicellular complexity. They are also an important resource for cell biologist, who can use them to assign core proteins in the processes of interest.
In addition, the Dictyostelids genomes showed large and highly varied families of polyketide synthesases, enzymes which produce a large array of secondary metabolites. Secondary metabolites are often used as antibiotic or biocides in defence strategies of soil organisms. The dictyostelid polyketide synthases can potentially be exploited by pharmaceutical industries for synthesis of bioactive compounds
Sectors Chemicals,Healthcare,Pharmaceuticals and Medical Biotechnology

URL http://genomes.dictybase.org/pallidum
 
Description Three Dictyostelid genomes were sequenced, assembled and finished to a very high standard. These genomes are now being exploited by the research community to resolve questions in cell and developmental biology. The genomes are widely used in our own laboratory to identify genes involved in encystation and to understand the genetic changes that enabled the evolution of multicellularity. In addition we identified a deeply conserved gene DgcA inthe genomes, that synthesized c-di-GMP, that proved to be the long-elusive morphogen for positional induction of Dictyostelium stalk cell differentiation. My team has meanwhile gained significant expertise in genome annotation and as a result we have collaborated on several other genome sequencing projects
First Year Of Impact 2011
Sector Pharmaceuticals and Medical Biotechnology
Impact Types Societal

 
Description Project grant
Amount £184,308 (GBP)
Funding ID RPG-2012-746 
Organisation The Leverhulme Trust 
Sector Academic/University
Country United Kingdom
Start 09/2013 
End 09/2016
 
Description Senior Investigator Award
Amount £1,428,543 (GBP)
Funding ID 100293/Z/12/Z 
Organisation Wellcome Trust 
Sector Charity/Non Profit
Country United Kingdom
Start 01/2013 
End 01/2018
 
Title Completely sequenced and assembled genomes 
Description Completely sequenced and assembled genomes for the species Dictyostelium fasciculatum, Polysphondylium pallidum and Dictyostelium lacteum that with the earlier sequenced Dictyostelium discoideum represent all major groups of Dictyostelia 
Type Of Material Improvements to research infrastructure 
Year Produced 2011 
Provided To Others? Yes  
Impact The availabilty of the genomes allows researchers to identify conservation and evolutionary change in any genes of interest in their research 
URL http://sacgb.fli-leibniz.de/cgi/index.pl
 
Description Comparative genome sequencing of Dictyostelia 
Organisation Friedrich Schiller University Jena (FSU)
Country Germany 
Sector Academic/University 
PI Contribution Comparative genome sequencing of three Dictyostelid species. Genome sequencing, fosmid mapping and gene annotation of three Dictyostelium genomes Continued with comparative transcriptome analysis and sequencing of Protostelium genomes
Collaborator Contribution The Jena team performed most of the genome and transcriptome sequencing, sequence assembly and gene model prediction. The Jena and Cologne also contributed to protein annotation.
Impact The sequences of two genomes have been published doi:10.1101/gr.121137.111. The publication of sequence of the third Dictyostelium genome was incorporated into a broader evolutionary analysis of multicellularity genes (doi: 10.1038/ncomms12085). The comparative transcriptomic analysis was also published (DOI: 10.1186/s12864-016-3223-z) and the data were also used for improved gene model annotation (doi: 10.1186/s12864-017-3505-0) A manuscript for one Protostelium genome is in preparation. The available genome sequences form a cornerstone of our evolutionary work and are also widely used by the Dictyostelium community.
Start Year 2013
 
Description Comparative genome sequencing of Dictyostelia 
Organisation University of Cologne
Country Germany 
Sector Academic/University 
PI Contribution Comparative genome sequencing of three Dictyostelid species. Genome sequencing, fosmid mapping and gene annotation of three Dictyostelium genomes Continued with comparative transcriptome analysis and sequencing of Protostelium genomes
Collaborator Contribution The Jena team performed most of the genome and transcriptome sequencing, sequence assembly and gene model prediction. The Jena and Cologne also contributed to protein annotation.
Impact The sequences of two genomes have been published doi:10.1101/gr.121137.111. The publication of sequence of the third Dictyostelium genome was incorporated into a broader evolutionary analysis of multicellularity genes (doi: 10.1038/ncomms12085). The comparative transcriptomic analysis was also published (DOI: 10.1186/s12864-016-3223-z) and the data were also used for improved gene model annotation (doi: 10.1186/s12864-017-3505-0) A manuscript for one Protostelium genome is in preparation. The available genome sequences form a cornerstone of our evolutionary work and are also widely used by the Dictyostelium community.
Start Year 2013
 
Description Sequencing and annotation of the Acanthamoeba castellani genome 
Organisation University College Dublin
Country Ireland 
Sector Academic/University 
PI Contribution My team contributed the annotation of cyclic nucleotide signalling genes to the Acanthamoeba genome project
Collaborator Contribution Sequencing, assembly and gene model prediction and annotation of the genome of Acanthamoeba castellani, an opportunistic pathogen
Impact A manuscript was published in 2013. doi:10.1186/gb-2013-14-2-r11. Acanthamoeba is an opportunistic pathogen and the availability of the genome will strongly support understanding of its pathogenesis and identification of drug targets
Start Year 2011
 
Description Sequencing and annotation of the Dictyostelium purpureum genome 
Organisation Baylor College of Medicine
Country United States 
Sector Hospitals 
PI Contribution Annotation of cyclic nucleotide signalling genes in the Dictyostelium purpureum genome
Collaborator Contribution Sequencing, assemly, gene model prediction and protein annotion
Impact The genome sequence was published in 2011. doi:10.1186/gb-2011-12-2-r20
Start Year 2009
 
Description Sequencing and annotation of the Physarum polycephalum genome 
Organisation The Otto-von-Guericke University Magdeburg
Country Germany 
Sector Academic/University 
PI Contribution Annotation of signal transduction genes in genome of Physarum polycephalum
Collaborator Contribution Sequencing assembly and gene model prediction in the Physarum genome
Impact no outcomes yet
Start Year 2013
 
Description Sequencing and annotation of the Physarum polycephalum genome 
Organisation U.S. Department of Energy
Country United States 
Sector Public 
PI Contribution Annotation of signal transduction genes in genome of Physarum polycephalum
Collaborator Contribution Sequencing assembly and gene model prediction in the Physarum genome
Impact no outcomes yet
Start Year 2013
 
Description RSE Masterclass 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact High school pupils participated with enthousiasm and obtained a certificate for successfully completing experiments

The Masterclasses continue to be highly popular with schools in the regions
Year(s) Of Engagement Activity Pre-2006,2006,2007,2008,
URL http://www.royalsoced.org.uk/cms//files/youngpeople/sciencemasterclass/DundeeAutum14flyer.pdf
 
Description Sharing science 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact The exhibit received a lot of visitors, mostly parents and their children, who performed experiments with great enthousiasm

No notable impact
Year(s) Of Engagement Activity 2007