Overview

TEAM @ Stony Brook University
Lead: Prof. Jackie L. Collier, School of Marine and Atmospheric Sciences; jackie.collier@stonybrook.edu
Co-Leads:
Prof. Joshua Rest, Department of Ecology & Evolution; joshua.rest@stonybrook.edu
Prof. Gábor Balázsi, Department of Biomedical Engineering
Students:
Mariana Rius, School of Marine and Atmospheric Sciences
Previous Participants:
Prof. Daiske Honda, Department of Biology, Konan University, Kobe, Japan; dhonda@konan-u.ac.jp
Prof. Thorsten Reusch, Helmholtz Centre for Ocean Research Kiel (GEOMAR), Kiel, Germany; treusch@geomar.de
Kylen Bao and Sarah Kelso, Simons Summer Research Fellows

THE PROJECT
For this project, we are developing genetic engineering tools in labyrinthulomycetes.
Following are links to data and results for the main goals of this project.
1) to assess a range of antibiotics as selectable markers and fluorescent proteins (FPs) as reporters,
2) to develop gene expression constructs
composed of regulatory elements (promoters and terminators) that function broadly across labyrinthulomycetes,
…and to evaluate how stringently codon biases must be matched to permit strong expression of encoded proteins,
3) to identify the most efficient and widely applicable method(s) for transforming DNA into a diverse array of labyrinthulomycetes, and
4) to support future work by taking initial steps in developing the CRISPR/Cas9 system as a standard tool for both forward and reverse genetic work in labyrinthulomycetes.
Leveraging the fact that transformation has been achieved for a few thraustochytrids of biotechnological interest, we have focused our efforts on developing transformation tools and protocols across a wide taxonomic breadth of labyrinthulomycetes, focusing initially on three representatives each of the labyrinthulids, aplanochytrids, and oblongichytrids.

Figure 1. Neighbor-joining phylogenetic tree constructed from 18S rDNA sequences of cultivated labyrinthulomycetes. Numbers before nodes indicate bootstrap support (%) in 500 replicate trees. Abbreviations ‘Th’ and ‘Thraust’ indicate organisms named ‘Thraustochytrium’ and illustrate taxonomic issues that remain to be resolved for this polyphyletic genus name.

ORGANISMS
The labyrinthulomycetes are a diverse group of marine heterokont protists with fungus-like lifestyles. They have been found in every marine habitat examined, and some are associated with devastating diseases of marine metazoans and metaphytes including bivalves and eelgrass [1,2]. There are representatives of four distinct labyrinthulomycete subgroups (labyrinthulids, aplanochytrids, oblongichytrids, and thraustochytrids; Figures 1 and 2) in cultivation, and environmental sequences indicate that additional distinct groups remain to be cultivated [3,4]. The thraustochytrids are frequently identified at the level of class (Thraustochytriaceae), and are clearly monophyletic, while the other three groups are usually placed in a second class (Labyrinthulaceae) although the monophyly of this grouping is less certain.
Recent efforts to resolve inconsistencies [5] in the classification of thraustochytrids have recognized numerous genera in which isolates share ~98% or greater 18S rRNA sequence identity [6,7], and there is typically <80% 18S rRNA sequence identity between thraustochytrid genera. Different labyrinthulid isolates, so far all assigned to the single genus Labyrinthula, can be less than 85% identical. 18S rRNA sequences within both the aplanochytrids (right now the single genus Aplanochytrium) and oblongichytrids (right now the single genus Oblongichytrium) generally have >95% identity, with <90% identity between the two groups. The recently sequenced genomes of three labyrinthulomycetes (work done in collaboration with the Joint Genome Institute) reveal extensive structural and gene content differences between thraustochytrids and aplanochytrids (Collier and others, work in progress).

Figure 2. Closer view of the Labyrinthulaceae subtree from Figure 1. Strains ‘GSBS’ and ‘PBS’ are isolates growing in the Collier lab from around Long Island, NY, USA.

Cultivation-dependent work has suggested differences in substrate and habitat preferences among major groups of labyrinthulomycetes, particularly labyrinthulids vs thraustochytrids, and cultivation-independent 18S rDNA sequence-based approaches now make it possible to investigate differences in the ecology of previously unresolved genera and species. While thraustochytrids have historically been a major focus, evidence is accumulating that aplanochytrids and oblongichytrids may be more abundant in most marine habitats (e.g., [3,4,8-11]). It appears likely that the 18S sequence diversity of labyrinthulomycetes reflects diversity in their ecology; for example, different 18S sequence types of Labyrinthula (likely different species) show distinct distributions [12] and there also appear to be ecological differences among aplanochytrids and oblongichytrids ([13]; unpublished data of Collier and Honda groups).
The ubiquity of labyrinthulomycetes means that they could be used to gain insights into ocean biogeochemistry and ecology everywhere from the sunlit surface to deep (oxic and anoxic) zones of the water column, and from littoral to deep ocean sediments. Importantly, their lifestyle offers a complement to that of diatoms, so having genetic tools for both would enable genetically-empowered investigations of marine food webs and elemental cycles from formation to remineralization of organic matter. That vision would be brought closer to reality by developing the ability to genetically manipulate a wide variety of labyrinthulomycetes. Progress has already been made in the genetic manipulation of some thraustochytrids (see below). Thus, we will prioritize our efforts by focusing initially on representatives of Labyrinthula, Aplanochytrium, and Oblongichytrium. As time and other resources allow, we will broaden our efforts to more strains in these three groups, and extend our study into the diverse thraustochytrid genera that have not yet been transformed, including as a first target the organism known as ‘QPX’, cause of QPX disease in the hard clam (quahog) Mercenaria mercenaria [14,15].

Our group has previously conducted work on the ecology and genomics of labyrinthulomycetes [3,6,7,11,12,14,15,17,19,20] and in transforming eukaryotic microbes [23].

REFERENCES

  1. Raghukumar S (2002) Ecology of the marine protists, the Labyrinthulomycetes (Thraustochytrids and Labyrinthulids). European Journal of Protistology 38: 127-145.
  2. Raghukumar S, Damare VS (2011) Increasing evidence for the important role of Labyrinthulomycetes in marine ecosystems. Botanica Marina 54: 3-11.
  3. Collado Mercado E, Radway JC, Collier JL (2010) Novel uncultivated labyrinthulomycetes revealed by 18S rDNA sequences from seawater and sediment samples. Aquatic Microbial Ecology 58: 215-228.
  4. Li Q, Wang X, Liu X, Jiao N, Wang G (2013) Abundance and novel lineages of thraustochytrids in Hawaiian waters. Microbial Ecology 66: 823-830.
  5. Chamberlain AHL, Moss ST (1988) The thraustochytrids: a protist group with mixed affinities. BioSystems 21: 341-349.
  6. Yokoyama R, Honda D (2007) Taxonomic rearrangement of the genus Schizochytrium sensu lato based on morphology, chemotaxonomic characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Schizochytrium and erection of Aurantiochytrium and Oblongichytrium gen. nov. Mycoscience 48: 199-211.
  7. Yokoyama R, Salleh B, Honda D (2007) Taxonomic rearrangement of the genus Ulkenia sensu lato based on morphology, chemotaxonomical characteristics, and 18S rRNA gene phylogeny (Thraustochytriaceae, Labyrinthulomycetes): emendation for Ulkenia and erection of Botryochytrium, Parietichytrium, and Sicyoidochytrium gen. nov. Mycoscience 48: 329-341.
  8. Damare V, Raghukumar S (2006) Morphology and physiology of the marine straminipilan fungi, the aplanochytrids isolated from the equatorial Indian Ocean. Indian Journal of Marine Sciences 35: 326-340.
  9. Damare VS, Damare S, Ramanujam P, Meena RM, Raghukumar S (2013) Preliminary studies on the association between zooplankton and the stramenopilan fungi, aplanochytrids. Microbial Ecology 65: 955-963.
  10. Nakai R, Nakamura K, Jadoon WA, Kashihara K, Naganuma T (2013) Genus-specific quantitative PCR of thraustochytrid protists. Marine Ecology Progress Series 486: 1-12.
  11. Ueda M, Nomura Y, Doi K, Nakajima M, Honda D (2015) Seasonal dynamics of culturable thraustochytrids (Labyrinthulomycetes, Stramenopiles) in estuarine and coastal waters. Aquatic Microbial Ecology 74: 187–204.
  12. Bockelmann A-C, Beining K, Reusch TBH (2012) Widespread occurrence of endophytic Labyrinthula spp. in northern European eelgrass Zostera marina beds. Marine Ecology Progress Series 445: 109-116.
  13. Sidik SM, Hackett CG, Tran F, Westwood NJ, Lourido S (2014) Efficient genome engineering of Toxoplasma gondii using CRISPR/Cas9. PLoS ONE 9: e100450.
  14. Liu Q, Allam B, Collier JL (2009) Quantitative real-time PCR assay for QPX (Thraustochytriidae), a parasite of the hard clam (Mercenaria mercenaria). Applied and Environmental Microbiology 75: 4913-4918.
  15. Qian H, Liu Q, Allam B, Collier JL (2007) Molecular genetic variation within and among isolates of QPX (Thraustochytridae), a parasite of the hard clam Mercenaria mercenaria. Diseases of Aquatic Organisms 77: 159-168.
  16. Raghukumar S (2008) Thraustochytrid marine protists: production of PUFAs and other emerging technologies. Marine Biotechnology 10: 631-640.
  17. Abe E, Ikeda K, Nutahara E, Hayashi M, Yamashita A, et al. (2014) Novel lysophospholipid acyltransferase PLAT1 of Aurantiochytrium limacinum F26-b responsible for generation of palmitate-docosahexaenoate-phosphatidylcholine and phosphatidylethanolamine. PLoS ONE 9: e102377.
  18. Lippmeier JC, Crawford KS, Owen CB, Rivas AA, Metz JG, et al. (2009) Characterization of both polyunsaturated fatty acid biosynthetic pathways in Schizochytrium sp. Lipids 44: 621-630.
  19. Matsuda T, Sakaguchi K, Hamaguchi R, Kobayashi T, Abe E, et al. (2012) Analysis of D-12-fatty acid desaturase function revealed that two distinct pathways are active for the synthesis of PUFAs in T. aureum ATCC 34304. Journal of Lipid Research 53: 1210–1222.
  20. Sakaguchi K, Matsuda T, Kobayashi T, Ohara J-i, Hamaguchi R, et al. (2012) Versatile transformation system that is applicable to both multiple transgene expression and gene targeting for thraustochytrids. Applied and Environmental Microbiology 78: 3193-3202.
  21. Chamberlain AHL (1980) Cytochemical and ultrastructural studies on the cell walls of Thraustochytrium spp. Botanica Marina 23: 669-677.
  1. Hsu PD, Lander ES, Zhang F (2014) Development and applications of CRISPR-Cas9 for genome engineering. Cell 157: 1262-1278.
  2. Rest, JS, Morales CM, Waldron JB, Opulente DA, Fisher J, et al. (2013) Nonlinear fitness consequences of variation in expression level of a eukaryotic gene. Molecular Biology and Evolution 30: 448-456.