Genome Sequence and Transcriptome Analyses of Chrysochromulina tobin: Metabolic Tools for Enhanced Algal Fitness in the Prominent Order Prymnesiales (Haptophyceae)

Abstract

Haptophytes are recognized as seminal players in aquatic ecosystem function. These algae are important in global carbon sequestration, form destructive harmful blooms, and given their rich fatty acid content, serve as a highly nutritive food source to a broad range of eco-cohorts. Haptophyte dominance in both fresh and marine waters is supported by the mixotrophic nature of many taxa. Despite their importance the nuclear genome sequence of only one haptophyte, Emiliania huxleyi (Isochrysidales), is available. Here we report the draft genome sequence of Chrysochromulina tobin (Prymnesiales), and transcriptome data collected at seven time points over a 24-hour light/dark cycle. The nuclear genome of C. tobin is small (59 Mb), compact (∼ 40% of the genome is protein coding) and encodes approximately 16,777 genes. Genes important to fatty acid synthesis, modification, and catabolism show distinct patterns of expression when monitored over the circadian photoperiod. The C. tobin genome harbors the first hybrid polyketide synthase/non-ribosomal peptide synthase gene complex reported for an algal species, and encodes potential anti-microbial peptides and proteins involved in multidrug and toxic compound extrusion. A new haptophyte xanthorhodopsin was also identified, together with two "red" RuBisCO activases that are shared across many algal lineages. The Chrysochromulina tobin genome sequence provides new information on the evolutionary history, ecology and economic importance of haptophytes.

Fig 1. Chrysochromulina tobin cell structure.

(A) Scanning electron micrograph of C. tobin. Two flagella are visible (marked F) along with the prominent coiled haptonema (white arrow). Scale bar represents 2.5 microns. (B) Electron micrograph of whole cell: Lipid body (LB); Mitochondrion (M); Chloroplast (C). Scale bar represents 500 nanometers.

Fig 2. Chrysochromulina tobin displays photoperiod controlled cell division and lipid metabolism.

(A) Cell division is observed to be highest during the light to dark transition. (B) Change in lipid body size is correlated to photoperiod when detected by BODIPY 505/515 dye incorporation (green); chloroplast auto-fluorescence (red).

Fig 3. Heatmap of highly expressed genes over 7 time points during the 12 hour light: 12 hour dark photoperiod.

Groups represent clusters of genes with similar expression patterns. A total of 1000 genes were included in this heatmap analysis. Color bar represents change in RNA abundance relative to average abundance of the 7 time point samples collected over 24 hours. Highest abundance in red and lowest abundance in blue.

Fig 4. Gene expression heatmap group 1, 2 and 7 GO term overrepresentation.

Black bars represent the percentage of genes with the associated GO term (X-axis) within groups 1, 2 and 7. Gray bars represent the percentage of all annotated genes in the C. tobin genome that have the corresponding GO term. For complete list of overrepresented GO terms in each group, see S2 Dataset.

Fig 5. Cell growth, lipid synthesis and lipase activity in a Chrysochromulina tobin culture maintained on a 12 hr light: 12 hr dark photoperiod.

Cell count (green; n = 3); neutral lipid quantity measured by BODIPY 505/515 dye incorporation (red; n = 2); lipase/esterase activity measured using fluorescein diacetate (blue; n = 3).

Fig 6. Identification of fatty acid synthesis genes of Chrysochromulina tobin and corresponding RNA transcript FPKM over a 12 hr light: 12 hr dark photoperiod.

The genes identified as fatty acid synthesis genes and the corresponding chloroplast and endoplasmic reticulum pathway schematic (from [37]).

Fig 7. Fatty acid content across haptophyte (blue text) and cryptophyte (green text) algal species.

Data collected using GC/MS analysis [41] of total fatty acids. Circle graph wedges represent the proportion of individual fatty acid types. The Bayesian species tree is inferred from an 857 bp alignment of psbA nucleotide sequence (S4 Dataset), with posterior probability and maximum likelihood bootstrap support shown for each node.

Fig 8. Type I Polyketide synthase and PKS-NRPS hybrid domains found in the C. tobin genome.

These gene clusters each represent one of three polyketide synthesis domains found on a single assembled contig of over 50,000 bp in length. Domains 1 and 2 are separated by a single stop codon whereas domain 3 is separated by a stop codon and is frame shifted. Brackets indicate individual PKS and NRPS modules. Modules contain the following components: adenylation domain (A); AMP binding domain (AMP); Acyl carrier proteins (ACP); acyltransferase (AT); condensation domain (CD); dehydratase (DH); enoyl reductase (ER); crotonase/Enoyl-Coenzyme A (CoA) hydratase family (HT); ketosynthase (KS) methyltransferase (MT); stop codon (S); HxxPF repeat domain (X).

Fig 9. Xanthorhodopsin structural modeling.

(A) Comparative model of C. tobin xanthorhodopsin (in color, blue as N-terminus and red as C-terminus) overlayed on the structural template (PDB code 3DDL, grey). A docked retinal molecule (magenta) and conjugated retinal (grey) in structural template are also shown. (B) Enlarged view of the ligand pocket in the aligned structures. Docked retinal molecule (magenta) and its first shell amino acids (in colors) are shown along with conjugated retinal (grey) and its first shell amino acids (in grey) confirm existence of a retinal compatible pocket in C. tobin xanthorhodopsin. Labels are based on C. tobin xanthorhodopsin sequence (bold characters) and 3DDL structural template (italics and in parentheses).

Fig 10. Phylogenetic placement of haptophyte, dinoflagellate, and cryptophyte xanthorhodopsins.

Bayesian phylogeny inferred from a 231 amino acid alignment of rhodopsins, with posterior probabilities and maximum-likelihood bootstrap support values shown at key nodes. Clades of xanthorhodopsins, novel cryptophyte-specific rhodopsins, sensory type I, II, and III rhodopsins, bacteriorhodopsins, xenorhodopsins, halorhodopsins and the proteorhodopsin outgroup are indicated.

Fig 11. Phylogeny of chloroplast and nuclear CbbX proteins from rhodophytes, cryptophytes, stramenopiles, and haptophytes in a background of cyanobacterial and proteobacterial CbbX proteins.

Bayesian phylogeny inferred from 257 amino acids, with posterior probabilities and maximum likelihood bootstrap support shown at key nodes. Alignments in S6 Dataset.