Plastid Genome Evolution of Two Colony-Forming Benthic Ochrosphaera neapolitana Strains (Coccolithales, Haptophyta).

Coccolithophores are well-known haptophytes that produce small calcium carbonate coccoliths, which in turn contribute to carbon sequestration in the marine environment. Despite their important ecological role, only two of eleven haptophyte plastid genomes are from coccolithophores, and those two belong to the order Isochrysidales. Here, we report the plastid genomes of two strains of Ochrosphaera neapolitana (Coccolithales) from Spain (CCAC 3688 B) and the USA (A15,280). The newly constructed plastid genomes are the largest in size (116,906 bp and 113,686 bp, respectively) among all the available haptophyte plastid genomes, primarily due to the increased intergenic regions. These two plastid genomes possess a conventional quadripartite structure with a long single copy and short single copy separated by two inverted ribosomal repeats. These two plastid genomes share 110 core genes, six rRNAs, and 29 tRNAs, but CCAC 3688 B has an additional CDS (ycf55) and one tRNA (trnL-UAG). Two large insertions at the intergenic regions (2 kb insertion between ycf35 and ycf45; 0.5 kb insertion in the middle of trnM and trnY) were detected in the strain CCAC 3688 B. We found the genes of light-independent protochlorophyllide oxidoreductase (chlB, chlN, and chlL), which convert protochlorophyllide to chlorophyllide during chlorophyll biosynthesis, in the plastid genomes of O. neapolitana as well as in other benthic Isochrysidales and Coccolithales species, putatively suggesting an evolutionary adaptation to benthic habitats.

Independent evolution of the thioredoxin system in photosynthetic Paulinella species.

Redox regulation allows phytoplankton to monitor and stabilize metabolic pathways under changing conditions1. In plastids, the thioredoxin (TRX) system is linked to photosynthetic electron transport and fine tuning of metabolic pathways to fluctuating light levels. Expansion of the number of redox signal transmitters and their protein targets, as seen in plants, is believed to increase cell robustness2. In this study, we searched for genes related to redox regulation in the photosynthetic amoeba Paulinella micropora KR01 (hereafter, KR01). The genus Paulinella includes testate filose amoebae, in which a single clade acquired a photosynthetic organelle, the chromatophore, from an alpha-cyanobacterial donor3. This independent primary endosymbiosis occurred relatively recently (∼124 million years ago) when compared to Archaeplastida (>1 billion years ago), making photosynthetic Paulinella a valuable model for studying the early stages of primary endosymbiosis4. Our comparative analysis demonstrates that this lineage has evolved a TRX system similar to other algae, relying, however, on genes with diverse phylogenetic origins (including the endosymbiont, host, bacteria, and red algae). One TRX of eukaryotic provenance is targeted to the chromatophore, implicating host-endosymbiont coordination of redox regulation. A chromatophore-targeted glucose-6-phosphate dehydrogenase (G6PDH) of red algal origin suggests that Paulinella exploited the existing redox regulation system in Archaeplastida to foster integration. Our study elucidates the independent evolution of the TRX system in photosynthetic Paulinella, whose parts derive from the existing genetic toolkit in diverse organisms.

Evolutionary History of Mitochondrial Genomes in Discoba, Including the Extreme Halophile Pleurostomum flabellatum (Heterolobosea).

Data from Discoba (Heterolobosea, Euglenozoa, Tsukubamonadida, and Jakobida) are essential to understand the evolution of mitochondrial genomes (mitogenomes), because this clade includes the most primitive-looking mitogenomes known, as well some extremely divergent genome information systems. Heterolobosea encompasses more than 150 described species, many of them from extreme habitats, but only six heterolobosean mitogenomes have been fully sequenced to date. Here we complete the mitogenome of the heterolobosean Pleurostomum flabellatum, which is extremely halophilic and reportedly also lacks classical mitochondrial cristae, hinting at reduction or loss of respiratory function. The mitogenome of P. flabellatum maps as a 57,829-bp-long circular molecule, including 40 coding sequences (19 tRNA, two rRNA, and 19 orfs). The gene content and gene arrangement are similar to Naegleria gruberi and Naegleria fowleri, the closest relatives with sequenced mitogenomes. The P. flabellatum mitogenome contains genes that encode components of the electron transport chain similar to those of Naegleria mitogenomes. Homology searches against a draft nuclear genome showed that P. flabellatum has two homologs of the highly conserved Mic60 subunit of the MICOS complex, and likely lost Mic19 and Mic10. However, electron microscopy showed no cristae structures. We infer that P. flabellatum, which originates from high salinity (313‰) water where the dissolved oxygen concentration is low, possesses a mitochondrion capable of aerobic respiration, but with reduced development of cristae structure reflecting limited use of this aerobic capacity (e.g., microaerophily).

Amoeba Genome Reveals Dominant Host Contribution to Plastid Endosymbiosis.

Eukaryotic photosynthetic organelles, plastids, are the powerhouses of many aquatic and terrestrial ecosystems. The canonical plastid in algae and plants originated >1 Ga and therefore offers limited insights into the initial stages of organelle evolution. To address this issue, we focus here on the photosynthetic amoeba Paulinella micropora strain KR01 (hereafter, KR01) that underwent a more recent (∼124 Ma) primary endosymbiosis, resulting in a photosynthetic organelle termed the chromatophore. Analysis of genomic and transcriptomic data resulted in a high-quality draft assembly of size 707 Mb and 32,361 predicted gene models. A total of 291 chromatophore-targeted proteins were predicted in silico, 208 of which comprise the ancestral organelle proteome in photosynthetic Paulinella species with functions, among others, in nucleotide metabolism and oxidative stress response. Gene coexpression analysis identified networks containing known high light stress response genes as well as a variety of genes of unknown function ("dark" genes). We characterized diurnally rhythmic genes in this species and found that over 49% are dark. It was recently hypothesized that large double-stranded DNA viruses may have driven gene transfer to the nucleus in Paulinella and facilitated endosymbiosis. Our analyses do not support this idea, but rather suggest that these viruses in the KR01 and closely related P. micropora MYN1 genomes resulted from a more recent invasion.

Oceaniradius stylonematis gen. nov., sp. nov., isolated from a red alga, Stylonema cornu-cervi.

A Gram-stain-negative, strictly aerobic bacterium, designated StC1T, was isolated from a marine alga, Stylonema cornu-cervi, in the Republic of Korea. Cells were oxidase- and catalase-positive rods that were motile by a single lateral flagellum. Growth of strain StC1T was observed at 30-45 °C(optimum, 37 °C), pH 6.0-11.0 (pH 7.0) and in the presence of 1.0-8.0 % (w/v) NaCl (2 %). Strain StC1T contained summed feature 8 (comprising C18 : 1ω7c/C18 : 1 ω6c) and 11-methyl-C18 : 1ω7c as the major fatty acids. Sulfoquinovosyl diacylglycerol, phosphatidylethanolamine and phosphatidylglycerol and ubiquinone-10 were identified as the major polar lipids and the sole isoprenoid quinone, respectively. The G+C content of the genomic DNA was 64.7 mol%. Strain StC1T was most closely related to Oricola cellulosilytica CC-AMH-OT, Nitratireductor basaltis J3T, Aquamicrobiumahrensii 905/1T and Mesorhizobium tamadayense Ala-3T with 97.3 , 96.9 , 96.8  and 96.6 % 16S rRNA gene sequence similarities, respectively, but it formed a distinct phylogenic lineage within the family Phyllobacteriaceae. On the basis of phenotypic, chemotaxonomic and molecular properties, strain StC1T represents a novel genus of the family Phyllobacteriaceae, for which the name Oceaniradius stylonematis gen. nov., sp. nov. is proposed. The type strain is StC1T (=KACC 19231T=JCM 32050T).

Evolutionary dynamics of the chromatophore genome in three photosynthetic Paulinella species.

The thecate amoeba Paulinella is a valuable model for understanding plastid organellogenesis because this lineage has independently gained plastids (termed chromatophores) of alpha-cyanobacterial provenance. Plastid primary endosymbiosis in Paulinella occurred relatively recently (90-140 million years ago, Mya), whereas the origin of the canonical Archaeplastida plastid occurred >1,500 Mya. Therefore, these two events provide independent perspectives on plastid formation on vastly different timescales. Here we generated the complete chromatophore genome sequence from P. longichromatophora (979,356 bp, GC-content = 38.8%, 915 predicted genes) and P. micropora NZ27 (977,190 bp, GC-content = 39.9%, 911 predicted genes) and compared these data to that from existing chromatophore genomes. Our analysis suggests that when a basal split occurred among photosynthetic Paulinella species ca. 60 Mya, only 35% of the ancestral orthologous gene families from the cyanobacterial endosymbiont remained in chromatophore DNA. Following major gene losses during the early stages of endosymbiosis, this process slowed down significantly, resulting in a conserved gene content across extant taxa. Chromatophore genes faced relaxed selection when compared to homologs in free-living alpha-cyanobacteria, likely reflecting the homogeneous intracellular environment of the Paulinella host. Comparison of nucleotide substitution and insertion/deletion events among different P. micropora strains demonstrates that increases in AT-content and genome reduction are ongoing and dynamic processes in chromatophore evolution.

Diversity of the Photosynthetic Paulinella Species, with the Description of Paulinella micropora sp. nov. and the Chromatophore Genome Sequence for strain KR01.

The thecate filose amoeba Paulinella chromatophora is a good model organism for understanding plastid organellogenesis because its chromatophore was newly derived from an alpha-cyanobacterium. Paulinella chromatophora was the only known photosynthetic Paulinella species until recent studies that suggested a species level of diversity. Here, we described a new photosynthetic species P. micropora sp. nov. based on morphological and molecular evidence from a newly established strain KR01. The chromatophore genome of P. micropora KR01 was fully determined; the genome was 976,991bp in length, the GC content was 39.9%, and 908 genes were annotated. A pairwise comparison of chromatophore genome sequences between strains KR01 and FK01, representing two different natural populations of P. micropora, showed a 99.85% similarity. Differences between the two strains included single nucleotide polymorphisms (SNPs) in CDSs, which resulted in 357 synonymous and 280 nonsynonymous changes, along with 245 SNPs in non-coding regions. Indels (37) and microinversions (14) were also detected. Species diversity for photosynthetic Paulinella was surveyed using samples collected from around the world. We compared our new species to two photosynthetic species, P. chromatophora and P. longichromatophora. Phylogenetic analyses using four gene markers revealed three distinct lineages of photosynthetic Paulinella species including P. micropora sp. nov.