Ahmed Moustafa

Genomic Footprints of a Cryptic Plastid Endosymbiosis in Diatoms

Green for Diatoms Diatoms account for 20% of global carbon fixation and, together with other chromalveolates (e.g., dinoflagellates and coccolithophorids), represent many thousands of eukaryote taxa in the worlds oceans and on the tree of life. Moustafa et al. (p. 1724; see the Perspective by Dagan and Martin) have discovered that the genomes of diatoms are highly chimeric, with about 10% of their nuclear genes being of foreign algal origin. Of this set of 1272 algal genes, 253 were, as expected, from a distant red algal secondary endosymbiont, but more than 1000 of the genes were derived from green algae and predated the red algal relationship. These protist taxa are important not only for genetic and genomic investigations but also for their potential in biofuel and nanotechnology applications and in global primary productivity in relation to climate change. The genomes of early plant representatives are composites, with a substantial number of foreign genes from red and green algae. Diatoms and other chromalveolates are among the dominant phytoplankters in the world’s oceans. Endosymbiosis was essential to the success of chromalveolates, and it appears that the ancestral plastid in this group had a red algal origin via an ancient secondary endosymbiosis. However, recent analyses have turned up a handful of nuclear genes in chromalveolates that are of green algal derivation. Using a genome-wide approach to estimate the “green” contribution to diatoms, we identified >1700 green gene transfers, constituting 16% of the diatom nuclear coding potential. These genes were probably introduced into diatoms and other chromalveolates from a cryptic endosymbiont related to prasinophyte-like green algae. Chromalveolates appear to have recruited genes from the two major existing algal groups to forge a highly successful, species-rich protist lineage.

Genome structure and metabolic features in the red seaweed Chondrus crispus shed light on evolution of the Archaeplastida

Red seaweeds are key components of coastal ecosystems and are economically important as food and as a source of gelling agents, but their genes and genomes have received little attention. Here we report the sequencing of the 105-Mbp genome of the florideophyte Chondrus crispus (Irish moss) and the annotation of the 9,606 genes. The genome features an unusual structure characterized by gene-dense regions surrounded by repeat-rich regions dominated by transposable elements. Despite its fairly large size, this genome shows features typical of compact genomes, e.g., on average only 0.3 introns per gene, short introns, low median distance between genes, small gene families, and no indication of large-scale genome duplication. The genome also gives insights into the metabolism of marine red algae and adaptations to the marine environment, including genes related to halogen metabolism, oxylipins, and multicellularity (microRNA processing and transcription factors). Particularly interesting are features related to carbohydrate metabolism, which include a minimalistic gene set for starch biosynthesis, the presence of cellulose synthases acquired before the primary endosymbiosis showing the polyphyly of cellulose synthesis in Archaeplastida, and cellulases absent in terrestrial plants as well as the occurrence of a mannosylglycerate synthase potentially originating from a marine bacterium. To explain the observations on genome structure and gene content, we propose an evolutionary scenario involving an ancestral red alga that was driven by early ecological forces to lose genes, introns, and intergenetic DNA; this loss was followed by an expansion of genome size as a consequence of activity of transposable elements.

Chlamydiae Has Contributed at Least 55 Genes to Plantae with Predominantly Plastid Functions

Background The photosynthetic organelle (plastid) originated via primary endosymbiosis in which a phagotrophic protist captured and harnessed a cyanobacterium. The plastid was inherited by the common ancestor of the red, green (including land plants), and glaucophyte algae (together, the Plantae). Despite the critical importance of primary plastid endosymbiosis, its ancient derivation has left behind very few “footprints” of early key events in organelle genesis. Methodology/Principal Findings To gain insights into this process, we conducted an in-depth phylogenomic analysis of genomic data (nuclear proteins) from 17 Plantae species to identify genes of a surprising provenance in these taxa, Chlamydiae bacteria. Previous studies show that Chlamydiae contributed many genes (at least 21 in one study) to Plantae that primarily have plastid functions and were postulated to have played a fundamental role in organelle evolution. Using our comprehensive approach, we identify at least 55 Chlamydiae-derived genes in algae and plants, of which 67% (37/55) are putatively plastid targeted and at least 3 have mitochondrial functions. The remainder of the proteins does not contain a bioinformatically predicted organelle import signal although one has an N-terminal extension in comparison to the Chlamydiae homolog. Our data suggest that environmental Chlamydiae were significant contributors to early Plantae genomes that extend beyond plastid metabolism. The chlamydial gene distribution and protein tree topologies provide evidence for both endosymbiotic gene transfer and a horizontal gene transfer ratchet driven by recurrent endoparasitism as explanations for gene origin. Conclusions/Significance Our findings paint a more complex picture of gene origin than can easily be explained by endosymbiotic gene transfer from an organelle-like point source. These data significantly extend the genomic impact of Chlamydiae on Plantae and show that about one-half (30/55) of the transferred genes are most closely related to sequences emanating from the genome of the only environmental isolate that is currently available. This strain, Candidatus Protochlamydia amoebophila UWE25 is an endosymbiont of Acanthamoeba and likely represents the type of endoparasite that contributed the genes to Plantae.

Multiple Genes of Apparent Algal Origin Suggest Ciliates May Once Have Been Photosynthetic

Plantae (as defined by Cavalier-Smith, 1981) plastids evolved via primary endosymbiosis whereby a heterotrophic protist enslaved a photosynthetic cyanobacterium. This primary plastid spread into other eukaryotes via secondary endosymbiosis. An important but contentious theory in algal evolution is the chromalveolate hypothesis that posits chromists (cryptophytes, haptophytes, and stramenopiles) and alveolates (ciliates, apicomplexans, and dinoflagellates) share a common ancestor that contained a red-algal-derived secondary plastid. Under this view, the existence of several later-diverging plastid-lacking chromalveolates such as ciliates and oomycetes would be explained by plastid loss in these lineages. To test the idea of a photosynthetic ancestry for ciliates, we used the 27,446 predicted proteins from the macronuclear genome of Tetrahymena thermophila to query prokaryotic and eukaryotic genomes. We identified 16 proteins of possible algal origin in the ciliates Tetrahymena and Paramecium tetraurelia. Fourteen of these are present in other chromalveolates. Here we compare and contrast the likely scenarios for algal-gene origin in ciliates either via multiple rounds of horizontal gene transfer (HGT) from algal prey or symbionts, or through endosymbiotic gene transfer (EGT) during a putative photosynthetic phase in their evolution.

Origin of Saxitoxin Biosynthetic Genes in Cyanobacteria

Background Paralytic shellfish poisoning (PSP) is a potentially fatal syndrome associated with the consumption of shellfish that have accumulated saxitoxin (STX). STX is produced by microscopic marine dinoflagellate algae. Little is known about the origin and spread of saxitoxin genes in these under-studied eukaryotes. Fortuitously, some freshwater cyanobacteria also produce STX, providing an ideal model for studying its biosynthesis. Here we focus on saxitoxin-producing cyanobacteria and their non-toxic sisters to elucidate the origin of genes involved in the putative STX biosynthetic pathway. Methodology/Principal Findings We generated a draft genome assembly of the saxitoxin-producing (STX+) cyanobacterium Anabaena circinalis ACBU02 and searched for 26 candidate saxitoxingenes (named sxtA to sxtZ) that were recently identified in the toxic strain Cylindrospermopsis raciborskii T3. We also generated a draft assembly of the non-toxic (STX−) sister Anabaena circinalis ACFR02 to aid the identification of saxitoxin-specific genes. Comparative phylogenomic analyses revealed that nine putative STX genes were horizontally transferred from non-cyanobacterial sources, whereas one key gene (sxtA) originated in STX+ cyanobacteria via two independent horizontal transfers followed by fusion. In total, of the 26 candidate saxitoxin-genes, 13 are of cyanobacterial provenance and are monophyletic among the STX+ taxa, four are shared amongst STX+ and STX-cyanobacteria, and the remaining nine genes are specific to STX+ cyanobacteria. Conclusions/Significance Our results provide evidence that the assembly of STX genes in ACBU02 involved multiple HGT events from different sources followed presumably by coordination of the expression of foreign and native genes in the common ancestor of STX+ cyanobacteria. The ability to produce saxitoxin was subsequently lost multiple independent times resulting in a nested relationship of STX+ and STX− strains among Anabaena circinalis strains.

Phytoplankton–bacterial interactions mediate micronutrient colimitation at the coastal Antarctic sea ice edge

Significance The coastal Southern Ocean is a critical climate system component and home to high rates of photosynthesis. Here we show that cobalamin (vitamin B12) and iron availability can simultaneously limit phytoplankton growth in late Austral summer coastal Antarctic sea ice edge communities. Unlike other growth-limiting nutrients, the sole cobalamin source is production by bacteria and archaea. By identifying microbial gene expression changes in response to altered micronutrient availability, we describe the molecular underpinnings of limitation by both cobalamin and iron and offer evidence that this limitation is driven by multiple delicately balanced phytoplankton–bacterial interactions. These results support a growing body of research suggesting that relationships between bacteria and phytoplankton are key to understanding controls on marine primary productivity. Southern Ocean primary productivity plays a key role in global ocean biogeochemistry and climate. At the Southern Ocean sea ice edge in coastal McMurdo Sound, we observed simultaneous cobalamin and iron limitation of surface water phytoplankton communities in late Austral summer. Cobalamin is produced only by bacteria and archaea, suggesting phytoplankton–bacterial interactions must play a role in this limitation. To characterize these interactions and investigate the molecular basis of multiple nutrient limitation, we examined transitions in global gene expression over short time scales, induced by shifts in micronutrient availability. Diatoms, the dominant primary producers, exhibited transcriptional patterns indicative of co-occurring iron and cobalamin deprivation. The major contributor to cobalamin biosynthesis gene expression was a gammaproteobacterial population, Oceanospirillaceae ASP10-02a. This group also contributed significantly to metagenomic cobalamin biosynthesis gene abundance throughout Southern Ocean surface waters. Oceanospirillaceae ASP10-02a displayed elevated expression of organic matter acquisition and cell surface attachment-related genes, consistent with a mutualistic relationship in which they are dependent on phytoplankton growth to fuel cobalamin production. Separate bacterial groups, including Methylophaga, appeared to rely on phytoplankton for carbon and energy sources, but displayed gene expression patterns consistent with iron and cobalamin deprivation. This suggests they also compete with phytoplankton and are important cobalamin consumers. Expression patterns of siderophore- related genes offer evidence for bacterial influences on iron availability as well. The nature and degree of this episodic colimitation appear to be mediated by a series of phytoplankton–bacterial interactions in both positive and negative feedback loops.

Metabolic Effectors Secreted by Bacterial Pathogens: Essential Facilitators of Plastid Endosymbiosis?

Under the endosymbiont hypothesis, over a billion years ago a heterotrophic eukaryote entered into a symbiotic relationship with a cyanobacterium (the cyanobiont). This partnership culminated in the plastid that has spread to forms as diverse as plants and diatoms. However, why primary plastid acquisition has not been repeated multiple times remains unclear. Here, we report a possible answer to this question by showing that primary plastid endosymbiosis was likely to have been primed by the secretion in the host cytosol of effector proteins from intracellular Chlamydiales pathogens. We provide evidence suggesting that the cyanobiont might have rescued its afflicted host by feeding photosynthetic carbon into a chlamydia-controlled assimilation pathway.

Genomes and gene expression across light and productivity gradients in eastern subtropical Pacific microbial communities

Transitions in community genomic features and biogeochemical processes were examined in surface and subsurface chlorophyll maximum (SCM) microbial communities across a trophic gradient from mesotrophic waters near San Diego, California to the oligotrophic Pacific. Transect end points contrasted in thermocline depth, rates of nitrogen and CO2 uptake, new production and SCM light intensity. Relative to surface waters, bacterial SCM communities displayed greater genetic diversity and enrichment in putative sulfur oxidizers, multiple actinomycetes, low-light-adapted Prochlorococcus and cell-associated viruses. Metagenomic coverage was not correlated with transcriptional activity for several key taxa within Bacteria. Low-light-adapted Prochlorococcus, Synechococcus, and low abundance gamma-proteobacteria enriched in the>3.0-μm size fraction contributed disproportionally to global transcription. The abundance of these groups also correlated with community functions, such as primary production or nitrate uptake. In contrast, many of the most abundant bacterioplankton, including SAR11, SAR86, SAR112 and high-light-adapted Prochlorococcus, exhibited low levels of transcriptional activity and were uncorrelated with rate processes. Eukaryotes such as Haptophytes and non-photosynthetic Aveolates were prevalent in surface samples while Mamielles and Pelagophytes dominated the SCM. Metatranscriptomes generated with ribosomal RNA-depleted mRNA (total mRNA) coupled to in vitro polyadenylation compared with polyA-enriched mRNA revealed a trade-off in detection eukaryotic organelle and eukaryotic nuclear origin transcripts, respectively. Gene expression profiles of SCM eukaryote populations, highly similar in sequence identity to the model pelagophyte Pelagomonas sp. CCMP1756, suggest that pelagophytes are responsible for a majority of nitrate assimilation within the SCM.

Evolution and Functional Diversification of Fructose Bisphosphate Aldolase Genes in Photosynthetic Marine Diatoms

Diatoms and other chlorophyll-c containing, or chromalveolate, algae are among the most productive and diverse phytoplankton in the ocean. Evolutionarily, chlorophyll-c algae are linked through common, although not necessarily monophyletic, acquisition of plastid endosymbionts of red as well as most likely green algal origin. There is also strong evidence for a relatively high level of lineage-specific bacterial gene acquisition within chromalveolates. Therefore, analyses of gene content and derivation in chromalveolate taxa have indicated particularly diverse origins of their overall gene repertoire. As a single group of functionally related enzymes spanning two distinct gene families, fructose 1,6-bisphosphate aldolases (FBAs) illustrate the influence on core biochemical pathways of specific evolutionary associations among diatoms and other chromalveolates with various plastid-bearing and bacterial endosymbionts. Protein localization and activity, gene expression, and phylogenetic analyses indicate that the pennate diatom Phaeodactylum tricornutum contains five FBA genes with very little overall functional overlap. Three P. tricornutum FBAs, one class I and two class II, are plastid localized, and each appears to have a distinct evolutionary origin as well as function. Class I plastid FBA appears to have been acquired by chromalveolates from a red algal endosymbiont, whereas one copy of class II plastid FBA is likely to have originated from an ancient green algal endosymbiont. The other copy appears to be the result of a chromalveolate-specific gene duplication. Plastid FBA I and chromalveolate-specific class II plastid FBA are localized in the pyrenoid region of the chloroplast where they are associated with β-carbonic anhydrase, which is known to play a significant role in regulation of the diatom carbon concentrating mechanism. The two pyrenoid-associated FBAs are distinguished by contrasting gene expression profiles under nutrient limiting compared with optimal CO2 fixation conditions, suggestive of a distinct specialized function for each. Cytosolically localized FBAs in P. tricornutum likely play a role in glycolysis and cytoskeleton function and seem to have originated from the stramenopile host cell and from diatom-specific bacterial gene transfer, respectively.

A Novel Protein, Ubiquitous in Marine Phytoplankton, Concentrates Iron at the Cell Surface and Facilitates Uptake

Numerous cellular functions including respiration require iron. Plants and phytoplankton must also maintain the iron-rich photosynthetic electron transport chain, which most likely evolved in the iron-replete reducing environments of the Proterozoic ocean [1]. Iron bioavailability has drastically decreased in the contemporary ocean [1], most likely selecting for the evolution of efficient iron acquisition mechanisms among modern phytoplankton. Mesoscale iron fertilization experiments often result in blooms dominated by diatoms [2], indicating that diatoms have adaptations that allow survival in iron-limited waters and rapid multiplication when iron becomes available. Yet the genetic and molecular bases are unclear, as very few iron uptake genes have been functionally characterized from marine eukaryotic phytoplankton, and large portions of diatom iron starvation transcriptomes are genes encoding unknown functions [3-5]. Here we show that the marine diatom Phaeodactylum tricornutum utilizes ISIP2a to concentrate Fe(III) at the cell surface as part of a novel, copper-independent and thermodynamically controlled iron uptake system. ISIP2a is expressed in response to iron limitation several days prior to the induction of ferrireductase activity, and it facilitates significant Fe(III) uptake during the initial response to Fe limitation. ISIP2a is able to directly bind Fe(III) and increase iron uptake when heterologously expressed, whereas knockdown of ISIP2a in P. tricornutum decreases iron uptake, resulting in impaired growth and chlorosis during iron limitation. ISIP2a is expressed by diverse marine phytoplankton, indicating that it is an ecologically significant adaptation to the unique nutrient composition of marine environments.