Insights into the red algae and eukaryotic evolution from the genome of Porphyra umbilicalis (Bangiophyceae, Rhodophyta).

Porphyra umbilicalis (laver) belongs to an ancient group of red algae (Bangiophyceae), is harvested for human food, and thrives in the harsh conditions of the upper intertidal zone. Here we present the 87.7-Mbp haploid Porphyra genome (65.8% G + C content, 13,125 gene loci) and elucidate traits that inform our understanding of the biology of red algae as one of the few multicellular eukaryotic lineages. Novel features of the Porphyra genome shared by other red algae relate to the cytoskeleton, calcium signaling, the cell cycle, and stress-tolerance mechanisms including photoprotection. Cytoskeletal motor proteins in Porphyra are restricted to a small set of kinesins that appear to be the only universal cytoskeletal motors within the red algae. Dynein motors are absent, and most red algae, including Porphyra, lack myosin. This surprisingly minimal cytoskeleton offers a potential explanation for why red algal cells and multicellular structures are more limited in size than in most multicellular lineages. Additional discoveries further relating to the stress tolerance of bangiophytes include ancestral enzymes for sulfation of the hydrophilic galactan-rich cell wall, evidence for mannan synthesis that originated before the divergence of green and red algae, and a high capacity for nutrient uptake. Our analyses provide a comprehensive understanding of the red algae, which are both commercially important and have played a major role in the evolution of other algal groups through secondary endosymbioses.

Red algal parasites: models for a life history evolution that leaves photosynthesis behind again and again.

Many of the most virulent and problematic eukaryotic pathogens have evolved from photosynthetic ancestors, such as apicomplexans, which are responsible for a wide range of diseases including malaria and toxoplasmosis. The primary barrier to understanding the early stages of evolution of these parasites has been the difficulty in finding parasites with closely related free-living lineages with which to make comparisons. Parasites found throughout the florideophyte red algal lineage, however, provide a unique and powerful model to investigate the genetic origins of a parasitic lifestyle. This is because they share a recent common ancestor with an extant free-living red algal species and parasitism has independently arisen over 100 times within this group. Here, we synthesize the relevant hypotheses with respect to how these parasites have proliferated. We also place red algal research in the context of recent developments in understanding the genome evolution of other eukaryotic photosynthesizers turned parasites.

Differential genetic expression within reward-specific ensembles in mice.

Maladaptive reward seeking is a hallmark of cocaine use disorder. To develop therapeutic targets, it is critical to understand the neurobiological changes specific to cocaine-seeking without altering the seeking of natural rewards, e.g., sucrose. The prefrontal cortex (PFC) and the nucleus accumbens core (NAcore) are known regions associated with cocaine- and sucrose-seeking ensembles, i.e., a sparse population of co-activated neurons. Within ensembles, transcriptomic alterations in the PFC and NAcore underlie the learning and persistence of cocaine- and sucrose-seeking behavior. However, transcriptomes exclusively driving cocaine seeking independent from sucrose seeking have not yet been defined using a within-subject approach. Using Ai14:cFos-TRAP2 transgenic mice in a dual cocaine and sucrose self-administration model, we fluorescently sorted (FACS) and characterized (RNAseq) the transcriptomes defining cocaine- and sucrose-seeking ensembles. We found reward- and region-specific transcriptomic changes that will help develop clinically relevant genetic approaches to decrease cocaine-seeking behavior without altering non-drug reward-based positive reinforcement.

Analysis of Porphyra membrane transporters demonstrates gene transfer among photosynthetic eukaryotes and numerous sodium-coupled transport systems.

Membrane transporters play a central role in many cellular processes that rely on the movement of ions and organic molecules between the environment and the cell, and between cellular compartments. Transporters have been well characterized in plants and green algae, but little is known about transporters or their evolutionary histories in the red algae. Here we examined 482 expressed sequence tag contigs that encode putative membrane transporters in the economically important red seaweed Porphyra (Bangiophyceae, Rhodophyta). These contigs are part of a comprehensive transcriptome dataset from Porphyra umbilicalis and Porphyra purpurea. Using phylogenomics, we identified 30 trees that support the expected monophyly of red and green algae/plants (i.e. the Plantae hypothesis) and 19 expressed sequence tag contigs that show evidence of endosymbiotic/horizontal gene transfer involving stramenopiles. The majority (77%) of analyzed contigs encode transporters with unresolved phylogenies, demonstrating the difficulty in resolving the evolutionary history of genes. We observed molecular features of many sodium-coupled transport systems in marine algae, and the potential for coregulation of Porphyra transporter genes that are associated with fatty acid biosynthesis and intracellular lipid trafficking. Although both the tissue-specific and subcellular locations of the encoded proteins require further investigation, our study provides red algal gene candidates associated with transport functions and novel insights into the biology and evolution of these transporters.

Author Correction: The Role of European Starlings (Sturnus vulgaris) in the Dissemination of Multidrug-Resistant Escherichia coli among Concentrated Animal Feeding Operations.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

The Role of European Starlings (Sturnus vulgaris) in the Dissemination of Multidrug-Resistant Escherichia coli among Concentrated Animal Feeding Operations.

Antimicrobial use in livestock production is a driver for the development and proliferation of antimicrobial resistance (AMR). Wildlife interactions with livestock, acquiring associated AMR bacteria and genes, and wildlife's subsequent dispersal across the landscape are hypothesized to play an important role in the ecology of AMR. Here, we examined priority AMR phenotypes and genotypes of Escherichia coli isolated from the gastrointestinal tracts of European starlings (Sturnus vulgaris) found on concentrated animal feeding operations (CAFOs). European starlings may be present in high numbers on CAFOs (>100,000 birds), interact with urban environments, and can migrate distances exceeding 1,500 km in North America. In this study, 1,477 European starlings from 31 feedlots in five U.S. states were sampled for E. coli resistant to third generation cephalosporins (3G-C) and fluoroquinolones. The prevalence of 3G-C and fluoroquinolone-resistant E. coli was 4% and 10%, respectively. Multidrug resistance in the E. coli isolates collected (n = 236) was common, with the majority of isolates displaying resistance to six or more classes of antibiotics. Genetic analyses of a subset of these isolates identified 94 genes putatively contributing to AMR, including seven class A and C ß-lactamases as well as mutations in gyrA and parC recognized to confer resistance to quinolones. Phylogenetic and subtyping assessments showed that highly similar isolates (≥99.4% shared core genome, ≥99.6% shared coding sequence) with priority AMR were found in birds on feedlots separated by distances exceeding 150 km, suggesting that European starlings could be involved in the interstate dissemination of priority AMR bacteria.

Use of a Sibling Subtraction Method for Identifying Causal Mutations in Caenorhabditis elegans by Whole-Genome Sequencing.

Whole-genome sequencing (WGS) is an indispensable tool for identifying causal mutations obtained from genetic screens. To reduce the number of causal mutation candidates typically uncovered by WGS, Caenorhabditis elegans researchers have developed several strategies. One involves crossing N2-background mutants to the polymorphic Hawaiian (HA) strain, which can be used to simultaneously identify mutant strain variants and obtain high-density mapping information. This approach, however, is not well suited for uncovering mutations in complex genetic backgrounds, and HA polymorphisms can alter phenotypes. Other approaches make use of DNA variants present in the initial background or introduced by mutagenesis. This information is used to implicate genomic regions with high densities of DNA lesions that persist after backcrossing, but these methods can provide lower resolution than HA mapping. To identify suppressor mutations using WGS, we developed an approach termed the sibling subtraction method (SSM). This method works by eliminating variants present in both mutants and their nonmutant siblings, thus greatly reducing the number of candidates. We used this method with two members of the C. elegans NimA-related kinase family, nekl-2 and nekl-3 Combining weak aphenotypic alleles of nekl-2 and nekl-3 leads to penetrant molting defects and larval arrest. We isolated ∼50 suppressors of nekl-2; nekl-3 synthetic lethality using F1 clonal screening methods and a peel-1-based counterselection strategy. When applied to five of the suppressors, SSM led to only one to four suppressor candidates per strain. Thus SSM is a powerful approach for identifying causal mutations in any genetic background and provides an alternative to current methods.

Regulation of germ cell development by ARI1 family ubiquitin ligases in C. elegans.

RING-between-RING (RBR) E3 ubiquitin ligases are implicated in various developmental processes, and mutations in genes encoding RBR proteins HHARI/ARIH1 and Parkin are associated with human diseases. Here we show by phylogenetic analysis that the ARI1 family has undergone a dramatic expansion within the Caenorhabditis clade in recent history, a characteristic shared by some genes involved in germline development. We then examined the effects of deleting all ARI1 family members in the nematode Caenorhabditis elegans, which to our knowledge represents the first complete knockout of ARI1 function in a metazoan. Hermaphrodites that lacked or had strongly reduced ARI1 activity had low fecundity and were partially defective in initiation of oocyte differentiation. We provide evidence that the C. elegans ARI1s likely function downstream or in parallel to FBF-1 and FBF-2, two closely related RNA-binding proteins that are required for the switch from spermatogenesis to oogenesis during late larval development. Previous studies have shown that the E2 enzymes UBC-18/UBCH7 and UBC-3/CDC34 can functionally collaborate with ARI1 family members. Our data indicated that UBC-18, but not UBC-3, specifically cooperates with the ARI1s in germline development. These findings provide new insights into the functions of RING-between-RING proteins and Ariadne E3s during development.

MAJOR DEVELOPMENTAL REGULATORS AND THEIR EXPRESSION IN TWO CLOSELY RELATED SPECIES OF PORPHYRA (RHODOPHYTA)(1).

Little is known about the genetic and biochemical mechanisms that underlie red algal development, for example, why the group failed to evolve complex parenchyma and tissue differentiation. Here we examined expressed sequence tag (EST) data from two closely related species, Porphyra umbilicalis (L.) J. Agardh and P. purpurea (Roth) C. Agardh, for conserved developmental regulators known from model eukaryotes, and their expression levels in several developmental stages. Genes for most major developmental families were present, including MADS-box and homeodomain (HD) proteins, SNF2 chromatin-remodelers, and proteins involved in sRNA biogenesis. Some of these genes displayed altered expression correlating with different life history stages or cell types. Notably, two ESTs encoding HD proteins showed eightfold higher expression in the P. purpurea sporophyte (conchocelis) than in the gametophyte (blade), whereas two MADS domain-containing paralogs showed significantly different patterns of expression in the conchocelis and blade respectively. These developmental gene families do not appear to have undergone the kinds of dramatic expansions in copy number found in multicellular land plants and animals, which are important for regulating developmental processes in those groups. Analyses of small RNAs did not validate the presence of miRNAs, but homologs of Argonaute were present. In general, it appears that red algae began with a similar molecular toolkit for directing development as did other multicellular eukaryotes, but probably evolved altered roles for many key proteins, as well as novel mechanisms yet to be discovered.

Porphyra (Bangiophyceae) Transcriptomes Provide Insights Into Red Algal Development And Metabolism.

The red seaweed Porphyra (Bangiophyceae) and related Bangiales have global economic importance. Here, we report the analysis of a comprehensive transcriptome comprising ca. 4.7 million expressed sequence tag (EST) reads from P. umbilicalis (L.) J. Agardh and P. purpurea (Roth) C. Agardh (ca. 980 Mbp of data generated using 454 FLX pyrosequencing). These ESTs were isolated from the haploid gametophyte (blades from both species) and diploid conchocelis stage (from P. purpurea). In a bioinformatic analysis, only 20% of the contigs were found to encode proteins of known biological function. Comparative analysis of predicted protein functions in mesophilic (including Porphyra) and extremophilic red algae suggest that the former has more putative functions related to signaling, membrane transport processes, and establishment of protein complexes. These enhanced functions may reflect general mesophilic adaptations. A near-complete repertoire of genes encoding histones and ribosomal proteins was identified, with some differentially regulated between the blade and conchocelis stage in P. purpurea. This finding may reflect specific regulatory processes associated with these distinct phases of the life history. Fatty acid desaturation patterns, in combination with gene expression profiles, demonstrate differences from seed plants with respect to the transport of fatty acid/lipid among subcellular compartments and the molecular machinery of lipid assembly. We also recovered a near-complete gene repertoire for enzymes involved in the formation of sterols and carotenoids, including candidate genes for the biosynthesis of lutein. Our findings provide key insights into the evolution, development, and biology of Porphyra, an important lineage of red algae.

Cyanophora paradoxa genome elucidates origin of photosynthesis in algae and plants.

The primary endosymbiotic origin of the plastid in eukaryotes more than 1 billion years ago led to the evolution of algae and plants. We analyzed draft genome and transcriptome data from the basally diverging alga Cyanophora paradoxa and provide evidence for a single origin of the primary plastid in the eukaryote supergroup Plantae. C. paradoxa retains ancestral features of starch biosynthesis, fermentation, and plastid protein translocation common to plants and algae but lacks typical eukaryotic light-harvesting complex proteins. Traces of an ancient link to parasites such as Chlamydiae were found in the genomes of C. paradoxa and other Plantae. Apparently, Chlamydia-like bacteria donated genes that allow export of photosynthate from the plastid and its polymerization into storage polysaccharide in the cytosol.

Porphyra: a marine crop shaped by stress.

The marine red alga Porphyra is an important marine crop, worth ∼US$1.3 billion per year. Cultivation research now includes farm ecology, breeding, strain conservation and new net-seeding technologies. The success of cultivation is due, in part, to the high stress tolerance of Porphyra. Many species of Porphyra lose 85-95% of their cellular water during the daytime low tide, when they are also exposed to high light and temperature stress. Antioxidant and mycosporine-like amino acid activities have been partially characterized in Porphyra, but, as we discuss here, the Porphyra umbilicalis genome project will further elucidate proteins associated with stress tolerance. Furthermore, phylogenomic and transcriptomic investigations of Porphyra sensu lato could elucidate tradeoffs made during physiological acclimation and factors associated with life-history evolution in this ancient lineage.