Ocean-wide comparisons of mesopelagic planktonic community structures.

For decades, marine plankton have been investigated for their capacity to modulate biogeochemical cycles and provide fishery resources. Between the sunlit (epipelagic) layer and the deep dark waters, lies a vast and heterogeneous part of the ocean: the mesopelagic zone. How plankton composition is shaped by environment has been well-explored in the epipelagic but much less in the mesopelagic ocean. Here, we conducted comparative analyses of trans-kingdom community assemblages thriving in the mesopelagic oxygen minimum zone (OMZ), mesopelagic oxic, and their epipelagic counterparts. We identified nine distinct types of intermediate water masses that correlate with variation in mesopelagic community composition. Furthermore, oxygen, NO3- and particle flux together appeared as the main drivers governing these communities. Novel taxonomic signatures emerged from OMZ while a global co-occurrence network analysis showed that about 70% of the abundance of mesopelagic plankton groups is organized into three community modules. One module gathers prokaryotes, pico-eukaryotes and Nucleo-Cytoplasmic Large DNA Viruses (NCLDV) from oxic regions, and the two other modules are enriched in OMZ prokaryotes and OMZ pico-eukaryotes, respectively. We hypothesize that OMZ conditions led to a diversification of ecological niches, and thus communities, due to selective pressure from limited resources. Our study further clarifies the interplay between environmental factors in the mesopelagic oxic and OMZ, and the compositional features of communities.

The cyanobacterium Prochlorococcus has divergent light-harvesting antennae and may have evolved in a low-oxygen ocean.

Marine picocyanobacteria of the genus Prochlorococcus are the most abundant photosynthetic organisms in the modern ocean, where they exert a profound influence on elemental cycling and energy flow. The use of transmembrane chlorophyll complexes instead of phycobilisomes as light-harvesting antennae is considered a defining attribute of Prochlorococcus Its ecology and evolution are understood in terms of light, temperature, and nutrients. Here, we report single-cell genomic information on previously uncharacterized phylogenetic lineages of this genus from nutrient-rich anoxic waters of the eastern tropical North and South Pacific Ocean. The most basal lineages exhibit optical and genotypic properties of phycobilisome-containing cyanobacteria, indicating that the characteristic light-harvesting antenna of the group is not an ancestral attribute. Additionally, we found that all the indigenous lineages analyzed encode genes for pigment biosynthesis under oxygen-limited conditions, a trait shared with other freshwater and coastal marine cyanobacteria. Our findings thus suggest that Prochlorococcus diverged from other cyanobacteria under low-oxygen conditions before transitioning from phycobilisomes to transmembrane chlorophyll complexes and may have contributed to the oxidation of the ancient ocean.

Wireless, battery-free, fully implantable multimodal and multisite pacemakers for applications in small animal models.

Small animals support a wide range of pathological phenotypes and genotypes as versatile, affordable models for pathogenesis of cardiovascular diseases and for exploration of strategies in electrotherapy, gene therapy, and optogenetics. Pacing tools in such contexts are currently limited to tethered embodiments that constrain animal behaviors and experimental designs. Here, we introduce a highly miniaturized wireless energy-harvesting and digital communication electronics for thin, miniaturized pacing platforms weighing 110 mg with capabilities for subdermal implantation and tolerance to over 200,000 multiaxial cycles of strain without degradation in electrical or optical performance. Multimodal and multisite pacing in ex vivo and in vivo studies over many days demonstrate chronic stability and excellent biocompatibility. Optogenetic stimulation of cardiac cycles with in-animal control and induction of heart failure through chronic pacing serve as examples of modes of operation relevant to fundamental and applied cardiovascular research and biomedical technology.

Proteomic analysis of cell cycle progression in asynchronous cultures, including mitotic subphases, using PRIMMUS.

The temporal regulation of protein abundance and post-translational modifications is a key feature of cell division. Recently, we analysed gene expression and protein abundance changes during interphase under minimally perturbed conditions (Ly et al., 2014, 2015). Here, we show that by using specific intracellular immunolabelling protocols, FACS separation of interphase and mitotic cells, including mitotic subphases, can be combined with proteomic analysis by mass spectrometry. Using this PRIMMUS (PRoteomic analysis of Intracellular iMMUnolabelled cell Subsets) approach, we now compare protein abundance and phosphorylation changes in interphase and mitotic fractions from asynchronously growing human cells. We identify a set of 115 phosphorylation sites increased during G2, termed 'early risers'. This set includes phosphorylation of S738 on TPX2, which we show is important for TPX2 function and mitotic progression. Further, we use PRIMMUS to provide the first a proteome-wide analysis of protein abundance remodeling between prophase, prometaphase and anaphase.

Multi-omics Analyses of Starvation Responses Reveal a Central Role for Lipoprotein Metabolism in Acute Starvation Survival in C. elegans.

Starvation causes comprehensive metabolic changes, which are still not fully understood. Here, we used quantitative proteomics and RNA sequencing to examine the temporal starvation responses in wild-type Caenorhabditis elegans and animals lacking the transcription factor HLH-30. Our findings show that starvation alters the abundance of hundreds of proteins and mRNAs in a temporal manner, many of which are involved in central metabolic pathways, including lipoprotein metabolism. We demonstrate that premature death of hlh-30 animals under starvation can be prevented by knockdown of either vit-1 or vit-5, encoding two different lipoproteins. We further show that the size and number of intestinal lipid droplets under starvation are altered in hlh-30 animals, which can be rescued by knockdown of vit-1. Taken together, this indicates that survival of hlh-30 animals under starvation is closely linked to regulation of intestinal lipid stores. We provide the most detailed poly-omic analysis of starvation responses to date, which serves as a resource for further mechanistic studies of starvation.

Comparative genetic, proteomic and phosphoproteomic analysis of C. elegans embryos with a focus on ham-1/STOX and pig-1/MELK in dopaminergic neuron development.

Asymmetric cell divisions are required for cellular diversity and defects can lead to altered daughter cell fates and numbers. In a genetic screen for C. elegans mutants with defects in dopaminergic head neuron specification or differentiation, we isolated a new allele of the transcription factor HAM-1 [HSN (Hermaphrodite-Specific Neurons) Abnormal Migration]. Loss of both HAM-1 and its target, the kinase PIG-1 [PAR-1(I)-like Gene], leads to abnormal dopaminergic head neuron numbers. We identified discrete genetic relationships between ham-1, pig-1 and apoptosis pathway genes in dopaminergic head neurons. We used an unbiased, quantitative mass spectrometry-based proteomics approach to characterise direct and indirect protein targets and pathways that mediate the effects of PIG-1 kinase loss in C. elegans embryos. Proteins showing changes in either abundance, or phosphorylation levels, between wild-type and pig-1 mutant embryos are predominantly connected with processes including cell cycle, asymmetric cell division, apoptosis and actomyosin-regulation. Several of these proteins play important roles in C. elegans development. Our data provide an in-depth characterisation of the C. elegans wild-type embryo proteome and phosphoproteome and can be explored via the Encyclopedia of Proteome Dynamics (EPD) - an open access, searchable online database.

Deep Proteome Analysis Identifies Age-Related Processes in C. elegans.

Effective network analysis of protein data requires high-quality proteomic datasets. Here, we report a near doubling in coverage of the C. elegans adult proteome, identifying >11,000 proteins in total with ∼9,400 proteins reproducibly detected in three biological replicates. Using quantitative mass spectrometry, we identify proteins whose abundances vary with age, revealing a concerted downregulation of proteins involved in specific metabolic pathways and upregulation of cellular stress responses with advancing age. Among these are ∼30 peroxisomal proteins, including the PRX-5/PEX5 import protein. Functional experiments confirm that protein import into the peroxisome is compromised in vivo in old animals. We also studied the behavior of the set of age-variant proteins in chronologically age-matched, long-lived daf-2 insulin/IGF-1-pathway mutants. Unexpectedly, the levels of many of these age-variant proteins did not scale with extended lifespan. This indicates that, despite their youthful appearance and extended lifespans, not all aspects of aging are reset in these long-lived mutants.

Global Membrane Protein Interactome Analysis using In vivo Crosslinking and Mass Spectrometry-based Protein Correlation Profiling.

We present a methodology using in vivo crosslinking combined with HPLC-MS for the global analysis of endogenous protein complexes by protein correlation profiling. Formaldehyde crosslinked protein complexes were extracted with high yield using denaturing buffers that maintained complex solubility during chromatographic separation. We show this efficiently detects both integral membrane and membrane-associated protein complexes,in addition to soluble complexes, allowing identification and analysis of complexes not accessible in native extracts. We compare the protein complexes detected by HPLC-MS protein correlation profiling in both native and formaldehyde crosslinked U2OS cell extracts. These proteome-wide data sets of both in vivo crosslinked and native protein complexes from U2OS cells are freely available via a searchable online database (www.peptracker.com/epd). Raw data are also available via ProteomeXchange (identifier PXD003754).

The cytotoxic T cell proteome and its shaping by the kinase mTOR.

We used high-resolution mass spectrometry to map the cytotoxic T lymphocyte (CTL) proteome and the effect of the metabolic checkpoint kinase mTORC1 on CTLs. The CTL proteome was dominated by metabolic regulators and granzymes, and mTORC1 selectively repressed and promoted expression of a subset of CTL proteins (~10%). These included key CTL effector molecules, signaling proteins and a subset of metabolic enzymes. Proteomic data highlighted the potential for negative control of the production of phosphatidylinositol (3,4,5)-trisphosphate (PtdIns(3,4,5)P3) by mTORC1 in CTLs. mTORC1 repressed PtdIns(3,4,5)P3 production and determined the requirement for mTORC2 in activation of the kinase Akt. Our unbiased proteomic analysis thus provides comprehensive understanding of CTL identity and the control of CTL function by mTORC1.

Micro-proteomics with iterative data analysis: Proteome analysis in C. elegans at the single worm level.

Proteomics studies typically analyze proteins at a population level, using extracts prepared from tens of thousands to millions of cells. The resulting measurements correspond to average values across the cell population and can mask considerable variation in protein expression and function between individual cells or organisms. Here, we report the development of micro-proteomics for the analysis of Caenorhabditis elegans, a eukaryote composed of 959 somatic cells and ∼1500 germ cells, measuring the worm proteome at a single organism level to a depth of ∼3000 proteins. This includes detection of proteins across a wide dynamic range of expression levels (>6 orders of magnitude), including many chromatin-associated factors involved in chromosome structure and gene regulation. We apply the micro-proteomics workflow to measure the global proteome response to heat-shock in individual nematodes. This shows variation between individual animals in the magnitude of proteome response following heat-shock, including variable induction of heat-shock proteins. The micro-proteomics pipeline thus facilitates the investigation of stochastic variation in protein expression between individuals within an isogenic population of C. elegans. All data described in this study are available online via the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd), an open access, searchable database resource.

Global Proteomics Analysis of the Response to Starvation in C. elegans.

Periodic starvation of animals induces large shifts in metabolism but may also influence many other cellular systems and can lead to adaption to prolonged starvation conditions. To date, there is limited understanding of how starvation affects gene expression, particularly at the protein level. Here, we have used mass-spectrometry-based quantitative proteomics to identify global changes in the Caenorhabditis elegans proteome due to acute starvation of young adult animals. Measuring changes in the abundance of over 5,000 proteins, we show that acute starvation rapidly alters the levels of hundreds of proteins, many involved in central metabolic pathways, highlighting key regulatory responses. Surprisingly, we also detect changes in the abundance of chromatin-associated proteins, including specific linker histones, histone variants, and histone posttranslational modifications associated with the epigenetic control of gene expression. To maximize community access to these data, they are presented in an online searchable database, the Encyclopedia of Proteome Dynamics (http://www.peptracker.com/epd/).

Reassessment of the toxin profile of Cylindrospermopsis raciborskii T3 and function of putative sulfotransferases in synthesis of sulfated and sulfonated PSP toxins.

The toxigenic freshwater cyanobacterium Cylindrospermopsis raciborskii T3 has been used as a model to study and elucidate the biosynthetic pathway of tetrahydropurine neurotoxins associated with paralytic shellfish poisoning (PSP). There are nevertheless several inconsistencies and contradictions in the toxin profile of this strain as published by different research groups, and claimed to include carbamoyl (STX, NEO, GTX2/3), decarbamoyl (dcSTX), and N-sulfocarbamoyl (C1/2, B1) derivatives. Our analysis of the complete genome of another PSP toxin-producing cyanobacterium, Raphidiopsis brookii D9, which is closely related to C. raciborskii T3, resolved many issues regarding the correlation between biosynthetic pathways, corresponding genes and the T3 toxin profile. The putative sxt gene cluster in R. brookii D9 has a high synteny with the T3 sxt cluster, with 100% nucleotide identity among the shared genes. We also compared the PSP toxin profile of the strains by liquid chromatography coupled to mass spectrometry (LC-MS/MS). In contrast to published reports, our reassessment of the PSP toxin profile of T3 confirmed production of only STX, NEO and dcNEO. We gained significant insights via correlation between specific sxt genes and their role in PSP toxin synthesis in both D9 and T3 strains. In particular, analysis of sulfotransferase functions for SxtN (N-sulfotransferase) and SxtSUL (O-sulfotransferase) enzymes allowed us to propose an extension of the PSP toxin biosynthetic pathway from STX to the production of the derivatives GTX2/3, C1/2 and B1. This is a significantly revised view of the genetic mechanisms underlying synthesis of sulfated and sulfonated STX analogues in toxigenic cyanobacteria.

The smallest known genomes of multicellular and toxic cyanobacteria: comparison, minimal gene sets for linked traits and the evolutionary implications.

Cyanobacterial morphology is diverse, ranging from unicellular spheres or rods to multicellular structures such as colonies and filaments. Multicellular species represent an evolutionary strategy to differentiate and compartmentalize certain metabolic functions for reproduction and nitrogen (N(2)) fixation into specialized cell types (e.g. akinetes, heterocysts and diazocytes). Only a few filamentous, differentiated cyanobacterial species, with genome sizes over 5 Mb, have been sequenced. We sequenced the genomes of two strains of closely related filamentous cyanobacterial species to yield further insights into the molecular basis of the traits of N(2) fixation, filament formation and cell differentiation. Cylindrospermopsis raciborskii CS-505 is a cylindrospermopsin-producing strain from Australia, whereas Raphidiopsis brookii D9 from Brazil synthesizes neurotoxins associated with paralytic shellfish poisoning (PSP). Despite their different morphology, toxin composition and disjunct geographical distribution, these strains form a monophyletic group. With genome sizes of approximately 3.9 (CS-505) and 3.2 (D9) Mb, these are the smallest genomes described for free-living filamentous cyanobacteria. We observed remarkable gene order conservation (synteny) between these genomes despite the difference in repetitive element content, which accounts for most of the genome size difference between them. We show here that the strains share a specific set of 2539 genes with >90% average nucleotide identity. The fact that the CS-505 and D9 genomes are small and streamlined compared to those of other filamentous cyanobacterial species and the lack of the ability for heterocyst formation in strain D9 allowed us to define a core set of genes responsible for each trait in filamentous species. We presume that in strain D9 the ability to form proper heterocysts was secondarily lost together with N(2) fixation capacity. Further comparisons to all available cyanobacterial genomes covering almost the entire evolutionary branch revealed a common minimal gene set for each of these cyanobacterial traits.

Toxicity phenotype does not correlate with phylogeny of Cylindrospermopsis raciborskii strains.

Cylindrospermopsis raciborskii is a species of freshwater, bloom-forming cyanobacterium. C. raciborskii produces toxins, including cylindrospermopsin (hepatotoxin) and saxitoxin (neurotoxin), although non toxin-producing strains are also observed. In spite of differences in toxicity, C. raciborskii strains comprise a monophyletic group, based upon 16S rRNA gene sequence identities (greater than 99%). We performed phylogenetic analyses; 16S rRNA gene and 16S-23S rRNA gene internally transcribed spacer (ITS-1) sequence comparisons, and genomic DNA restriction fragment length polymorphism (RFLP), resolved by pulsed-field gel electrophoresis (PFGE), of strains of C. raciborskii, obtained mainly from the Australian phylogeographic cluster. Our results showed no correlation between toxic phenotype and phylogenetic association in the Australian strains. Analyses of the 16S rRNA gene and the respective ITS-1 sequences (long L, and short S) showed an independent evolution of each ribosomal operon. The genes putatively involved in the cylindrospermopsin biosynthetic pathway were present in one locus and only in the hepatotoxic strains, demonstrating a common genomic organization for these genes and the absence of mutated or inactivated biosynthetic genes in the non toxic strains. In summary, our results support the hypothesis that the genes involved in toxicity may have been transferred as an island by processes of gene lateral transfer, rather than convergent evolution.

Precise excision of the large pathogenicity island, SPI7, in Salmonella enterica serovar Typhi.

The large pathogenicity island (SPI7) of Salmonella enterica serovar Typhi is a 133,477-bp segment of DNA flanked by two 52-bp direct repeats overlapping the pheU (phenylalanyl-tRNA) gene, contains 151 potential open reading frames, and includes the viaB operon involved in the synthesis of Vi antigen. Some clinical isolates of S. enterica serovar Typhi are missing the entire SPI7, due to its precise excision; these strains have lost the ability to produce Vi antigen, are resistant to phage Vi-II, and invade a human epithelial cell line more rapidly. Excision of SPI7 occurs spontaneously in a clinical isolate of S. enterica serovar Typhi when it is grown in the laboratory, leaves an intact copy of the pheU gene at its novel join point, and results in the same three phenotypic consequences. SPI7 is an unstable genetic element, probably an intermediate in the pathway of lateral transfer of such pathogenicity islands among enteric gram-negative bacteria.