Biology subjects

Allen, A. E.

Publications and source records attributed to Allen, A. E..

31 records · Page 2Linked to original sources

bioRxiv2021

Proteomic traits vary across taxa in a coastal Antarctic phytoplankton bloom

Production and use of proteins is under strong selection in microbes, but its unclear how proteome-level traits relate to ecological strategies. We identified and quantified proteomic traits of eukaryotic and prokaryotic microbes through an Antarctic phytoplankton bloom using in situ metaproteomics. To do this, we first used simulations, cultures, and bioinformatic methods to rigorously assess our inferences about various proteomic traits and use these assessments to provide several practical recommendations for researchers using metaproteomics. Different taxa, rather than different environmental conditions, formed distinct clusters based on their ribosomal and photosynthetic proteomic proportions, and we propose that these characteristics relate to ecological differences. We defined and used a proteomic proxy for regulatory cost, which showed that SAR11 had the lowest regulatory cost of any taxa we observed at our summertime Southern Ocean study site. Haptophytes had lower regulatory cost than diatoms, which may underpin haptophyte-to-diatom bloom progression in the Ross Sea. Using metaproteomics, we have quantified several proteomic traits (ribosomal and photosynthetic proteomic proportions, regulatory cost) in eukaryotic and prokaryotic taxa, which can then be incorporated into trait-based models of microbial communities that reflect resource allocation strategies.

microbiology↗

bioRxiv2021

GNPS Dashboard: Collaborative Analysis of Mass Spectrometry Data in the Web Browser

Access to web-based platforms has enabled scientists to perform research remotely. A critical aspect of mass spectrometry data analysis is the inspection, analysis, and visualization of the raw data to validate data quality and confirm statistical observations. We developed the GNPS Dashboard, a web-based data visualization tool, to facilitate synchronous collaborative inspection, visualization, and analysis of private and public mass spectrometry data remotely.

bioinformatics↗

bioRxiv2020

The Phaeodactylum tricornutum Diaminopimelate Decarboxylase was Acquired via Horizontal Gene Transfer from Bacteria and Displays Substrate Promiscuity

Diatoms are predicted to synthesize certain amino acids within the chloroplast, including L-lysine via a diaminopimelate-dependent pathway. Herein, we report that the model diatom, Phaeodactylum tricornutum, possesses a chimeric lysine biosynthetic pathway, which coalesces bacterial and plant genes, and is terminated by a chloroplast-localized diaminopimelate decarboxylase (DAPDC, PtLYSA). We show that while RNAi ablation of PtLYSA is either synthetically lethal or concomitant with a slower growth rate, Cas9-mediated mutagenesis of PtLYSA results in recovery of heterozygous cells lines, suggesting that PtLYSA is an essential gene. Previously characterized DAPDCs are unique within the PLP-dependent decarboxylases where catalysis occurs at the D-stereocenter of the substrate and display a strict stereochemical preference for a (D,L)- or meso-substrate and not the D,D- or L,L-isomers of diaminopimelate (DAP) to synthesize L-lysine. Using decarboxylation assays and differential scanning calorimetry analyses, we validate that PtLYSA is a bona fide DAPDC and uncover its unexpected stereopromiscuous behavior in substrate specificity. The crystal structure of PtLYSA confirms the enzyme is an obligate homodimer in which both protomers reciprocally participate in the active site. The structure underscores features unique to the PtLYSA clan of DAPDC and provides structural insight into the determinants responsible for the substrate-promiscuity observed in PtLYSA.

biochemistry↗

bioRxiv2020

Colwellia and Marinobacter metapangenomes reveal species-specific responses to oil and dispersant exposure in deepsea microbial communities

Over 7 million liters of Corexit EC9500A and EC9527A were applied to the Gulf of Mexico in response to the Deepwater Horizon oil spill. The impacts of dispersants remain under debate and negative, positive, and inconclusive impacts have been reported. Here, metatrancriptomics was applied in the context of metapangenomes to microcosms that simulated environmental conditions comparable to the hydrocarbon-rich 1,100 m deep plume. Within this microcosm study, negative effects of dispersants on microbial hydrocarbon degradation were previously reported based on activity measurements and geochemical data. Transcriptional enrichment of Colwellia, a potential dispersant degrader, followed variable time-dependent trajectories due to interactions between oil, dispersants, and nutrients. The Colwellia metapangenome captured a mixture of environmental responses linked to the Colwellia psychrerythraea 34H genome and to the genomes of other members of the Colwellia genus. The activation of genes involved in lipid degradation, nitrogen metabolism, and membrane composition under oil or nutrient availability, suggested an opportunistic growth strategy for Colwellia. In contrast, transcripts of Marinobacter, a natural hydrocarbon degrader, increased only in oil treatments. Marinobacter transcripts largely recruited to the accessory metapangenome of Marinobacter sp. C18, the closest genomic reference. A complex response involving carbon and lipid metabolism, chemotaxis and a type IV secretion system suggested active energy-dependent processes in Marinobacter. These findings highlight chemistry-dependent responses in the metabolism of key hydrocarbon-degrading bacteria and underscore that dispersant-driven selection could temper the ability of the community to respond to hydrocarbon injection.

microbiology↗

bioRxiv2020

Proteomic analysis of metabolic pathways shows chloroplast-mitochondria cross-talk in a Cu-limited diatom

Diatoms are one of the most successful phytoplankton groups in our oceans, being responsible for over 20% of the Earths photosynthetic productivity. Their chimeric genomes have genes derived from red algae, green algae, bacteria and heterotrophs, resulting in multiple isoenzymes targeted to different cellular compartments with the potential for differential regulation under nutrient limitation. The resulting interactions between metabolic pathways are not yet fully understood. We previously showed how acclimation to Cu limitation enhanced susceptibility to overreduction of the photosynthetic electron transport chain and its reorganization to favor photoprotection over light-harvesting in the oceanic diatom Thalassiosira oceanica (Hippmann et al., 2017). In order to gain a better understanding of the overall metabolic changes that help alleviate the stress of Cu limitation, we have further analyzed the comprehensive proteomic datasets generated in that study to identify differentially expressed proteins involved in carbon, nitrogen and oxidative stress-related metabolic pathways. Metabolic pathway analysis showed integrated responses to Cu limitation. The up-regulation of ferredoxin (Fdx) was correlated with up-regulation of plastidial Fdx-dependent isoenzymes involved in nitrogen assimilation as well as enzymes involved in glutathione synthesis, thus integrating nitrogen uptake and metabolism with photosynthesis and oxidative stress resistance. The differential expression of glycolytic isoenzymes located in the chloroplast and mitochondria may enable them to channel both excess electrons and/or ATP between these compartments. Additional evidence for chloroplast-mitochondrial cross-talk is the increased expression of chloroplast and mitochondrial proteins involved in the proposed malate shunt under Cu limitation. One sentence summaryDiatoms adapt to Cu limitation by regulating their large repertoire of isoenzymes to channel electrons away from the chloroplast, enhance nitrogen uptake, and integrate the oxidative stress response. 123

microbiology↗

bioRxiv2020

Molecular underpinnings and biogeochemical consequences of enhanced diatom growth in a warming Southern Ocean

The Southern Ocean (SO) harbours some of the most intense phytoplankton blooms on Earth. Changes in temperature and iron availability are expected to alter the intensity of SO phytoplankton blooms, but little is known about how environmental change will influence community composition and downstream biogeochemical processes. We performed experimental manipulations on surface ocean microbial communities from McMurdo Sound in the Ross Sea, with and without iron addition, at −0.5 °C, 3 °C, and 6 °C. We then examined nutrient uptake patterns as well as the growth and molecular responses of two dominant diatoms, Fragilariopsis and Pseudo-nitzschia, to these conditions. We found that nitrate uptake and primary productivity were elevated at increased temperature in the absence of iron addition, and were even greater at high temperature with added iron. Pseudo-nitzschia became more abundant under increased temperature without added iron, while Fragilariopsis required additional iron to benefit from warming. We attribute the apparent advantage Pseudo-nitzschia shows under warming to upregulation of iron-conserving photosynthetic processes, utilization of iron-economic nitrogen assimilation mechanisms, and increased iron uptake and storage. These data identify important molecular and physiological differences between dominant diatom groups and add to the growing body of evidence for Pseudo-nitzschia’s increasingly important role in warming SO ecosystems. This study also suggests that temperature-driven shifts in SO phytoplankton assemblages may increase utilization of the vast pool of excess nutrients in iron-limited SO surface waters, and thereby influence global nutrient distributions and carbon cycle.Significance Statement Phytoplankton assemblages contribute to the Southern Ocean’s ability to absorb atmospheric CO2, form the base of marine food webs, and shape the global distribution of macronutrients. Anthropogenic climate change is altering the SO environment, yet we do not fully understand how resident phytoplankton will react to this change. By comparing the responses of two prominent SO diatom groups to changes in temperature and iron in a natural community, we find that one group, Pseudo-nitzschia, grows better under warmer low-iron conditions by managing cellular iron demand and efficiently increasing photosynthetic capacity. This ability to grow and draw down nutrients in the face of warming, regardless of iron availability, may have major implications for ocean ecosystems and global nutrient and carbon cycles.Competing Interest StatementThe authors have declared no competing interest.View Full Text

ecology↗

bioRxiv2020

Daytime light enhances the amplitude of circadian output in a diurnal mammal

Mammalian circadian rhythms are orchestrated by a master pacemaker in the hypothalamic suprachiasmatic nuclei (SCN), which receives information about the 24 h light:dark cycle from the retina. The accepted function of this light signal is to reset circadian phase in order to ensure appropriate synchronisation with the celestial day. Here, we ask whether light also impacts another key property of the circadian oscillation, its amplitude. To this end, we measured rhythms in behavioural activity and body temperature, and SCN electrophysiological activity in the diurnal murid rodent Rhabdomys pumilio following stable entrainment to 12:12 light:dark cycles at 4 different daytime intensities (ranging from 12.77 to 14.80 log melanopsin effective photons/cm2/s). Rhabdomys showed strongly diurnal activity and body temperature rhythms in all conditions, but measures of rhythm robustness were positively correlated with daytime irradiance under both entrainment and subsequent free run. Whole-cell and extracellular recordings of electrophysiological activity in ex vivo SCN revealed substantial differences in electrophysiological activity between dim and bright light conditions. At lower daytime irradiance, daytime peaks in SCN spontaneous firing rate and membrane depolarisation were substantially depressed, leading to an overall marked reduction in the amplitude of circadian rhythms in spontaneous activity. Our data reveal a previously unappreciated impact of daytime light intensity on SCN physiology and the amplitude of circadian rhythms, and highlight the potential importance of daytime light exposure for circadian health.

neuroscience↗

bioRxiv2020

Using a bistable animal opsin for switchable and scalable optogenetic inhibition of neurons

There is no consensus on the best optogenetic tool for neuronal inhibition. Lamprey parapinopsin ( Lamplight) is a Gi/o-coupled bistable animal opsin that can be activated and deactivated by short and long wavelength light, respectively. Since native mechanisms of neuronal inhibition frequently employ Gi/o signalling, we asked here whether Lamplight could be used for optogenetic silencing. We show that short (405nm) and long (525nm) wavelength pulses repeatedly switch Lamplight between stable signalling active and inactive states, and that combining these wavelengths can be used to achieve intermediate levels of activity. We demonstrate that these properties can be applied to produce switchable and scalable neuronal hyperpolarisation, and suppression of spontaneous spike firing in the mouse hypothalamic suprachiasmatic nucleus. We show that expressing Lamplight in (predominantly) ON bipolar cells can photosensitise retinas following advanced photoreceptor degeneration, and that 405 and 525nm stimuli can produce responses of opposite sign in output neurons of the retina. Lamplight-driven responses to both activating (405nm) and deactivating (525nm) light can occur within 500ms and be elicited by intensities at least 10x below threshold for available inhibitory optogenetic tools. We conclude that Lamplight can co-opt endogenous signalling mechanisms to allow optogenetic inhibition that is scalable, sustained and rapidly reversible.

neuroscience↗

bioRxiv2020

Infra-slow modulation of fast beta/gamma oscillations in the mouse visual system.

Infra-slow (<0.02 Hz) and fast beta/gamma (20 - 100 Hz) oscillations in neurophysiological activity have been widely found in the subcortical visual system. While it is well established that fast beta/gamma oscillations are involved in visual processing, the role (if any) of infra-slow oscillations is currently unknown. One possibility is that infra-slow oscillations exert influence by modulating the amplitude of fast oscillations, yet the extent to which these different oscillations arise independently and interact remains unknown. We addressed these questions by recording in vivo spontaneous activity from subcortical visual system of visually intact mice, and animals whose retinal network was disrupted by advanced rod/cone degeneration (rd/rd cl) or melanopsin loss (Opn4-/- We found many neurons expressing only one type of oscillation, and indeed fast oscillations were absent in rd/rd cl. Conversely, neurons co-expressing the two oscillations were also common, and were encountered more often than expected by chance in visually intact but not Opn4-/- mice. Finally, where they co-occurred we found that beta/gamma amplitude was modulated by the infra-slow rhythm. Our data thus reveal that: 1.) infra-slow and beta-gamma oscillations are separable phenomena; and 2.) that they actively co-occur in a subset of neurones in which the phase of infra-slow oscillations define beta-gamma oscillation amplitude. These findings suggest that infra-slow oscillations could influence vision by modulating beta-gamma oscillations, and raise the possibility that disruptions in these oscillatory behaviours contribute to vision dysfunction in retinal dystrophy. KEY POINTS SUMMARYO_LINeurophysiological activity in the subcortical visual system fluctuates in both infra-slow and fast oscillatory ranges, however the level of co-occurrence and potential functional interaction of these rhythms is unknown. C_LIO_LIAnalyzing dark-adapted spontaneous activity in the mouse subcortical visual system, we find that these two types of oscillation interact uniquely through a population of neurons expressing both rhythms. C_LIO_LIGenetic ablation of rod/cone signaling potentiates infra-slow and abolishes fast beta/gamma oscillations while genetic ablation of melanopsin substantially diminishes the interaction between these two rhythms. C_LIO_LIOur results indicate that in an intact visual system the phase of infra-slow modulates fast beta/gamma oscillations. C_LIO_LIThus one possible impact of infra-slow oscillations in vision is to guide visual processing by interacting with fast narrowband oscillations. C_LI

neuroscience↗

bioRxiv2019

Phytotransferrin endocytosis mediates a direct cell surface-to-chloroplast iron trafficking axis in marine diatoms

Iron is a biochemically critical metal cofactor in enzymes involved in photosynthesis, cellular respiration, nitrate assimilation, nitrogen fixation, and reactive oxygen species defense. Marine microeukaryotes have evolved a phytotransferrin-based iron uptake system to cope with iron scarcity, a major factor limiting primary productivity in the global ocean. Diatom phytotransferrin is endocytosed, however proteins downstream of this environmentally ubiquitous iron receptor are unknown. We applied engineered ascorbate peroxidase APEX2-based subcellular proteomics to catalog proximal proteins of phytotransferrin in the model marine diatom Phaeodactylum tricornutum. Proteins encoded by poorly characterized iron-sensitive genes were identified including three that are expressed from a chromosomal gene cluster. Two of them showed unambiguous colocalization with phytotransferrin adjacent to the chloroplast. Further phylogenetic, domain, and biochemical analyses suggest their involvement in intracellular iron processing. Proximity proteomics holds enormous potential to glean new insights into iron acquisition pathways and beyond in these evolutionarily, ecologically, and biotechnologically important microalgae.

cell biology↗

bioRxiv2019

Evolved resistance to GAPDH inhibition results in loss of the Warburg Effect but retains a different state of glycolysis

Aerobic glycolysis or the Warburg Effect (WE) is characterized by increased glucose uptake and incomplete oxidation to lactate. Although ubiquitous, the biological role of the WE remains controversial and whether glucose metabolism is functionally different during fully oxidative glycolysis or during the WE is unknown. To investigate this question, we evolved resistance to koningic acid (KA), a natural product shown to be a specific inhibitor of glyceraldehyde-3-phosphate dehydrogenase (GAPDH), a rate-controlling glycolytic enzyme during the WE. We find that KA-resistant cells lose the WE but conduct glycolysis and surprisingly remain dependent on glucose and central carbon metabolism. Consequentially this altered state of glycolysis leads to differential metabolic activity and requirements including emergent activities in and dependencies on fatty acid metabolism. Together, these findings reveal that, contrary to some recent reports, aerobic glycolysis is a functionally distinct entity from conventional glucose metabolism and leads to distinct metabolic requirements and biological functions.

systems biology↗

bioRxiv2017

Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum functional genome and evolutionary origin of diatoms

Diatoms are one of the most successful and ecologically important groups of eukaryotic phytoplankton in the modern ocean. Deciphering their genomes is a key step towards better understanding of their biological innovations, evolutionary origins, and ecological underpinnings. Here, we have used 90 RNA-Seq datasets from different growth conditions combined with published expressed sequence tags and protein sequences from multiple taxa to explore the genome of the model diatom Phaeodactylum tricornutum, and introduce 1,489 novel genes. The new annotation additionally permitted the discovery for the first time of extensive alternative splicing (AS) in diatoms, including intron retention and exon skipping which increases the diversity of transcripts to regulate gene expression in response to nutrient limitations. In addition, we have used up-to-date reference sequence libraries to dissect the taxonomic origins of diatom genomes. We show that the P. tricornutum genome is replete in lineage-specific genes, with up to 47% of the gene models present only possessing orthologues in other stramenopile groups. Finally, we have performed a comprehensive de novo annotation of repetitive elements showing novel classes of TEs such as SINE, MITE, LINE and TRIM/LARD. This work provides a solid foundation for future studies of diatom gene function, evolution and ecology.

bioinformatics↗

bioRxiv2016

Diatom Centromeres Suggest a Novel Mechanism for Nuclear Gene Acquisition

Centromeres are essential for cell division and growth in all eukaryotes, and knowledge of their sequence and structure guides the development of artificial chromosomes for functional cellular biology studies. Centromeric proteins are conserved among eukaryotes; however, centromeric DNA sequences are highly variable. We combined forward and reverse genetic approaches with chromatin immunoprecipitation to identify centromeres of the model diatom Phaeodactylum tricornutum. Diatom centromere sequences contain low GC content regions and an abundance of long contiguous AT windows, but lack repeats or other conserved sequence features. Native and foreign sequences of similar GC content can maintain episomes and recruit the diatom centromeric histone protein CENP-A, suggesting non-native sequences can also function as diatom centromeres. Thus, simple sequence requirements enable DNA from foreign sources to incorporate into the nuclear genome repertoire as stable extra-chromosomal episomes, revealing a potential mechanism for bacterial and foreign eukaryotic DNA acquisition.

molecular biology↗