Gene expression under multiple stressors in Daphnia pulex

Freshwater organisms are constantly under pressure from an array of stressors with complex affects. In this thesis I first review the interactive effects on Daphnia pulex of three prevalent anthropogenic stressors: climate change, calcium decline, and toxic metal exposure. Then, I examine gene expression levels of five Daphnia genes related to carapace building and calcium homeostasis to understand the effects and interaction of low calcium and predator presence. Finally, I use phylogenetic reconstruction to explore the evolutionary history of one of the tested genes, the Sarco(endo)plasmic Calcium ATPase. My results indicate that both stressors tested and their interaction affects the expression patterns of all the tested genes, often in surprising ways. The results from the phylogenetic reconstruction suggest that many ancient and recent gene duplication events have shaped the evolution of this gene

Comparative Transcriptomics of Cold Growth and Adaptive Features of a Eury- and Steno-Psychrophile

Permafrost subzero environments harbor diverse, active communities of microorganisms. However, our understanding of the subzero growth, metabolisms, and adaptive properties of these microbes remains very limited. We performed transcriptomic analyses on two subzero-growing permafrost isolates with different growth profiles in order to characterize and compare their cold temperature growth and cold-adaptive strategies. The two organisms, Rhodococcus sp. JG3 (-5 to 30°C) and Polaromonas sp. Eur3 1.2.1 (-5 to 22°C), shared several common responses during low temperature growth, including induction of translation and ribosomal processes, upregulation of nutrient transport, increased oxidative and osmotic stress responses, and stimulation of polysaccharide capsule synthesis. Recombination appeared to be an important adaptive strategy for both isolates at low temperatures, likely as a mechanism to increase genetic diversity and the potential for survival in cold systems. While Rhodococcus sp. JG3 favored upregulating iron and amino acid transport, sustaining redox potential, and modulating fatty acid synthesis and composition during growth at -5°C compared to 25°C, Polaromonas sp. Eur3 1.2.1 increased the relative abundance of transcripts involved in primary energy metabolism and the electron transport chain, in addition to signal transduction and peptidoglycan synthesis at 0°C compared to 20°C. The increase in energy metabolism may explain why Polaromonas sp. Eur3 1.2.1 is able to sustain growth rates at 0°C comparable to those at higher temperatures. For Rhodococcus sp. JG3, flexibility in use of carbon sources, iron acquisition, control of membrane fatty acid composition, and modulating redox and co-factor potential may be ways in which this organism is able to sustain growth over a wider range of temperatures. Increasing our understanding of the microbes in these habitats helps us better understand active pathways and metabolisms in extreme environments. Identifying novel, thermolabile, and cold-active enzymes from studies such as this is also of great interest to the biotechnology and food industries

Geomicrobiological heterogeneity of lithic habitats in the extreme environment of Antarctic nunataks: a potential early Mars analog

Nunataks are permanent ice-free rocky peaks that project above ice caps in polar regions, thus being exposed to extreme climatic conditions throughout the year. They undergo extremely low temperatures and scarcity of liquid water in winter, while receiving high incident and reflected (albedo) UVA-B radiation in summer. Here, we investigate the geomicrobiology of the permanently exposed lithic substrates of nunataks from Livingston Island (South Shetlands, Antarctic Peninsula), with focus on prokaryotic community structure and their main metabolic traits. Contrarily to first hypothesis, an extensive sampling based on different gradients and multianalytical approaches demonstrated significant differences for most geomicrobiological parameters between the bedrock, soil, and loose rock substrates, which overlapped any other regional variation. Brevibacillus genus dominated on bedrock and soil substrates, while loose rocks contained a diverse microbial community, including Actinobacteria, Alphaproteobacteria and abundant Cyanobacteria inhabiting the milder and diverse microhabitats within. Archaea, a domain never described before in similar Antarctic environments, were also consistently found in the three substrates, but being more abundant and potentially more active in soils. Stable isotopic ratios of total carbon (δ 13C) and nitrogen (δ 15N), soluble anions concentrations, and the detection of proteins involved in key metabolisms via the Life Detector Chip (LDChip), suggest that microbial primary production has a pivotal role in nutrient cycling at these exposed areas with limited deposition of nutrients. Detection of stress-resistance proteins, such as molecular chaperons, suggests microbial molecular adaptation mechanisms to cope with these harsh conditions. Since early Mars may have encompassed analogous environmental conditions as the ones found in these Antarctic nunataks, our study also contributes to the understanding of the metabolic features and biomarker profiles of a potential Martian microbiota, as well as the use of LDChip in future life detection missions.This project has been funded by the Spanish Ministry of Science and Innovation (MICINN)/European Regional Development Fund (FEDER) project no. RTI2018-094368-B-I00; the European Research Council Consolidator grant no. 818602; and the Spanish State Research Agency (AEI) project no. MDM-2017-0737, Unidad de Excelencia “María de Maeztu” to Centro de Astrobiología

The impact of an active soil microbial community on greenhouse gas emissions in Arctic cryosols

Anthropogenic climate change is thought to have a disproportionately larger impact on polar regions, resulting in permafrost thaw and microorganism mediated greenhouse gas (GHG) emission. Permafrost soils contain between 25-50 % of the total soil organic carbon pool and as permafrost thaws, this carbon will become accessible to microbial degradation. Carbon dioxide (CO2), methane (CH4), and nitrous oxide (N2O) are the most important GHGs and their flux from permafrost affected soils contributes to a positive feedback loop of climatic warming. However, our understanding of how microorganisms contribute to the biogeochemical cycling and flux of these gases in Arctic soils remains limited. Topography of the Arctic landscape has a significant impact on GHG emissions as evidenced by the flux at the ice-wedge polygon (IWP) terrain. The wetter tough soils exhibited higher emissions of CO2 and N2O, but lower uptake of CH4, compared to the drier polygon interior soils. The elevated CO2 and N2O fluxes, and the lower CH4 uptake from troughs is concerning from a climate warming perspective since parts of the Arctic are predicted to become warmer and wetter. Topography also affected the composition of the overall microbial community, with the trough soils having a higher proportion of Betaproteobacteria, Deltaproteobacteria, and Bacterioidetes but a lower proportion of Acidobacteria compared to polygon interior soils. The community of nitrogen fixers, methanotrophs, and denitrifiers was also affected by the topography with all three groups showing unique structures. Overall, members of the nitrogen-fixing and denitrifying bacteria included Rhizobiaceae, Nostocaceae, Cyanothecaceae, Rhodobacteraceae, Burkholderiaceae, Chloroflexaceae, Azotobacteraceae, and Ectothiorhodospiraceae. Moreover, these organisms appear to be active in the soils, as metatranscriptomic RNA analysis was also able to detect these microbial clades. The active methanotrophs in these soils are likely part of the USCα cluster of currently uncultured high-affinity methanotrophs, as evidenced by stable isotope probing (SIP) of soils exhibiting CH4 uptake. SIP analysis coupled with metagenome binning lead to the identification of several intermediate-high quality MAGs (metagenome assemble genomes). One Alphaproteobacterial MAG was identified that contained many of the methane cycling genes including a soluble methane monooxygenase (mmoX) and genes involved in the serine cycle for assimilating formaldehyde characteristic of type II methanotrophs. This MAG also contained genes for ammonia assimilation, biopolymer production, and mercury detoxification. In addition to identifying non-culturable members of the community through metagenome binning, sequencing of culturable isolates reveal presence of carbon cycling genes involved in fermentation, CO2 fixation, denitrification, polysaccharide and aromatic compound metabolism. Suggesting that the microbial community at the IWP terrain is poised to degrade the thawing carbon stores in permafrost. In addition to topography affecting the microbial community structure, key microbial members across the IWP terrain also appear to have positive and negative impacts on other microbial species. This was determined by developing a novel hybrid network analysis to determine species interactions within of the microbial community. Overall, members of Proteobacteria, Candidatus Rokubacteria, and Actinobacteria phyla tended to have a positive impact, while members of Verrucomicrobia and Acidobacteria had a negative impact on other microbials members. These results indicate that both environmental abiotic parameters and biotic interactions impact the microbial community structure and possibly GHG fluxes from soils.Les changements climatiques d'origine anthropogénique ont un impact plus important dans les régions polaires, provoquant la fonte du pergélisol et l'émission de gaz à effet de serre (GES) par les microorganismes. Le pergélisol contient entre 25 et 50 pourcents du réservoir total de carbone organique et, avec la fonte du pergélisol, ce carbone devient accessible à la dégradation microbienne. Le dioxyde de carbone (CO2), le méthane (CH4) et le protoxyde d'azote (N2O) sont les GES les plus importants et leurs flux provenant de la fonte du pergélisol contribuent à une boucle de rétroaction positive sur le réchauffement du climat. Toutefois, notre compréhension du rôle des microorganismes sur les cycles biogéochimiques et sur les flux de ces gaz dans les sols arctiques est limitée. La topographie des paysages arctiques a elle aussi un impact important sur l'émission de GES, comme le démontrent les flux de gaz en terrain « ice-wedge polygon » (IWP), topographie retrouvée dans l'Arctique. Les sols des arêtes des polygones sont plus creux et plus humides. Ils présentent un plus haut taux d'émission de CO2 et de N2O, mais une plus faible absorption de CH4 que les sols plus secs de l'intérieur des polygones. Les flux de CO2 et de N2O élevés ainsi que la faible absorption de CH4 provenant des arêtes des polygones sont particulièrement inquiétants dans une perspective de changements climatiques puisque plusieurs régions arctiques devraient devenir plus chaudes et humides. La topographie affecte aussi la composition des communautés microbiennes. Les communautés de fixateurs d'azote, de méthanotrophes et de microorganismes dénitrifiants sont aussi affectées par la topographie, puisque chacun de ces groupes présente une structure unique. De plus, ces organismes semblent être actifs dans les sols, puisque ces clades ont pu être détectés grâce à une analyse de métatranscriptomique. Les méthanotrophes actifs dans ces sols font partie du groupe USCα de microorganismes incultivés, soupçonné d'être des méthanotrophes ayant une haute affinité pour le méthane, démontré par la méthode de sonde à isotope stable (SIP), utilisant des sols démontrant une absorption de CH4. L'analyse SIP couplée au binning de métagénome a conduit à l'identification de plusieurs MAG (metagenome assembled genomes) de qualité moyenne à supérieure. Nous avons identifié un MAG Alphaproteobacterial qui contient plusieurs gènes associés au cycle du méthane, incluant une monooxygénase soluble de méthane (mmoX) et des gènes impliqués dans le cycle de la serine, pour assimiler le formaldéhyde, caractéristique des méthanotrophes de Type II. En plus d'identifier des membres non cultivables de la communauté microbienne par binning de métagénome, le séquençage des membres cultivables a révélé la présence de gènes du cycle du carbone impliqués dans la fermentation, la fixation du CO2, la dénitrification, ainsi que dans le métabolisme des polysaccharides et le métabolisme des composés aromatiques. Ceci suggère que la communauté microbienne des terrains IWP est prête à dégrader les réserves de carbone disponible suite à la fonte du pergélisol. En plus de la topographie qui affecte la structure des communautés microbiennes, certains microorganismes clés du terrain IWP semblent aussi avoir un impact, positif ou négatif, sur les autres espèces microbiennes de la communauté. Ces relations ont été déterminé grâce au développement d'un nouveau modèle hydride d'analyse de réseau de la communauté microbienne. Dans l'ensemble, les membres des phyla Proteobacteria, Candidatus Rokubacteria, et Actinobacteria ont tendance à avoir un impact positif, alors que des membres Verrucomicrobia et des Acidobacteria des ont un impact négatif sur les autres microorganismes. Ces résultats indiquent que les paramètres environnementaux abiotiques et les interactions biotiques modifient la structure de la communauté microbienne et ont possiblement un impact sur les flux de GES provenant des sols

Characterizing biofilms and their associated biosignatures in an Arctic hypersaline cold spring Mars analog

The last surface-level aqueous environments on Mars were likely sulfurous brines that formed as the climate cooled and large bodies of water receded during the transition from the wet Noachian to the dry Hesperian (4.1 – 3.0 Gya). To understand the diversity of microorganisms that could have inhabited such environments and their associated biosignatures, we turn to analogous environments on Earth. Here we investigated biofilm communities and their associated biosignatures at Gypsum Hill, (GH), a perennial cold spring system located at nearly 80°N on Axel Heiberg Island in the Canadian high Arctic. The biofilms develop during the summer months alongside the oligotrophic and sulphur rich GH brines and spread out along the flood plains formed by meltwater and spring run-off. Our objective was to link the microbial community structure of the biofilms to geochemical changes across the GH site as an analog to the micro-niches that could have formed during the recession of an ancient Martian Ocean. We collected 14 morphologically distinct biofilms over two field season and found that minor variations in chemistry between proximal sites impacted community structure. 16S amplicon sequencing revealed that biofilms closest to outflow channels were dominated by sulfur oxidizing bacteria, suggesting that primary production may be driven by chemolithoautotrophy. The community structure shifted towards more heterotrophic and phototrophic populations the further the biofilms appeared from a spring source. Microbial eukaryotes at the GH site were investigated for the first time through 18S sequencing with diatoms and photoautotrophic algae dominating all biofilms. Lastly, we linked the biofilm communities to potential biosignatures by examining lipid profiles to help guide the search and identification of potential remnants of hypothetical ancient Martian life.

Characterizing biofilms and their associated biosignatures in an Arctic hypersaline cold spring Mars analog

The last surface-level aqueous environments on Mars were likely sulfurous brines that formed as the climate cooled and large bodies of water receded during the transition from the wet Noachian to the dry Hesperian (4.1 – 3.0 Gya). To understand the diversity of microorganisms that could have inhabited such environments and their associated biosignatures, we turn to analogous environments on Earth. Here we investigated biofilm communities and their associated biosignatures at Gypsum Hill, (GH), a perennial cold spring system located at nearly 80°N on Axel Heiberg Island in the Canadian high Arctic. The biofilms develop during the summer months alongside the oligotrophic and sulphur rich GH brines and spread out along the flood plains formed by meltwater and spring run-off. Our objective was to link the microbial community structure of the biofilms to geochemical changes across the GH site as an analog to the micro-niches that could have formed during the recession of an ancient Martian Ocean. We collected 14 morphologically distinct biofilms over two field season and found that minor variations in chemistry between proximal sites impacted community structure. 16S amplicon sequencing revealed that biofilms closest to outflow channels were dominated by sulfur oxidizing bacteria, suggesting that primary production may be driven by chemolithoautotrophy. The community structure shifted towards more heterotrophic and phototrophic populations the further the biofilms appeared from a spring source. Microbial eukaryotes at the GH site were investigated for the first time through 18S sequencing with diatoms and photoautotrophic algae dominating all biofilms. Lastly, we linked the biofilm communities to potential biosignatures by examining lipid profiles to help guide the search and identification of potential remnants of hypothetical ancient Martian life.

Correction to: A longitudinal census of the bacterial community in raw milk correlated with Staphylococcus aureus clinical mastitis infections in dairy cattle

BACKGROUND: Staphylococcus aureus is a common cause of clinical mastitis (CM) in dairy cattle. Optimizing the bovine mammary gland microbiota to resist S. aureus colonization is a growing area of research. However, the details of the interbacterial interactions between S. aureus and commensal bacteria, which would be required to manipulate the microbiome to resist infection, are still unknown. This study aims to characterize changes in the bovine milk bacterial community before, during, and after S. aureus CM and to compare bacterial communities present in milk between infected and healthy quarters. METHODS: We collected quarter-level milk samples from 698 Holstein dairy cows over an entire lactation. A total of 11 quarters from 10 cows were affected by S. aureus CM and milk samples from these 10 cows (n = 583) regardless of health status were analyzed by performing 16S rRNA gene amplicon sequencing. RESULTS: The milk microbiota of healthy quarters was distinguishable from that of S. aureus CM quarters two weeks before CM diagnosis via visual inspection. Microbial network analysis showed that 11 OTUs had negative associations with OTU0001 (Staphylococcus). A low diversity or dysbiotic milk microbiome did not necessarily correlate with increased inflammation. Specifically, Staphylococcus xylosus, Staphylococcus epidermidis, and Aerococcus urinaeequi were each abundant in milk from the quarters with low levels of inflammation. CONCLUSION: Our results show that the udder microbiome is highly dynamic, yet a change in the abundance in certain bacteria can be a potential indicator of future S. aureus CM. This study has identified potential prophylactic bacterial species that could act as a barrier against S. aureus colonization and prevent future instances of S. aureus CM. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1186/s42523-022-00211-x

Sulfur-cycling chemolithoautotrophic microbial community dominates a cold, anoxic, hypersaline Arctic spring

Abstract Background Gypsum Hill Spring, located in Nunavut in the Canadian High Arctic, is a rare example of a cold saline spring arising through thick permafrost. It perennially discharges cold (~ 7 °C), hypersaline (7–8% salinity), anoxic (~ 0.04 ppm O2), and highly reducing (~ − 430 mV) brines rich in sulfate (2.2 g.L−1) and sulfide (9.5 ppm), making Gypsum Hill an analog to putative sulfate-rich briny habitats on extraterrestrial bodies such as Mars. Results Genome-resolved metagenomics and metatranscriptomics were utilized to describe an active microbial community containing novel metagenome-assembled genomes and dominated by sulfur-cycling Desulfobacterota and Gammaproteobacteria. Sulfate reduction was dominated by hydrogen-oxidizing chemolithoautotrophic Desulfovibrionaceae sp. and was identified in phyla not typically associated with sulfate reduction in novel lineages of Spirochaetota and Bacteroidota. Highly abundant and active sulfur-reducing Desulfuromusa sp. highly transcribed non-coding RNAs associated with transcriptional regulation, showing potential evidence of putative metabolic flexibility in response to substrate availability. Despite low oxygen availability, sulfide oxidation was primarily attributed to aerobic chemolithoautotrophic Halothiobacillaceae. Low abundance and transcription of photoautotrophs indicated sulfur-based chemolithoautotrophy drives primary productivity even during periods of constant illumination. Conclusions We identified a rare surficial chemolithoautotrophic, sulfur-cycling microbial community active in a unique anoxic, cold, hypersaline Arctic spring. We detected Mars-relevant metabolisms including hydrogenotrophic sulfate reduction, sulfur reduction, and sulfide oxidation, which indicate the potential for microbial life in analogous S-rich brines on past and present Mars. Video Abstrac

Synergistic interactions of biotic and abiotic environmental stressors on gene expression

Understanding the response of organisms to multiple stressors is critical for predicting if populations can adapt to rapid environmental change. Natural and anthropogenic stressors often interact, complicating general predictions. In this study, we examined the interactive and cumulative effects of two common environmental stressors, lowered calcium concentration, an anthropogenic stressor, and predator presence, a natural stressor, on the water flea Daphnia pulex. We analyzed expression changes of five genes involved in calcium homeostasis — cuticle proteins (Cutie, Icp2), calbindin (Calb), and calcium pump and channel (Serca and Ip3R) — using real-time quantitative PCR (RT-qPCR) in a full factorial experiment. We observed strong synergistic interactions between low calcium concentration and predator presence. While the Ip3R gene was not affected by the stressors, the other four genes were affected in their transcriptional levels by the combination of the stressors. Transcriptional patterns of genes that code for cuticle proteins (Cutie and Icp2) and a sarcoplasmic calcium pump (Serca) only responded to the combination of stressors, changing their relative expression levels in a synergistic response, while a calcium-binding protein (Calb) responded to low calcium stress and the combination of both stressors. The expression pattern of these genes (Cutie, Icp2, and Serca) were nonlinear, yet they were dose dependent across the calcium gradient. Multiple stressors can have complex, often unexpected effects on ecosystems. This study demonstrates that the dominant interaction for the set of tested genes appears to be synergism. We argue that gene expression patterns can be used to understand and predict the type of interaction expected when organisms are exposed simultaneously to natural and anthropogenic stressors. </jats:p

The Evolutionary History of Sarco(endo)plasmic Calcium ATPase (SERCA)

<div><p>Investigating the phylogenetic relationships within physiologically essential gene families across a broad range of taxa can reveal the key gene duplication events underlying their family expansion and is thus important to functional genomics studies. P-Type II ATPases represent a large family of ATP powered transporters that move ions across cellular membranes and includes Na⁺/K⁺ transporters, H⁺/K⁺ transporters, and plasma membrane Ca²⁺ pumps. Here, we examine the evolutionary history of one such transporter, the Sarco(endo)plasmic reticulum calcium ATPase (SERCA), which maintains calcium homeostasis in the cell by actively pumping Ca²⁺ into the sarco(endo)plasmic reticulum. Our protein-based phylogenetic analyses across Eukaryotes revealed two monophyletic clades of SERCA proteins, one containing animals, fungi, and plants, and the other consisting of plants and protists. Our analyses suggest that the three known SERCA proteins in vertebrates arose through two major gene duplication events after the divergence from tunicates, but before the separation of fishes and tetrapods. In plants, we recovered two SERCA clades, one being the sister group to Metazoa and the other to Apicomplexa clade, suggesting an ancient duplication in an early eukaryotic ancestor, followed by subsequent loss of one copy in Opisthokonta, the other in protists, and retention of both in plants. We also report relatively recent and independent gene duplication events within invertebrate taxa including tunicates and the leech <em>Helobdella robusta</em>. Thus, it appears that both ancient and recent gene duplication events have played an important role in the evolution of this ubiquitous gene family across the eukaryotic domain.</p> </div