Responses of triacylglycerol synthesis in Skeletonema marinoi to nitrogen and phosphate starvations

Skeletonema marinoi is one of the most widespread marine planktonic diatoms in temperate coastal regions and sometimes can form massive blooms. Yet, the molecular mechanisms of triacylglycerol (TAG) synthesis in nutrient-deficient conditions for this species are still unknown. This study aimed to investigate how the TAG biosynthetic pathway of S. marinoi reacts to the culture age and nitrogen (N) or phosphorus (P) deficiency at molecular levels. Meanwhile, we also described the physiological and biochemical changes of S. marinoi in response to N or P starvation over time. To obtain reliable qRT-PCR data, six putative reference genes were identified for assessing expression stability using geNorm and BestKeeper software, and Actin exhibited the most stable expression across 45 tested S. marinoi samples. We found that the expression of TAG biosynthesis-related genes and ACCase enzyme activity varied in response to the different nutrient conditions and culture age. Taken together, we speculated that the capacity of TAG biosynthesis in S. marinoi is induced by N or P stress, and increases with culture age. Furthermore, TAG biosynthesis appears to respond more strongly to P deficiency than to N deficiency. Our study provides important insights into how diatoms regulate the TAG biosynthetic pathway when stressed by nutrient limitation. Besides, the data obtained from this study also provide useful clues for further exploring genes that can be used for metabolic engineering to enhance lipid production.     

Resting Stages of Skeletonema marinoi Assimilate Nitrogen From the Ambient Environment Under Dark,Anoxic Conditions

The planktonic marine diatom Skeletonema marinoi forms resting stages, which can survive for decades buried in aphotic, anoxic sediments and resume growth when re-exposed to light, oxygen, and nutrients. The mechanisms by which they maintain cell viability during dormancy are poorly known. Here, we investigated cell-specific nitrogen (N) and carbon (C) assimilation and survival rate in resting stages of three S. marinoi strains. Resting stages were incubated with stable isotopes of dissolved inorganic N (DIN), in the form of ¹⁵N-ammonium (NH₄⁺) or -nitrate (NO₃⁻) and dissolved inorganic C (DIC) as ¹³C-bicarbonate (HCO₃⁻) under dark and anoxic conditions for 2 months. Particulate C and N concentration remained close to the Redfield ratio (6.6) during the experiment, indicating viable diatoms. However, survival varied between <0.1% and 47.6% among the three different S. marinoi strains, and overall survival was higher when NO₃⁻ was available. One strain did not survive in the NH₄⁺ treatment. Using secondary ion mass spectrometry (SIMS), we quantified assimilation of labeled DIC and DIN from the ambient environment within the resting stages. Dark fixation of DIC was insignificant across all strains. Significant assimilation of ¹⁵N-NO₃⁻ and ¹⁵N-NH₄⁺ occurred in all S. marinoi strains at rates that would double the nitrogenous biomass over 77–380 years depending on strain and treatment. Hence, resting stages of S. marinoi assimilate N from the ambient environment at slow rates during darkness and anoxia. This activity may explain their well-documented long survival and swift resumption of vegetative growth after dormancy in dark and anoxic sediments.     

Microbes mediating the sulfur cycle in the Atlantic Ocean and their link to chemolithoautotrophy

Only about 10%–30% of the organic matter produced in the epipelagic layers reaches the dark ocean. Under these limiting conditions, reduced inorganic substrates might be used as an energy source to fuel prokaryotic chemoautotrophic and/or mixotrophic activity. The aprA gene encodes the alpha subunit of the adenosine-5′-phosphosulfate (APS) reductase, present in sulfate-reducing (SRP) and sulfur-oxidizing prokaryotes (SOP). The sulfur-oxidizing pathway can be coupled to inorganic carbon fixation via the Calvin–Benson–Bassham cycle. The abundances of aprA and cbbM, encoding RuBisCO form II (the key CO₂ fixing enzyme), were determined over the entire water column along a latitudinal transect in the Atlantic from 64°N to 50°S covering six oceanic provinces. The abundance of aprA and cbbM genes significantly increased with depth reaching the highest abundances in meso- and upper bathypelagic layers. The contribution of cells containing these genes also increased from mesotrophic towards oligotrophic provinces, suggesting that under nutrient limiting conditions alternative energy sources are advantageous. However, the aprA/cbbM ratios indicated that only a fraction of the SOP is associated with inorganic carbon fixation. The aprA harbouring prokaryotic community was dominated by Pelagibacterales in surface and mesopelagic waters, while Candidatus Thioglobus, Chromatiales and the Deltaproteobacterium_SCGC dominated the bathypelagic realm. Noticeably, the contribution of the SRP to the prokaryotic community harbouring aprA gene was low, suggesting a major utilization of inorganic sulfur compounds either as an energy source (occasionally coupled with inorganic carbon fixation) or in biosynthesis pathways.     

Nodule-enhanced expression of a sucrose phosphate synthase gene member (MsSPSA) has a role in carbon and nitrogen metabolism in the nodules of alfalfa (Medicago sativa L.)

Sucrose phosphate synthase (SPS) catalyzes the first step in the synthesis of sucrose in photosynthetic tissues. We characterized the expression of three different isoforms of SPS belonging to two different SPS gene families in alfalfa (Medicago sativa L.), a previously identified SPS (MsSPSA) and two novel isoforms belonging to class B (MsSPSB and MsSPSB3). While MsSPSA showed nodule-enhanced expression, both MsSPSB genes exhibited leaf-enhanced expression. Alfalfa leaf and nodule SPS enzymes showed differences in chromatographic and electrophoretic migration and differences in V _max and allosteric regulation. The root nodules in legume plants are a strong sink for photosynthates with its need for ATP, reducing power and carbon skeletons for dinitrogen fixation and ammonia assimilation. The expression of genes encoding SPS and other key enzymes in sucrose metabolism, sucrose phosphate phosphatase and sucrose synthase, was analyzed in the leaves and nodules of plants inoculated with Sinorhizobium meliloti. Based on the expression pattern of these genes, the properties of the SPS isoforms and the concentration of starch and soluble sugars in nodules induced by a wild type and a nitrogen fixation deficient strain, we propose that SPS has an important role in the control of carbon flux into different metabolic pathways in the symbiotic nodules.     

Assessing the influence of physical,geochemical and biological factors on anaerobic microbial primary productivity within hydrothermal vent chimneys

Chemosynthetic primary production supports hydrothermal vent ecosystems, but the extent of that productivity and its governing factors have not been well constrained. To better understand anaerobic primary production within massive vent deposits, we conducted a series of incubations at 4, 25, 50 and 90 °C using aggregates recovered from hydrothermal vent structures. We documented in situ geochemistry, measured autochthonous organic carbon stable isotope ratios and assessed microbial community composition and functional gene abundances in three hydrothermal vent chimney structures from Middle Valley on the Juan de Fuca Ridge. Carbon fixation rates were greatest at lower temperatures and were comparable among chimneys. Stable isotope ratios of autochthonous organic carbon were consistent with the Calvin–Benson–Bassham cycle being the predominant mode of carbon fixation for all three chimneys. Chimneys exhibited marked differences in vent fluid geochemistry and microbial community composition, with structures being differentially dominated by gamma (γ) or epsilon (ε) proteobacteria. Similarly, qPCR analyses of functional genes representing different carbon fixation pathways showed striking differences in gene abundance among chimney structures. Carbon fixation rates showed no obvious correlation with observed in situ vent fluid geochemistry, community composition or functional gene abundance. Together, these data reveal that (i) net anaerobic carbon fixation rates among these chimneys are elevated at lower temperatures, (ii) clear differences in community composition and gene abundance exist among chimney structures, and (iii) tremendous spatial heterogeneity within these environments likely confounds efforts to relate the observed rates to in situ microbial and geochemical factors. We also posit that microbes typically thought to be mesophiles are likely active and growing at cooler temperatures, and that their activity at these temperatures comprises the majority of endolithic anaerobic primary production in hydrothermal vent chimneys.     

Time series metagenomic sampling of the Thermopyles,Greece, geothermal springs reveals stable microbial communities dominated by novel sulfur-oxidizing chemoautotrophs

Geothermal springs are essentially unaffected by environmental conditions aboveground as they are continuously supplied with subsurface water with little variability in chemistry. Therefore, changes in their microbial community composition and function, especially over a long period, are expected to be limited but this assumption has not yet been rigorously tested. Toward closing this knowledge gap, we applied whole metagenome sequencing to 17 water samples collected between 2010 and 2016 from the Thermopyles sulfur-rich geothermal springs in central Greece. As revealed by 16S rRNA gene fragments recovered in the metagenomes, Epsilonproteobacteria-related operational taxonomic units (OTUs) dominated most samples and grouping of samples based on OTU abundances exhibited no apparent seasonal pattern. Similarities between samples regarding functional gene content were high, with all samples sharing >70% similarity in functional pathways. These community-wide patterns were further confirmed by analysis of metagenome-assembled genomes (MAGs), which showed that novel species and genera of the chemoautotrophic Campylobacterales order dominated the springs. These MAGs carried different pathways for thiosulfate or sulfide oxidation coupled to carbon fixation pathways. Overall, our study showed that even in the long term, functions of microbial communities in a moderately hot terrestrial spring remain stable, presumably driving the corresponding stability in community structure.     

Metagenomic and 14C tracing evidence for autotrophic microbial CO2 fixation in paddy soils

Autotrophic carbon dioxide (CO₂) fixation by microbes is ubiquitous in the environment and potentially contributes to the soil organic carbon (SOC) pool. However, the multiple autotrophic pathways of microbial carbon assimilation and fixation in paddy soils remain poorly characterized. In this study, we combine metagenomic analysis with ¹⁴C-labelling to investigate all known autotrophic pathways and CO₂ assimilation mechanisms in five typical paddy soils from southern China. Marker genes of six autotrophic pathways are detected in all soil samples, which are dominated by the cbbL genes (67%–82%) coding the ribulose-bisphosphate carboxylase large chain in the Calvin cycle. These marker genes are associated with a broad range of phototrophic and chemotrophic genera. Significant amounts of ¹⁴C-CO₂ are assimilated into SOC (74.3–175.8 mg ¹⁴C kg⁻¹) and microbial biomass (5.2–24.1 mg ¹⁴C kg⁻¹) after 45 days incubation, where more than 70% of ¹⁴C-SOC was concentrated in the relatively stable humin fractions. These results show that paddy soil microbes contain the genetic potential for autotrophic carbon fixation spreading over broad taxonomic ranges, and can incorporate atmospheric carbon into organic components, which ultimately contribute to the stable SOC pool.     

Metagenomic assessment of a sulfur-oxidizing enrichment culture derived from marine sediment

The biological oxidation of reduced sulfur compounds is a critically important process in global sulfur biogeochemistry. In this study, we enriched from marine sediments under denitrifying conditions, chemolithotrophic sulfur oxidizers that could oxidize a variety of reduced sulfur compounds: thiosulfate, tetrathionate, sulfide, and polysulfide. Two major phylotypes of 16S rRNA gene (>99% identity in each phylotype) were detected in this enrichment culture. In order to characterize sulfide oxidation, we sequenced and characterized one fosmid clone (43.6 kb) containing the group I sulfide-quinone reductase (sqr) gene. Interestingly, four putative rhodanese genes were found in this clone. Furthermore, comparative alignment with the closest genome of Thiomicrospira crunogena XCL2 revealed that three homologous genes were located within the vicinity of the sqr gene. Fosmid clones harboring carbon fixation (cbbL and cbbM) and denitrification (narG) genes were screened, and the phylogeny of the functional genes was analyzed. Along with the comparison between the sqr-containing fosmid clones and the relevant -proteobacteria, our phylogenetic study based on the 16S rRNA gene and carbon fixation genes suggest the prevalence of chemolithotrophic -proteobacteria in the denitrifying cultures. The findings of this study imply that a combination of cultivation and metagenomic approaches might provide us with a glimpse into the characteristics of sulfur oxidizers in marine sediments.     

Heterotrophic carbon metabolism and energy acquisition in Candidatus Thioglobus singularis strain PS1, a member of the SUP05 clade of marine Gammaproteobacteria

A hallmark of the SUP05 clade of marine Gammaproteobacteria is the ability to use energy obtained from reduced inorganic sulfur to fuel autotrophic fixation of carbon using RuBisCo. However, some SUP05 also have the genetic potential for heterotrophic growth, raising questions about the roles of SUP05 in the marine carbon cycle. We used genomic reconstructions, physiological growth experiments and proteomics to characterize central carbon and energy metabolism in Candidatus Thioglobus singularis strain PS1, a representative from the SUP05 clade that has the genetic potential for autotrophy and heterotrophy. Here, we show that the addition of individual organic compounds and 0.2 μm filtered diatom lysate significantly enhanced the growth of this bacterium. This positive growth response to organic substrates, combined with expression of a complete TCA cycle, heterotrophic pathways for carbon assimilation, and methylotrophic pathways for energy conversion demonstrate strain PS1's capacity for heterotrophic growth. Further, our inability to verify the expression of RuBisCO suggests that carbon fixation was not critical for growth. These results highlight the metabolic diversity of the SUP05 clade that harbours both primary producers and consumers of organic carbon in the oceans and expand our understanding of specific pathways of organic matter oxidation by the heterotrophic SUP05.