Report
Teaser, summary, work performed and final results
Periodic Reporting for period 2 - APPELS (A Probe of the Periodic Elements for Life in the Sea)
Teaser
\"The problem: Metals are a fundamental component of life. Biological processing of oxygen, nitrogen, sulphur and carbon all rely on metalloproteins. But less than half of all metalloproteins are characterised and the genome remains opaque to inference of metal binding and...
Summary
\"The problem: Metals are a fundamental component of life. Biological processing of oxygen, nitrogen, sulphur and carbon all rely on metalloproteins. But less than half of all metalloproteins are characterised and the genome remains opaque to inference of metal binding and optimal concentrations for growth. APPELS aims to unveil, via cutting-edge techniques, a comprehensive view of the essential elements to life in the ocean, at the heart of the carbon cycle and global change, and will expand the dictionary of metal-binding motifs, applicable across the tree of life.
The objectives:
The overarching objective of APPELS is to probe the expanse of the periodic table to provide a comprehensive definition of the elements required by the modern marine phytoplankton (the metallome) and the marine metalloproteome. APPELS will revolutionise our understanding of metal-binding by proteins and the required elements that control the efficacy of biological pumping of carbon to depth in the ocean:
• Explore which, as yet untested, elements are biologically important to 5 “model†marine phytoplankton, representative of contrasting marine chemotypes. Elements of interest include those suspected of importance (B, Si, V, Se, Cd, I), those thought to be of use to some species (Ti, Sn, Br) and those emerging from novel approaches to be part of life’s metallome (Sr, Ba, W, As, U, Pb, Ge);
• At the physiological level, delineate the concentrations for maximal growth for all “suspected†elements, and identify any limitation implying requirement/use and the toxic threshold hence defining the “elemental sweet-spotâ€
• Provide a complete elemental fingerprint of the metalloproteome of our 5 model organisms by coupling liquid chromatographic (LC) separation techniques with protein quantification and Quad- ICPMS elemental analysis,
• Refine separation towards individual metalloproteins, particularly of unusual chemistry, to identify and characterise the interlinking between the selected metal, with coordination sites and protein function. Use combined peaks in protein abundance, and unusual element content to select cellular
fractions worthy of refined separation to the individual protein level via LC, SDS- and nondenaturing PAGE gel. Identify the single proteins via high throughput tandem mass-spectrometry HT MS/MS with Mascot analysis
• Assess concentration thresholds for metal competition and toxic interference (Cd, and Pb) within the metalloproteome by analysis of model organisms grown within a range of chemistries
• Recombinant expression of novel metalloproteins within E. coli to confirm metal-selection at the active site, and to explore metal isotopic fractionation during enzyme assembly
Importance to Society:
Trace metals lie at the heart of human health but are an underappreciated component of the human diet. Increasingly, getting the \"\"right amount\"\" of trace metals is being recognised as necessary for human health with elements such as in iron, selenium and iodine being identified as at most risk of deficiency whereas anthropogenically perturbed metals in the food chain such as arsenic, mercury and thallium pose a toxic risk. This ambitious, project aims to define the metallome and metalloproteome for marine life, ultimately the ancient ancestor of life on land and human beings. The probing of the farther reaches of the periodic table for biological use within APPELS will deliver an expansive definition of the metallome for marine life and the range of elemental concentrations that allow maximum growth of diverse phytoplankton chemotypes. It will pinpoint limiting and toxic thresholds for photosynthesisers, key to the chemical controls on the biological pump of atmospheric pCO2, and with implications for marine productivity in a future perturbed ocean and how elements will enter the marine foodchain. These “elemental sweetspots†allow powerful insight into the geological past in terms of how evolving chemistry d\"
Work performed
1) We have developed and optimised a novel method for direct analysis of 32 elements simultaneously in small volume of cell lysate in buffers with a high salt matrix (800µL, up to 30% TDS). The method allows a high throughput analysis of complex samples (see Zhang et al., 2018: Direct measurement of multi-elements in high matrix samples with a flow injection ICP-MS: application to the extended Emiliania huxleyi Redfield ratio Q Zhang, JT Snow, P Holdship, D Price, P Watson, REM Rickaby, Journal of Analytical Atomic Spectrometry) which was presented as a poster at ICOBTE 2017, and in an invited talk in a Young Scientists Forum in Zhuhai, China. This has a wide application to multiple biological systems including humans.
2) A wide range of organisms (>15 species) have been analyzed for whole cell and intracellular elemental composition and can show the exotic chemistry that organisms accumulate when grown in a complex matrix of seawater chemistry.
3) We have identified 4 candidate metals for novel metalloproteins
3) We have further worked towards compiling data for the ‘dose-response’ curves across the realm of marine microbes, thinking carefully about what a ‘metal requirement’ encapsulates in the context of phytoplankton and other marine microbes. The bulk of this has centred around iron (Fe), of which most data is available, compiling datasets of dose-response curves for a range of different microorganisms, and not just limited to photosynthetic microbes but also other marine microbes such as heterotrophic bacteria which have a key role in the carbon cycle. However, we have also began compiling the available data for Cu, Co and Zn. This compilation of data indicates that cellular metal quotas should be measured at the point where microorganisms just reach maximum growth – this prevents measurement of a value reflecting intracellular storage and instead reflects the true metallome and we are continuing to amass this data from the literature.
4) We have been able to demonstrate an interaction between the nutrient status of a cell, and its susceptibility to metal toxicity. We have demonstrated a nutrient dependent toxicity where the toxic effects of V and As (both observed to accumulate in T.oceanica) are dependent on the cellular sufficiency of Phosphate. Under P deplete conditions the concentration at which both element becomes toxic is vastly lower than at P replete conditions. This is because there is competition between uptake of these chemically similar metals such that at low P availability, when the cell upregulates transport of P, there is enhanced mistaken uptake and cellular accumulation of V and As aggravating the toxicity within the cell.
Final results
1)) Ammonia-oxidising archaea (AOA) mediate the rate limiting step of nitrification, the central component of the marine nitrogen cycle that converts ammonia to nitrite then nitrate. Competition with phytoplankton for ammonium and light inhibition are considered to restrict AOA activity to below the photic zone, but observations of surface nitrification now demand a further understanding of the factors driving AOA distribution and activity. Pico- to nanomolar concentrations of iron (Fe) limit the growth of microorganisms in a significant portion of the world’s surface oceans, yet there is no examination of the role of Fe in AOA growth despite the process of ammonia oxidation being considered to rely on the micronutrient. Here we investigate the Fe requirements and Fe uptake strategies of the Nitrosopumilus maritimus strain SCM1, a strain representative of globally abundant marine AOA. Using trace metal clean culturing techniques, we found that N. maritimus growth is controlled by Fe availability, displaying a free inorganic Fe (Fe´) half saturation constant 1-2 orders of magnitude greater for cell growth than numerous marine phytoplankton and heterotrophic bacteria driven by a reduced affinity for Fe´. In addition we discovered that, whilst unable to produce siderophores to enhance access to Fe, N. maritimus is able to use the exogenous siderophore desferrioxamine B (DFB), likely through a reductive uptake pathway analogous to that demonstrated in phytoplankton. Our work suggests AOA growth in surface waters may be Fe limited and advances our understanding of AOA physiology at the cellular level with implications for ecosystem dynamics and the biogeochemical N-cycle. This work is under review at ISME journal.
2) We have found that Cr, U, Ni and Tl can promote growth of some microorganisms when added beyond current environmental concentrations, elements normally considered toxic at such high concentrations. These elements are our candidates to separate and identify the proteins which bind these metals and find novel biological use in the coming years of the project.
3) At the Palaeozoic-Mesozoic boundary, the dominance of marine eukaryotic algae shifted from the green (chlorophyll b) to the red (chlorophyll c) superfamily. Selection pressures caused by the bioavailability of trace metals associated with an increasing oxygenation of the ocean may have played a key role in this algal revolution. From a broad scan of elemental compositions, a significant difference in the cellular Cr/P quota was found between the two superfamilies. The different responses to Cr exposure reveal contrasting strategies for metal uptake and homeostasis in these algal lineages. At high Cr(VI) concentrations, red lineages experience growth inhibition through reduced photosynthetic capability, while green lineages are completely unaffected. Moreover, Cr(VI) has a more significant impact on the metallomes of red lineage algae, in which metal/P ratios increased with increasing Cr(VI) concentration for many trace elements. Green algae have higher specificity transporters to prevent Cr(VI) from entering the cell, and more specific intracellular stores of Cr within the membrane fraction than the red algae, which accumulate more Cr mistakenly in the cytosol fraction via lower affinity transport mechanisms. Green algal approaches require greater nutrient investments in the more numerous transport proteins required and management of specific metals, a strategy better adapted to the resource-rich coastal waters. By contrast the red algae are nutrient efficient with fewer and less discriminate metal transporters which can be fast and better adapted in the oligotrophic, oxygenated open ocean which has prevailed since the deepening of the oxygen minimum zones at the start of the Mesozoic. This work is in revision for publication in Limnology and Oceanography.
4) Thallium (Tl) is the most toxic metal of all and is present at concentrations of concer
Website & more info
More info: https://www.earth.ox.ac.uk/research-groups/ocean-biogeochemistry/research-projects/.