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Abstract: Candidatus Vesicomyosocius okutanii is a currently uncultured endosymbiotic bacterium of the clam Pheragena okutanii, which lives in deep-sea vent environments. The genome of Ca. V. okutanii encodes a sulfur-oxidizing (Sox) enzyme complex, presumably generating biological energy for the host from inorganic sulfur compounds. Here, Ca. V. okutanii SoxX (VoSoxX), a mono-heme cytochrome c component of the Sox complex, was shown to be phylogenetically related to its homologous counterpart (HcSoxX) from a free-living deep-sea vent bacterium, Hydrogenovibrio crunogenus. Both proteins were heterologously expressed in Escherichia coli cells with co-expressing cytochrome c maturation genes. Biochemical analysis using the recombinant proteins showed that VoSoxX had a significantly lower thermal stability than HcSoxX, possibly due to structural differences. For example, the Asn-60 residue in VoSoxX may be hydrophobically disadvantageous compared with the spatially corresponding Val-73 residue in HcSoxX. This study represents the first successful case of heterologous expression of genes from Ca. V. okutanii, suggesting that the endosymbiotic VoSoxX protein does not require stabilization, unlike the free-living HcSoxX protein.
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Aug 2022
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I03-Macromolecular Crystallography
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Diamond Proposal Number(s):
[14692]
Open Access
Abstract: Marine cyanobacteria are critical players in global nutrient cycles that crucially depend on trace metals in metalloenzymes, including zinc for CO2 fixation and phosphorus acquisition. How strains proliferating in the vast oligotrophic ocean gyres thrive at ultra-low zinc concentrations is currently unknown. Using Synechococcus sp. WH8102 as a model we show that its zinc-sensor protein Zur differs from all other known bacterial Zur proteins in overall structure and the location of its sensory zinc site. Uniquely, Synechococcus Zur activates metallothionein gene expression, which supports cellular zinc quotas spanning two orders of magnitude. Thus, a single zinc sensor facilitates growth across pico- to micromolar zinc concentrations with the bonus of banking this precious resource. The resultant ability to grow well at both ultra-low and excess zinc, together with overall lower zinc requirements, likely contribute to the broad ecological distribution of Synechococcus across the global oceans.
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Jun 2022
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I08-Scanning X-ray Microscopy beamline (SXM)
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Diamond Proposal Number(s):
[20839]
Open Access
Abstract: Minerals are widely proposed to protect organic carbon from degradation and thus promote the persistence of organic carbon in soils and sediments, yet a direct link between mineral adsorption and retardation of microbial remineralisation is often presumed and a mechanistic understanding of the protective preservation hypothesis is lacking. We find that methylamines, the major substrates for cryptic methane production in marine surface sediment, are strongly adsorbed by marine sediment clays, and that this adsorption significantly reduces their concentrations in the dissolved pool (up to 40.2 ± 0.2%). Moreover, the presence of clay minerals slows methane production and reduces final methane produced (up to 24.9 ± 0.3%) by a typical methylotrophic methanogen—Methanococcoides methylutens TMA-10. Near edge X-ray absorption fine structure spectroscopy shows that reversible adsorption and occlusive protection of methylamines in clay interlayers are responsible for the slow-down and reduction in methane production. Here we show that mineral-OC interactions strongly control methylotrophic methanogenesis and potentially cryptic methane cycling in marine surface sediments.
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May 2022
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I13-2-Diamond Manchester Imaging
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Sebastian J.
Hennige
,
Uwe
Wolfram
,
Leslie
Wickes
,
Fiona
Murray
,
J. Murray
Roberts
,
Nicholas A.
Kamenos
,
Sebastian
Schofield
,
Alexander
Groetsch
,
Ewa M.
Spiesz
,
Marie-Eve
Aubin-Tam
,
Peter J.
Etnoyer
Diamond Proposal Number(s):
[19794, 20412]
Open Access
Abstract: Ocean acidification is a threat to the net growth of tropical and deep-sea coral reefs, due to gradual changes in the balance between reef growth and loss processes. Here we go beyond identification of coral dissolution induced by ocean acidification and identify a mechanism that will lead to a loss of habitat in cold-water coral reef habitats on an ecosystem-scale. To quantify this, we present in situ and year-long laboratory evidence detailing the type of habitat shift that can be expected (in situ evidence), the mechanisms underlying this (in situ and laboratory evidence), and the timescale within which the process begins (laboratory evidence). Through application of engineering principals, we detail how increased porosity in structurally critical sections of coral framework will lead to crumbling of load-bearing material, and a potential collapse and loss of complexity of the larger habitat. Importantly, in situ evidence highlights that cold-water corals can survive beneath the aragonite saturation horizon, but in a fundamentally different way to what is currently considered a biogenic cold-water coral reef, with a loss of the majority of reef habitat. The shift from a habitat with high 3-dimensional complexity provided by both live and dead coral framework, to a habitat restricted primarily to live coral colonies with lower 3-dimensional complexity represents the main threat to cold-water coral reefs of the future and the biodiversity they support. Ocean acidification can cause ecosystem-scale habitat loss for the majority of cold-water coral reefs.
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Sep 2020
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I18-Microfocus Spectroscopy
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Diamond Proposal Number(s):
[15802]
Open Access
Abstract: Bacteria acquire phosphate (Pi) by maintaining a periplasmic concentration below environmental levels. We recently described an extracellular Pi buffer which appears to counteract the gradient required for Pi diffusion. Here, we demonstrate that various treatments to outer membrane (OM) constituents do not affect the buffered Pi because bacteria accumulate Pi in the periplasm, from which it can be removed hypo-osmotically. The periplasmic Pi can be gradually imported into the cytoplasm by ATP-powered transport, however, the proton motive force (PMF) is not required to keep Pi in the periplasm. In contrast, the accumulation of Pi into the periplasm across the OM is PMF-dependent and can be enhanced by light energy. Because the conventional mechanism of Pi-specific transport cannot explain Pi accumulation in the periplasm we propose that periplasmic Pi anions pair with chemiosmotic cations of the PMF and millions of accumulated Pi pairs could influence the periplasmic osmolarity of marine bacteria.
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May 2020
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[16989]
Abstract: Coccolithophores are single-celled marine algae that produce calcified scales called coccoliths. Each scale is composed of anvil-shaped single crystals of calcite that are mechanically interlocked, constituting a remarkable example of the multi-level construction of mineralized structures. Coccolith formation starts with the nucleation of rhombohedral crystals on an organic substrate called base plate. The crystals then grow preferentially along specific directions to generate the mature structure, which is then transported to the outside of the cells. Here, we extracted forming coccoliths from Pleurochrysis carterae cells and used cryo-electron tomography to characterize, in their native, hydrated state, the three-dimensional morphology and arrangement of the crystals. Comparing the crystal morphology across three different stages of coccolith formation, we show that competition for space between adjacent crystals contributes significantly to regulation of morphology by constraining growth in certain directions. We further demonstrate that crystals within a coccolith ring develop at different rates and that each crystalline unit rests directly in contact with the base plate and overgrow the rim of the organic substrate during development.
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Feb 2020
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Krios I-Titan Krios I at Diamond
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Diamond Proposal Number(s):
[16989]
Open Access
Abstract: Coccolithophores are marine phytoplankton that are among the most prolific calcifiers widespread in Earth’s oceans, playing a crucial role in the carbon cycle and in the transport of organic matter to the deep sea. These organisms produce highly complex mineralized scales that are composed of hierarchical assemblies of nano-crystals of calcium carbonate in the form of calcite. Coccolith formation in vivo occurs within compartmentalized mineralisation vesicles derived from the Golgi body, which contain coccolith-associated polysaccharides (‘CAPs’) providing polymorph selection and mediating crystal growth kinetics, and oval organic mineralisation templates, also known as base plates, which promote heterogenous nucleation and further mechanical interlocking of calcite single crystals. Although the function of coccolith base plates in controlling crystal nucleation have been widely studied, their 3D spatial organization and the chemical functional groups present on the crystal nucleation sites, which are two crucial features impacting biomineralization, remain unsolved. Utilising cryo-electron tomography we show that base plates derived from an exemplary coccolithophore Pleurochrysis carterae (Pcar) in their native hydrated state have a complex 3-layered structure. We further demonstrate, for the first time, the edge and rim of the base plate – where the crystals nucleate - are rich in primary amine functionalities that provide binding targets for negatively charged complexes composed of synthetic macromolecules and Ca2+ ions. Our results indicate that electrostatic interactions between the negatively charged biogenic CAPs and the positively charged rim of the base plate are sufficient to mediate the transport of Ca2+ cations to the mineralization sites.
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Aug 2019
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I08-Scanning X-ray Microscopy beamline (SXM)
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Alastair J. M.
Lough
,
Douglas P.
Connelly
,
William B.
Homoky
,
Jeffrey A.
Hawkes
,
Valerie
Chavagnac
,
Alain
Castillo
,
Majid
Kazemian
,
Ko-Ichi
Nakamura
,
Tohru
Araki
,
Burkhard
Kaulich
,
Rachel A.
Mills
Diamond Proposal Number(s):
[12738]
Open Access
Abstract: Iron (Fe) limits primary productivity and nitrogen fixation in large regions of the world’s oceans. Hydrothermal supply of Fe to the global deep ocean is extensive; however, most of the previous work has focused on examining high temperature, acidic, focused flow on ridge axes that create “black smoker” plumes. The contribution of other types of venting to the global ocean Fe cycle has received little attention. To thoroughly understand hydrothermal Fe sources to the ocean, different types of vent site must be compared. To examine the role of more diffuse, higher pH sources of venting, a hydrothermal plume above the Von Damm vent field (VDVF) was sampled for Total dissolvable Fe (unfiltered, TDFe), dissolved Fe (<0.2 μm, dFe) and soluble Fe (<0.02 μm, sFe). Plume particles sampled in situ were characterized using scanning electron microscopy and soft X-ray spectromicroscopy. The VDVF vents emit visibly clear fluids with particulate Fe (TDFe-dFe, >0.2 μm) concentrations up to 196 nmol kg–1 comparable to concentrations measured in black smoker plumes on the Mid-Atlantic Ridge. Colloidal Fe (cFe) and sFe increased as a fraction of TDFe with decreasing TDFe concentration. This increase in the percentage of sFe and cFe within the plume cannot be explained by settling of particulates or mixing with background seawater. The creation of new cFe and sFe within the plume from the breakdown of pFe is required to close the Fe budget. We suggest that the proportional increase in cFe and sFe reflects the entrainment, breakdown and recycling of Fe bearing organic particulates near the vents. Fe plume profiles from the VDVF differ significantly from previous studies of “black smoker” vents where formation of new pFe in the plume decreases the amount of cFe. Formation and removal of Fe-rich colloids and particles will control the amount and physico-chemical composition of dFe supplied to the deep ocean from hydrothermal systems. This study highlights the differences in the stabilization of hydrothermal Fe from an off-axis diffuse source compared to black smokers. Off-axis diffuse venting represent a potentially significant and previously overlooked Fe source to the ocean due to the difficulties in detecting and locating such sites.
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Jul 2019
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I08-Scanning X-ray Microscopy beamline (SXM)
|
Daniela
Medas
,
Ilaria
Carlomagno
,
Carlo
Meneghini
,
Giuliana
Aquilanti
,
Tohru
Araki
,
Diana
Bedolla
,
Carla
Buosi
,
Maria Antonietta
Casu
,
Alessandra
Gianoncelli
,
Andrei C.
Kuncser
,
V. Adrian
Maraloiu
,
Giovanni
De Giudici
Diamond Proposal Number(s):
[16496]
Abstract: Zinc incorporation into marine bivalve shells belonging to different genera (Donax, Glycymeris, Lentidium, and Chamelea) grown in mine-polluted seabed sediments (Zn up to 1% w/w) was investigated using x-ray diffraction (XRD), chemical analysis, soft x-ray microscopy combined with low-energy x-ray fluorescence (XRF) mapping, x-ray absorption spectroscopy (XAS), and transmission electron microscopy (TEM). These bivalves grew their shells, producing aragonite as the main biomineral and they were able to incorporate up to 2.0–80 mg/kg of Zn, 5.4–60 mg/kg of Fe and 0.5–4.5 mg/kg of Mn. X-ray absorption near edge structure (XANES) analysis revealed that for all the investigated genera, Zn occurred as independent Zn mineral phases, i.e., it was not incorporated or adsorbed into the aragonitic lattice. Overall, our results indicated that Zn coordination environment depends on the amount of incorporated Zn. Zn phosphate was the most abundant species in Donax and Lentidium genera, whereas, Chamelea shells, characterized by the highest Zn concentrations, showed the prevalence of Zn-cysteine species (up to 56% of total speciation). Other Zn coordination species found in the investigated samples were Zn hydrate carbonate (hydrozincite) and Zn phosphate. On the basis of the coordination environments, it was deduced that bivalves have developed different biogeochemical mechanisms to regulate Zn content and its chemical speciation and that cysteine plays an important role as an active part of detoxification mechanism. This work represents a step forward for understanding bivalve biomineralization and its significance for environmental monitoring and paleoreconstruction.
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Oct 2018
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I02-Macromolecular Crystallography
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Diamond Proposal Number(s):
[11945]
Open Access
Abstract: Atmospheric nitrogen fixation by photosynthetic cyanobacteria (diazotrophs) strongly influences oceanic primary production and in turn affects global biogeochemical cycles. Species of the genus Trichodesmium are major contributors to marine diazotrophy, accounting for a significant proportion of the fixed nitrogen in tropical and subtropical oceans. However, Trichodesmium spp. are metabolically constrained by the availability of iron, an essential element for both the photosynthetic apparatus and the nitrogenase enzyme. Survival strategies in low-iron environments are typically poorly characterized at the molecular level, as these bacteria are recalcitrant to genetic manipulation. Here, we studied a homolog of the iron deficiency-induced A (IdiA)/ferric uptake transporter A (FutA) protein, Tery_3377, which has been used as an in situ iron-stress biomarker. IdiA/FutA has an ambiguous function in cyanobacteria, with its homologs hypothesized to be involved in distinct processes depending on their cellular localization. Using signal sequence fusions to GFP and heterologous expression in the model cyanobacterium Synechocystis sp. PCC 6803 we show that Tery_3377 is targeted to the periplasm by the twin-arginine translocase and can complement the deletion of the native Synechocystis ferric-iron ABC transporter periplasmic binding protein (FutA2). EPR spectroscopy revealed that purified recombinant Tery_3377 has specificity for iron in the Fe3+ state, and an X-ray crystallography determined structure uncovered a functional iron substrate-binding domain, with Fe3+ penta-coordinated by protein and buffer ligands. Our results support assignment of Tery_3377 as a functional FutA subunit of an Fe3+ ABC-transporter, but do not rule out that it also has dual IdiA function.
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Sep 2018
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