Antioxidants, another use for DMSP and its associates

DMSP is known to act as an osmoprotectant, cryoprotectant and in some cases a grazing deterrent. Interestingly, DMSP and its derivatives may have another use, as antioxidants which protect against oxidative stressors such as high light levels. Sunda et al., (2002) investigated this by testing DMSP reactivity with the free radical OH^. and production under various oxidative stressors in Emiliania huxleyi and the diatom Thalassiosira pseudonana.

OH^. radicals were used to oxidise DMS to form DMSO, MSNA and MSA in a step-wise manner. DMSP also oxidised in a similar way. Reaction rates with OH^. and cellular concentrations were worked out for DMSP and MSNA and compared to information from the literature. It was discovered that DMSP reacts with OH^., but Acrylate and DMS are 20-60x more reactive. DMS also reacts with singlet oxygen and is lipid soluble, so could protect photosynthetic membranes. Therefore, cleavage of DMSP by DMSP lyase could increase protection. DMSO and MSNA also reacted, and with its low solubility DMSO could concentrate to high levels in the cell.

For the response of production in the face of oxidative stressors, CO₂ and Iron limitation experiments were firstly conducted in bottles with nutrient enriched seawater. CO₂ limitation cultures were grown to high cell volumes to reduce CO₂ by 40 times. For Iron limitation, cells were transferred from a medium containing iron to one without. Cultures were grown and harvested regularly for measurements of DMSP, DMS, total cell volume, chlorophyll a and APX activities (an enzyme associated with antioxidant defence) in T. pseudonana. Under both CO₂ and iron limitation, T. pseudonana showed a 20-60x increase in DMSP, increased DMS/cell-volume ratios, and elevated APX activities with reduced growth and chlorophyll a levels, indicating they were under oxidative stress. E. hux showed a similar trend. It would also have been interesting to directly quantify synthesis of DMSP lyase, to examine if it was upregulated.

For experiments using UV light, the control culture was grown under fluorescent light (no UV), whereas the UV treatments were grown in 30% sunlight and exposed to full UV, UV with (more harmful) UV-B filtered or with (almost) all UV-A and UV-B filtered. Total DMS, total DMSP, cell volume and cell chlorophyll a were measured. DMSP and DMS production was higher in sunlit treatments relative to controls, due to exposure to UV light. Interestingly, DMS and DMSP production peaked when UVB was filtered but UVA was unfiltered, rather than at full exposure. The authors put this down to production being underestimated, as increased production may be balanced by consumption of DMSP and DMS and also photolysis of DMS. 

DMS and DMSP production in response to UV highlights a negative feedback mechanism which directly benefits algae. Increased UV exposure will lead to higher DMS release, causing increased cloud formation above the bloom, so shielding the bloom from UV light and oxidative stress. Although, I think the lower DMS and DMSP levels found at the highest exposures should examined closely for the idea to be fully vindicated. It could also be interesting to look at more recent publications to see if Symbiodinium in coral show similar responses and whether this impacts the ability of coral to cope with oxidative stress and so bleaching. 

Sunda, W. K. D. J., Kieber, D. J., Kiene, R. P., & Huntsman, S. (2002). An antioxidant function for DMSP and DMS in marine algae. Nature, 418, 317-320.

Feeling anaemic? Have some siderophores!

Iron is a fundamental element involved in many biochemical processes including oxidative phosphorylation, photosynthesis, nitrogen fixation, and the detoxification of free radicals. However, dissolved inorganic Fe is scarce in the marine environment due to its propensity to precipitate in the presence of oxygen and relatively high oceanic pH levels. Fe is rapidly scavenged onto particles under such conditions leading to mean global concentrations of just 0.07 nmol kg^-1. Of this dissolved iron 99.9% exists bound to disparate organic ligands which are present in high numbers. So does this mean that organically bound iron is unavailable to marine heterotrophic bacteria for growth?

Siderophores are small molecules with a high affinity for chelating and forming complexes with Fe³⁺ and are produced in response to iron depravation by microorganisms. Siderophores are released extracellularly where they bind inorganic iron and ‘steal’ iron from other organic complexes with lower affinities. Siderophores are then internalised and the iron assimilated to help fulfil cellular iron requirements for growth. This study investigated how heterotrophic marine bacteria acquired iron for growth and the extent to which siderophores played a role in iron acquisition.

Seven gram-negative heterotrophic marine bacteria strains isolated from the Gulf of Mexico, Sargasso Sea, and NE subarctic Pacific were grown under iron-replete (8.4 µM) and iron-deplete (12.5 nM) conditions. Cellular iron uptake rates and iron quotas were quantified using the radioactive isotope  ⁵⁵Fe. The growth rates of all 7 strains were reduced on average by 50% on the iron-deplete growth medium and iron quotas were significantly reduced.

Separate culture medium was used for the isolation of siderophores and their iron binding strength determined. Iron chelating compounds were produced by 4 of the 7 strains with one siderophore isolated from the Gulf of Mexico having an extremely high affinity for Fe, binding >99% of available iron. Importantly siderophore production only occurred under iron limiting conditions. However all strains utilised Fe bound to at least one siderophore whether or not that strain produced its own.

 The importance of siderophores in the rate of iron uptake was quantified by comparing Fe uptake of all 7 strains on iron-deplete medium with and without the addition of siderophores isolated from 2 of the 7 strains, and a terrestrial fungal siderophore. The results showed that one marine siderophore and the fungal siderophore increased Fe uptake in the majority of strains, while the other isolated marine siderophore inhibited Fe uptake in 5 of the 7 strains whilst significantly increasing Fe uptake in the producing strain.

Two representative strains were used to elucidate the significance of organic vs inorganic iron uptake. The measured Fe uptake rates of inorganic Fe were tiny compared to the expected uptake rates based on diffusive flux. An ecological implication being that marine heterotrophic bacteria are not optimised to acquire inorganic iron for growth. Instead they rely on organically bound iron, unlike many eukaryotic phytoplankton species which require inorganic iron for growth.

The utilisation of siderophores of differing provenance (terrestrial as well as marine) suggest that perhaps only a finite amount of iron chelating compounds exist and that bacteria have evolved to be able to utilise iron bound to siderophores that it does not/cannot synthesise. Conversely a bacterium may monopolise iron in certain situations by specialising in the production of an uncommon ligand with high iron affinity that binds much of the available iron, thus inhibiting the growth of neighbouring bacterial heterotrophs.

I found the paper a valuable contribution to the in vitro study of siderophores and iron acquisition by bacteria. Further characterisation of marine siderophores and cellular uptake mechanisms need to be investigated to better understand the significance of siderophores in ocean biogeochemical cycles. For example; in this study differing bacteria were isolated from distinctly different water masses and it would be purposeful to see if differences in siderophores and siderophore specificity reflects their ecology and iron acquisition strategy.

Reference

Granger, Julie, and Neil M. Price. "The importance of siderophores in iron nutrition of heterotrophic marine bacteria." Limnology and Oceanography 44.3 (1999): 541-555.

http://m.avto.aslo.info/lo/toc/vol_44/issue_3/0541.pdf

Proposed method of DMSP cleavage into DMS via DddQ lyase

Dimethylsulfide (DMS) is a gas that plays a key role in the global biogeochemical sulphur cycle. Synthesis of this gas is due to breakdown of dimethylsulfoniopropionate (DMSP) by specialised lyases which can be released via turbulence, grazing or viral lysis. Approximately 300 Tg of DMS is created and 10% of this is transferred to the atmosphere via ocean microbial processes. The authors state the importance of DMS in its influence on global weather patterns. DMSO (a form of oxidised DMS) can form cloud condensation nuclei’s (CCN’s) which are tiny hygroscopic particles on which water vapour can condense and cause formation of a cloud. DMSO can also affect cloud reflectivity resulting in a decreased amount of damaging solar radiation. With more reflected energy, this would decrease the global mean temperature. Could altering these interactions result in some control over the amount of exposure from this damaging energy? 

This paper explores the crystal structure of a DddQ lyase extracted from Ruegeria lacuscaerulensis ITI_1157 and the molecular mechanism of the catalytic effect it has on the cleavage of DMSP into DMS. This was achieved via structural analyses, molecular dynamics simulations and mutational assays.

In terms of structure, the authors ascertained that DddQ lyase folds into a β-barrel structure and contains six hydrophilic residues, essential to catalysis. To determine their importance, site-directed mutations were created; Tyr120Ala, His123Ala, His125Ala, Glu129Ala, Glu129Gln, and His163Ala. They discovered that these mutations inhibited the ability of DddQ towards DMSP therefore confirming the key role these residues play in DMSP cleavage. The mechanism they proposed is thus; DMSP enters the DddQ active site and oxygen from DMSP binds to Zn²⁺ ion, replacing Tyr131 (one of the six highly conserved hydrophilic residues in DddQ lyase) and resulting in a β-elimination reaction.

This paper is of high interest especially in the dynamic global climate. By exploring the key processes that affect the cleavage of DMSP into DMS and the role this biogenic gas plays, therein lies a greater ability to reveal its interactions with the environment.

Li C., Wei, T., Zhang, X., Gao, X., Wang, P., Xie, B., Su, H., Qin, Q., Zhang, X., Yu, J., Zhang, H., Zhou, B., Yang, G. & Zhang, Y. (2013). Molecular insight into bacterial cleavage of oceanic dimethylsulfoniopropionate into dimethyl sulfide. PNAS. 111 (3), 1026–1031. doi: 10.1073/pnas.1312354111

Cyanobacteria: more than meets the eye...

Shown to play key roles in quorum sensing, virulence and horizontal gene transfer, vesicles are key to survival and are found in all domains of life. Despite their ubiquity, their function in the marine environment is relatively understudied. A study by Biller et al. 2014 addresses this knowledge gap. Observation of an axenic Prochlorococcus culture revealed the presence of vesicles, despite extensive study they were not previouly observed. It was soon realised with the vast abundance of Prochlorococcus in the world's oceans, these vesicles could have impacts on the functioning of marine systems.  

Examination of Prochlorococcus and their vesicles by transmission electron microscopy (TEM), micrographs and nanoparticle tracking, derived their structure and concentration respectively. They were found in greatest abundance during the exponential and stationary phases of growth - here they were around 10x that of Prochlorococcus cells. As stated by the authors, this represents vesicle production under lab conditions, the numbers produced naturally may vary. They hypothesised 10^27 - 10^28 to be produced per day. Responsible for the transport of molecules/substances, the contents of the vesicles were then analysed. Vesicles are supramolecular structures, consisting mainly of lipids, hence a number of lipids, fatty acids and lipopolysaccharides were identified. A diverse range of proteins and enzymes were also found following proteome analysis; nutrient transporters porins and hydrolases were among some of the identifiable proteins - are these another means by which cyano-bacteria can obtain nutrients in an oligotrophic environment? DNA and RNA were also encapsulated in these vesicles, amplification and sequencing showed that around 50% of the chromosomal sequence was bound in these vesicles. Transformation is one method of horizontal gene transfer (HGT), these vesicles may be the source of genetic material fueling HGT. Comparison to natural ocean samples was undertaken and similar findings to the lab study were found. Samples at varying depths were taken and prochlorococcal vesicles were present at each depth. 

It has been hypothesised that Prochlorococcus may be responsible for the release of dissolved organic carbon (DOC) in to the oceans, owing to the diverse vesicle contents as determined by this study, a mechanism of DOC release could potentially be proposed. Additionally, the vesicles may act as a source of nitrogen and phosphorus. Heterotrophic bacteria (in culture) were successfully grown with Prochlorococcus-derived vesicles as the only carbon source, thus testing the above hypothesis. It is rather odd however, that Prochlorococcus should release these useful substances, particularly as it occupies a nutrient-poor environment. Further study revealed that these vesicles may function in defense against phages - could this be a primitive immune system?

It is evident that further study and consideration needs to be given to microbial 'organelles', as mechanisms underpinning defense, biogeochemistry and nutrient flow may not be as understood as initially thought. This study is particularly interesting from an evolutionary perspective. 


Jack 

Biller, S. J., F. Schubotz, S. E. Roggensack, A. W. Thompson, R. E. Summons, and S. W.Chisholm. (2014) Bacterial Vesicles in Marine Ecosystems, Science 343, : 183-186

A missing link in the nitrogen cycle

The Black Sea is stratified due to the cooler and less saline Mediterranean water that fills the basin, producing a significant anoxic layer from 80m. Phytoplankton live and die in the upper oxic layer, transferring fixed nitrogen to the anoxic deep water. There is however, a discrepancy between the concentration of ammonium in the deep water and the concentration of inorganic nitrogen in the suboxic .

Kuypers et al (2003) set out to investigate whether anaerobic ammonia oxidation (anammox) was the reason for this discrepancy. If nitrate is converted to nitrite in the suboxic, the nitrite can be used as the electron donor to convert ammonium from the sediments to nitrogen gas and water – resulting in a net loss of inorganic nitrogen.

The author tested the hypothesis using various methods. Firstly, a nutrient depth profile was created using a CTD to give the authors a picture of where the anammox reactions might be occurring. The profile showed that nitrate peaked at 65m and nitrite at 80m, but are both absent past 90m. Oxygen was absent past 80m, ruling out aerobic nitrification. Water samples were also incubated from different depths with radioactively labelled (N¹⁵) ammonium and (N¹⁴) nitrite knowing that one mole of each would produce one mole nitrogen gas, confirming if anammox was occurring; it was.

Knowing that the bacteria Planctomycetes undergo the anammox reaction, using a membrane bound organelle largely composed of ladderane lipids, the authors did a lipid analysis of water samples to identify if these lipids were present, and at what depth. Three lipids were identified, and all at similar depths to where nitrate and nitrite disappear.

Next they extracted DNA from water samples where the lipids were at the highest concentration, and used molecular cloning methods to create a phylogenetic tree. This confirmed the bacteria isolated from the Black Sea were related to bacteria capable of anammox, and were 98% similar to bacteria sequenced from bioreactor (sewage treatment).

Molecular cloning allowed them to create a specific oligonucleotide probe, which be used identify Planctomycete using FISH microscopy.

Using a diffusion model they estimated that anaerobic ammonium oxidation was occurring the Black Sea suboxic at 0.007  µM day^-1. When compared to what was known for bioreactors, it estimated that 300-3000 anammox cells ml^-1 would have to be present for this amount of ammonia oxidation per day. Luckily having already created an oligonucleotide for the Planctomycete, the FISH probe was able to analyse water samples and showed that there was 1900 (±) 800 anammox cells per ml.

Based on the area of the basin, they went on to predict that 0.3Tg of inorganic nitrogen per year may be lost through anammox reactions – a significant amount considering 14Tg of fixed nitrogen of produced through photosynthesis.

The results presented here are really valuable for the nitrogen cycle in these environmental conditions, showing a net loss of nitrogen in basins with a suboxic zone. There could be similarities in fjords for example, which are also stratified in a similar way due to different water densities.

I thought the authors were thorough in their methodology, using this variety of methods and knowledge of the subject. They built up a picture of what was occurring by using different analyses, giving very conclusive and reliable results.

Reference:

Kuypers, M. M., Sliekers, A. O., Lavik, G., Schmid, M., Jørgensen, B. B., Kuenen, J. G., ... & Jetten, M. S. (2003). Anaerobic ammonium oxidation by anammox bacteria in the Black Sea. Nature, 422(6932), 608-611.