Phytoplankton proteins "concentrate" to increase iron uptake.

In 1982, it was hypothesised that phytoplankton in the oligotrophic open ocean overcame the problem of iron uptake by first binding it to their membranes before metabolising it. They suggested it was a hypothetical protein “phytotransferrin” that carried out this process but lacked the technology to test for such a protein. In 2014, Morrissey et al. identified novel proteins deemed “ubiquitous in marine phytoplankton” that concentrate dissolved iron around the surface of the cell, resolving the hypothesis that was put forward some 30 years ago.

The main finding of this study is the discovery of the Iron Starvation Induced Proteins (ISIPs), in the diatom Phaeodactylum tricornutum. The study focused on ISIP2a, which metatranscriptomics show to be “ubiquitous” in marine phytoplankton, being expressed in species of Pelagophyceae, Haptophyta, Cryptophyta, Chlorophyta, Dinoflagellata, Rhodophyta and Bacillariophyta. These proteins are transmembrane and composed of two subunits, ISIP-N and ISIP-C. ISIP-N and C are highly conserved in brown and green macroalgae but it’s the combination and arrangement of the two in phytoplankton that makes them novel.

ISIP2a functions by binding to ferric iron (Fe³⁺) in the water column, facilitating the activity of other proteins, such as ferrireductases that metabolise the iron once it is localised to the cell surface. Quantitative PCR found that ISIP2a is sharply upregulated when the cell first experiences iron deprivation. Ferrireductase and other “more conventional” iron uptake proteins are expressed a while after ISIP2a, suggesting its expression is an immediate response to low iron levels.

ISIP2a is also inhibited by chelating agents (another way phytoplankton are able to acquire dissolved iron from the water column) of the same oxidative state of iron (Fe³⁺) I think this is interesting, why is this protein inhibited by chelated ferric iron? Perhaps it is not energetically viable for the diatom to express proteins that acquire the same nutrient simultaneously. Or, seeing as ISIP2a binds to iron, chelated ferric iron competitively inhibits its function. It would be interesting to see how the expression of ISIP2a relates to expression of chelating agents for ferric iron as I would have thought the diatoms would want to be able to acquire as much iron as possible and would express both.

This study presents a novel protein that upon investigation is revealed to be prevalent in many 

phytoplankton and gives new insight into phytoplankton response to iron deprivation. Whilst it may not be a novel idea that they are able to concentrate iron around their cell surface, this may well be the first evidence to support that hypothesis. Another paper I have recently read (and will blog about) states that different phytoplankton use the same proteins for different purposes, so I would be interested to see studies on the function of the “ubiquitous” ISIP2a in the rest of the phytoplankton it is expressed in, there may be analogues that concentrate other oxidation states of iron, or other nutrients entirely.

Reference:

Morrissey, J. Sutak, R. Paz-Yepez, J. Tanaka, A. Moustafa, A. Veluchamy, A. McQuaid, J.B. Tirichine, L. Allen, A.E. Lesuisse, E. and Bowler, C. (2015) A Novel Protein, Ubiquitous in Marine Phytoplankton, Concentrates Iron at the Cell Surface and Facilitates Uptake. Current Biology, 25, 364-374.

The Unstoppable Vibrios

Fibrinal blood clots are essential defences that present a barrier to pathogens in order to protect the organism’s internal environment. The blood clot is also vital in activating many other innate immune responses within an organism. A common bacterial species that infects marine organisms is Vibrio harveyi, and is immediately captured within clots. However, this bacterium has been shown by Chaikeeratisak et al. (2014) to utilise serine- and metallo-proteases in order to escape clots in just over two hours after entrapment.

The infection of the Pacific white shrimp, Litopenaeus vannamei, by V. harveyi was studied, with the behaviour of the Vibrio noted. As this species of shrimp is very susceptible to infection from Vibrios, and is an important part of the aquacultural industry, it was deemed essential to record the performance of the blood clot in the defence against infection.

Freshly drawn haemolymph from the shrimp was mixed with washed, free swimming V. harveyi cells and allowed to clot in order to estimate the blood clots’ efficiency. The result showed that 84% of the Vibrio cells were entrapped during the clotting process (30 mins, room temperature), with cytolysis not playing a significant role in bacterial removal. This suggests that the clot is the principal process for removing bacteria.

Microscopic analysis revealed that immediately after entrapment, there was no flagellular movement of the Vibrio cells. However, after only one hour increasing numbers of the bacteria were resuming the flagellar swimming by fibrinolysis; after two hours the area of fibrinolysis achieved by the bacteria had risen from 0.015 – 1.448 mm². When the bacteria had extended the fibrinolysis area past the clot, the cells were able to escape completely.

In order to understand how the Vibrios may achieve such efficient fibrinolysis, a cocktail of protease inhibitors was added to the bacterial blood clots to determine which of the many enzymes secreted is vital for fibrinolysis. It was found that inhibitors to the serine- and metallo-proteases suppressed the breakdown of the clot, and prevented the bacteria from escaping. It was thought that as the blood clots of arthropods are resistant to many proteolytic attacks, the bacteria need both of these proteases in order to successfully lyse the clots.

This study has been able to show that the infection of V. harveyi in an economically important shrimp may be hindered by the host’s immune system for only two hours before the parasite can overcome it. The clot itself is an effective defence within the first moments of a wound occurring, however the suite of proteases released by the bacteria may act as virulence factors, allowing the survival of the Vibrio and so successful infection of the shrimp.

I believe this study has prompted many questions as to what extent the proteases play a role in virulence, and if any other Vibrios may infect organisms in this way. It would be economically important to extend this line of research so that possible methods of resistance to parasites in aquaculture could be produced.

Reference: Chaikeeratisak, V., Tassanakajon, A., & Armstrong, P. B. (2014). Interaction of pathogenic vibrio bacteria with the blood clot of the Pacific white shrimp, Litopenaeus vannamei. The Biological Bulletin, 226(2), 102-110.

http://www.biolbull.org/content/226/2/102.short

Brace yourself - Cholera is coming

Cholera is a bacterial infectious disease caused by pathogenic strains of the gram-negative bacteria Vibrio cholera. Firstly described in the 19^th century origin, functionality and consequences of Cholera are well investigated. Still thousands of cases of human cholera infection are reported each year. In the 70s of the 20^th century Rita Colwell discovered that V. cholerae strains occur naturally in coastal waters. She assumed that outbreaks of cholera epidemics are closely related to the distribution of natural plankton hosts in coastal areas. Today it is known that V. cholerae is spread and passed on not only by plankton but also by a wide range of other marine organisms. Hence, distribution of V. cholerae depends on oceanographic variables such as temperature, pH, salinity, plankton blooms and so forth. Regarding these variables it would be possible to characterize distribution areas and predict potential outbreaks. However, because of climate change it is also necessary to consider shifting of these variables. For instance, it has been established that increasing abundance and concentration of V. cholerae is linked to increased temperature. 

The study by Escobar et al. (2015) identified significant environmental variables linked to the occurrence of V. cholerae. The oceanographic variables were used to create a model which describes potential V. cholerae distribution areas. Further, the model was applied on current and future climate scenarios.

Firstly, they performed a literature review to accumulate all sites of V. cholerae presence. Twelve environmental variables were taken into account to build Environmental niche models (ENM). ENM are mathematical models which help to predict the distribution of species in geographic space considering their known distribution in environmental space. Principal components analysis (PCA) and jackknife test were used to identify and evaluate highly correlated environmental variables and their model performance.

Literature review yielded 15 sites in marine areas with V. cholerae reports worldwide. Most significant environmental variables which were used for the final ENM were Chlorophyll-a, maximum temperature, pH and mean salinity. Cholorphyll-a was the most powerful variable for explaining presence of V. cholerae. Appearance of V. cholerae is closely related to plankton blooms. The bacteria in estuaries and coastal waters are associated with plankton. Increased Chlorophyll-a concentration indicates increased potential for infection of humans.

Several regions were identified to be suitable for V. cholerae occurrence. Future models showed a pregnant increase of suitable regions worldwide. This may suggest that there is a higher level of risk of V. cholerae distribution in a future of global warming.

Since this is only a model, results of these findings have yet to be proven realistic. More information about distribution sites and environmental variables including not only abiotic factors, but also biotic factors are necessary to improve the model.

Although this is not a typical hands-on research paper, I think it gives an important overview about the future of Cholera disease. With increasing water temperature especially in coastal regions it is important to know which areas could be populated by the toxic bacteria in future events. Future studies should focus on the identification of new distribution areas. Has V. cholerae already moved somewhere else? Further, migration pathways should be more investigated to get a better picture of future spreading. 

Escobar L., Ryan S., Ibarra A., Finkelstein J., King C., Qiao H., Polhemus M (2015), Acta Tropica. A global map of suitability for coastal Vibrio cholerae under current and future climate conditions. doi:10.1016/j.actatropica.2015.05.028 

Microbes Play Part in Oil Spill Clean Up

Hydrocarbons naturally enter the marine environment at cold seeps along continental shelves. Natural seeps are sporadic and can be diffuse or intense. The Gulf of Mexico is one of the most oil rich basins in the world, the 2010 Macondo blow out leaked 15 times more oil and gas than the Gulfs natural seeps. Microbial hydrocarbon degraders in marine environments (e.g. Alcanivorax spp. and Marinobacter spp.) work together to acts as a metabolic network: primary oil degraders produce surfactants to emulsify oil, secondary consumers intercept intermediate molecules (e.g. alcohols and alkenes) increasing molecules bioavailability to other organisms. This slick led to unprecedented changes in the marine environment and microbial populations. This review paper explores microbial responses to the deep water oil plume, surface slicks and sediment composition changes.

Native microorganisms responded rapidly until limited by environmental factors; increased archaea changed nitrifying microorganism community structure. During the lifetime of the oil plume the deep water bacterial community shifted and hydrocarbon degradation gene presence increased. 16s PCR showed the blow out caused gammaproteobacteria alkane degraders Oceanspirillales to dominate, between 2 and 4 weeks later communities shifted to mostly PAH degraders, Cycloclasticus and Colwellia. After 6 months, communities near the well head diversified to include alkane, PAH and methyl degraders (Methtlophilacaea, Methylococcaceae and Methylophaga). Despite the blowout being offshore and the majority of hydrocarbons being weathered before reaching coastal waters oil contamination on beaches affected microbial communities in a similar way to those in deep water. Oil contaminated sands contained up to 4 times the microbes in clean sands and followed similar assemblage changes to the deep water. Taxonomic diversity decreased in response to the pollution but rebounded a year later.

Sediments significantly changed, within a few weeks large aggregations of marine snow were observed which sunk and settled on the sea floor. Laboratory experiments showed the huge aggregations were due to increased cell densities, activities of carbohydrate degrading enzymes with the entrapment of oil droplets in an extensive matrix of polysaccharides characteristic of aromatics and alkane reducing bacteria. When deposited, aerobic oil degrading communities on the detritus were gradually replaced by anaerobic decomposers. 8 months after the spill the top 2cm of sediments local to the well showed 10-20% less organic carbon and nitrogen compared to underlying sediment nutrients are likely to have been consumed by pelagic and benthic microbiota.

This review article exploits a unique situation to link studies and build a view of microbial effects and habitats. In addition, lapses in knowledge can be identified: oil and gas are rarely accurately measured in the marine environment making it difficult to quantify oil degradation; marine snow provides a net to capture oil droplets, but we need to investigate what happens to them in the water column and on the seabed further; microbes are known to break down many hydrocarbon compounds (e.g. alkanes, PAHs, methane) but we need to quantify and assess more persistent compounds (e.g. tar).

Joye, S. B., Teske, A. P., Kostka, J. E. (2014) Microbial Dynamics Following the Macondo Oil Well Blowout across Gulf of Mexico Environments. BioScience. 64 (9): 766-777. http://bioscience.oxfordjournals.org/content/64/9/766.full

Algae and Bacteria, a toxic relationship

Gymnodinium catenatum is a species of dinoflagellate which, like many other dinoflagellates, produces paralytic shellfish toxins (PST), a range of compounds responsible for paralytic shellfish poisoning. Many in depth studies on PST-producing dinoflagellates have been carried out over the years, and have revealed that these algae live in association with complex bacterial communities, which have a large effect on the cellular toxicity of the algae.

Using G. catenatum, and a bacterial community replacement method, Albinsson et al. (2014) studied how changing the associated bacterial community composition affected the toxicity, and how large the effect would be.  

Previous studies of the ‘normal’ bacterial community associated with G. catenatum have been carried out, finding that the algae-bacteria association is obligate for the algae, and shown that Alcanivorax and Marinobacter are constant and dominant in these cultures. Not only that, but both Alcanivorax and Marinobacter are capable of maintaining and supporting G. catenatum cells alone. Therefore, building on this information, Albinsson et al., took lab-grown offspring cultures of G. catenatum and grew them in the presence of various bacterial community compositions to then measure the range and amount of PST produced.

The method of offspring production (a sexual cross method), and the interest in the role of Alcanivorax and Marinobacter determined the different bacterial communities tested on the algae in this experiment. Sterile offspring were grown in the presence of Alcanivorax only, Marinobacter only, the communities associated with each of the algal parent strains, and 2 controls, one being completely sterile and another being the combined parental communities.

As expected, the algal cells grown in the sterile control did not survive, as stated earlier, they have an obligate relationship with their bacterial community. Additionally, there were no significant changes in the types of PST produced by the offspring cultures, only changes in the levels of net PST production. Interestingly, this study showed that there was significantly lower toxin content in the cultures grown in the presence of Alcanivorax or Marinobacter than in the cultures grown with mixed bacterial communities. Additionally, although non-significant, the net toxin production rates in offspring cultures were almost 8-fold lower than that of the parents, which indicates that lab conditions are likely having some effect on the results.

Though there are multiple reasons for the changes in toxicity of an algal cell relating to its bacterial community, such as the actual bacteria present, a reduction in community complexity or a combination of both, these findings lean more towards a reduction in bacterial complexity being the reason behind the reduction in toxicity.

The results from this work, combined with other studies showing that Marinobacter doesn’t produce PST toxins suggests that it is likely that the interactions between the bacteria themselves, and between the bacteria and the algae have more influence over the toxicity of the algae, rather than the individual activities of bacteria.

I feel that this paper contributes some interesting findings to the field of bacterial associations and interactions, and emphasises the need for further studies on how bacterial interactions (with other bacteria and other organisms) play an important role in not only toxin production, but pathogenicity, diseases and other behaviours.

http://journals.plos.org/plosone/article?id=10.1371/journal.pone.0104623 

M. E. Albinsson, A. P. (2014). Bacterial Community Affects Toxin Production by Gymnodinium catenatum. PLoS One.

Microbes take an easy ride on marine plastics

Global plastic production has accelerated dramatically over the past century to over 250 million tonnes produced per year. Most marine litter originated from land based sources and direct human disposal. Plastics threaten all trophic levels with injuries caused by ingestion, entanglement and by toxins or microbial communities on the plastic surface. In particular, micro-organisms ability to adapt to new niches and provide key ecosystem functions through primary production and nutrient cycling means their presence on plastics could have profound influences on both macro and microbiota.

This study investigates the structure and taxonomic composition of microbial assemblages on plastic fragments in both coastal and offshore Northern European waters in respect to season, geographical location and plastic type. To study of assemblages’ spatial and seasonal variation in the North Sea PET water bottles along with glass slides, for reference, were attached to smart buoys for six weeks during winter, spring and summer. Two research cruises used trawled nets at 41 stations during January and July, 2012 to investigate plastic variation of assemblages on plastics already present in the oceans. Sampling stations encompassed the North Sea, English Channel, Celtic Sea and Bristol Channel. Samples were imaged using SEM and sequenced using PCR and Denaturing Gradient Gel Electrophoresis (DGGE), plastic samples were analysed using Fourier transform infrared spectroscopy (FTIR).

SEM images showed the presence of prokaryotic and eukaryotic growth on plastic debris, eukaryotes were attached by filamentous stalks. There was high variability in community structure and composition in relation to location, season, and polymer type despite some underlying similarities. PET bottle communities showed greatest difference in winter compared to those in spring and summer. A high abundance of Bacteriodetes, Cyanobacteriai, Proteobacteria, and the eukarya Stramenopiles were identified on all bottles. Bacteriodetes, including biofilm constituent Tenacbaculum, were abundant on PET bottles across all seasons and sampling. FTIR analysis did not reveal any difference in the surface structure of the PET bottles, however it is unclear whether this is due to lack of microbial action or because this new technique needs refining. There were clear differences between communities on the exposed PET bottles and the collected open water fragments. DGGE analysis on open water fragments showed Cyanobacteria (predominantly Phormidium and Pseudophormidium) were most dominant across all plastic types and sampling stations.

Through an investigation of multiple types of plastic in various locations over a three season time scale this study has highlighted the variation and hinted at the biodiversity on marine plastics. The majority of microbiota inhabiting marine plastics are commonly found in natural biofilms however the study appears to overlook the less abundant bacteria, which could have novel or harmful effects. Similarities were detected between assemblages on plastic and glass, implying links between natural and plastic biofilm colonisers. Plastic biofilms pose a higher risk to oceans as it floats and migrates within water transporting non-native biota. 

Obeckmann, S., Loeder, M. G. J., Gerdts, G., Osborn, M. (2014) Spatial and seasonal variation in diversity and structure of

microbial biofilms on marine plastics in Northern European

waters. Microbiology Ecology. 90: 478-492. http://onlinelibrary.wiley.com/doi/10.1111/1574-6941.12409/epdf 

Oysters with a side dish of... Probiotic bacteria?

Paralytic shellfish toxins (PSTs) are non-protein neurotoxins produced by saltwater dinoflagellates and freshwater cyanobacteria. They are a group of water-soluble carbamate alkaloids, which are either non-sulfated (saxitoxin (STX), neo-STX), singly-sulfated (gonyautoxins (GTX)) or doubly-sulfated (C-toxins). A high intake of PST leads to paralytic shellfish poisoning, where the toxins block the influx of sodium channels, restricting signal transmission along neurons therefore causing death from respiratory failure. The effect of long-term, low level exposure to PSTs is unknown, however most cases where humans are affected occur through contaminated seafood (such as mussels and oysters), algal dietary supplements and toxin-producing cyanobacterial cells. Human paralytic poisoning has been an increasing problem in the food industry, where seafood regulations set the maximum acceptable limit of PSTs in shellfish at 80 µg STC equivalents 100g^-1 tissue. There is no known antidote or cure for paralytic shellfish poisoning, and most methods involving removing PSTs have limitations. 

This study by Vasama et al. (2014) looks at using two probiotic lactic acid bacteria, Lactobacillus rhamnosus strains GG (GG) and LC-705 (LC-705) (in viable and non-viable form), in removing six PSTs from acidic and neutral solutions, mimicking the pH variation in the gastrointestinal tract. Both bacterial strains (GG and LC-705) in both viable and non-viable forms were cultured, and cyanobacterial extract containing PSTs (taken from a bloom of toxic Anabaena circinalis) were obtained and prepared to give two pH level solutions. Pure PST solution was prepared and each strain of bacteria was suspended in either Pure PST solution (as a control) or one of the two pH PST solutions (pH 7.3 or 2.0). High Performance liquid chromatography was then performed on the samples to detect each of the three classes of PSTs (STXs, GTXs and C-toxins). 

The results showed a strong removal of PSTs by non-viable bacteria indicating that PSTs are possibly removed by binding rather than metabolism. The highest degree of removal was observed for STX and neoSTX (77%-97.2%). The effect of non-viable and viable showed no significant difference therefore suggesting viable bacteria may also remove PSTs through binding, which is consistent with previous reports that specific bacterial strains remove a range of mycotoxins through binding. 

Overall, our knowledge on absorption of PSTs through mammalian intestinal epithelium is still limited, however this is the first study to show that by possibly altering the intestinal microflora composition using probiotics, the uptake of harmful compounds by the body when ingesting PSTs can be decreased and symptoms of sickness can be prevented. This can therefore lead to development for industrial applications or health benefits. 

Vasama, M., Kumar, H., Salminen, S. & Haskard, C. A., 2014. Removal of Paralytic Shellfish Toxins by Probiotic Lactic Acid Bacteria. Toxins, Volume 6, pp. 2172-2136. 

http://www.mdpi.com/2072-6651/6/7/2127/htm