DMSP-degrading bacteria - In control of Corals

Dimethylsulfoniopropionate (DMSP) is an organic sulfur molecule known for its role in climate regulation. Previous studies have shown that zooxanthellae (genus Symbiodinium) abundance found in cnidarian species are correlated to DMSP concentrations among the host, which are further available for coral-associated bacteria that metabolize it into dimethylsulfide (DMS) – the gas implicated in climate regulation. Two DMSP degradation pathways may occur, one involving phytoplankton or bacteria using enzymes (DMSP lyases) and the other involving only bacteria which carry out an initial demethylation of DMSP to methylmercaptopropionate (MMPA). This initial step involves a single gene, DMSP demethylase (dmdA). DMSP has been thought to act as an antioxidant towards marine algae, helping corals survive periods of thermal stress. Mucus-associated microbes have also been seen to play an important role in protecting the host coral against pathogens. The surface mucus is also subjected to high levels of DMSP which leaks from coral tissues. Furthermore, there is a possibility for DMSP degradation to take place in the surface mucus layer in corals, where coral associated DMSP-demethylating microbial communities perform this. 

This study by Frade et al. (2015) looks at the link between coral DMSP availability and the community dynamics of DMSP-demethylating bacteria found in the surface mucus of corals. This was done by first determining in situ DMSP concentrations in the tissue of three common reef-building coral species (Meandrina meandrites, Porites astreoides and Siderastrea sidereal) in relation to solar radiation intensity and desiccation stress (exposure to air). Genetic analysis was used to investigate taxonomic affiliation and relative abundance of mucus-associated bacterial assemblages harboring the gene dmdA. 

The results showed that DMSP varied with host species-specific traits such as zooxanthellae cell abundance. It was mostly seen that the occurrence of higher symbiont cell abundance as a cause for increased amounts of DMSP in the tissue. Exposure to air caused a doubling of their DMSP concentration, suggesting rapid de novo production of DMSP by the coral symbiont, either by the zooxanthellae or the coral host itself, possibly due to oxidative stress from thermal stress which further suggests the molecules role as an antioxidant. A higher availability of DMSP corresponded to a lower relative abundance of the dmdA gene, however this was not seen throughout all host species suggesting distinct DMSP microbial niches exist. 

Overall, this study is the first to provide quantification of dmdA gene assemblages in corals and link it to changes in community dynamics of DMSP-degrading bacteria to DMSP availability. It shows that DMSP is important in establishing links between coral hosts and associated bacterial communities, with the possibility of shifts in these communities and growth rates to depend on DMSP availability. Further study should be done into correcting the assumptions (such as whether changes in the relative abundance of dmdA genes relate to the degree of adaptation) which may allow a more definitive answer behind this study. 

Frade, P. R. et al., 2015. Dimethylsulfoniopropionate in corals and its interrelations with bacterial assemblages in coral surface mucus. Environmental Chemistry, pp. A-K.

http://www.publish.csiro.au/?paper=EN15023

There may be hope yet - Corals can adapt to climate change

Corals and their symbionts often live at the limit of their thermal tolerance, with small changes potentially resulting in mass bleaching. While many papers have shown that corals can bleach to adapt to change, it can take weeks/months for an effect to be seen. During this time, disease may kill the corals before they can recover.

However, a recent paper by Palumbi et al. (2014) showed that the transplantation of a fast growing, dominant coral species (Acropora hyacinthus) from cooler microclimes to areas experiencing highly variable (HV) temperatures (up to 35°C) adapted in less two years. Coral branches were transplanted from moderately variable (MV) sites, where the temperature rarely exceeds 32°C, to HV sites and grown in situ.

Coral branches from each site were bleached according to native conditions, with the retention of chlorophyll a in the symbionts used as a measure of resistance to thermal stress. The results showed that the MV corals had on average less heat resistance than the HV corals; the MV group retained only 45% of chlorophyll compared to 80% in HV.

To test for acclimatisation, the corals from MV were transported to HV conditions (vice versa) and then experimentally bleached at 12, 19 and 27 months. Of the 23 experiments, 22 showed that the corals acquired partial heat sensitivity of the pool they were transplanted to. This means that the corals from the MV group transported into HV conditions and bleached were able to retain more chlorophyll a in the new site than the native MV corals. What was interesting was that the HV corals that retain more chlorophyll a than the MV group when bleached actually dropped their levels to match the native MV group when transplanted.

An ANOVA using 16,728 genes revealed that 74 had changed significantly when comparing the genetically identical corals from each site. The gene expression had changed between sites, despite coming from genetic clones, purely based on environmental differences. Some genes were expressed purely based on the origin of the corals, regardless of where they were transplanted to. Based on these findings, there may be fixed constitutive expression levels for thermal resistance in both sites, and adapted gene expression acquired after transplantation.

Differences in the Symbiodinium clades present in each group after transplantation were measured by looking at transcriptome reads. Clades C and D are common in this species, with the HV group containing higher levels of D. Despite predictions that the clades may have changed, they determined that there was little shift in one clade over another even after transplantation.

Phenotypic change seen in the corals was also measured in order to put this acclimation effect in terms of evolution. This change is often studied by measuring the change in mean phenotype before versus after natural selection, divided by standard deviation. Estimates were made based on all data collected, which showed that the corals did in fact acclimate to higher temperatures.

Pooling all the data together, this study has shown “that acclimatisation can allow corals to acquire substantial high-temperature resistance more quickly than strong natural selection would produce”. Acclimation alone may not save corals world-wide, due to other increasing factors having an impact, however this paper could provide a method for preserving most corals. This is a good starting point for looking at how other species of coral found in shallow/deep waters may be affected by temperature change, and could show how they may adapt to changing conditions.

Palumbi, S. R., Barshis, D. J., Traylor-Knowles, N., & Bay, R. A. (2014). Mechanisms of reef coral resistance to future climate change. Science,344(6186), 895-898.

http://www.sciencemag.org/content/344/6186/895.full.pdf

How low can pH go? The effects of ocean acidification on biofilm microbial composition

Biofilms are made up of the early colonisers of rocky intertidal ecosystems; they act as the basis for succession and are vital to the grazing of organisms at higher trophic levels. Microbes are a large component of these biofilms and so it is important to study the diversity of such epilithic communities in order to monitor the effects of changing environmental conditions.

As such, in a study by Taylor et al, molecular techniques were used to examine microbial community composition at three sites along a pH gradient caused by a large CO₂ seep beneath Levante Bay in Italy. The seep itself produces around 3.6 tonnes of CO₂ per day which causes localised seawater acidification around it, leading to a pH of 5.6 that increases to normal Mediterranean sea pH as you move away from the seep. This can therefore act as an environmental model for the effects of global ocean acidification on intertidal epilithic microbial diversity.

Comparison of OTUs found at each site showed that community composition differed significantly between all three sites. And, while primary producers such as Proteobacteria, Bacteroidetes and Cyanobacteria were found to be dominant in all the sites, the diversity and abundance of such groups individually varied significantly between sites. The ecological processes driving these differences are currently unknown however it is thought that decreasing pH may incur improved carbon turnover and therefore create competition, niche modification and cross-feeding that will result in altered community composition.  In other words, changes in community composition the sites may indicate the identity of the potential ‘winners’ and ‘losers’ of the future changing environmental parameters caused by ocean acidification.

The importance of this is the use of a seep site; by using in situ sampling as opposed to laboratory manipulations it is possible to examine communities that have experienced ‘real’ environmental selection. This therefore lends a predictive advantage to such a study. However, there is a drawback to this technique in that it cannot possibly take into account all conceivable variables that may have led to this change in community composition. It is therefore my opinion that it is only the amalgamation of in situ environmental sampling around CO₂ seeps and the use of highly selective mesocosm-based laboratory experiments that will provide the most accurate model for the predicted future impacts of ocean acidification on microbial diversity and in turn ecosystem functioning.

Taylor, J. D, Ellis, R, Milazzo, M, Hall-Spencer, J. M. and Cunliffe, M.. (2014). Intertidal epilithic bacteria diversity changes along a naturally occurring carbon dioxide and pH gradient. FEMS Microbial Ecology. 89, 670-678.

Battling Iron- a specialised approach

Iron is an essential element to almost all organisms, particularly those containing iron-rich cytochromes and iron-sulfur proteins which are essential for photosynthesis and nitrogen fixation. Fe(III) has very low solubility resulting in very low iron bioavailability. Many organisms have developed iron reducing mechanisms to overcome iron limitation: alagal species such as Chlorella vulgaris, plants and fungi use ferric reductase proteins within cell membranes for FE(III) reduction before internalisation whereas, microorganisms including cyanobacteria have developed siderophores- FE(III) binding chelating agents which prevent oxidation and transport into the cell. There are however, exceptions: the cyanobacteria Synechocystis lacks the genes for siderophore production and has been observed reducing Fe(III) extracellularly before transportation of FE(II) into the plasma where Fe(II) and Fe(III) transport systems (FutABC and FeoB) have been identified.  

Relatively little is known about iron reduction involving ferricyanide and its limiting factors and much that is, e.g. effects of light, is up for dispute. Previous studies have investigated bio-applications which make use of the electrochemical cell as a fuel source e.g. interactions of Synechocystic sp. 6803 with ferricyanide to produce hydrogen however, these have been largely abandoned due to unsustainability. Despite this, ferricyanide is still an important probe for cell membrane activity due to the strong binding of its iron atom which is not released during reduction to ferrocyanide.

This study investigated the ability of Synechocystis sp. PCC 6803 to reduce Fe(III) in ferricyanide with varied concentrations of ferric cyanide and cells present. Using electrochemical techniques: rotating disk electrochemistry (RDE), a technique using varied electrode rotation speeds to pull the solution towards the planar disk electrode giving laminar flow across the electrode; and chronoamperometry at a static macromolecule.

Typical measurements using a static electrode showed light induced a small reduction in current compared to dark conditions for cultures with both cells and ferricyanide present. Control media and solutions with cells but no ferrocyanide had a very low amplitude while a control with no cells and 1mM ferrocyanide was slightly higher (~1 µA). To prevent discrepancies in the data, variable concentration experiments were carried out in the dark and control measurements were taken during all experiments and subtracted from the data. Increased rate of ferricyanide reduction per cell with ferricyanide increase and constant rate of ferricyanide reduction per cell with constant ferricyanide and cell concentration manipulations implies Synechocystis cells reduce ferricyanide at a constant rate while the mass transport of ferricyanide to the cells is the limiting factor of reduction.

I thought this paper was an interesting read finding light causes ~10-20% difference in current and Synechocystis Fe(III) reduction is mainly limited by Fe concentration, which will be of high importance to anyone investigating Fe(III) reduction or Synechocystis. Unfortunately, much of their static electrode experiment findings are difficult to accept as more than trends due to the chance cells settled on the electrode, it would be interesting to repeat these with the mentioned dialysis membrane over the electrode to prevent cell settling. Overall, I thought this was a well composed study further developing our understanding of Synechocystis membrane activity and electrochemical techniques. 

Reference:
Thorne, R. J., Schneider, K., Hu, H., Cameron, P. J. (2015) Iron reduction by the cyanobacterium Synechocystis sp. PCC 6803. Bioelectrochemistry. 105: 103-109.

Life After Death for Tubeworm Endosymbionts

Riftia pachyptila, better known as tubeworms, are deep sea invertebrates that live in symbiosis with what seems to be a single species of symbiotic bacteria: Candidatus Endoriftia persephone, better known as Endoriftia, that live in a specially created organ in the tubeworm called a trophosome. While the worm is alive, it provides these Endoriftia with substrates for chemosynthesis (the fixation of 1-carbon compounds into organic carbon using chemical energy) and in return the symbiont gives the host organic carbon. But what happens when the worm dies? The general consensus was that the symbionts die too, but how can this be when this symbiosis has lasted for so long? This study by Klose et al. investigates the fate of the symbionts once the tubeworm has died.

The researchers found that 97% of the endosymbionts in their experimental setup did in fact escape the dead tubeworm tissue in a process similar to how they initially enter the host (through the skin); estimating 7 million symbionts released per 10 dead worms, though this is an interesting figure as later in the paper it is stated that 3g of trophosome in a 20g tubeworm can house 1.11x10^10 symbionts, what is causing this loss? Potentially autolytic enzymes or loss of symbionts that are not able to pass back through the skin of the worm. Furthermore, the symbiont metagenome revealed the capacity for flagellar motility. I wonder if these Endoriftia are able to leave their host pre-death should conditions become unfavourable to them, similar to a coral bleaching event.

Though they live in symbiosis, Endoriftia are also able to live in a planktonic state, but the distance to travel to a new hydrothermal vent must be unachievable for these micro-organisms. A thought of mine is that once these symbionts escape their dead host and establish a free living population, they resume chemosynthesis. This production of organic carbon might attract larger organisms or even their next host to ingest them or otherwise pick them up and carry them to another vent site. Ocean current dispersal is a much more feasible idea however.
In order to acquire the data, special recruitment plates were built with 0.4g tubeworm trophosome at the bottom and 5 glass cover slides stationed at distances away from the flesh to investigate the post-death dispersion of the Endoriftia. The plates were kept under simulated environmental conditions from where the worms were collected from and incubated for up to six days. after the experimental period and then analysed with FiSH and TEM.

We knew before that these symbionts were transmitted horizontally but this paper further shows how and on what magnitude  they are released into the environment once their host dies or can no longer provide for them, giving us insight into the life history of these symbionts and their potential impact on Carbon chemistry of the local water after their host dies.

Reference:

Klose, J. Polz, M. F. Wagner, M. Schimak, M.P. Gollner,S. and Bright, M. (2015) Endosymbionts escape dead hydrothermal vent tubeworms to enrich the free living population. PNAS, 112 (36), 11300-11305

Available at: 

http://www.pnas.org.plymouth.idm.oclc.org/content/112/36/11300.full 

Microbes, Corals and Oil: The deep dark secret to cleaning up spills

The Deepwater Horizon oil spill (DWH) spilled an estimated 210 gallons of oil and gas into the Gulf of Mexico in April 2010. Many studies have looked at the detrimental impacts on megafauna, however few have looked at how the release of oil and gas has influenced the microbial community structures, particularly in the deep sea where many corals thrive. Oil degrading microbes, which include γ-Proteobacteria, have been linked via meta-genomic, -transcriptomic and metabolomics studies to the degradation of the aliphatic components of oil and aromatic components of oil. Previous studies have shown varying microbial community when comparing initial responses to 4-5 months after the spill. This study by Simiser et al. (2015) focuses on providing a further insight into the microbial community structure and the diversity of microbial genes involved in oil-degradation pathways in the deep-sea. 

Samples, including flocculent material from coral surfaces (floc) and surface-sediment, were collected at 1370m depth from a site of the Macondo well blowout, known to be impacted by the DWH oil spill. These samples were tested using molecular techniques such as 16S rRNA sequencing which involved constructing and analyzing bacteria and archaea clone libraries from full-length 16S rRNA sequences, Illumina MiSeq 16S rRNA amplicon sequencing and analysis and functional gene sequencing (constructing and analyzing genes associated with oil-compound degradation processes). Functional genes included those for alkane hydroxylase (alkB) and alkylsuccinate synthase/benzylsuccinate synthase (assA/bssA).

The results from the Archaeal 16S rRNA Illumina sequencing were dominated by Thaumarchaeota, however in both sediment samples and coral floc, majority of full length 16S rRNA archaeal sequences are affiliated to the ammonia oxidizing archaea (AOA), which does not correspond to laboratory cultures where ammonia oxidation and nitrification were inhibited by oil, suggesting further study needs to be done to fully understand the impact of oil on archaeal diversity, community structure and function. The results for most abundant bacterial and archaeal OTUs showed evidence of oil degrading species being dominant in sediment samples, such as Cycloclasticus sp. that were enriched in the subsurface plume and surface slick samples. They are also known degraders of petroleum hydrocarbons including PAHs. The next most abundant OTUs for both sediment samples are affiliated within the δ-Proteobacteria, particularly to sulfate reducing bacteria (SRBs). At hydrocarbon sites in the GoM, high abundances of these bacteria are found and can be directly involved in the anaerobic degradation of propane and butane. Sequencing results for functional genes involved in oil degradation exhibited broad diversity within the Proteobacteria, with many OTUs aligned closely to γ-Proteobacteria. The functional gene for aerobic degradation of oil (alkB) were detected in all samples, with the functional gene for anaerobic degradation of oil (assA/bssA) detected in sediment samples. This suggests aerobic oil degradation dominates in the floc.

Overall, this study presents a microbial approach using genetic techniques to look at the impacts of oil spills on the marine environment, and the response by which bacteria can potentially degrade various compounds in oil. This could potentially lead to further studies and monitoring in the microbial community, especially if similar events to the DWH occur in the future. The DWH oil spill has presented an in situ study where data collected now, can be compared to pre-spill data (though this is limited and poses challenges) and data found at deep-sea sediments, natural hydrocarbon seep sites and sediments. 

Reference: 

Simister, R. L., Antzis, E. W. & White, H. K., 2015. Examining the diversity of microbes in a deep-sea coral community impacted by the Deepwater Horizon oil spill. Deep Sea Research II.

http://www.sciencedirect.com/science/article/pii/S0967064515000211