Bacteria are not the primary cause of bleaching in the Mediterranean coral Oculina patagonica

After recently reviewing the paper by Rosenberg, E., et al. published in 2009, titled ‘The role of microorganisms in coral bleaching’ which held the view that high temperatures act upon the coral microorganisms as well as the host, causing a change in microbial community that can either directly or indirectly lead to coral bleaching; I have read a paper from 2008 that opposes the idea that bacteria are the primary cause of coral bleaching.

It is well documented that during times when the sea surface temperature is warm, symbiont photosynthesis is reduced due to an amplified susceptibility to photo-inhibition, which leads directly to active oxygen production and results in the breakdown of the symbiosis. Alternative recent studies have proposed that bacterial pathogens are the primary source of bleaching in reef-building corals. The study explained within this paper aimed to investigate the in situ bacterial involvement, and in situ coral microbial ecology, in ecological patterns of bleaching O. patagonica across the Israeli coastline.

Extensive monitoring of O. patagonica during the bleaching event of 2005 (14/06/2005-22/08/2005) took place along the Israeli Mediterranean Sea. Both bleached and non bleached corals were monitored with samples taken every 2 weeks at Sdot Yam as well as Ashkelon, Bat Yam and Acziv; giving a total of over 140 samples. From each colony 3 replicate core samples were taken from each tissue region. Regions of sampling were designated either bleached or unbleached tissue- and bleached tissue cores were taken around the bleached lesion to ensure both bleached tissue and the active region of bleaching were sampled. Flourescent In Situ Hybridization (FISH) was used with oligonucleotide probes and coupled with spectral imaging to explore identity and structural complexity of the microbial communities. The authors also used transmission electron microscopy to examine intracellular bacterial proliferation. They decalcified the coral cores in 20% EDTA and stained 1μm thin tissue sections with 1% Toluidine blue. Samples were photographed using standard light microscopy, following which ultra-thin sections were viewed in transmission electron microscope at acceleration voltage 90kV and images taken.

The results showed that V. sholoi is not associated with O. patagonica bleaching. No evidence of bacterial populations was found associated with any of the 48 bleached coral samples, 48 samples of unbleached tissue or 48 samples of healthy corals during their study. Each sample was probed with a general probe mix and a Vibrio sp probe, with a resulting 144 FISH experiments conducted on each region type; FISH was even repeated on arbitrarily selected samples. Bacterial populations penetrating or multiplying were not found within either the epithelium or gastrodermis of the bleached regions. The only microbial communities found interacting with and in close association with the tissue layers of field bleached corals were members of the endolithic community. Endolithic communities were found not only on the bleached corals but also healthy corals. They therefore suggest that there is no evidence to support a primary role of bacteria in causing coral bleaching as in the basis of the ‘Bacterial Bleaching Hypothesis’.

If bacteria do not play a primary but rather secondary role during coral bleaching or some diseases (being corals are susceptible to microbial attack during stress) it needs to be determined if the use of microbial remedies on a local or regional scale could reduce the impact of disease events.

A review of:

Ainsworth, T. D., Fine, M., Roff, G., and Hoegh-Guldberg, O. (2008). Bacteria are not the primary cause of bleaching in the Mediterranean coral Oculina patagonica. The ISME Journal. Vol. 2. pp. 67-73.

Quorum sensing in coral associated vibrios

Corals are hosts to a diverse population of microbes associated with their tissue and mucus, these microbes are different from those found in the water column and usually distinct to the coral species. This has led to the idea that corals are ‘holbionts’, consisting of the coral itself and the associated zooxanthellae and microbes. This relationship is thought to have an important impact on the health of the coral ecosystem and the ability of the corals to deal with environmental stress.

Shifts in the coral associated bacteria have been seen during coral disease. It is thought that Vibrios are a main cause of coral disease, and a shift of the coral associated bacteria to a population dominated by Vibrios has been observed during times of disease. There is an idea that the bacterial population shifts to one dominated by Vibrios during times when the holobiont is under stress, although this is an idea that has been criticised. The paper gives several examples of coral diseases that a caused by Vibrio species being associated in times of thermal stress, one of these is taken from the paper by Ben Haim et al., 2003, where coral bleaching by V.coralliilyticus was associated with elevated sea temperatures.

The aim of the paper was the asses the idea that coral diseases may be attributed to the Vibrios utilising quorum sensing to outcompete the coral associated bacteria in times of environmental stress. A range of Vibrio strains from healthy and diseased coral were analysed for the quorum sensing molecules AHL’s (N-acylhomoserine lactones) and AI-2 (autoinducer – 2) at a range of different temperatures.

While increases in temperature have been shown to cause a shift towards Vibrio dominated populations, it was not known if increases in temperature caused an increase in AHL production. For V.harveyi R-21466 and V.Shiloi LMG 19703 AHL production decreased with increasing temperature. The AHL production remained constant at the different temperatures for the other Vibrio strains.

As quorum sensing is known to control the production of enzymes in Vibrios, so the effect of temperature on enzymes activity was also investigated. Generally enzyme activity was higher at 25⁰c and 30⁰c than at 18⁰c. There was only one strain where enzyme activity was correlated with AHL production, this had a negative correlation.

This is apparently the first paper that has tried to analyse the effect of temperature on quorum sensing in Vibrios, although the results from the paper did not seem to indicate any significant results, which may explain the lack of a discussion it is an important building block. Furthermore the authors state that many Vibrio species, such as V.harveyi and V.cholorea among others converge their various signals the transcriptional activator LuxO. Therefore investigation into LuxO would be an ideal development of this study.

The final part of the study was concerned with the inhibition of AHL production by V.harveyi R-21446, although this part of the paper is interesting I feel that the reasoning behind conducting this part of the experiment was poorly explained and is the first mention of it throughout the paper. A purple pigment violacein is regulated by QS. I assumed that the pigment was produced by the C. violaceum as the names are similar; the author only stated that it was produced by a strain, not saying which one. It was found that when R-21446 was overlaid with C. violaceum a non-pigmented zone was created round the R-21446 grown at higher temperatures. Similar results were seen with two other species, this suggests that R-21446 is capable of interfering with QS signals from other species. It has been shown that other bacteria can both produce and degrade signal molecules with this is the first time it has been shown in Vibrios.

The study concludes that the inhibitory activity of V.harveyi may give the species a competitive advantage and may explain why it is represented among Vibrios in the holobiont during times of thermal stress.

A review of: Tait, K., Hutchinson, Z., Thompson, F.L., Munn, C.B. 2010. Quorum sensing signal production and inhibition by coral-associated vibrios. Environmental Microbiology Reports. 2(1), pp. 145-150.

The energy-diversity relationship of complex bacterial communities in Arctic deep-sea sediments

This paper describes and discusses the links between energy, bacterial activity and bacterial diversity at different taxonomic levels, as well as identifying the bacterial taxa which are most likely affected by changes in energy availability. Their reasons for conducting this research was down to the rarity of studies linking energy availability to the bacterial diversity patterns, and in recent years advances in methods such as high-throughput fingerprinting has made them much more available. The authors believe that it is important to unravel the relationships between environmental conditions, organism diversity and ecosystem functions if we are to understand the effects of global change.

They chose the natural energy gradient of the Arctic continental slope (as it covers a range of phytodetritus fluxes and represents both mesotrophic and oligotrophic deep-sea settings) to place depth transects across; minimising any confounding factors by sampling across different ocean provinces. Their study began in September 1993 during RV Polarstern cruise ARK IX/4, in which they took samples from 17 stations from the outer Laptev Sea shelf into the deep Eurasian basin. Sediment cores were sliced 1cm thick horizontally, and sediment samples from the same stations were used to measure environmental-parameters, potential enzyme activity and DNA extraction.

For community structure analysis, DNA was extracted using ‘UltraClean Soil DNA Isolation Kits’ from 1g of sediment, and stored in Tris-EDTA buffer. 42 samples made up of various sediment horizons (0-1, 1-2, 4-5cm) were analysed using Automated Ribosomal Intergenic Spacer Analysis (ARISA), a technique developed for the rapid estimation of microbial diversity and community composition. A set of 10 samples were also chosen for 454 Massively Parallel Tag Sequencing (MPTS). In order to keep analysis over different taxonomic levels consistent, they used a subset of the 454 MPTS dataset for further analysis, in which only sequences with a complete assignment up to genus level were retained. A high Spearman’s rank correlation between dissimilarity matrices of the reduced and original dataset confirmed that ecological patterns were consistent in both.

The results shown prove a strong relationship between changes in alpha-diversity (sample richness) and beta-diversity (changes in community structure between sites) with changes in pigment concentrations. OTU (defined by ARISA) richness and pigments concentrations showed a strong positive, linear relationship until around 2µgcm^-3 sediment was reached, after this the relationship started to level off. The two molecular techniques ARISA and 454 MPTS, exposed similar ecological patterns; when 454 MPTS was applied to the subset of samples, a similar linear relationship was found, appearing to level off at higher pigment concentrations (3µgcm^-3).

Many more results following statistical analysis of varying methods show similar correlations and with regards to change in richness with increasing energy availability in the form of phytodetritus, suggest an overall positive response of bacterial OTU which was strongest at oligotrophic conditions (defined by low levels of pigment concentrations 2-3µgcm^-3); as well as benthic meiofauna and megafauna. An increased phytodetritus input sustains an increase in bacterial abundance and biomass, in line with the ‘more individuals’ hypothesis of the species energy theory.

Further studies of natural and experimental systems are required in order to decipher the mechanisms which can be held responsible for the establishment and preservation of energy-diversity relationships in bacterial communities- and if these can be extended to a global scale. Beyond energy availability-diversity relationships for complex bacterial communities, this study showed strongly implies that any environmental changes affecting primary productivity and particle export will cause shifts in bacterial community structure and function in the Arctic, which in turn may affect key processes such as the carbon cycle.

A review of:

Bienhold, C., Boetius, A., and Ramette, A., (2011). The energy-diversity relationship of complex bacterial communities in Arctic deep-sea sediments. The ISME Journal. pp. 1-9.

Symbiotic light, fantastic! : The Characterisation of Host and Symbiont Proteomes.

Within hours of hatching the Hawaiian bobtail squid, Euprymna scolopes, is colonised with the bioluminescent bacterium Vibrio fischeri in a specialised light organ. At night the epithelial crypts of the light organ contain the highest density of bacteria (up to 10^9cfu) which emit light that is used to avoid predation. However, a daily routine is established where the contents of the light organ, including 95% of the symbiont population, are vented at dawn when E.scolopes buries in the substrate. This cycle is completed where the remaining population of V.fischeri repopulate the crypts to similar levels as previously by nightfall. It is this relationship between colonisation, venting and growth in a host/symbiont system and the different genes that are expressed at each stage in both E.scolopes and V.fischeri that the authors have studied to determine which genes are required to establish a host/symbiont relationship.

The analysis of the host proteins expressed in the light organ showed the most abundant proteins are involved with the innate immune system, oxidative stress (superoxide dismutase) and other signalling pathways including NFκB (involved in DNA transcription under stress responses such as infection). Host proteins involved with iron sequestration and peptidoglycan recognition were also detected in the light organ and could explain the specificity for V.fischeri colonisation. Reactive oxygen species (ROS) and reactive nitrogen species (RNS) intermediates were found in high quantities in the light organ which play a key role in the continuation of the squid/Vibrio symbiosis. V.fischeri must adapt to this environment to allow it to colonise the light organ which again allows for specificity of colonisers and allows some protection of the squid tissues.

Proteins expressed between V.fischeri found in the light organ of E.scolopes and V.fischeri in culture were compared and identified and indicates that the 25 most abundant V.fischeri proteins were proteins such as luciferase (key to fluorescence), alkyl hydroperoxide reductases, and a host of cold shock proteins. These analyses identify a range of different stress responses such as oxidative stress where the expression of catalases such as katA and peroxiredoxins may indicate a mechanism for V.fischeri to protect itself from ROS and RNS produced by the host tissues. Quorum sensing between bacteria is identified through the abundance of immunoglobulin like domains allowing the adhesion of bacteria to host surfaces which is vital for the original colonisation. Motility proteins are also abundant in the colonising V.fischeri community prior to the expulsion of the symbiont through venting with the regeneration of flagella a few hours before. V.fischeri also expresses several haem utilising genes such as HutA to combat iron sequestration by the host. Symbiont metabolism also changes where, at night whilst at high concentrations in the light organ, V.fischeri ferments chitin for energy however, after expulsion, the remaining bacteria anaerobically respire glycerol.

This study, by using advanced proteomics methods, allowed for more accurate identification of host and symbiont protein expression than previous studies. These results allow greater insight and greater options to further understanding through future research into host/symbiont relationships such as the mechanism of flagellar regeneration prior to venting and the mechanisms leading to the specificity for specific bacteria to form a symbiosis with a specific host.

A review of : Shleicher,T.R.; Nyholm,S.V.; 2011; Characterizing the Host and Symbiont Proteomes in the Association between the Bobtail Squid, Euprymna scolopes, and the Bacterium, Vibrio fischeri; PLoS ONE 6(10):e25649. doi: 10.1371/journal.pone.0025649

Keeping Oxygen away from Nitrogenase

Approximately 50% of natural nitrogen fixation occurs in the oceans. Although this may not sound like a particularly large amount as our oceans cover 71% of the Earth, it occurs almost exclusively where the surface temperature is 25^oC or higher. The concentrations of nitrate are higher below the euphotic zone due to sedimentation but they may be pushed to the surface in upwelling zones and therefore be available to the cyanobacteria.

The paper discusses some of the ways that cyanobacteria may be well adapted to protect nitrogenase from oxygen inactivation so that they can perform the oxygenic process of photosynthesis as well as N₂ fixation. One group takes an ‘avoidance’ strategy, only fixing N₂ in anoxic conditions and inhibiting phoytosynthesis.

The second group is able to confineN₂ fixation to differentiated cells known as heterocysts. The heterocyst is able to generate energy using PS-I which is anoxygenic but has no PS-II so no oxygen is reduced. It is not able to fix CO₂ but receives sucrose from neighbouring non-heterocysts and also gives fixed nitrogen to neighbouring cells. The heterocysts cannot differentiate between O₂ and N₂ so must allow some O₂ in but this is quickly respired to keep the cell anoxic. It is thought that heterocyst cells may have dynamic pores so would be able to close during the night when nitrogenase activity is low to keep O₂ out and maintain an anoxic cell but even if this were the case it would only apply to some species of heterocystic cyanobacteria as some have been found to fix a lot of nitrogen at night.

The least is known about the third group which separates the process of N₂ fixation from photosynthesis by performing them at different time. There has been a lot of research into Trichodesmium and it has been found that it may also have differentiated cells, similar to heterocysts, although another study was unable to replicate these results. Another theory is that the cells are able to perform both and individually switch between the two.

Whichever group the cyanobacteria are in they must be able to respire any O₂ that enters with the N₂. If the temperature is high enough the nitrogenase activity is controlled by the O₂ flux into the cell (Transport Control) but at lower temperatures the respiration rate is not fast enough to maintain anoxic conditions so nitrogenase activity is largely reduced (Reaction Control). Different species of cyanobacteria switch from transport to reaction control at different temperature. Although these temperatures do reflect the environment each cyanobacteria comes from it must be noted that the values are absolute limits and in order for the cyanobacteria to compete they must be in higher temperatures.

This paper doesn't seem to come to a conclusion as to why the N₂ fixing cyanobacteria are mostly limited to temperatures of 25^oC or higher but after reading the paper it seems that the decreased rate in respiration with temperature may play a part in this as anoxic conditions cannot be maintained so N₂ Fixation cannot occur.

Reference: Stal, L. J. (2009), Is the distribution of nitrogen-fixing cyanobacteria in the oceans related to temperature?. Environmental Microbiology, 11: 1632–1645

The role of microorganisms in coral bleaching

Coral bleaching is the disturbance of the symbiotic relationship between the coral and its endosymbiotic zooxanthellae (of the genus Symbiodinium). The severity of the disease is often correlated with high seawater temperature; and in recent years the main hypothesis to explain coral bleaching is that the high temperature of the water causes irreversible damage to the symbiotic algae, resulting in loss of pigment/ algae from the holobiont- the results of this is that the coral tissue becomes transparent, showing the calcium carbonate skeleton underneath; other signs of bleaching include reduction in mucus and often inhibition of sexual reproduction. If bleaching is not reversed then corals will die. This paper discusses evidence for an alternative hypothesis- the microbial hypothesis of coral bleaching.

Although the running hypothesis is mainly based on a raise in seawater temperature, other findings have proven that salinity, cyanide exposure, sediments and seawater temperature decrease have all been causes of bleaching as well as the often disregarded microbial pathogens.

Coral bleaching was discovered in the Eastern Mediterranean Sea, as it occurs every summer amongst the species Oculina patagonica. It was found that an infection by Vibrio shiloi was the cause using ‘Koch’s postulates’ (a criteria for establishing a causal relationship between a causative microbe and a disease), but the effects could only occur if both the causative agent was present and an elevated temperature of above 25^oC. The pathogenic bacteria are chemotactic to the coral mucus and adhere to the β-galactoside-containing receptor, on the coral surface; they penetrate through into the epidermal layer and then multiply intracellularly (reaching 10⁸- 10⁹ cells per cm³). The V. shiloi produce an extracellular peptide toxin (PYPVYPPPVVP) which inhibits photosynthesis in the alagae. All of the reactions are temperature dependent, relying on the conditions being 25-30^oC, explaining why it only occurs in the summer and not all year round.

The authors consider the ideas of coral bleaching being due to ultraviolet radiation, however, they discuss a paper that argues against this, by using samples of corals from high ultraviolet radiation points (water 0-80cm in depth) with results showing that UV radiation actually inhibited coral bleaching by killing the pathogen.

O. patagonica is not actually a reef coral, so to test the theory of microbial bleaching, it was important to check the hypothesis on a reef coral; for this they chose Pocillopora damicornis, from the Zanzibar coral reef. It was shown to have also been bleached by a pathogenic species of Vibrionacea known as Vibrio coralliilyticus.

There are two main and differing viewpoints on the matter of coral bleaching.

Most coral biologists take the view that high temperatures and light act directly on the symbiotic algae to inhibit photosynthesis and produce reactive oxygen species. According to this idea, microbes play no role in the bleaching of the corals, and the change in microbial community is a result and not a cause.

The second hypothesis which is possible (and is taken to be the view of the authors of this paper) is that high temperatures act on the coral microorganisms as well as the host, causing a change in microbial community that can either directly or indirectly lead to bleaching.

Clearly there is not enought evidence to rule out one hypothesis and further research needs to be conducted, combining coral microbiology together with coral host physiology is required to clarify the bleaching process.

A review of:

Rosenberg, E., Kushmaro, A., Kramarsky-Winter, E., Banin, E., and Yossi, L. (2009). The role of microorganisms in coral bleaching. The ISME Journal. Vol. 3. pp. 139-146