Building bacterial biofilms

Biofilms are complex structures which are created by the attachment and growth of microorganisms on available substrates. Biofilms are formed in succession, starting with pioneers and later colonisers; but very little is known about a biofilms early formation; despite it being relevant to many sectors of marine ecology, such as larval recruitment, settlement and dynamics of microbial communities. It is thought that a biofilm commences with the adsorption of a film of polysaccharides, proteins, lipids, nucleic acids and aromatic amino acids, and is modified to create a stable climax community by secondary microorganisms/ colonisers, following the reproduction, growth and death of the pioneers. The coral surface mucus layer (SML) provides the perfect surface for the formation of the biofilm. Microbial biofilms can be established and maintained in three ways:

1) Microbes continually settle or are trapped by the SML, but not form a stable community due to the constant sloughing off of the layer.

2) A semi-established community may form in the SML of coral species which periodically shed their mucus as a tunic (e.g. Porites spp.)

3) Microbes may settle in the SML and/or coral tissues and become established, forming a distinct community from that of the water column.

Specific physical and chemical properties of the various mucus’ created by different corals may effect, and therefore explain, differences in microbial communities found in biofilms of different species; there is also an idea that differences may occur between communities due to the settlement surface offered.

While many studies have employed the use of flat settlement surfaces, this project used artificial corals coated in agar to test for different effects of surface shape and chemical composition on the development of a microbial biofilm community over 96 hours. The results were compared to the surrounding water column and a major reef building coral A. muricata; in both summer and winter.

The initial experiment used 4 types of agar coating for the artificial coral: plain agar, agar plus mucus, agar plus exudates from healthy coral and agar plus exudates from stressed coral; however there was no significant difference between the 16s rRNA gene bacterial assemblages settling, therefore, only plain agar was used for further temporal analysis.

Microscope slides were used to compare a smooth surface to the artificial coral nubbins, and gave a significant difference in microbial assemblage, with the nubbins giving a much more diverse community; tested for using DGGE; however, they found that 22% of variance could be put down to season alone, but after finding no ribotypes exclusive to one season meant significant differences were due to shifts in dominance of particular ribotypes. Large fluctuations in diversity between replicates in the first 12h, indicated that the initial settlement period is highly dynamic, but after this time becomes a lot more stable.

Vibrio species seemed to be opportunistic bacteria, as they appeared in early periods around 2h but were absent by 6h, apparently outcompeted. The idea of the Vibrio species being opportunistic ties in well with their role in coral bleaching; and as they are out competed it would be interesting to see if Flavobacteria sp., Glaciecola sp., and Klebsiella sp. etc. would prevent coral bleaching?

This study showed that there is a strong correlation between early colonisers of the SML and the surface type for settlement; and as shown before, a greater bacterial diversity was found on a more textured surface when compared to a smooth one. One of the reasons suggested for this is because a textured surface provides shelter from hydrodynamic processes such as wave action, and to further this investigation, it would be fascinating to see what effect would arise using the same principles, in a different marine environment, where hydrodynamic processes will not play much of a role, such as a sheltered lagoon.

A review of:

Sweet, M.J., Croquer, A., and Bythell, J.C. (2011). Development of Bacterial Biofilms on Artificial Corals in Comparison to Surface-Associated Microbes of Hard Corals. PLoS ONE.

Vol. 6. (6): e21195.doi:10.1371/journal.pone.0021195

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