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

Marvellous Mucus

Many coral reefs are found in nutrient poor zones, but are none-the-less high primary production ecosystems. Mucus secreted by some corals (mucopolysaccharide) carry energy and nutrients to a range of planktonic and benthic consumers, and supports a large microbial growth within the reef system.The conductors of this study decided to investigate which bacteria in seawater are favoured by the release of freshly detached mucus material. Short-term changes in organic carbon and nitrogen, and microbial community composition were simultaneously analysed in mucus enriched seawater.

Coral mucus and seawater were collected from the lagoon of Dahab, in the southern part of the Gulf of Aqaba, northern Red Sea, in May 2004. Polyps of one of the most common Red Sea scleractinian corals (Fungia sp.) with a diameter of 5-10cm were collected from depths of 3-8m, and then exposed to air for 3 minutes in order to trigger mucus production. Mucus produced in the first minute was discarded to reduce contamination of bacteria from the coral surface and mucus produced in the further 2 minutes was aseptically collected and bottled. The coral mucus was homogenised and mixed with seawater (1:10) obtained from the same site within 60 minutes and bottled; bottles of seawater which hadn’t been enriched with mucus were kept for control. All bottles were kept incubated for 50 hours at in situ temperature (24^oC) and light conditions (500-800 µmol quanta m^-2day^-1) at a depth of 1m.

Each bottle was portioned and filtered onto precombusted GF/F filters. The concentrations of particulate organic C (POC) and particulate N (PN) and stable isotope ratios of C to N were determined using dried filters with THERMO NA 2500 elemental analyser coupled to THERMO/Finnigan MAT Delta plus isotope ratio mass spectrometer. The POC and PN concentrations were two- to threefold higher in the mucus amended incubations when compared with the controls.

15ml samples were taken after 0, 2, 4, 6, 10, 26 and 50 hours for bacterial cell counting and fluorescent in situ hybridisation (FISH). Portions were filtered onto polycarbonate membrane filters, after 24h of fixation in buffered paraformaldyhide solution. Total cell numbers were determined by staining with 4’, 6-diamino-2-phenylindole and automated epifluorescence microscopy. The initial cell numbers in the mucus-seawater mixture were marginally higher than the control cell numbers. Additions of mucus lead to an almost fourfold increase in bacterial abundance after 10 hours; whereas the control approximately doubled over the whole 50 hours.

Addition of mucus to the seawater clearly favoured the growth of the Gammaproteobacteria. The relative abundance of these microorganisms initially developed similarly in the mucus and control incubations. Between 4 and 10h, the proportions of Gammaproteobacteria in the mucus incubations rose steeply, to around 60%after 10h and around 70% toward the end of the experiment. The Gammaproteobacteria never exceeded 40% of the total cell counts in the controls.

A review of:

Allers, E., Niesner, C., Wild, C., and Pernthaler, J. (2008). Microbes Enriched in Seawater after Addition of Coral Mucus. Applied and Environmental Biology. Vol. 74 (10). p. 3274-3278

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.

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