Signalling-mediated cross-talk modulates swarming and biofilm formation in coral pathogen Serratia marcescens

Symbiotic relationships are formed between corals, dianoflagellates and associated bacteria. The stability and effectiveness of this symbiotic relationship determines the health of a whole reef ecosystem and its resistance to stress and diseases. Coral mucus is excreted onto a coral surface and mucus binds to bacterial receptors, this directly controls the composition of associated microbiota. The fate of mucus and its role in microbial communities are becoming clearer however less is known about microbe-microbe interactions within coral mucus. It has been suggested that microbe to microbe interactions within coral communities may have important functions relating to coral health and interactions with pathogens, some evidence has emerged showing that microbes associated with specific corals produce antimicrobials. One known mechanism in microbe-microbe interactions is exchange of small molecules. Cell to cell signalling and the resulting changes in the bacteria are known as quorum sensing. QS controls surface spreading, production of antibodies and exoenzymes, attachment to surfaces and timing of virulence gene expression. The aim of this study was to test whether bacteria associated with marine invertebrates and their endosymbiotic dinoflagellates, produce cell-to-cell signals capable of affecting behaviours in opportunistic pathogens by manipulating inversely regulated multicellular behaviours.

300 bacterial isolates from the mucus were screened with Chromeobacterium violaceum. Three luminescent reporters based on AHL receptors were used to analyse isolates which were capable of stimulating or inhibiting QS-mediated pigment. It was found that 4% of the tested bacteria were capable of affecting at least one reporter. In a similar study by Golberg et al. (2011), it was found that 30% of isolates affected reporters.  Thin-layer chromatography was used to further characterise 13 isolates. This was done to separate inhibitory from stimulatory QS activities produced by bacteria. Most of the tested strains produced one or two activities which co-migrated with AHLs of medium lengths. This shows a lack of complexity in the signals which is differs from other research.

The next few experiments focused on testing the behaviours of the isolates in the dual-species microbial consortia consisting of the coral pathogen S. marcescens PDL 100 and the isolate of interest. The hypothesis that marine isolates capable of affecting QS-reporters will also modulate behaviours from S. marcescens was tested. Ten out of the thirteen strains produced QS signals detectable with an Argobacterium tumefaciens reporter. This suggested that either QS activities detected with the Agrobacterium reporter are not AHLs or that in addition to QS signals; bacteria produce compounds which specifically disrupt swarming. The isolates were then inoculated with a wild-type Serratia strain and the PDL 100 strain. Five species inhibited swarming in the wild-type but not in PDL100. These results suggest that in addition to producing compounds that trigger responses in QS reporters, tested strains secrete other substances that inhibit swarming in S. marcescens. This is done by interfering with the regulation of the flagellar regulon or disrupting the synthesis of the surfactant.  It also suggests that mutant strains partially restore the ability to swarm and so will probably have similar signals; however the signals are usually strain specific.

The next hypothesis stated that in the absence of an interaction between a secreted compound from associated bacteria affecting global regulatory systems then there should be a measurable effect on biofilm formation by S. marcescens in the presence of the tested isolates. The results showed that a number of strains stimulated swarming and also inhibited biofilm formation by PDL100, even though growth of the pathogen in the suspension cultures was not affected. This suggested that the compound secreted by these organisms may target a global regulatory switch involved in the regulation of swarming and biofilm formation, without affecting growth of another organism. Not all strains showed the same results, some stimulated both swarming and biofilm formation, however some reduced both.

Finally, to test whether the isolates capable of affecting biofilm formation and swarming in the model opportunistic pathogen S. marcescens PDL 100, individual polyps were inoculated under laboratory conditions with the pathogen with or without the antagonistic marine isolates The results show that, PDL 100 can completely degrade the polyp within 3 -5 days. Pre-inoculation of the polyps with either a cocktail of isolates or a monoculture of  α-proteobacterium reduced the appearance of the disease symptoms in the polyps infected with the white pox pathogen. This indicates that the native microbiota associated with the invertebrates or their endosymbiotic dinoflagellates are capable of producing activities that reduce susceptibility of marine invertebrates to opportunistic pathogens.

Overall, there was no strong correlation between QS activities and the effect on swarming and/or biofilm formation by S. marcescens PDL100.

We found that was a significant paper as they used novel method, such as using the dialysis tubing (which isn’t mentioned here we had too much to talk about). The last experiment was the most significant as it showed some bacteria fully repressed the effect of pathogen damaging coral or anemones. However we found that the paper incorporated a number of methods, some of which are unnecessary. This made it confusing; also some methods are just mentioned in the results section and not the methods section. Sorry for such a long blog post, it was a very long paper!

This is a joint post by Georgia Smith and Sophie McKeeman relating to our seminar paper.

Alagely, A., Krediet, C. J., Ritchie, K. B., and Teplitski, M. (2011). Signaling-mediated cross-talk modulates swarming and biofilm formation in a coral pathogen Serratia marcescens. The ISME journal 5, 1609-1620.

http://www.ncbi.nlm.nih.gov/pubmed/21509042