Marine Fouling mediated by Quorum Sensing

Quorum sensing is the mechanisms where bacteria cells are able to communicate and co-ordinate their behaviour within population densities. This paper looks at the possible use of quorum sensing in marine fouling, the main aim of this paper is to determine the presence of AHLs and verifying quorum sensing systems within the bacteria species. The quorum sensing systems in Gram-negative bacteria are regulated by N-acyl homoserine lactones (N-AHLs) whereas quorum sensing systems in Gram-positive bacteria are mediated by small peptides. AHLs are signal molecules called acyl homoserine lactone and can vary in their acyl group chain. Their general structure is shown in figure 1. These signal molecules can accumulate into high concentrations and cause a group response such as bioluminescence within bacterial communities. This paper explores the proposal that AHLs or N-AHLs can mediate fouling.

Gram negative bacteria strains were isolated from biofilms present on four different hosts. Among these hosts were two sponges A. insularis and A.lacunose, the shell of Donax sp and submerged phytagel dishes. All bacteria were collected from the Santa Marta Bay.

The authors used gram negative staining, morphological description and 16s rRNA gene sequence analyses. These methods identified the isolated bacteria from the biofilm coating.

The bacteria identified from the biofilm were:

·         Ochrobactrum sp

·         Vibrio sp. (23-6PIN)

·         Vibrio campellii

·         Vibrio sp (11-6DEP)

·         Ochrobactrum pseudogringnonense

·         Shewanella sp

·         Vibrio harveyi

·         Alteromonas sp

Growth curves were used to ensure proper incubation conditions for each of the bacterial strains.

The bacteria Escherichia coli was used as a biosensor, this (as well as the bacteria strains P.putida IsoF and P.putida F117) was supplied from Dr Katrin Riedel from the Microbiology department of the biology Plant institute, Zurich, Switzerland. A cross streaking assay was performed with E.coli as a biosensor to detect the presence of quorum sensing in the bacteria. In a modified Marine medium (mMM) the E.coli (pSB401) biosensor was streaked with each bacterium isolate as well as the positive control strain (P.putida 117) and the negative control (a mutant P.putida 117) strain.

The results of the cross streaking assay are found in figure 2, this figure shows that the positive control and Ochrobactrum sp induced bioluminescence in the biosensor strain.

Vibrio sp (23-6PIN) showed no bioluminescence activity in the bio assay. The possible reasons given for this were firstly, that this genus has many species with recognised quorum sensing systems with Luxl-type typical proteins; this can be a dumb strain with an orphan luxR gene. Alternatively it produces the AHLs in very low concentrations or because it produces other AHLs that are not detected by the biosensor.

The marine bacteria, the positive (P.putida IsoF) and negative controls (P.putida F117) were cultured and filtered for N-acyl homoserine lactone extraction, this extract was needed to establish all the AHLs present in each bacterial strain. To confirm if all AHLs were present in the supernatant the extracts were analysed by RP-TLC revealing with a biosensor. Since four AHLs were identified in the positive control (P.putida IsoF) it was used for comparison with the other extracts, a demonstration of the four AHLs are shown in (J) from figure 3. The RP-TLC analysis of the bioluminescence of each extract showed that all the bacteria isolated produced AHLs.

Within both analyses there were exceptions, for instance Vibrio harveyi produce luminescence in the cross streaking assay, however it did not in the RP-TLC analysis, a possible reason for this is C4-AHL is very water soluble and so during organic extraction it is likely to not completely recover. Vibrio sp (23-6PIN) did the opposite, it was not luminescent in the cross streaking assay but was in the RP-TLC analysis, the reason for this is a long chain AHL. This is evidenced by its low motility in the TLC plates and the low motility would make it difficult to detect in the cross streaking assay.

The authors concluded that they found quorum sensing systems present in the different bacteria isolated. Using a biosensor for C4AHL, C6-3-oxo-AHL, C6-AHL, C8-3-oxo-AHL and C8-AHL, they could establish that the main AHLs in those bacterial communities were C4AHL and C6AHL. There was also evidence of inter-species communication; this was shown by the same AHLs identified in different bacteria found on the same surface. This inter-species communication can lead to stratified and stable biofilms.

Quorum sensing systems regulates biofilm maturation and is essential in the macro fouling process. The identified AHLs in this study have been previously recorded as settlement inductors for marine macro-organisms. There is evidence of fouling promotion by several AHLs for instance the C6-AHL isolated from the natural biofilm of Vibrio anguillarum induces the zoospores settlement of the green algae Enteromorpha, as well as the settlement of Hydroides elegans larvae. C4-AHL however induces the settlement of spores from the algae Arochaetium sp. In some cases the absence of certain AHLs can inhibit the process of bio-fouling.

The study showed Ochrobactrum sp and Alteromonas sp produced the highest amounts of AHLs. New AHLs were also found to be produced by the sherwanella genus.

My previous post about quorum sensing explained the main points and its purpose related to specific bacteria. This paper provides evidence of the quorum sensing mechanism and its applications to marine fouling. It is remarkable that the different bacteria can use a variety of AHLs to induce bioluminescence or create a biofilm. The impact of specific AHLs to facilitate or inhibit fouling could be useful in for many people; for example controlling the surface area covered. Since the absence of some AHL can inhibit the initial settlement of algae, this will reduce the accumulation of macro fouling and could be adapted for protecting the undersides of boats instead of using pollutant paints, like a biological control.

Cuadrado-silva C.T, Castellanos L, Arevalo-Ferro C, Osorno O.E (2012) Detection of Quorum sensing systems of bacteria isolated from fouled marine organisms,Biochemical systematics and ecology (2013) 46 pages 101-107