Antifouling compounds from the sponge-associated marine bacterium Winogradskyella poriferorum.

摘要

Biofouling, the undesired colonization of submerged surfaces by aquatic organisms, is a major problem for all marine industries; hence, substantial resources are now directed towards finding natural anti-foulants. In this study, I screened 7 novel sponge-associated marine bacterial extracts for their anti-bacterial and anti-larval-settlement activity to search for non-toxic or less toxic bioactive anti-foulants. The organic extracts of one of the bacterial species studied, Winogradskyella poriferorum, effectively inhibited both the larval settlement of two model macrofouling organisms---Hydroides elegans and Balanus amphitrite --- and the biofilm formation of two test bacterial species. Then, I investigated the combined effects of 4 factors (pH, salinity, temperature and agitation) on the growth, secondary metabolite profile, and bioactivity of W. poriferorum using the Box-Behnken design. My results indicated the optimal culture conditions with respect to bacterial bioactivity as pH of 8, salinity of 35ppt, temperature of 30 °C, and agitation at 188 RPM. In order to purify and identify the active components present in the extract, I fermented W. poriferorum on a large scale under optimal conditions and systematically separated the crude extracts using bioassay- guided fractionation. I purified and characterized a group of bioactive yet non-toxic poly-ethers with EC50 20 mugml-1 from these extracts. I also studied the natural course of production of these poly-ethers using nuclear magnetic resonance spectroscopy.;In order to understand how the poly-ethers inhibit biofilms, I studied the effect of one of the most active poly-ether on the biofilm formation of a marine biofilm isolate Vibrio sp. 010. Flow cytometry analysis showed that cell viability could not completely explain the differential biofilm formation. Therefore, I explored the biochemical changes occurring during the process based on metabolome and proteome profiling. The metabolites were profiled by ultra performance liquid chromatography-mass spectrometry (UPLC-MS) analysis followed by unsupervised principal component analysis (PCA) of the chromatograms, which statistically narrowed down the differentially expressed metabolites to tri-peptides, fatty acids and quorum sensing molecules. The 2D-gel based proteome analysis showed 50 up-regulated proteins and 9 down-regulated proteins in the treated biofilms. Mass spectrometric analysis of the spots revealed the differential expression of proteins related to the production of exopolymeric substances. In order to study the effect of this poly-ether on the macrofouler B.amphitrite, I challenged their competent cyprids with the active poly-ether B. Substantial changes in protein expression and phosphorylation status were observed. Interestingly, most of the differentially phosphorylated proteins and differentially expressed proteins identified by MS were relevant to oxidative stress, corroborating the involvement of redox pathway proteins in larval settlement.;The findings of this thesis showed that 1) sponge-associated bacteria are indeed a good source of compounds with interesting bioactive potential; 2) the isolated poly-ethers are highly active and non- toxic, hence acceptable for different industrial applications; 3) the inhibition of microfouling and macrofouling by these poly-ethers is through the modulation of reduction-oxidation pathway proteins.