Screening of a genomic library of Pseudoalteromonas tunicata for the identification of genes involved in the production of bioactive compounds.

The thesis is the result of one year project carried out in the laboratory of the Centre for Marine Biofouling and Bioinnovation, University of New South Wales, Sydney, Australia. The project main aim was to develop an hight-throughput assay for the identification of bioactive compounds using the screening of a genomic library of E. coli clones.

The bacterial genus Pseudoalteromonas contains numerous marine species, which synthesize biologically active molecules. Many Pseudoalteromaonas species have been demostrated to produce an array of low and high molecular weight compounds with antimicrobial, algicidal, neurotoxic and other pharmaceutically relevant activities.

P. tunicata is the best studied species within the genus. It lives in association with sessile eukaryotes such as algae and tunicates, and is a known producer of several bioactive compounds with activities towards other surface colonisers including bacteria, fungi, invertebrate larvae, diatoms, algal spores and protozoa.

The aim of this study is to identify the gene(s) involved in the synthesis of bioactive compounds in the marine bacterium P. tunicata that act against eukaryotic organisms.

The nematode Caenorhabditis elegans was used in this study as a model eukaryote for screening bioactive compounds produced by P. tunicata. The development of a screen of a genomic library of P. tunicata DNA allowed for the identification of genes encoding for compounds acting against C. elegans.

Three positive clones with anti-nematode activity were found and there gene contents where analysed by genomic analysis and through random transposon mutagenesis.
The genes involved in the anti-nematode activity encode for a novel fast-killling protein, moreover a gene cluster for the biosynthesis of a small molecule was found out to be involved in the slow-killing activity.