Novel Nematode-Killing Protein-1 (Nkp-1) from a Marine Epiphytic Bacterium Pseudoalteromonas tunicata

Drug resistance among parasitic nematodes has resulted in an urgent need for the development of new therapies. However, the high re-discovery rate of anti-nematode compounds from terrestrial environments necessitates a new repository for future drug research. Marine epiphytes are hypothesised to produce nematicidal compounds as a defence against bacterivorous predators, thus representing a promising yet underexplored source for anti-nematode drug discovery. The marine epiphytic bacterium Pseudoalteromonas tunicata is known to produce several bioactive compounds. Screening heterologously expressed genomic libraries of P. tunicata against the nematode Caenorhabditis elegans, identified as an E. coli clone (HG8), shows fast-killing activity. Here we show that clone HG8 produces a novel nematode-killing protein-1 (Nkp-1) harbouring a predicted carbohydrate-binding domain with weak homology to known bacterial pore-forming toxins. We found bacteria expressing Nkp-1 were able to colonise the C. elegans intestine, with exposure to both live bacteria and protein extracts resulting in physical damage and necrosis, leading to nematode death within 24 h of exposure. Furthermore, this study revealed C. elegans dar (deformed anal region) and internal hatching may act as a nematode defence strategy against Nkp-1 toxicity. The characterisation of this novel protein and putative mode of action not only contributes to the development of novel anti-nematode applications in the future but reaffirms the potential of marine epiphytic bacteria as a new source of novel biomolecules.

Marine Bacteria from Danish Coastal Waters Show Antifouling Activity against the Marine Fouling Bacterium Pseudoalteromonas sp. Strain S91 and Zoospores of the Green Alga Ulva australis Independent of Bacteriocidal Activity

ABSTRACTThe aims of this study were to determine if marine bacteria from Danish coastal waters produce antifouling compounds and if antifouling bacteria could be ascribed to specific niches or seasons. We further assess if antibacterial effect is a good proxy for antifouling activity. We isolated 110 bacteria with anti-Vibrioactivity from different sample types and locations during a 1-year sampling from Danish coastal waters. The strains were identified asPseudoalteromonas,Phaeobacter, andVibrionaceaebased on phenotypic tests and partial 16S rRNA gene sequence similarity. The numbers of bioactive bacteria were significantly higher in warmer than in colder months. While some species were isolated at all sampling locations, others were niche specific. We repeatedly isolatedPhaeobacter gallaeciensisat surfaces from one site andPseudoalteromonas tunicataat two others. Twenty-two strains, representing the major taxonomic groups, different seasons, and isolation strategies, were tested for antiadhesive effect against the marine biofilm-forming bacteriumPseudoalteromonassp. strain S91 and zoospores of the green algaUlva australis. The antiadhesive effects were assessed by quantifying the number of strain S91 orUlvaspores attaching to a preformed biofilm of each of the 22 strains. The strongest antifouling activity was found inPseudoalteromonasstrains. Biofilms ofPseudoalteromonas piscicida,Pseudoalteromonas tunicata, andPseudoalteromonas ulvaepreventedPseudoalteromonasS91 from attaching to steel surfaces.P. piscicidakilled S91 bacteria in the suspension cultures, whereasP. tunicataandP. ulvaedid not; however, they did prevent adhesion by nonbactericidal mechanism(s). SevenPseudoalteromonasspecies, includingP. piscicidaandP. tunicata, reduced the number of settlingUlvazoospores to less than 10% of the number settling on control surfaces. The antifoulingalpPgene was detected only inP. tunicatastrains (with purple and yellow pigmentation), so other compounds/mechanisms must be present in the otherPseudoalteromonasstrains with antifouling activity.

Identification of Compounds with Bioactivity against the Nematode Caenorhabditis elegans by a Screen Based on the Functional Genomics of the Marine Bacterium Pseudoalteromonas tunicata D2

ABSTRACT Marine bacteria are a rich, yet underexplored, resource of compounds with inhibitory bioactivity against a range of eukaryotic target organisms. Identification of those inhibitors, however, requires a culturable or genetically tractable producer strain, a prerequisite that is not often fulfilled. This study describes a novel functional genomic screen that is based on expression of inhibitors in a heterogeneous recombinant host (i.e., Escherichia coli). Functional libraries were screened by selective grazing by the nematode Caenorhabditis elegans, in a simple, rapid, high-throughput manner. We applied our approach to discover inhibitors of C. elegans produced by the marine bacterium Pseudoalteromonas tunicata D2, a model organism for exploring a range of antagonistic activities between bacteria and eukaryotes and a known producer of several toxic compounds. Expression of P. tunicata DNA in E. coli and grazing selection by the nematode Caenorhabditis elegans identified two clones, with slow- and fast-killing modes of action. Genomic analysis of the slow-killing clone revealed that the activity was due to a small molecule, tambjamine, while the fast-killing activity involved a gene encoding for a novel protein. Microscopic analysis showed substantial colonization of the intestinal lumen, or rapid death of the nematode without colonization, for the two activities, respectively. The novel functional genomic screen presented here therefore detects new eukaryotic inhibitors with different chemical structures, kinetics, and predicted modes of actions.