Purification and characterization of a novel antibacterial protein from the marine bacterium D2

A biofilm-forming marine bacterium, D2, isolated from the surface of the tunicate Ciona intestinalis, was found to produce a novel, 190-kDa protein with antibacterial activity. The protein contained at least two subunits of 60 and 80 kDa, joined together by noncovalent bonds, and was shown to be released by D2 cells into the surrounding medium during stationary phase. N-terminal sequence analysis revealed no close similarity of this protein to any other proteins within the Swiss Prot database. Bacteriocidal activity against a wide variety of marine and medical bacterial isolates was observed, 77% of the strains tested being sensitive to the protein. Bacterial strains varied in their resistance to the D2 protein, with D2 itself being among the most sensitive with an MBC in liquid suspension of 4 micrograms/ml. An apparent increased resistance of D2 to the protein as the cells progressed further into stationary phase was observed and seen as a possible explanation for its survival despite the production of an autoinhibitory factor. The ability of the D2 bacterium to produce an antibacterial factor in addition to its inhibitory effects on marine invertebrates and algae (S. Egan et al., unpublished data) indicates that D2 has the potential to greatly affect the survival of a range of colonizers of the marine surface environment.