Inhibiting sulfate-reducing bacteria in biofilms by expressing the antimicrobial peptides indolicidin and bactenecin

To identify novel, less-toxic compounds capable of inhibiting sulfate-reducing bacteria (SRB), Desulfovibrio vulgaris and Desulfovibrio gigas in suspension cultures were exposed to several antimicrobial peptides, The bacterial peptide antimicrobials gramicidin S, gramicidin D, and polymyxin B as well as the cationic peptides indolicidin and bactenecin from bovine neutrophils decreased the viability of both SRB by 90% after a 1-h exposure at concentrations of 25-100 mu g ml(-1). To reduce corrosion by inhibiting SRB in biofilms, the genes for indolicidin and bactenecin were expressed in Bacillus subtilis BE1500 and B. subtilis WB600 under the control of the constitutive alkaline protease (apr) promoter, and the antimicrobials were secreted into the culture medium using the apr signal sequence. Bactenecin was also synthesized and expressed as a fusion to the pro-region of barnase from Bacillus amyloliquefaciens. Concentrated culture supernatants of B. subtilis BE1500 expressing bactenecin at 3 mu g ml(-1) decreased the viability of Escherichia coli BK6 by 90% and the reference SRB D. vulgaris by 83% in suspension cultures, B. subtilis BE1500 and B. subtilis WB600 expressing bactenecin in biofilms also inhibited the SRB-induced corrosion of 304 stainless steel six to 12-fold in continuous reactors as evidenced by the lack of change in the impedance spectra (resistance polarization) upon addition of SRB and by the reduction in hydrogen sulfide and iron sulfide in batch fermentations with mild steel. A 36-fold decrease in the population of D. vulgaris in a B. subtilis BE1500 biofilm expressing bactenecin was also observed, This is the first report of an antimicrobial produced in a biofilm for in vivo applications and represents the first application of a beneficial, genetically-engineered biofilm for combating corrosion.