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

To identify novel, less-toxic compounds capable of inhibiting sulfate-reduc ing bacteria (SRB), Desulfovibrio vulgaris and Desulfovibrio gigas in suspe nsion cultures were exposed to several antimicrobial peptides, The bacteria l peptide antimicrobials gramicidin S, gramicidin D, and polymyxin B as wel l as the cationic peptides indolicidin and bactenecin from bovine neutrophi ls decreased the viability of both SRB by 90% after a 1-h exposure at conce ntrations of 25-100 mu g ml(-1). To reduce corrosion by inhibiting SRB in b iofilms, the genes for indolicidin and bactenecin were expressed in Bacillu s subtilis BE1500 and B. subtilis WB600 under the control of the constituti ve alkaline protease (apr) promoter, and the antimicrobials were secreted i nto the culture medium using the apr signal sequence. Bactenecin was also s ynthesized and expressed as a fusion to the pro-region of barnase from Baci llus amyloliquefaciens. Concentrated culture supernatants of B. subtilis BE 1500 expressing bactenecin at 3 mu g ml(-1) decreased the viability of Esch erichia coli BK6 by 90% and the reference SRB D. vulgaris by 83% in suspens ion cultures, B. subtilis BE1500 and B. subtilis WB600 expressing bacteneci n in biofilms also inhibited the SRB-induced corrosion of 304 stainless ste el six to 12-fold in continuous reactors as evidenced by the lack of change in the impedance spectra (resistance polarization) upon addition of SRB an d by the reduction in hydrogen sulfide and iron sulfide in batch fermentati ons 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, genetica lly-engineered biofilm for combating corrosion.