Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin

Entianin, a Novel Subtilin-Like Lantibiotic from Bacillus subtilis subsp. spizizenii DSM 15029 T with High Antimicrobial Activity

“…Chromogenic subtilin autoinduction plate assay for identification of lantibiotic producer strains. For the identification of lantibiotic-producing strains, the reporter strain B. subtilis ATCC 6633 ⌬spaS carrying P spaS -lacZ was streaked out onto a TY (0.8% tryptone and 0.5% yeast extract) agar plate supplemented with 0.3 M NaCl, 1.5% (wt/vol) agar, and 40 g/ml X-gal (5-bromo-4-chloro-3-indolyl-␤-D-galactopyranoside) in such a way that the plate was bisected into identical parts (6). The B. subtilis strains to be investigated were streaked out perpendicularly to the reporter strain, starting from the side of the plate and ending inside the reporter strain growth zone.…”

“…After centrifugation at room temperature for 20 min at 10,000 rpm, the culture supernatants were diluted with equal amounts of methanol and were loaded onto StrataX solid-phase extraction (SPE) cartridges (particle size, 33 m; Phenomenex, Aschaffenburg, Germany). After a wash with 70% methanol-0.1% trifluoroacetic acid (TFA), which led to elution of the truncated S-entianin fragment comprising amino acids 1 to 19 (fragment [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19], intact entianin, succinylated entianin (S-entianin), and succinylated subtilin (S-subtilin) were eluted with 99.9% methanol-0.1% TFA, dried, and stored at Ϫ20°C.…”

“…The protein concentrations of these stock solutions were determined by the micro-bicinchoninic acid (micro-BCA) method (35) using a bovine albumin standard of known concentration for quantification. Additionally, solutions of S-entianin and S-entianin [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] were quantified without previous dilution to identical HPLC peak integrals. The concentrations in the stock solutions were determined to be 3.7 M S-subtilin and 3.2 M S-entianin; 3.4 M S-entianin and 3.5 M entianin; and 2.9 M S-entianin and 15.3 M S-entianin [1][2][3][4][5][6][7][8][9][10][11][12][13][14][15][16][17][18][19] .…”

“…The specific autoinduction of entianin and S-entianin was determined by means of a quantitative chromogenic microtiter plate bioassay developed by Burkard et al (6). The reporter strain ATCC 6633 ⌬spaS P spaS -lacZ was cultivated overnight in 5 ml modified TY medium containing 0.3 M NaCl and was subsequently diluted with the same modified medium to an optical density at 600 nm (OD 600 ) of 0.1.…”

“…After the fusion of the promoter of the subtilin structural gene spaS with the ␤-galactosidase reporter gene, it was possible to establish a microtiter plate-based autoinduction bioassay for the detection of subtilin (6). This autoinduction bioassay allowed for the identification of the new subtilinlike lantibiotic entianin.…”