Isolation of a gramicidin S hyperproducing strain of Bacillus brevis by use of a fluorescence activated cell sorting system.
A gramicidin S (GS) hyperproducing mutant of Bacillus brevis was isolated by using a protein-staining fluorescence dye (fluorescein isothiocyanate, FITC), and a fluorescence-activated cell sorting system (FACS). By flow cytometry (FCM) analysis after staining with FITC, higher producing cells of the wild-type had higher fluorescence signals than cells with low productivity or cells from a GS non-producing mutant. Staining with FITC did not affect the viability of cells under the conditions chosen for FCM analysis. This enabled us to recover viable cells after sorting. After wild-type cells were mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine, mutants with higher fluorescence than the parental strain were obtained by cell sorting. Among them, strain 18 was chosen as a GS hyperproducer; it produced 590 micrograms GS/ml compared to 350 micrograms/ml by the wild-type strain. This method has the advantage of being able to screen large numbers of cells in a short time. Furthermore, use of the fluorescence dye technique will expand the use of FACS to the improvement of other cultures that produce metabolites that do not have a specific fluorescence or strong enough fluorescence for normal cell sorting.
Azuma
T
T
Department of Biology, Massachusetts Institute of Technology, Cambridge 02139.
Harrison
G I
GI
Demain
A L
AL
D001407:Bacillus / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D005434:Flow Cytometry; D016650:Fluorescein-5-isothiocyanate; D006096:Gramicidin / Q000096:biosynthesis*; D009154:Mutation; D013194:Staining and Labeling
D006096:Gramicidin; D016650:Fluorescein-5-isothiocyanate