Development and application of a microtiter plate-based autoinduction bioassay for detection of the lantibiotic subtilin.
Production of the lantibiotic subtilin in Bacillus subtilis ATCC 6633 is regulated in a quorum sensing-like mechanism with subtilin acting as autoinducer and signal transduction via the subtilin-specific two-component regulation system SpaRK. Here, we report the construction and application of a subtilin reporter strain in which subtilin induced lacZ gene expression in a B. subtilis ATCC 6633 spaS gene deletion mutant is monitored and visualized by the beta-galactosidase in a chromogenic plate assay. A quantitative microtiter plate subtilin bioassay was developed and optimized. Maximal sensitivity of the system was achieved after 6 h of incubation of the reporter strain together with subtilin in a medium containing 300 mM NaCl. This sensitive and unsusceptible method was applied to identify subtilin producing B. subtilis wild type strains from both, culture collections and soil samples. The B. subtilis lantibiotic ericin S with four amino acid exchanges compared to subtilin induces the subtilin reporter strain, in contrast to the structurally closely related Lactococcus lactis lantibiotic nisin. These observations suggest a certain substrate specificity of the histidine kinase SpaK, which however, also would allow the identification of subtilin-isoform producing microorganisms.
Burkard
Michael
M
Center of Excellence, Macromolecular Complexes, Johann Wolfgang Goethe-University, Institute of Molecular Biosciences, Max-von-Laue-Strasse 9, 60438 Frankfurt am Main, Germany.
Entian
Karl-Dieter
KD
Stein
Torsten
T
D000595:Amino Acid Sequence; D019677:Artificial Gene Fusion; D001412:Bacillus subtilis / Q000235:genetics / Q000502:physiology*; D001430:Bacteriocins / Q000032:analysis* / Q000235:genetics; D001681:Biological Assay / Q000379:methods*; D017353:Gene Deletion; D015964:Gene Expression Regulation, Bacterial; D017930:Genes, Reporter; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009561:Nisin / Q000032:analysis; D010455:Peptides / Q000032:analysis* / Q000235:genetics; D012680:Sensitivity and Specificity; D001616:beta-Galactosidase / Q000096:biosynthesis*
D001430:Bacteriocins; D010455:Peptides; D009561:Nisin; D001616:beta-Galactosidase; C002836:subtilin