Development and Characterization of a Subtilin-Regulated Expression System in Bacillus subtilis : Strict Control of Gene Expression by Addition of Subtilin

Abstract

ABSTRACT A system for subtilin-regulated gene expression (SURE) in Bacillus subtilis that is based on the regulatory module involved in cell-density-dependent control of the production of subtilin is described. An integration vector for introduction of the essential sensor-regulator couple spaRK into the amyE locus of the B. subtilis chromosome and a B. subtilis 168-derived production host in which the spaRK genes were functionally introduced were constructed. Furthermore, several expression plasmids harboring the subtilin-inducible wild-type spaS promoter or a mutated derivative of this promoter were constructed, which facilitated both transcriptional and translational promoter-gene fusions. Functional characterization of both spaS promoters and the cognate expression host could be performed by controlled overproduction of the β-glucuronidase (GusA) and green fluorescent protein (GFP) reporters. Both spaS promoters exhibited very low levels of basal expression, while extremely high levels of expression were observed upon induction with subtilin. Moreover, the level of expression depended directly on the amount of inducer (subtilin) used. The wild-type spaS promoter appeared to be more strictly controlled by the addition of subtilin, while the highest levels of expression were obtained when the mutated spaS promoter was used. Induction by subtilin led to 110- and 80-fold increases in GusA activity for the spaS promoter and its mutant derivative, respectively. Since the SURE system has attractive functional characteristics, including promoter silence under noninducing conditions and a controlled and high level of expression upon induction, and since it is not subject to catabolite control, we anticipate that it can provide a suitable expression system for various scientific and industrial applications.