Construction of a Knockout Mutant of the Streptothricin-Resistance Gene in Streptomyces albulus by Electroporation

Abstract

Streptothricins (STs) produced by Streptomyces strains are broad-spectrum antibiotics. All STs consist of a carbamoylated D-gulosamine to which the β-lysine homopolymer (1 to 7 residues) and the amide form of the unusual amino acid streptolidine (streptolidine lactam) are attached. In a previous study, we successfully isolated a novel ST-resistance gene (sttH) from Streptomyces albulus by shotgun cloning with a heterologous host. In vitro analysis of SttH demonstrated that this enzyme catalyzes the hydrolysis of the amide bond of streptolidine lactam, thereby conferring ST resistance. We suggested that the true role of SttH may not be its involvement in resistance against STs; instead, it may catalyze the hydrolysis of naturally occurring cyclic amide compounds in the metabolism of S. albulus. In this study, we therefore constructed an sttH gene-knockout mutant to better understand the true biological role of SttH in S. albulus and to clarify whether or not S. albulus possesses different ST-resistance gene(s). The minimum inhibitory concentration MIC of STs in the sttH gene-knockout mutant was determined to be 6.25 μg/ml, whereas the MIC of STs in the S. albulus wild strain was determined to be >400 μg/ml. This result clearly demonstrated that sttH is the sole gene responsible for ST resistance in the S. albulus strain. Moreover, we developed a transformation system for S. albulus by electroporation in this study.