A new system to control the barnase expression by a NifA-dependent promoter

Barnase is a potent ribonuclease widely used as a cytotoxic agent, tightly regulated by barstar to maintain cell viability. In this report, we describe a new composite regulatory system to control barnase cytotoxicity and expression, involving barstar and lacI genes under control of the NifA-, sigma54-dependent Sinorhizobium meliloti nifH promoter, and the barnase gene under control of the LacI-repressible ptac promoter. In this system, expression of thenifH promoter, activated by constitutively expressed NifA, resulted in constitutive synthesis of the LacI and barstar proteins. LacI, in turn, represses transcription of the barnase gene and barstar inhibits any ribonuclease activity. Full expression of the barnase gene induced by IPTG led to cell death. Control of barnase synthesis and activity could be achieved by regulating nifA expression and NifA protein activity by specific environmental signals.