Construction of in vitro transcription system for Corynebacterium glutamicum and its use in the recognition of promoters of different classes

To facilitate transcription studies in Corynebacterium glutamicum, we have developed an in vitro transcription system for this bacterium used as an industrial producer of amino acids and a model organism for actinobacteria. This system consists of C. glutamicum RNA polymerase (RNAP) core (alpha 2, beta, beta'), a sigma factor and a promoter-carrying DNA template, that is specifically recognized by the RNAP holoenzyme formed. The RNAP core was purified from the C. glutamicum strain with the modified rpoC gene, which produced His-tagged beta' subunit. The C. glutamicum sigA and sigH genes were cloned and overexpressed using the Escherichia coli plasmid vector, and the sigma subunits sigma(A) and sigma(H) were purified by affinity chromatography. Using the reconstituted C. glutamicum holo-RNAPs, recognition of the sigma(A)- and sigma(H)-dependent promoters and synthesis of the specific transcripts was demonstrated. The developed in vitro transcription system is a novel tool that can be used to directly prove the specific recognition of a promoter by the particular sigma factor(s) and to analyze transcriptional control by various regulatory proteins in C. glutamicum.