Mining transcriptome data: Utilization of environmentally regulated promoters for protein expression and purification in Clostridium perfringens

Abstract

Clostridium perfringens is a Gram-positive pathogen with low GC content. To identify genes that are transcribed at higher levels when the bacteria grow on a surface, we used RNA-seq in a previous study to measure global transcript levels of cells grown in three types of media on both plates and in liquid culture. We found the arcABDC-argR operon is induced >1000-fold when the cells were grown on plates than in liquid brain heart infusion (BHI). In addition, the pyrBICFZDE operon was transcribed >1000-fold higher in liquid BHI than on plates. Biochemical analysis of C. perfringens proteins is usually accomplished by cloning and expressing the relevant genes in Escherichia coli, a Gram-negative bacterium. Here we utilize both the arcA and pyrB promoters to express and purify proteins from C. perfringens plate and liquid-grown cultures, respectively. Three mg of the His-tagged cytoplasmic protein PilM were obtained when the pilM gene was expressed in cells grown on 10 BHI plates using the arcA promoter. Using the pyrB promoter, 0.85 mg of the C. perfringens His-tagged secreted toxin collagenase was purified from the culture supernatant of 500 ml of cells grown in liquid BHI. In the process of constructing clones, we found we can transform C. perfringens strain HN13 directly with DNA from an in vitro ligation mix, bypassing E. coli. These environmentally regulated promoters can be used to express clostridial or other low GC content genes for protein purification without the addition of an inducer molecule.