Functional Characterization of CRISPR-Cas System in the Ethanologenic Bacterium Zymomonas mobilis ZM4

CRISPR-Cas (clustered regularly interspaced short palindromic repeats—CRISPR associated proteins) is a RNA-guided defense immune system that prevents some genetic elements such as plasmids and virus from getting into the bacterial cells. Zymomonas mobilis is an ethanologenic bacterium, which encodes a subtype I-F CRISPR-Cas system containing three CRISPR loci and a far distant cas gene cluster. Reverse transcription (RT)-PCR analysis revealed that the CRISPR loci were transcribed on both strands. The Cas proteins were suggested to be expressed based on the previous transcriptomic analysis. Challenging with the invader plasmids containing the artificial protospacer with the protospacer adjacent motif (PAM) of NGG or GG exhibited immune interference activity. However, PAM motif of GG seems more effective than NGG in interference activity. Further, the artificial CRISPR arrays with the spacer sequences targeting to the specific genome sites could also lead to strong immune activity, resulting in almost no transformant grown on the agar plates. It was suggested that bacteria like Z. mobilis ZM4 are lack of the rejoining function to heal the double breakage of genomic DNA made by the CRISPR system. Conclusively, the Type I-F CRISPRCas system in Z. mobilis ZM4 is active to functionally defense the invading DNA elements.