Cas1 and Cas2 From the Type II-C CRISPR-Cas System of Riemerella anatipestifer Are Required for Spacer Acquisition.

Yang He,Ling Zhang,Yanling Yu,Xinxin Zhao,Dekang Zhu,Shaqiu Zhang,Qiao Yang,Shun Chen,Anchun Cheng,Li Huang,Ying Wu,Yunya Liu,Haibo Yi,Chaoyue Liu,Renyong Jia,Mafeng Liu,Xin Zhang,Mingshu Wang

doi:10.3389/fcimb.2018.00195

Abstract

Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) proteins provide acquired genetic immunity against the entry of mobile genetic elements (MGEs). The immune defense provided by various subtypes of the CRISPR-Cas system has been confirmed and is closely associated with the formation of immunological memory in CRISPR arrays, called CRISPR adaptation or spacer acquisition. However, whether type II-C CRISPR-Cas systems are also involved in spacer acquisition remains largely unknown. This study explores and provides some definitive evidence regarding spacer acquisition of the type II-C CRISPR-Cas system from Riemerella anatipestifer (RA) CH-2 (RA-CH-2). Firstly, introducing an exogenous plasmid into RA-CH-2 triggered spacer acquisition of RA CRISPR-Cas system, and the acquisition of new spacers led to plasmid instability in RA-CH-2. Furthermore, deletion of cas1 or cas2 of RA-CH-2 abrogated spacer acquisition and subsequently stabilized the exogenous plasmid, suggesting that both Cas1 and Cas2 are required for spacer acquisition of RA-CH-2 CRISPR-Cas system, consistent with the reported role of Cas1 and Cas2 in type I-E and II-A systems. Finally, assays for studying Cas1 nuclease activity and the interaction of Cas1 with Cas2 contributed to a better understanding of the adaptation mechanism of RA CRISPR-Cas system. This is the first experimental identification of the naïve adaptation of type II-C CRISPR-Cas system.

Highlights

Riemerella anatipestifer (RA) is a gram-negative bacterium that causes infection with high morbidity and mortality (Segers et al, 1993)
The higher band gained intensity as the growth cycles increased; in contrast, the lower band became increasingly weaker. This finding suggested that R. anatipestifer CH-2 (RA-CH-2) was able to acquire new spacers after the introduction of an exogenous plasmid
Spacer acquisition of the cas1 or cas2 deletion mutant was rescued by the introduction of the shuttle plasmid expressing both Cas1 and Cas2. These results suggested that Cas1 and Cas2 were required for spacer acquisition of RA-CH-2 Clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system

Summary

Introduction

Riemerella anatipestifer (RA) is a gram-negative bacterium that causes infection with high morbidity and mortality (Segers et al, 1993). GenBank database in recent years (Wang X. et al, 2012; Wang et al, 2014; Zhu et al, 2016) Among these strains, R. anatipestifer CH-2 (RA-CH-2) is highly virulent and is one of the common serotype 2 strains in China (Wang et al, 2014). Studies on the physiology and pathogenic mechanism of RA are rare

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Conclusion