The Restriction Activity Investigation of Rv2528c, an Mrr-like Modification-Dependent Restriction Endonuclease from Mycobacterium tuberculosis

Author:

Liu Tong1,Wei Wei2,Xu Mingyan1,Ren Qi1,Liu Meikun1,Pan Xuemei1,Feng Fumin1ORCID,Han Tiesheng1ORCID,Gou Lixia3

Affiliation:

1. Hebei Province Key Laboratory of Occupational Health and Safety for Coal Industry, School of Public Health, North China University of Science and Technology, Tangshan 063210, China

2. Centers for Disease Control and Prevention of He Xi District, Tianjin 300210, China

3. School of Life Science, North China University of Science and Technology, Tangshan 063210, China

Abstract

Mycobacterium tuberculosis (Mtb), as a typical intracellular pathogen, possesses several putative restriction–modification (R-M) systems, which restrict exogenous DNA’s entry, such as bacterial phage infection. Here, we investigate Rv2528c, a putative Mrr-like type IV restriction endonuclease (REase) from Mtb H37Rv, which is predicted to degrade methylated DNA that contains m6A, m5C, etc. Rv2528c shows significant cytotoxicity after being expressed in Escherichia coli BL21(DE3)pLysS strain. The Terminal deoxynucleotidyl transferase dUTP Nick-End Labeling (TUNEL) assay indicates that Rv2528c cleaves genomic DNA in vivo. The plasmid transformation efficiency of BL21(DE3)pLysS strain harboring Rv2528c gene was obviously decreased after plasmids were in vitro methylated by commercial DNA methyltransferases such as M.EcoGII, M.HhaI, etc. These results are consistent with the characteristics of type IV REases. The in vitro DNA cleavage condition and the consensus cleavage/recognition site of Rv2528c still remain unclear, similar to that of most Mrr-family proteins. The possible reasons mentioned above and the potential role of Rv2528c for Mtb were discussed.

Funder

National Natural Science Foundation of China

Natural Science Foundation of Hebei Province

Publisher

MDPI AG

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