Abstract
The first reported Type IV restriction endonuclease (REase) GmrSD consists of GmrS and GmrD subunits. In most bacteria, however, the gmrS and gmrD genes are fused together to encode a single-chain protein. The fused coding sequence for ECSTEC94C_1402 from E. coli strain STEC_94C was expressed in T7 Express. The protein designated as Eco94GmrSD displays modification-dependent ATP-stimulated REase activity on T4 DNA with glucosyl-5-hydroxymethyl-cytosines (glc-5hmC) and T4gt DNA with 5-hydroxymethyl-cytosines (5hmC). A C-terminal 6xHis-tagged protein was purified by two-column chromatography. The enzyme is active in Mg2+ and Mn2+ buffer. It prefers to cleave large glc-5hmC- or 5hmC-modified DNA. In phage restriction assays, Eco94GmrSD weakly restricted T4 and T4gt, whereas T4 IPI*-deficient phage (Δip1) were restricted more than 106-fold, consistent with IPI* protection of E. coli DH10B from lethal expression of the closely homologous E. coli CT596 GmrSD. Eco94GmrSD is proposed to belong to the His-Asn-His (HNH)-nuclease family by the identification of a putative C-terminal REase catalytic site D507-H508-N522. Supporting this, GmrSD variants D507A, H508A, and N522A displayed no endonuclease activity. The presence of a large number of fused GmrSD homologs suggests that GmrSD is an effective phage exclusion protein that provides a mechanism to thwart T-even phage infection.
Highlights
{ Current address: College of Science, Thomas H
The first reported Type IV restriction endonuclease (REase) GmrSD consists of GmrS and GmrD subunits
GmrSD endonuclease activity is stimulate by ATP/GTP, there is no predicted ATPase/GTPase domain in the protein by NCBI BlastP analysis
Summary
{ Current address: College of Science, Thomas H. The gmrS and gmrD genes are fused together to encode a single-chain protein. The enzyme is active in Mg21 and Mn21 buffer It prefers to cleave large glc-5hmC- or 5hmC-modified DNA. The goal of this work was to evaluate the endonuclease activity of such a single-chain GmrSD homolog found in the genome of E. coli strain STEC_94C and to develop methods for simple purification of the target protein. This property differs from the two-chain GmrS/ GmrD enzyme complex that only cleaved glc-5hmC DNA Despite this difference in in vitro substrate sensitivity we found that the phage restriction activity of Eco94GmrSD is very similar to that of the two-chain GmrS/GmrD: Eco94GmrSD only weakly restricted WT T4 and T4gt (deficient in a-, b-glucosyltransferase (gt) phages), but strongly restricted T4Dip[1] phage (about a million fold). The possible involvement of GmrSD-like enzyme in the bacterial immigration control region (ICR) is discussed
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