EcoBLMcrX, a classical modification-dependent restriction enzyme in Escherichia coli B: Characterization in vivo and in vitro with a new approach to cleavage site determination

Abstract

Here we characterize the modification-dependent restriction enzyme (MDE) EcoBLMcrX in vivo, in vitro and in its genomic environment. MDE cleavage of modified DNAs protects prokaryote populations from lethal infection by bacteriophage with highly modified DNA, and also stabilizes lineages by reducing gene import when sparse modification occurs in the wrong context. The function and distribution of MDE families are thus important. Here we describe the properties of EcoBLMcrX, an enzyme of the E. coli B lineage, in vivo and in vitro. Restriction in vivo and the genome location of its gene, ecoBLmcrX, were determined during construction and sequencing of a B/K-12 hybrid, ER2566. In classical restriction literature, this B system was named r6 or rglAB. Like many genome defense functions, ecoBLmcrX is found within a genomic island, where gene content is variable among natural E. coli isolates. In vitro, EcoBLMcrX was compared with two related enzymes, BceYI and NhoI. All three degrade fully cytosine-modified phage DNA, as expected for EcoBLMcrX from classical T4 genetic data. A new method of characterizing MDE specificity was developed to better understand action on fully-modified targets such as the phage that provide major evolutionary pressure for MDE maintenance. These enzymes also cleave plasmids with m5C in particular motifs, consistent with a role in lineage-stabilization. The recognition sites were characterized using a site-ranking approach that allows visualization of preferred cleavage sites when fully-modified substrates are digested. A technical constraint on the method is that ligation of one-nucleotide 5' extensions favors G:C over A:T approximately five-fold. Taking this bias into account, we conclude that EcoBLMcrX can cleave 3' to the modified base in the motif Rm5C|. This is compatible with, but less specific than, the site reported by others. Highly-modified site contexts, such as those found in base-substituted virulent phages, are strongly preferred.

Fig 1. Illumina data analysis strategy.

Fig 2. ECD_02034 is EcoBLMcrX.

Products of in vitro translation (IVT) were incubated with three experimental substrates with complete base substitution of C. ^hm5C-T4: T4gt, sensitive to restriction in vivo; WT-T4: wild type T4, glucosylated ^hm5C, resistant in vivo; XP12: ^m5C-substituted phage, as described in Materials and Methods. Triangles signify 3-fold dilutions of IVT products. V: IVT product of empty vector. ECD1: IVT product of ECD_02033, ECD2: IVT product of ECD_02034; ECD1+2: mixture of the separately translated products; ECD1-2: IVT with cotranscribed, cotranslated genes.

Fig 3. Three MDE action on densely modified targets.

BceYI, EcoBLMcrX and NhoI MDE were tested in 3-fold serial dilution (left side of wedge = highest amount of enzyme) on pBR322 modified in vitro with (top panel) M.SssI (^m5CG) or (bottom panel) M.CviPI (G^m5C). 1kb: NEB 1 kb ladder. Controls: negative,— = no enzyme; Fnu4H: digested with the modification-protected enzyme Fnu4HI (GCNGC). Few of the 42 Fnu4HI sites in pBR322 overlap CpG sites, so Fnu4HI cuts extensively, but GpC modification protects fully. Positive control for Fnu4HI: pBR322 Non-Meth Fnu4HI = unmodified pBR322 digested with Fnu4HI.

Fig 4. Control WebLogo—Type II enzyme on unmodified target.

Fig 5. MDE WebLogos with C-replacement targets.

Fig 6. MDE site rank and ligation preference.

On the X-axis, sites are numbered according to rank in BceYI digestion as in determined in S5 Table, and are listed in Table 2. On the Y-axis the normalized number of reads per site were ranked relative to the most-frequently-recovered site (Relative Efficiency, diamond symbols) as described in Materials and Methods for sites detected with at least 11 reads. The number of ^m5C residues in both strands is also shown (square symbols, right-hand axis). Panel A, BceYI; panel C, NhoI; panel D, EcoBLMcrX. Panel B displays the effect of base composition at the adaptor ligation junction for BceYI. Sequence pairs (Table 3) that differ at the position of adaptor ligation (R = A or G; Y = T or C) were ordered according to Relative Efficiency as in panel A, and reads/site for S (R = G or Y = C; diamonds) and W (R = A or Y = T; crosses) were tabulated as paired values and displayed.