Genetic and sequence organization of the mcrBC locus of Escherichia coli K-12

Abstract

The mcrB (rglB) locus of Escherichia coli K-12 mediates sequence-specific restriction of cytosine-modifled DNA. Genetic and sequence analysis shows that the locus actually comprises two genes, mcrB and mcrC. We show here that in vivo, McrC modifies the specificity of McrB restriction by expanding the range of modified sequences restricted. That is, the sequences sensitive to McrB+-dependent The locus known as mcrB was one of the first restriction systems to be discovered (33), by virtue of its action on special variants of T-even bacteriophage that incorporate 5-hydroxymethylcytosine (hm`C) into their DNA without further modification (see reference 50 for a review). This locus, formerly known as rglB (or r2,4) (48), was rediscovered because of difficulties encountered in cloning the genes for site-specific modification methylases associated with type II restriction-modification systems (7,26,40,49). In addition to hm5C-DNA, many but not all sequences methylated by site-specific cytosine modification methylases are restricted by the system in vivo, and the consensus recognition sequence 5'GmC was proposed (49). McrB is thus a sequence-specific, modification-requiring restriction system. We show here that the mcrB locus described above actually comprises two genes and that both are required for restriction of most the sequences previously characterized as sensitive. Thus, we will refer to the complete system as the McrBC system.The genes encoding the system are contained within the immigration control region of the Escherichia coli K-12 genome. Three restriction systems are encoded within 14 kilobases (kb) here (48). The well-studied hsdRMS locus (20, 31, 55) encodes the multisubunit type I system EcoK, which recognizes a seven-base sequence and cleaves the target when the sequence is not modified. The other two systems are the flanking loci mcrBC, described above, and mrr, which mediates site-specific restriction of adenine-modified DNA (22). The sequence organization of this region, judged by Southern blot analysis of chromosomal DNA, is highly variable in enteric bacteria (12), both in the hsd genes specifically and in the flanking sequences. Sequence analysis presented here is consistent with recent acquisition of the mcrBC genes by E. coli, possible accounting for some of the observed variability. * Corresponding author.At the molecular level, restriction systems consist of sequence-specific double-stranded endonucleases, usually accompanied by a sequence-specific modification methylase. So far, four classes of endonucleases have been described. The simplest are the type II enzymes, in which the endonuclease and protective methylase activities reside in separate enzymes. These endonucleases typically act as dimers of identical subunits and require only Mg2" for activity (38).One group of type II isoschizomers, typified by DpnI, recognizes a modified site (28), as McrBC appears to do. In contrast, type I and type III enzymes have separate specificity subunits that recognize the DNA site and require ATP in a...

Isolation of temperature-sensitive McrA and McrB mutations and complementation analysis of the McrBC region of Escherichia coli K-12

“…The DNA encoding the McrBC system has been cloned and sequenced, and the gene products have been analyzed (4,19,(22)(23)(24)(25)(26). Although these reports differ in their conclusions with respect to the number of polypeptides identified as mcrBC products and the identity of those needed for the restriction of T4gt phage, they agree that the mcrBC locus can be further subdivided genetically into mcrB and mcrC genes (4,24).…”

“…Since APl-10 was mutagenized with nitrosoguanidine, this is not an unreasonable assumption. Dila et al (4) have shown that the mcrB gene encodes two proteins of 29 and 49 kDa, as detected in an in vitro translation system, but there are no data on the functional activities of the two gene products. It is possible that one of these polypeptides is responsible for restriction of HMC-containing DNA, while the second one is responsible for restriction of methylcytosine-containing DNA.…”

“…This mutation could affect the presumptive MrcC protein-binding domain of McrB protein since it was suggested that interaction of McrC protein with McrB protein can allow recognition of additional methylated sites and cytosine modifications (4).…”

“…Cloning of the E. coli K-12 region that codes for McrBC activity (4,16,(22)(23)(24)(25)(26) facilitated the study of its genetic structure. However, the conclusions reached by several different research groups are contradictory.…”

Nomenclature relating to restriction of modified DNA in Escherichia coli

“…Genetic loci (mcrA, mcrBC, and mrr) required for expression of the three known modification-specific restriction systems of Escherichia coli have been mapped (7,15,18,19), cloned (5,15,21,23,24), and, for mcrA and mcrBC, correlated with previously described loci, rglA and rglB (11,15,16). At a recent meeting (2nd New England Biolabs Workshop on Biological DNA Modification, Berlin, 2 to 7 September 1990), preliminary reports of more genes involved in modification-specific restriction, and of more mutations affecting specificity of restriction by existing genes, suggested an incipient terminological crisis, particularly for the many people outside the field who use E. coli as a tool for cloning modified DNA but are unfamiliar with the details of the evolving story.…”