Multispecific DNA methyltransferases (Mtases) of temperate Bacillus subtilis phages SPR and phi 3T methylate the internal cytosine of the sequence GGCC. They differ in their capacity to methylate additional sequences. These are CCGG and CC(A/T)GG in SPR and GCNGC in phi 3T. Introducing unique restriction sites at equivalent locations within the two genes facilitated the construction of chimeric genes. These expressed Mtase activity at a level comparable to that of the parental genes. The methylation specificity of chimeric enzymes was correlated with the location of chimeric fusions. This analysis, which also included the use of mutant genes, showed that domains involved in the recognition of target sequences unique to each enzyme [CCGG, CC(A/T)GG or GCNGC] are represented by the central non‐conserved parts of the proteins, whilst recognition of the sequence (GGCC), which is a target for both enzymes, is determined by an adjacent conserved region.

Several chimeric pBR322/328 derivatives containing genes for cytosine-specific DNA methyltransferases (Mtases) can be transformed into the Escherichia coli K12/E. coli B hybrid strains HB101 and RR1 but not into other commonly used E. coli K12 strains. In vitro methylation of cytosine residues in pBR328 and other unrelated plasmids also reduces their potential to transform such methylation sensitive strains, albeit to a lesser degree than observed with plasmids containing Mtase genes. The extent of reduced transformability depends on the target specificity of the enzyme used for in vitro modification. The role of a host function in the discrimination against methylated plasmids was verified by the isolation of K12 mutants which tolerate cytosine methylated DNA. The mutations map in the vicinity of the serB locus. This and other data indicate that the host rglB function is involved in the discrimination against modified DNA.

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SPR, a temperate Bacillus subtilis phage, codes for a DNA methyltransferase that can methylate the sequences GGCC (or GGCC) and CCGG at the cytosines indicated. We show here that it can also methylate the sequence CC(A/T)GG and protect it from cleavage with EcoRII and ApyI. This methylation can be seen in vivo as well as in vitro with purified SPR methyltransferase. SPR19 and SPR83 are two mutant phages, defective in GGCC or CCGG methylation, respectively. These mutants have not lost their ability to methylate CC(A/T)GG sites. Mutation SPR26 has lost the ability to methylate all three sites. Thus the SPR methyltransferase codes for three genetically distinguishable methylation abilities.

Temperate Bacillus subtilis phages SPR, q3T, e l l and SPB code for DNA methyltransferases, each having multiple sequence specificities. The SPR wild-type and various mutant methyltransferases were overproduced 1 000-fold in Escherichia coli and were purified by three consecutive chromatographic steps.The stable form of these multispecific enzymes in solution are monomers with a relative molecular mass (M,) of about 50000. The methyl-transfer kinetics of the SPR wild-type and mutant enzymes were determined with DNA substrates carrying either none or one of the three recognition sequences (GGCC, CCGG, CC,^CG).Evaluation of the catalytic properties for DNA and S-adenosylmethionine binding suggested that the N H1-terminal part of the protein is important for both non-sequence-specific DNA binding and S-adenosylmethonine binding as well as transfer of methyl groups. On the other hand, mutations in the COOH-terminal part lead to weaker site-specific interactions of the enzyme.Antibodies raised against the purified SPR enzyme specifically immunoprecipitated the q3T, el 1 and SPB methyltransferases, but failed to precipitate the chromosomally coded enzymes from B. subtilis (BsuRI) and B. sphaericus (BspRI). Immunoaffinity chromatography is an efficient purification step for the related phage methyltransferases.The temperate Bacillus subtilis phages SPR, q3T, el 1 and SPP have evolved a protection mechanism against host restriction enzymes by self-methylating their DNA at various sequences [l]. q3T, e l l and SPP methyltransferases have the potential to methylate the sequences GGCC (HaeIIIIBsuRI) and GCNGC (Fnu4HI) [2], while the SPR methyltransferase recognizes three sequences: GGCC [3], CCGG (HpaII/ MspIIBsuF) [4, 51 and C&GG (EcoRII) (U. Gunthert and L. Reiners, unpublished results). The asterisk denotes the position of methylation.Like the related type I1 modification enzymes, the phagederived methyltransferases are composed of a single polypeptide. As calculated from the DNA sequence, the SPR methyltransferase gene encodes a 439-residue protein with a M , = 49826 [6]. An approximate M , of 47000 for the q3T, e l l and SPP enzymes has been determined by minicell expression studies and in an in vitro transcription/translation system [7].A set of SPR methyltransferase mutants has been isolated and the positions of the mutations localized in the methyltransferase coding region [6] represented in this study by the SPR26 methyltransferase [3], is affected in its ability to methylate all three sequences. Another group of mutants is altered in their ability to interact with either the GGCC sequence (SPR19) [4] or the CCGG sequence (SPR83) [6]. A mutation which affects methylation at CC&G has not yet been identified. Another mutation (a frame-shift) has been isolated, which codes for a truncated protein of M, 28000 (SPRlOl) [6] showing reduced sitespecific methylation (U. Gunthert and L. Keiners, unpublished results).The goal of our research is the understanding of interactions between enzymes and DNA and these methyltrans€...