Families of restriction enzymes: an analysis prompted by molecular and genetic data for type ID restriction and modification systems

Abstract

Current genetic and molecular evidence places all the known type I restriction and modification systems of Escherichia coli and Salmonella enterica into one of four discrete families: type IA, IB, IC or ID. StySBLI is the founder member of the ID family. Similarities of coding sequences have identified restriction systems in E.coli and Klebsiella pneumoniae as probable members of the type ID family. We present complementation tests that confirm the allocation of EcoR9I and KpnAI to the ID family. An alignment of the amino acid sequences of the HsdS subunits of StySBLI and EcoR9I identify two variable regions, each predicted to be a target recognition domain (TRD). Consistent with two TRDs, StySBLI was shown to recognise a bipartite target sequence, but one in which the adenine residues that are the substrates for methylation are separated by only 6 bp. Implications of family relationships are discussed and evidence is presented that extends the family affiliations identified in enteric bacteria to a wide range of other genera.

Figure 1

Polylinker sequences of M13 vectors. The sequences of the polylinkers of mp8, mp10 and mp18 are aligned. The targets for type II endonucleases are identified in the sequence of mp18 and the numbers (1–15) correspond to bases 1–15 in Figure 2.

Figure 2

Base substitutions made to identify the nucleotide sequence recognised by StySBLI. The upper case letters in bold define the nucleotide sequence recognised by StySBLI and the nucleotides are numbered as in Figure 1. Substitutions were made for the bases identified by numbers 1–5 and 10–15. _ identifies a base change that is without effect on the recognition of the nucleotide sequence by StySBLI. x identifies a base change that destroys the target sequence. n.a., not applicable; n.d., not done. *When G is replaced with T at this position, the A residues within the sequence GATC become the substrate for the Dam methylase; methylation of the A residues blocks restriction by StySBLI.

Figure 3

An alignment of the amino acid sequences of the S subunits of StySBLI and EcoR9I. The alignment was made using PILEUP [Wisconsin Package Version 10, Genetics Computer Group (GCG), Madison, WI, USA]. Conserved amino acids are indicated by bold type. The alignments identify two variable regions (TRDs) flanking a central conserved region and a conserved C-terminus.