Tetra-amino-acid tandem repeats are involved in HsdS complementation in type IC restriction–modification systems Monika Adamczyk-Poplawska, Aneta Kondrzycka, Katarzyna Urbanek and Andrzej Piekarowicz Correspondence Andrzej Piekarowicz anpiek@biol.uw.edu.pl Institute of Microbiology, University of Warsaw, 02-096 Warsaw, Poland Received 19 May 2003 Revised 10 July 2003 Accepted 7 August 2003 All known type I restriction and modification (R–M) systems of Escherichia coli and Salmonella enterica belong to one of four discrete families: type IA, IB, IC or ID. The classification of type I systems from a wide range of other genera is mainly based on complementation and molecular evidence derived from the comparison of the amino acid similarity of the corresponding subunits. This affiliation was seldom based on the strictest requirement for membership of a family, which depends on relatedness as demonstrated by complementation tests. This paper presents data indicating that the type I NgoAV R–M system from Neisseria gonorrhoeae, despite the very high identity of HsdM and HsdR subunits with members of the type IC family, does not show complementation with E. coli type IC R–M systems. Sequence analysis of the HsdS subunit of several different potential type IC R–M systems shows that the presence of different tetra-amino-acid sequence repeats, e.g. TAEL, LEAT, SEAL, TSEL, is characteristic for type IC R–M systems encoded by distantly related bacteria. The other regions of the HsdS subunits potentially responsible for subunit interaction are also different between a group of distantly related bacteria, but show high similarity within these bacteria. Complementation between the NgoAV R–M system and members of the EcoR124 R–M family can be restored by changing the tetra-amino-acid repeat within the HsdS subunit. The authors propose that the type IC family of R–M systems could consist of several complementation subgroups whose specificity would depend on differences in the conserved regions of the HsdS polypeptide. INTRODUCTION Restriction and modification (R–M) systems are found in a wide variety of bacteria and are thought to protect the host bacterium from the uptake of foreign DNA (Bickle & Kruger, 1993; Noyer-Weidner & Trautner, 1993). R–M systems have been categorized on the basis of their subunit structures, cofactor requirements, substrate specificity and other properties (Bickle & Kruger, 1993; Noyer-Weidner & Trautner, 1993; Murray, 2000; Pingoud & Jeltsch, 2001). The type I R–M systems can be divided into four distinct families: types IA, IB, IC and ID (Fuller-Pace et al., 1985; Suri & Bickle, 1985; Price et al., 1987; Price & Bickle, 1988; Titheradge et al., 1996; Murray, 2000). The R–M systems belonging to types IA, IB and ID are chromosomally encoded (Barcus & Murray, 1995; Murray, 2000; Titheradge et al., 1996). Type IC systems are plasmid or chromosomally encoded (Bannister & Glover, 1968; Piekarowicz et al., 1985; Skrzypek & Piekarowicz, 1989; Redaschi & Bickle, 1996; Schouler et al., 1998a; Tyndall et al., 1994; Sitaraman & Dybvig, 1997). The strictest requirement for membership of a family depends on relatedness as demonstrated by complementation tests in which subunits from different enzymes associate to make a functional enzyme. These tests require partial diploids made in bacterial strains sensitive to tester phage. Complementation tests have been carried out for type IA, IB and ID R–M systems (Roulland-Dussoix & Boyer, 1969; Ryu et al., 1988; Fuller-Pace et al., 1985; Titheradge et al., 2001). However, complementation tests for type IC systems have been carried out only for the systems encoded by Escherichia coli or Salmonella (Bickle, 1987; Gubler et al., 1992; Price et al., 1987; Skrzypek & Piekarowicz, 1989). It was also shown that in Lactococcus the plasmid-encoded HsdS subunits interact with the chrom- osomally encoded HsdM subunits (Schouler et al., 1998b). More generally applicable tests rely on molecular evidence derived from hybridization tests using hsd sequences as Abbreviations: R–M: restriction and modification; r +/2 , restriction deficient/proficient; m +/2 , modification deficient/proficient; TRD, target recognition domain. Nomenclature. The nomenclature for restriction endonucleases and methyltransferases in this paper follows the recommendations of Roberts et al. (2003b). 0002-6497 G 2003 SGM Printed in Great Britain 3311 Microbiology (2003), 149, 3311–3319 DOI 10.1099/mic.0.26497-0