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

Institute of Microbiology, University of Warsaw, 02-096 Warsaw, Poland

Correspondence
Andrzej Piekarowicz
anpiek{at}biol.uw.edu.pl


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
REFERENCES
 
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.


Abbreviations: R–M: restriction and modification; r+/-, restriction deficient/proficient; m+/-, 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).


   INTRODUCTION
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ABSTRACT
INTRODUCTION
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RESULTS AND DISCUSSION
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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 chromosomally encoded HsdM subunits (Schouler et al., 1998b). More generally applicable tests rely on molecular evidence derived from hybridization tests using hsd sequences as probes or serological tests with antibodies raised against representatives of a known family of enzymes (Murray et al., 1982; Barcus et al., 1995). Currently, analysis of DNA and amino acid sequence homology is also used to place the known type I R–M systems found in the genomes of different bacteria into families (Sitaraman & Dybvig, 1997; Schouler et al., 1998a; Roberts et al., 2003a; http://rebase.neb.com/rebase/). However, as noted by Titheradge et al. (2001) such subdivision of type I R–M systems is an empirical one. High levels of identity at the level of nucleotide sequence are indicative of relatively recent divergence and conservation at the level of protein subunits. Any comparison between the Hsd subunits of two families of type I R–M systems identifies little sequence similarity even at the level of amino acid sequence: commonly only 20–30 % amino acid identity. A high level of identity is taken as proof that each representative belongs to the same family. Recently we have described a type I R–M system from Neisseria gonorrhoeae that, due to the very high level of identity with founder members of R–M systems referred as to type IC (EcoR124II, EcoDXXI), was classified also as a member of this group (Piekarowicz et al., 2001). In fact, as we previously described, the NgoAV hsdS gene is interrupted by a frameshift mutation and the expressed protein represents a natural truncated form of HsdS, i.e. has only 208 N-terminal amino acids instead of 410 (Fig. 1). Such truncated subunits of EcoDXXI and EcoR124II were made by genetic engineering (Abadijeva et al., 1993; Meister et al., 1993; MacWilliams & Bickle, 1996) and were active if a part of the central conserved domain was present. The HsdSNgoAV subunit mediates interactions with other subunits of the same system and recognizes the specific interrupted palindromic sequence 5'-GCA(N8)TGC-3' (Piekarowicz et al., 2001). In this paper we provide evidence, based on complementation tests, that the Hsd subunits of the NgoAV R–M system do not associate with the Hsd subunits of EcoR124II or EcoDXXI to make a functional enzyme. Complementation can be at least partially restored by changing the tetra-amino-acid tandem repeated sequence LEAT to the sequence TAEL present in the central conserved domain of the HsdS subunit of the EcoR124 family of type IC R–M systems. We propose that the NgoAV R–M system represents a complementation subgroup of type IC R–M systems.



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Fig. 1. Map of the HsdS subunit of EcoR124II (1) showing the variable and conserved regions in the amino acid sequence (adapted from Kneale, 1994). Below are shown the truncated form of the HsdS protein of EcoR124II (2) and the structure of the HsdS protein of NgoAV (3). V1 represents the N-terminal TRD; V2 is the C-terminal TRD. The amino acid sequences of regions A and B are compared in Table 4. A' is duplicated in the EcoR124II subunit and its truncated form. A' does not exist in wild-type NgoAV HsdS because of a frameshift mutation in the hsdS gene.

 

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Bacterial strains, phages and media.
The E. coli K-12 strain DH5{alpha}mcr (supE44 recA1 gyrA (NalR) thi-1 hsdR17 {Delta}mcr) was used throughout this work. This strain is {lambda}-sensitive and restriction-deficient. This strain and its derivatives were grown at 37 °C in Luria–Bertani medium (LB) (Sambrook et al., 1989). The strains carrying pSRNgoAV were grown in the presence of 1 % (final concentration) arabinose to induce the expression of the hsdS and hsdR genes. Construction of pNgoAV and pMS5 plasmids carrying HsdRMS and HsdMS subunits of NgoAV R–M system was described by Piekarowicz et al. (2001). Plasmid pRMS23, encoding HsdM and HsdR subunits of the NgoAV system, was obtained by introducing the chloramphenicol-resistance (Cm) cassette into the hsdS1 gene of the NgoAV R–M system. Plasmid pR was obtained by cloning the EcoRI–HindIII fragment of pNgoAV, carrying part of the hsdS1 gene and the intact hsdR gene, between the EcoRI and HindIII sites of pMPMT6{Omega} (Mayer, 1995). In this plasmid both genes were under control of the ara promoter and could be induced in the presence of arabinose. pMPMT6{Omega} is compatible with pUC19 and confers resistance to tetracycline. Plasmid pS17, carrying the hsdS1 gene, was constructed by cloning of the BstBI–StuI fragment into pACYC184 DNA, which confers resistance to chloramphenicol. Plasmid pMMW68 (MacWilliams & Bickle, 1996), carrying hsdRMS genes cloned into pACYC184, was the source of the R–M system EcoDXXI. Plasmid pHJ6 carrying hsdRMS genes cloned into pACYC184 was the source of the R–M system EcoR124II. Plasmid pJT22 carries hsdM and the 5' half of hsdS genes from the EcoDXXI R–M system cloned into pBluescript and confers resistance to ampicillin (Meister et al., 1993). Plasmid pMG3 is a derivative of pACYC177 which expresses the EcoR124I hsdR gene and confers chloramphenicol resistance (Gubler & Bickle, 1991). Plasmid pMG2 carries hsdR and hsdM from the EcoR124II R–M system (Gubler & Bickle, 1991) cloned into pBluescript. Plasmid pSAJ1 carries the 3' half of the hsdS gene from the EcoDXXI R–M system cloned into pACYC184 and confers chloramphenicol resistance. Plasmid pEKU19 carries the hsdM and hsdS genes of EcoR124I cloned into pACYC184 (Kulik & Bickle, 1996). Plasmid pMMW62 carries hsdM and the 5' half of hsdS genes from the EcoDXXI R–M system cloned into pACYC184 (MacWilliams & Bickle, 1996). All the clones, derivatives of the R–M systems EcoR124 and EcoDXXI were obtained from M. MacWilliams and T. Bickle, Division of Molecular Microbiology, University of Basel, Switzerland.

{lambda}vir, used to test restriction and modification, was propagated on E. coli strain DH5{alpha}mcr (unmodified phage, {lambda}vir.0) or propagated on the same strain containing the appropriate plasmids for modification. All tests dependent on plasmids were done with freshly transformed strains. The cultures were grown in the presence of appropriate antibiotics.

Mutant construction by PCR.
We produced the in-frame LEAT triplicate encoding sequence deletion mutant of the HsdS subunit of the NgoAV R–M enzyme by removing 36 bp in the central conserved region (pMS5-{Delta}). The same residues are exchanged to a TAEL repeat in pMS5-TAEL. Plasmids pMS5-{Delta} and pMS5-TAEL were constructed by PCR with primers annealing on both sides of the deleted sequence encoding LEAT, using purified plasmid pMS5 DNA as template. For pMS5-{Delta} construction, the forward primer was 5'-GCCCTGCGCAAACGCCAATACCGGTA-3' and the reverse primer 5'-TTCCAGCTCGGTGAATTTGTCAAGTA-3'. For pMS5-TAEL construction, the forward primer 5'-GCTGAGTTAACCGCGGAATTAGCCCTGCGCAAACGCCAATACC-3' and the reverse primer 5'-GGTTAACTCAGCGGTCAGCTCGGTGAATTTGTCAAGTATTTTTAC-3' were used. The underlined nucleotides represent the non-complementary sequences which, after ligation, encode TAEL repeats. The PCR was carried out with the Long PCR enzyme mix kit (Fermentas) using the manufacturer's recommended conditions and 5 min + 3 s per cycle extension time. After filling in the single-stranded overhangs with the Klenow fragment of DNA polymerase I, the DNA was phosphorylated with T4 polynucleotide kinase. Linear DNA was self-ligated with T4 DNA ligase in the presence of 5 % PEG 4000. Transformed DH5{alpha}mcr cells were selected for ampicillin resistance. The structure of new constructs was checked by DNA sequence analysis from pUC19 universal primer.

Enzymes and chemicals.
Long PCR enzyme mix kit, restriction enzymes, T4 DNA ligase, Klenow fragment and T4 polynucleotide kinase were purchased from Fermentas. The restriction enzymes FokI and SfaNI were obtained from New England Biolabs. All chemicals used were reagent grade or better and were obtained from Sigma, unless otherwise noted. Routine plasmid isolations were carried out according to Sambrook et al. (1989). The DNA clean-up and plasmid DNA miniprep kits were from A&A Biotechnology.

Sequence comparisons.
Alignments were made using the BLAST program available on the National Center for Biotechnology Information web site (http://www.ncbi.nlm.nih.gov/BLAST). The genomic sequence of N. gonorrhoeae strain FA 1090 was obtained from the University of Oklahoma's Advanced Center for Genome Technology (http://www.genome.ou.edu/gono.html). The sequences of HsdS subunits of different IC R–M systems were obtained from the REBASE server (http://rebase.neb.com/rebase/). The GenBank numbers for complete genomes or gene sequences were: S.EcoprrI-X52284, S.EcoR124II-X13145, S.EcoDXXI-X73984, S.Lla1403I-Nc_002662, S.Lla103I-AFO13595, S.Lla130I-AFO13596, S.MpnORF342P-NC000912, S.HpyCR38P-AF326625, S.HpyCR2P-AF326617, S.HpyCR29P-AF326623, S.NmeAORF1038P-NC_003112.


   RESULTS AND DISCUSSION
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ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Lack of complementation between the Hsd subunits of E. coli type IC and NgoAV R–M enzymes
The close relatedness of the best-known type IC R–M systems represented by EcoR124I, EcoR124II, EcoDXXI and EcoprrI was demonstrated by antigenic cross-reactivity and/or genetic complementation, i.e. the possibility of subunit exchange (Bickle, 1987; Price et al., 1987; Gubler et al., 1992; MacWilliams & Bickle, 1996; Skrzypek & Piekarowicz, 1989). It has been previously shown that even the truncated forms of the HsdS subunit within the E. coli type IC group of R–M systems can complement subunits of other members (Meister et al., 1993; MacWilliams & Bickle, 1996). The high identity of the HsdR (74 %) and HsdM (75 %) subunits of the NgoAV system compared to other members of the type IC family allowed it to be classified as a likely member of the IC family (Piekarowicz et al., 2001). To verify this hypothesis we carried out a complementation test in vivo between NgoAV and two members of the type IC family, EcoR124II and EcoDXXI. First, we showed that the HsdR, HsdM and HsdS subunits encoded by the genes of the NgoAV R–M system, cloned into compatible plasmids pACYC184 and pUC19, respectively, are able to create an active complex that restricts and modifies phage {lambda}vir (Table 1).


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Table 1. Complementation between subunits of NgoAV

 
When all the subunits of the EcoR124II and NgoAV R–M systems were together in the same bacterial strain, unmodified phage {lambda}vir or {lambda}vir carrying EcoR124II or NgoAV modification was restricted (Table 2). Similarly, EcoDXXI or NgoAV modification did not protect phage {lambda}vir from restriction when the subunits of the complete EcoDXXI and NgoAV R–M systems were present together (Table 3). However, the observed levels of restriction of unmodified phage were not the sum of the restriction of both systems. This indicated that when both systems were expressed, active interference between the Hsd subunits of the different R–M systems was possible. Such interference can be a result of an unequal concentration of subunits encoded by the hsd genes present on the high- and low-copy-number plasmids that can interfere with the formation of the stable R–M complexes. A similar observation was made when the complementation between the Hsd subunits of two members of type ID R–M systems, StySBLI and KpnAI, was tested (Titheradge et al., 2001). It was also shown that overproduction of the HsdS leads to altered R–M function in vivo (Hubacek et al., 1998; Weiserova et al., 1994). To test the complementation between the particular gene products of the NgoAV and EcoR124II or EcoDXXI systems, partial diploids were made by transforming the E. coli DH5{alpha}mcr {lambda}-sensitive strain with the compatible plasmids carrying the different sets of the hsd genes. In all cases, the particular Hsd subunits of the NgoAV R–M system could not substitute for the subunits of the EcoR124II or EcoDXXI and vice versa (Tables 2 and 3). A lack of complementation was observed between HsdS subunits of wild-type EcoR124II or EcoDXXI systems carried by plasmids pHJ6, pMG2 or pMMW68, or in truncated form on plasmids pJT22 or pMMW62 (5' half of HsdSDXXI) or pSAJ1 (3' half of HsdSDXXI). These results demonstrate that the Hsd subunits of the NgoAV R–M system are not sufficiently similar to the subunits of the other members of EcoR124-type IC R–M systems to meet the most demanding requirement for membership of the same family of type I R–M systems.


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Table 2. Complementation between subunits of NgoAV and EcoR124II

 

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Table 3. Complementation between subunits of NgoAV and EcoDXXI

 
Comparison of conserved domains in type IC R–M systems
It is believed that all type I R–M systems have a common origin (Sharp et al., 1992). Those belonging to the same family, even from distinct genera, show high similarity of HsdM and HsdR subunits. The presence of only small differences in their amino acid sequence, which probably appear following horizontal gene transfer, suggests the importance of the conservation of all their domains. Recently published (Titheradge et al., 2001) sequence comparisons between 18 different type I R–M systems, belonging to all four groups, showed that the HsdM polypeptides within the same group usually had >45 % identity, but those between different groups had <35 % homology. Similarly, the levels of identity found for HsdR were 37 % or greater and therefore higher than those (17–26 %) for interfamily comparisons. Although the similarity between HsdM and HsdR allows one to affiliate particular type I R–M systems to a general group, it does not determine whether they will show an ability to exchange subunits and make a functional complex. Although the HsdR and HsdM subunits of the NgoAV R–M system show very high similarity with the respective Hsd polypeptides of the EcoR124-type IC R–M system (Piekarowicz et al., 2001), they are not able to complement each other, as described above.

Complementation requires sufficient sequence conservation to permit subunits from one complex to substitute those in another (Fuller-Pace et al., 1985) and the HsdS subunit is critical for the correct assembly of polypeptides (Kneale, 1994; Murray, 2000; Weiserova et al., 2000). This means that the difference between two members of one family resides in the HsdS subunits that confer three functions: DNA binding, sequence specificity of the enzyme and the ability for interaction (i.e. complementation) with other subunits. The DNA specificity subunit is composed of two independent target recognition domains (TRDs), which are different for particular members of the same group and specify the recognition sequence (Gough & Murray, 1983; Dryden et al., 1999) as well as several regions whose amino acid sequence is conserved within an enzyme family (Argos, 1985; Gough & Murray, 1983; Kneale, 1994). Since the HsdS subunits within a family are interchangeable, their conserved regions are thought to mediate interactions with the other enzyme subunits (Abadijeva et al., 1994; Cooper & Dryden, 1994). This is also true for the truncated forms of the hsdS genes (Abadijeva et al., 1993; Meister et al., 1993; MacWilliams & Bickle, 1996). In type IC R–M systems, three conserved regions are present in the HsdS subunit: N-terminal, central and C-terminal (Fig. 1). A portion of the N-terminal region shows a high degree of similarity to part of the central region. The remainder of the central region is similar to part of the C-terminal region (Abadijeva et al., 1993; Meister et al., 1993; Kneale, 1994; Tyndall et al., 1994; MacWilliams & Bickle, 1996; Murray, 2000). The lack of complementation between Hsd subunits of NgoAV and EcoR124II or EcoDXXI R–M IC systems means that the conserved regions responsible for subunit interactions are probably different.

To test this prediction we analysed such regions within the HsdS subunit of NgoAV and other members of the IC family. These regions were located in the N-terminal region, up to 20–27 aa (region A) and between the beginning of the central conserved region and the beginning of the second (C-terminal) TRD region (141–210/230 aa) (region B) (Fig. 1) of the each protein. The conserved regions that participate in inter-subunit interactions show a high level of similarity between different members of a particular type IC family (Fuller-Pace & Murray, 1986; Kannan et al., 1989; Gubler et al., 1992; Tyndall et al., 1994). Within the central conserved region, there is a tandem repeat of tetra-amino-acid sequences that is characteristic for particular members of type I R–M systems (Price et al., 1989; Gubler & Bickle, 1991; Gubler et al., 1992; Piekarowicz et al., 2001). In the EcoR124 family the sequence TAEL is present in duplicate or triplicate. It was shown that the number of TAEL repeats governs the length of the recognition site spacer. Two repeats result in a 6 bp spacer for EcoR124I, while three repeats result in a 7 bp spacer for EcoR124II (Price et al., 1989; Gubler & Bickle, 1991; Gubler et al., 1992). Related repeat sequences were found in enzymes affiliated to the IC family (Roberts et al., 2003a; Sitaraman & Dybvig, 1997; Schouler et al., 1998a; Titheradge et al., 2001). The triplicate sequence LEAT is present in the HsdS subunit of the NgoAV R–M system.

A computer analysis of the aligned regions A and B indicates that the sequence of NgoAV differs markedly from those of EcoR124II, EcoDXXI or EcoprrI (Table 4). The identity in region A is more than 70 % similar and in the region B 50 % or more similar between the EcoR124 family members of type IC, while in both cases it is less than 35 % similar to NgoAV regions. This sequence comparison indicates the presence of several groups within the type IC R–M family that have an identity in region A of more than 70 % and in region B of 50 % or more. Each of these groups also has a different, characteristic, tetra-amino-acid sequence.


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Table 4. Sequence comparison of the repeated sequence present at the N-terminal and in the central conserved domain of HsdS proteins encoded by different type IC R–M systems

The R–M systems, identified by numbers 1–12, are listed in the left-hand column. The values represent the percentage identity of aligned amino acid sequences. Below the diagonal for the maximum of 27 aa (region A; see Fig. 1); above, between the beginning of the central conserved region and the beginning of the C-terminal TRD region (141–210/230 aa, region B; see Fig. 1). Values for comparisons within proposed subfamilies are given in bold.

 
The tetra-amino-acid repeat sequence in the HsdS subunit influences the intrafamily complementation ability
The changes in the length and the context of the tandem repeated region have a profound effect on the specificity (Gubler & Bickle, 1991) of EcoR124II R–M activity. This may indicate that this region has a double function: determining the length of the non-specific spacer in the recognized sequence and mediating (influencing) protein–protein interaction, at least with HsdR. We decided to examine the role of this sequence in interactions between subunits. Two classes of mutants (within the plasmid pMS5) were constructed; the first one (mutant pMS5-{Delta}) lacked the original tetra-amino-acid LEAT repeat of NgoAV (subunit HsdSNgoAV : : {Delta}) and the second (pMS5-TAEL) contained three repeats of the tetra-amino-acid sequence TAEL instead of the sequence LEAT (subunit HsdSNgoAV : : TAEL). Both these mutants retained the original amino and C-terminal sequences.

First, we checked the modification and restriction activity of mutant subunits. Phage {lambda}vir modified by the mutants was used to infect DH5{alpha}mcr cells expressing the wild-type EcoR124II, EcoR124I, EcoDXXI and NgoAV systems. The lack of the tetra-amino-acid repeats results in the loss of the specific NgoAV R–M system modification activity as the phage {lambda}vir grown on the cells carrying the HsdSNgoAV : : {Delta} subunit is restricted by the NgoAV R–M system (data not shown). Also, these mutants do not express restriction activity of unmodified {lambda}vir in the presence of the pR plasmid encoding the HsdR NgoAV subunit or in the presence of the HsdR subunit from the EcoR124II or EcoDXXI R–M systems (Table 5). On the other hand, the mutant pMS5-TAEL, encoding the chimeric HsdSNgoV : : TAEL, shows the methylase activity specific for the NgoAV R–M system and the ability to form the active restriction endonuclease in the presence of pR plasmid, as measured by the ability to modify and restrict the unmodified or NgoAV modified phage {lambda}vir (Table 5).


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Table 5. Complementation between mutant subunits of NgoAV and EcoR124II or EcoDXXI

 
To confirm these results, we analysed the effect of FokI restriction activity on purified DNA of pMS5, pMS5-{Delta} and pMS5-TAEL. All these constructs had only one NgoAV-specific sequence GCA(N)8TGC at position 2694–2707 bp, which overlaps the sequence recognized by FokI (5'-GGATG/CATCC-3') (2702 bp). This enzyme will not cleave DNA if this sequence contains a methylated adenine residue at position 2705 bp (Roberts et al., 2003a; http://rebase.neb.com/rebase/). After isolation, plasmids pMS5, pMS5-{Delta} and pMS5-TAEL were subjected to FokI digestions and the cleavage products were separated by agarose gel electrophoresis. Cleavage by FokI should generate 13 fragments, with all but three of them being smaller than 800 bp in size, the exceptions being fragments of 890, 929 and 1429 bp. If the site at 2702 bp is cleaved, the resulting fragments should be 890 and 200 bp in size. Protection from FokI cleavage at this site should result in the loss of these products and the appearance of a 1090 bp fragment. The data presented in Fig. 2 indicate that after digestions of pMS5 and pMS5-TAEL a new fragment of 1090 bp appears while such protection is not visible in the case of pMS5-{Delta}. The same observations were made when SfaNI digestion products were analysed: in pMS5 and pMS5-TAEL, the NgoAV-methylated site at 2705 bp was not cleaved and an undigested fragment of 1219 bp appeared (data not presented), indicating that only pMS5 and pMS5-TAEL encode proteins with NgoAV methylase activity. These results confirm previous observations that the lack of the tetra-amino-acid sequence results in the loss of both methylase and restriction activity (Gubler & Bickle, 1991). Moreover, they indicate that the exchange of one type of tetra-amino-acid sequence by a different type sequence of the same length does not change the specificity of the HsdS subunit (HsdSNgoAV : : TAEL). This also confirms the observation that only the length variations and the insertion of various amino acids into the ‘spacer’ region give recognition properties that vary in a non-predictable way (Gubler & Bickle, 1991).



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Fig. 2. Possible outcomes of FokI restriction digestion at an overlapping NgoAV recognition site. Unmodified at this site, pMS5 DNA would be cleaved by FokI at site 2702 as well as at flanking sites at 1819 and 2902 bp. This would result in two fragments of 890 bp and 200 bp. If cleavage at this site were inhibited by M.NgoAV methylation, a single 1090 bp fragment would result. The computer analysis indicates that unmodified pMS5 DNA cleaved by FokI would generate 13 fragments, with only three larger than 800 bp (1429, 929 and 890 bp), allowing us to distinguish an undigested fragment from all others. The theoretical digest of pMS5 is shown in lane A. To analyse the FokI cleavage products, pMS5 DNA (lane B), pMS5-TAEL (lane C) and pMS5-{Delta} (lane D) were digested with FokI, and the digests were electrophoresed on a 1·2 % agarose gel. Fermentas ladder molecular mass standards (lane E): 10 000, 8000, 6000, 5000, 4000, 3500, 3000, 2000, 1500, 1200, 1031, 900, 800, 700, 600, 500, 400, 300, 200 and 100 bp. The lack of an 890 bp FokI fragment and the presence of a 1090 bp fragment confirm that this enzyme cannot cleave at the methylated site 2702 that overlaps the recognition sequence of NgoAV. The arrow shows the 1090 bp fragment.

 
A complementation assay between HsdSNgoV : : TAEL and the founder systems of the type IC family showed that the pMS5-TAEL-encoded polypeptides are able to make an active restriction complex with the HsdR subunits from EcoR124II, EcoR124I and EcoDXXI R–M systems, when they are present together with their respective HsdM and HsdS subunits. When the EcoR124II HsdR subunit is present alone (plasmid pMG3), formation of the active NgoAV R–M system as a result of complementation shows a variable level in particular experiments as measured by the level of restriction, which varies from 4x10-1 to 1x10-3 (Table 5). The variability of the restriction function may be influenced by the different relative concentration of the particular Hsd subunits encoded by the genes of EcoR124II, EcoDXXI and M.NgoAV cloned into the plasmids present at different copy numbers inside the cells. It was previously shown that the different intracellular level of Hsd subunits may prevent correct assembly of the R–M complex (Dryden et al., 1993; Taylor et al., 1992; Weiserova et al., 1994, 2000; Hubacek et al., 1998; Weiserova & Firman, 1998). This could also explain why the level of restriction of phage {lambda}vir by E. coli cells carrying the wild-type R–M systems of EcoR124II or EcoDXXI and NgoAV is not the sum of the restriction level shown separately by each of these systems.

According to the HsdS model presented by Kneale (1994) the linker region does not make any specific contact with HsdM but is overlapped by it, and can be envisaged as an ‘elbow’ joint within the two conserved ‘arms’. Since the insertion of the extra amino acids into this sequence and the change to another ‘elbow’ is tolerated, the elbow joint must have a certain degree of flexibility matching the flexibility in the HsdM subunit that allows some tolerance in the positioning of the contact regions. This flexibility seems to be more ‘relaxed’ in the chimeric form of HsdSNgoAV : : TAEL than in the wild-type forms of HsdSNgoAV or HsdSEcoR124. While the hybrid HsdSNgoAV : : TAEL form cooperates with the Hsd subunits of both the NgoAV and EcoR124 R–M systems, the complementation between wild-type HsdS subunits belonging to different type IC subfamilies (as for example HsdSEcoR124II : : TAEL, HsdSEcoDXXI : : TAEL and HsdSNgoAV : : LEAT) has not been possible. Both the arms and the elbow will determine the flexibility and the ability of the HsdS subunit to cooperate and complement with the subunits of different R–M type IC systems.

We suggest that complementation can be achieved more easily within the members of each group or ‘subfamily’ containing the same specific tetra-amino-acid sequence and a high level of identity in the conserved amino and central regions. The complementation between the members of different ‘subfamilies’, as for example between NgoAV and EcoR124II or EcoDXXI, would be not possible. The observed complementation between type IC R–M systems of Lactococcus (Schouler et al., 1998b) would argue for this interpretation of our results. In this work, complementation between five different plasmid-encoded HsdS subunits, which are almost 85 % identical in the central conserved domain, and chromosomally encoded HsdM and/or HsdR subunits was observed. An alternative explanation might be that all these different type IC R–M systems present in distantly related bacteria reflect their phylogenetic differences.

In conclusion, it can be stated that the members of the particular family of type I R–M system should show not only high similarity in the amino acid sequence of the HsdM and HsdR subunits but also similarity in the regions responsible for interaction of the subunits. If this is true, then within the type IC family several subfamilies could be identified, such as EcoR124 and subfamilies from Helicobacter pylori, Lactococcus lactis and Neisseria.


   ACKNOWLEDGEMENTS
 
This work was supported by KBN grant no. 6 PO4A 037 18. We are very grateful to Tom Bickle and Maria MacWilliams, who sent us plasmid DNA carrying the genes encoding EcoR124 and EcoDXXI R–M systems.


   REFERENCES
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS AND DISCUSSION
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Received 19 May 2003; revised 10 July 2003; accepted 7 August 2003.



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