Institute of Microbiology, University of Warsaw, 02-096 Warsaw, Poland
Correspondence
Andrzej Piekarowicz
anpiek{at}biol.uw.edu.pl
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ABSTRACT |
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Nomenclature. The nomenclature for restriction endonucleases and methyltransferases in this paper follows the recommendations of Roberts et al. (2003b).
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INTRODUCTION |
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METHODS |
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vir, used to test restriction and modification, was propagated on E. coli strain DH5
mcr (unmodified phage,
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 RM enzyme by removing 36 bp in the central conserved region (pMS5-). The same residues are exchanged to a TAEL repeat in pMS5-TAEL. Plasmids pMS5-
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-
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
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 RM 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.
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RESULTS AND DISCUSSION |
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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 RM 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 RM 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 2027 aa (region A) and between the beginning of the central conserved region and the beginning of the second (C-terminal) TRD region (141210/230 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 RM 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 RM 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 RM 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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First, we checked the modification and restriction activity of mutant subunits. Phage vir modified by the mutants was used to infect DH5
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 RM system modification activity as the phage
vir grown on the cells carrying the HsdSNgoAV : :
subunit is restricted by the NgoAV RM system (data not shown). Also, these mutants do not express restriction activity of unmodified
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 RM systems (Table 5
). On the other hand, the mutant pMS5-TAEL, encoding the chimeric HsdSNgoV : : TAEL, shows the methylase activity specific for the NgoAV RM 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
vir (Table 5
).
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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 RM 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 RM 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 RM 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 RM 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 RM 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.
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ACKNOWLEDGEMENTS |
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Received 19 May 2003;
revised 10 July 2003;
accepted 7 August 2003.
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