The recognition and modification sites for the bacterial type I restriction systems KpnAI, StySEAI, StySENI and StySGI

Abstract

Using an in vivo plasmid transformation method, we have determined the DNA sequences recognized by the KpnAI, StySEAI, StySENI and StySGI R-M systems from Klebsiella oxytoca strain M5a1, Salmonella eastbourne, Salmonella enteritidis and Salmonella gelsenkirchen, respectively. These type I restriction-modification systems were originally identified using traditional phage assay, and described here is the plasmid transformation test and computer program used to determine their DNA recognition sequences. For this test, we constructed two sets of plasmids, pL and pE, that contain phage lambda and Escherichia coli K-12 chromosomal DNA fragments, respectively. Further, using the methylation sensitivities of various known type II restriction enzymes, we identified the target adenines for methylation (listed in bold italics below as A or T in case of the complementary strand). The recognition sequence and methylation sites are GAA(6N)TGCC (KpnAI), ACA(6N)TYCA (StySEAI), CGA(6N)TACC (StySENI) and TAAC(7N)RTCG (StySGI). These DNA recognition sequences all have a typical type I bipartite pattern and represent three novel specificities and one isoschizomer (StySENI). For confirmation, oligonucleotides containing each of the predicted sequences were synthesized, cloned into plasmid pMECA and transformed into each strain, resulting in a large reduction in efficiency of transformation (EOT).

Figure 1

pL series plasmid subclones derived from phage lambda DNA (A) and pE series plasmids derived from fragments of E.coli K-12 chromosomal DNA (B). Each number and the corresponding box represent the plasmid inserts. Shaded boxes contain KpnAI recognition site(s) (data from Figure 2). The triangles and arrows show the location and direction of the KpnAI sites predicted from this study. Restriction sites are also shown: HI, BamHI; RI, EcoRI; RV, EcoRV; HIII, HindIII; PI, PstI; NI, NdeI; BHII, BssHII; and SI, SphI. All chromosomal fragments in the pE series plasmids were cloned into the EcoRI site.

Figure 2

EOT values obtained from pL series plasmids (A) and pE series plasmids (B). Each plasmid was transformed into both strains M5a1 and M5a1R (control). Relative transformant numbers (EOT) were calculated. The number of KpnAI sites varies from 0 to 3. The range of EOT values for each KpnAI site are shown on the right. Standard deviations are shown as error bars.

Figure 3

Oligonucleotides used to confirm the recognition sequence (A) and to determine the methylation sites for KpnAI (B). Oligonucleotides B-1 and B-2 were used to determine the methylated adenine in the prime strand and B-3 was used for the complementary strand. The KpnAI sequence is shown in bold. Plasmids containing these oligonucleotides are designated, from top to bottom: pKpnAI, pKpnAIH1, pKpnAIH2 and pKpnAIH3.

Figure 4

Determination of the methylation sites of KpnAI. Plasmid DNA was digested with HindIII. Lane 1, 1 kb marker; lane 2, pMECA control; lane 3, pKpnAIH1 unmodified; lane 4, pKpnAIH1 modified; lane 5, pKpnAIH2 unmodified; lane 6, pKpnAIH2 modified; lane 7, pKpnAIH3 unmodified; lane 8, pKpnAIH3 modified. The arrow shows the position of the 209 bp fragments.

Figure 5

Design of oligonucleotides used for confirmation of recognition sequences and determination of methylation sites in Salmonella R-M systems. Oligonucleotides SEA-1, SEN-1 and SG-1 were used to determine the methylated adenine in the trinucleotide portion of each recognition sequence (marked in bold). SEA-2, SEN-2 and SG-2 represent the oligonucleotide used for the tetranucleotide component. Each oligonucleotide was inserted into the EcoRV site (or SspI for SG-1) in pMECA and the plasmids were designated from the top, pSEA-S1, pSEA-S2, pSEN-S1, pSEA-S2, pSG-S1 and pSG-T1. The type II restriction enzyme site is shown with double arrows below each oligonucleotide sequence. Each methylated adenine is shown in large font.