Four new type I restriction enzymes identified in Escherichia coli clinical isolates

Abstract

Using a plasmid transformation method and the RM search computer program, four type I restriction enzymes with new recognition sites and two isoschizomers (EcoBI and Eco377I) were identified in a collection of clinical Escherichia coli isolates. These new enzymes were designated Eco394I, Eco826I, Eco851I and Eco912I. Their recognition sequences were determined to be GAC(5N)RTAAY, GCA(6N)CTGA, GTCA(6N)TGAY and CAC(5N)TGGC, respectively. A methylation sensitivity assay, using various synthetic oligonucleotides, was used to identify the adenines that prevent cleavage when methylated (underlined). These results suggest that type I enzymes are abundant in E.coli and many other bacteria, as has been inferred from bacterial genome sequencing projects.

Figure 1

Plasmid R-M tests for Eco394I on a series of subclones identifying two Eco394I sites (A and B) on plasmid pL9. Positive plasmids (with Eco394I site) are darkened, whereas negative plasmids (no site) are hatched. Plasmid pL9 is a subclone of pL2 (Table 1). Plasmids pL31 to pL35 are PstI subclones of pL9. Plasmids pL49, pL50 and pL51 are derived from pL32, whereas plasmids pL54, pL55 and pL57 are derived from pL34. Two plasmids pL34-96 and pL34-97 were obtained from an ExoIII/S1 nested deletion experiment, whereas the pC2 is a plasmid containing an 18 bp oligonucleotide cloned into the pMECA EcoRV site. Only relevant DNA sequences are shown. Note that the Eco394I sequence described here at B site is a complementary sequence of the consensus sequence, GAC(5N)RTAAY described in Table 2.

Figure 2

Methylation sensitivity assay to determine the methylated adenine in the target sequence. (A) General design of the ‘reference’ plasmid. An oligonucleotide containing a type I recognition sequence was cloned into the EcoRV site of the MCS region of pMECA. The ‘reference’ plasmids were modified in the corresponding bacteria. (B) Restriction digestions before and after methylation. All plasmids, except for lane 2, were digested with ScaI and subjected to 0.8% agarose gel electrophoresis. Lane 1, 1 kb ladder; lanes 2 and 3, pMECA control digested with EcoRV and ScaI, respectively; lanes 4 and 5, pEco826 unmodified and modified, respectively; lanes 6 and 7, pEco851, unmodified and modified, respectively; lanes 8 and 9, pEco912, unmodified and modified, respectively. Modified plasmids were digested only once and linearized, whereas unmodified plasmids were cut in two places resulting in two bands, one ∼0.9 kb (arrow).

Figure 3

Frequencies of the length of known type I recognition sequences.

Figure 4

Frequencies of the patterns (number of bases in 5′, random and 3′ component) of type I recognition sequences.