Phenotypically cryptic EcoRI endonuclease activity specified by the ColE1 plasmid.

Abstract

(1), is encoded by genes located on a bacterial plasmid that has been designated pMB1 (2); a second site-specific enzyme, which methylates the adenosine at the center of the nucleotide sequence comprising the EcoRI endonuclease cleavage site (3) thereby rendering the site insusceptible to cleavage, is also specified by the pMB1 plasmid. It has been reported that a large segment of the pMB1 plasmid is homologous with another widely studied bacterial plasmid, ColEl (2). Since ColEl DNA is cleaved in vitro by the EcoRI endonuclease (4), and because bacterial cells carrying ColEl are not known to be associated with restriction activity, earlier investigators concluded that the EcoRI restriction and modification genes are located on a 1.95-kilobase segment of DNA that is present in pMB1 but not within ColEl (2). During in vitro studies of possible site-specific DNA cleavage associated with the Tn3 transposon, we observed that bacterial cells carrying the ColEl plasmid itself produce an endonuclease having EcoRI restriction specificity. However ColEl-containing cells do not express EcoRI restriction or modification functions in vivo and plasmid or phage DNA isolated from such cells is cleaved in vitro by the EcoRI endonuclease. The present report describes these findings.MATERIALS AND METHODS Bacterial Strains and Plasmids. E. coli strains RY13, C600, and HB101 (pMB4) were obtained from H. W. Boyer (University of California, San Francisco). E. coli strain 1200 [endo I-, RNase-, rK+mK+ (5)] was provided by M. Pearson. The ColEl-K30 plasmid was obtained from D. Helinski (6), and the Tn3 transposon (7) was introduced onto this plasmid fromThe costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U. S. C. §1734 solely to indicate this fact. 1265 pSC204 by the selected translocation procedure (8). The pMB3 plasmid and the Ri drd-19 plasmid were isolated from E. coli RY13 and purified by a cycle of transformation (9). The pMB3 and pMB4 (a spontaneous deletion mutant of pMB3) plasmids, which specify the EcoRI restriction and modification edzymes, are derived from the clinical isolate pMB1 (2). pCM100 is a spontaneously occurring mutant (isolated in our laboratory) of pMB3 that has lost the ability to restrict or modify DNA in vvo. Bacteriophages Xmr and XCI83 (10) and plasmid pSC355 (11) were used for detection of phenotypically expressed restriction and modification functions. Purified X DNA was isolated (12) from Xplac MS505S7 phage (13). Isolation of Enzyme. Bacterial cells used for isolation of endonuclease and methylase were grown in L broth (14) containing appropriate antibiotics, at concentrations of 25 Ag/ml for ampicillin, and 10,gg/ml for kanamycin, chloramphenicol, tetracycline, and streptomycin. Bacterial cultures of 500 ml or less were grown at 370 in shaking water baths to ODW0 = 1.0 and chilled by transfer of flasks to an ice water bath. Cells were collected by centrifugation, suspended (fin...

Genetic recombination during transformation in Bacillus subtilis: appearance of a deoxyribonucleic acid methylase

“…Shibata et al (33) showed the presence ofa site-specific endonuclease in B. subtilis Marburg (strain 168) that can cleave in vitro the DNA of bacteriophages grown on the same strain. This observation suggests lack of significant modification and restriction in vivo similar to that of the EcoRI enzyme system recently reported (24). In general, only a weak restriction type of nuclease activity has been demonstrated in B. subtilis (38).…”

Plasmid Diversity and Adaptation Analyzed by Massive Sequencing of Escherichia coli Plasmids

“…Less epidemiological attention is normally paid to small mobilizable plasmids. However, they are also carriers of antibiotic resistance genes (110)(111)(112)(113), as well as competition weapons, such as bacteriocins (113)(114)(115)(116) and restriction-modification enzymes (117)(118)(119)(120). They rely on conjugative plasmids as helpers for their conjugative transmission.…”