A Rapid and Efficient Method for Cloning Genes of Type II Restriction-Modification Systems by Use of a Killer Plasmid

Abstract

We present a method for cloning restriction-modification (R-M) systems that is based on the use of a lethal plasmid (pKILLER). The plasmid carries a functional gene for a restriction endonuclease having the same DNA specificity as the R-M system of interest. The first step is the standard preparation of a representative, plasmid-borne genomic library. Then this library is transformed with the killer plasmid. The only surviving bacteria are those which carry the gene specifying a protective DNA methyltransferase. Conceptually, this in vivo selection approach resembles earlier methods in which a plasmid library was selected in vitro by digestion with a suitable restriction endonuclease, but it is much more efficient than those methods. The new method was successfully used to clone two R-M systems, BstZ1II from Bacillus stearothermophilus 14P and Csp231I from Citrobacter sp. strain RFL231, both isospecific to the prototype HindIII R-M system.In the biology of microorganisms, the restriction-modification (R-M) systems play the important role of "molecular wardens" by protecting bacteria against phage infections. On the other hand, the enzymatic constituents of these systems have also made great contributions to the field of recombinant DNA technology and have served as attractive models for the study of DNA-protein interactions due to the exact mode of target recognition (33,34,37). However, practical applications are limited almost solely to the enzymes belonging to type II R-M systems. Each of these systems is composed of two entities that act independently: a restriction endonuclease (ENase) that recognizes and cleaves a specific short nucleotide sequence (4 to 8 bp) in DNA and a methyltransferase (MTase) that modifies the same sequence in order to protect the host genomic DNA against the action of the cognate restriction enzyme. As both genes coding for R-M systems are usually localized in close proximity (55), cloning an MTase gene often results in the cloning of a matching ENase gene.Until now, more than 400 type II R-M systems have been cloned (38) by the use of either (i) the "Hungarian trick" (45), which selects clones carrying the gene coding for a specific DNA MTase that confers resistance to digestion by the cognate restriction enzyme; (ii) methods based on the detection of SOS response induction in Escherichia coli (10, 32); or (iii) a direct search through genomic databases to identify open reading frames coding for conserved amino acid sequence motifs characteristic of DNA MTases, followed by PCR-based cloning (27).Our research is focused on the nature of the isospecificity phenomenon among type II R-M systems. We would like to know (i) how similar the genes coding for isospecific enzymes are, (ii) whether is it possible to map their functional domains, (iii) whether the systems recognize a cognate sequence in the same way, (iv) what their mode of action is, and (v) how those systems spread among bacteria.For the model in our study, we decided to use a group of systems isospecific to HindIII, an R-M s...

Natural C-independent expression of restriction endonuclease in a C protein-associated restriction-modification system

“…RFL231, by selecting for the MTase. We used a suicide plasmid that carried a functional REase gene (no MTase gene), from which cells would be protected if they had a MTase with corresponding specificity ( 59 ). The recognition sequence (5′-AAGCTT-3′) is the same as the prototype HindIII R-M system ( 60 ) as well as several other isospecific systems studied in our laboratory: EcoVIII from E. coli E1585–68 ( 47 , 48 , 61 ); LlaCI from Lactococcus lactis subsp.…”

“…The recognition sequence (5′-AAGCTT-3′) is the same as the prototype HindIII R-M system ( 60 ) as well as several other isospecific systems studied in our laboratory: EcoVIII from E. coli E1585–68 ( 47 , 48 , 61 ); LlaCI from Lactococcus lactis subsp. cremoris W15 ( 62 , 63 ) and BstZ1II from Bacillus stearothermophilus 14P ( 59 ).…”

“…According to the Sorokin classification C proteins fall into groups based on their distinct motifs in DNA sequence binding, which for C.Csp231I and its prototype C.EcoO109I (motif 8 – ACTAAGGA-T-TnCTTAGT) is a small fraction of the entire C protein number ( 43 , 44 ). C.Csp231I and C.EcoO109I share about 70% identity in amino acid sequence and are significantly larger proteins at about 11 kDa ( 59 ). They are also different structurally from other studied C proteins with motifs 1–6 (∼8–9 kDa).…”

Plasmid stability analysis based on a new theoretical model employing stochastic simulations

“…When necessary, appropriate antibiotics were used at the following concentrations: chloramphenicol (Cm) 30 μg/ml and kanamycin (Km) 50 μg/ml. Plasmid pIB8 (EcoVIII R + M + cer + Cm R , 5.2-kb) [ 42 ] was obtained from Dr. Iwona Mruk (University of Gdansk, Poland). Plasmid pRB1 (EcoVIII R + M + cer − Cm R , 4.6 kb) and pRB2 (EcoVIII R − M − cer − Cm R , 2.8 kb) were constructed in our laboratory and described in our previous report [ 35 ].…”

Isospecific adenine DNA methyltransferases show distinct preferences towards DNA substrates

“…1 ). The tested enzymes were: M.EcoVIII from Escherichia coli E1585-68 16 – 21 , M.HindIII from Haemophilus influenzae Rd 22 , 23 and M.LlaCI from Lactococcus lactis subs. cremonis W15 24 .…”