Two-step cloning and expression in Escherichia coli of the DNA restriction-modification system StyLTI of Salmonella typhimurium.
The StyLTI restriction-modification system is common to most strains of the genus Salmonella, including Salmonella typhimurium. We report here the two-step cloning of the genes controlling the StyLTI system. The StyLTI methylase gene (mod) was cloned first. Then, the companion endonuclease gene (res) was introduced on a compatible vector. A strain of S. typhimurium sensitive to the coliphage lambda was constructed and used to select self-modifying recombinant phages from a Res- Mod+ S. typhimurium genomic library in the lambda EMBL4 cloning vector. The methylase gene of one of these phages was then subcloned in pBR328 and transferred into Escherichia coli. In the second step, the closely linked endonuclease and methylase genes were cloned together on a single DNA fragment inserted in pACYC184 and introduced into the Mod+ E. coli strain obtained in the first step. Attempts to transform Mod- E. coli or S. typhimurium strains with this Res+ Mod+ plasmid were unsuccessful, whereas transformation of Mod+ strains occurred at a normal frequency. This can be understood if the introduction of the StyLTI genes into naive hosts is lethal because of degradation of host DNA by restriction activity; in contrast to most restriction-modification systems, StyLTI could not be transferred into naive hosts without killing them. In addition, it was found that strains containing only the res gene are viable and lack restriction activity in the absence of the companion mod gene. This suggests that expression of the StyLTI endonuclease activity requires at least one polypeptide involved in the methylation activity, as is the case for types I and III restriction-modification systems but not for type II systems.
De Backer
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Département de Biologie, Université Catholique de Louvain, Louvain-la-Neuve, Belgium.
Colson
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D003001:Cloning, Molecular; D015254:DNA Modification Methylases / Q000235:genetics* / Q000378:metabolism; D004262:DNA Restriction Enzymes / Q000235:genetics* / Q000378:metabolism; D015263:Deoxyribonucleases, Type III Site-Specific / Q000235:genetics* / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics*; D015964:Gene Expression Regulation, Bacterial; D005798:Genes, Bacterial; D010641:Phenotype; D010957:Plasmids; D015183:Restriction Mapping; D012486:Salmonella typhimurium / Q000201:enzymology / Q000235:genetics*; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific) / Q000235:genetics* / Q000378:metabolism; D014169:Transformation, Bacterial
D015254:DNA Modification Methylases; C067137:DNA modification methylase StyLTI; D015265:Site-Specific DNA-Methyltransferase (Adenine-Specific); D004262:DNA Restriction Enzymes; C067136:endodeoxyribonuclease StyLTI; D015263:Deoxyribonucleases, Type III Site-Specific