title: Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system.
abstract: Type II restriction-modification (R-M) systems encode a restriction endonuclease that cleaves DNA at specific sites, and a methyltransferase that modifies same sites protecting them from restriction endonuclease cleavage. Type II R-M systems benefit bacteria by protecting them from bacteriophages. Many type II R-M systems are plasmid-based and thus capable of horizontal transfer. Upon the entry of such plasmids into a naïve host with unmodified genomic recognition sites, methyltransferase should be synthesized first and given sufficient time to methylate recognition sites in the bacterial genome before the toxic restriction endonuclease activity appears. Here, we directly demonstrate a delay in restriction endonuclease synthesis after transformation of Escherichia coli cells with a plasmid carrying the Esp1396I type II R-M system, using single-cell microscopy. We further demonstrate that before the appearance of the Esp1396I restriction endonuclease the intracellular concentration of Esp1396I methyltransferase undergoes a sharp peak, which should allow rapid methylation of host genome recognition sites. A mathematical model that satisfactorily describes the observed dynamics of both Esp1396I enzymes is presented. The results reported here were obtained using a functional Esp1396I type II R-M system encoding both enzymes fused to fluorescent proteins. Similar approaches should be applicable to the studies of other R-M systems at single-cell level.
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia.
authors.forename: Natalia
authors.initials: N
authors.lastname: Morozova
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia.
authors.forename: Anton
authors.initials: A
authors.lastname: Sabantsev
authors.affiliation: Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
authors.forename: Ekaterina
authors.initials: E
authors.lastname: Bogdanova
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia Skolkovo Institute of Science and Technology, Skolkovo, 143026, Russia.
authors.forename: Yana
authors.initials: Y
authors.lastname: Fedorova
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia Skolkovo Institute of Science and Technology, Skolkovo, 143026, Russia.
authors.forename: Anna
authors.initials: A
authors.lastname: Maikova
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia.
authors.forename: Alexey
authors.initials: A
authors.lastname: Vedyaykin
authors.affiliation: Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia.
authors.forename: Andjela
authors.initials: A
authors.lastname: Rodic
authors.affiliation: Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia.
authors.forename: Marko
authors.initials: M
authors.lastname: Djordjevic
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia.
authors.forename: Mikhail
authors.initials: M
authors.lastname: Khodorkovskii
authors.affiliation: Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA severik@waksman.rutgers.edu K.Severinov@skoltech.ru.
authors.forename: Konstantin
authors.initials: K
authors.lastname: Severinov
mesh_terms: D015254:DNA Modification Methylases / Q000235:genetics / Q000378:metabolism; D015280:DNA Restriction-Modification Enzymes / Q000032:analysis / Q000235:genetics / Q000378:metabolism*; D015245:Deoxyribonuclease BamHI / Q000235:genetics / Q000378:metabolism; D004926:Escherichia coli / Q000235:genetics; D008164:Luminescent Proteins / Q000235:genetics / Q000378:metabolism; D008856:Microscopy, Fluorescence; D008954:Models, Biological; D011994:Recombinant Proteins / Q000235:genetics / Q000378:metabolism; D059010:Single-Cell Analysis / Q000379:methods*; D000097573:Red Fluorescent Protein
chemical_list: D015254:DNA Modification Methylases; D015280:DNA Restriction-Modification Enzymes; D015245:Deoxyribonuclease BamHI; D008164:Luminescent Proteins; D011994:Recombinant Proteins; D000097573:Red Fluorescent Protein