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Nucleic Acids Res. 2016 Jan 29;44(2):790-800. doi: 10.1093/nar/gkv1490. Epub 2015 Dec 19.

Temporal dynamics of methyltransferase and restriction endonuclease accumulation in individual cells after introducing a restriction-modification system.

Author information

1
Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia.
2
Waksman Institute of Microbiology, Rutgers, the State University of New Jersey, Piscataway, NJ 08854, USA.
3
Peter the Great St. Petersburg Polytechnic University, St. Petersburg, 195251, Russia Skolkovo Institute of Science and Technology, Skolkovo, 143026, Russia.
4
Faculty of Biology, University of Belgrade, 11000 Belgrade, Serbia.
5
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.

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.

PMID:
26687717
PMCID:
PMC4737168
DOI:
10.1093/nar/gkv1490
[Indexed for MEDLINE]
Free PMC Article

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