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

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.

Prediction of bacterial proteins carrying a nuclear localization signal and nuclear targeting of HsdM from Klebsiella pneumoniae.

Nuclear targeting of bacterial proteins is an emerging pathogenic mechanism whereby bacterial proteins can interact with nuclear molecules and alter the physiology of host cells. The fully sequenced bacterial genome can predict proteins that target the nuclei of host cells based on the presence of nuclear localization signal (NLS). In the present study, we predicted bacterial proteins with the NLS sequences from Klebsiella pneumoniae by bioinformatic analysis, and 13 proteins were identified as carrying putative NLS sequences. Among them, HsdM, a subunit of KpnAl that is a type I restriction-modification system found in K. pneumoniae, was selected for the experimental proof of nuclear targeting in host cells. HsdM carried the NLS sequences, (7)KKAKAKK(13), in the N-terminus. A transient expression of HsdM-EGFP in COS-1 cells exhibited exclusively a nuclear localization of the fusion proteins, whereas the fusion proteins of HsdM with substitutions in residues lysine to alanine in the NLS sequences, (7)AAAKAAA(13), were localized in the cytoplasm. HsdM was co-localized with importin o in the nuclei of host cells. Recombinant HsdM alone methylated the eukaryotic DNA in vitro assay. Although HsdM tested in this study has not been considered to be a virulence factor, the prediction of NLS motifs from the full sequenced genome of bacteria extends our knowledge of functional genomics to understand subcellular targeting of bacterial proteins.

Evidence for the existence of a restriction-modification system common to several species of the family Vibrionaceae.

A broad-host-range vibriophage KVP40 originally isolated on Vibrio parahaemolyticus 1010 was restricted and modified by strains of at least five Vibrio and one Photobacterium species. 1010 was a non-restricting host. An anti-restriction mutant KVP40 aar1 was isolated after propagating the phage on a restricting host, V. anguillarum VIB36. KVP40 aar1 grown on either 1010 or VIB36, as well as the parental phage grown on VIB36, showed much higher efficiencies of plating on all the restricting hosts as compared with the parental phage grown on 1010, indicating that these restricting hosts probably share a common restriction-modification system active in vivo on KVP40.

A general method to optimize the amount of enzyme in restriction and DNA modification reactions using the beta galactosidase blue-white plaques assay.

We propose a simple and economical method for assaying the activity of restriction and other modifying enzymes. The method involves assaying the use of the blue and white colored phenotypes of bacterial colonies obtained by digesting the polylinker sequence of M13 bacteriophage vectors followed by transformation in appropriate strains on X-gal/IPTG plates. In conjunction with restriction enzymes and DNA ligases, the method can evaluate polymerase activity and can be applied to test 3'...5' exonuclease activities such as that of T4 DNA polymerase, without having to use expensive radioisotopes. We describe its application in the assessment of restriction enzymes, DNA ligase and DNA polymerase activities.