A type III-like restriction endonuclease functions as a major barrier to horizontal gene transfer in clinical Staphylococcus aureus strains.

Staphylococcus aureus is an versatile pathogen that can cause life-threatening infections. Depending on the clinical setting, up to 50% of S. aureus infections are caused by methicillin-resistant strains (MRSA) that in most cases are resistant to many other antibiotics, making treatment difficult. The emergence of community-acquired MRSA drastically changed the picture by increasing the risk of MRSA infections. Horizontal transfer of genes encoding for antibiotic resistance or virulence factors is a major concern of multidrug-resistant S. aureus infections and epidemiology. We identified and characterized a type III-like restriction system present in clinical S. aureus strains that prevents transformation with DNA from other bacterial species. Interestingly, our analysis revealed that some clinical MRSA strains are deficient in this restriction system, and thus are hypersusceptible to the horizontal transfer of DNA from other species, such as Escherichia coli, and could easily acquire a vancomycin-resistance gene from enterococci. Inactivation of this restriction system dramatically increases the transformation efficiency of clinical S. aureus strains, opening the field of molecular genetic manipulation of these strains using DNA of exogenous origin.

The structure of the KlcA and ArdB proteins reveals a novel fold and antirestriction activity against Type I DNA restriction systems in vivo but not in vitro.

Plasmids, conjugative transposons and phage frequently encode anti-restriction proteins to enhance their chances of entering a new bacterial host that is highly likely to contain a Type I DNA restriction and modification (RM) system. The RM system usually destroys the invading DNA. Some of the anti-restriction proteins are DNA mimics and bind to the RM enzyme to prevent it binding to DNA. In this article, we characterize ArdB anti-restriction proteins and their close homologues, the KlcA proteins from a range of mobile genetic elements; including an ArdB encoded on a pathogenicity island from uropathogenic Escherichia coli and a KlcA from an IncP-1b plasmid, pBP136 isolated from Bordetella pertussis. We show that all the ArdB and KlcA act as anti-restriction proteins and inhibit the four main families of Type I RM systems in vivo, but fail to block the restriction endonuclease activity of the archetypal Type I RM enzyme, EcoKI, in vitro indicating that the action of ArdB is indirect and very different from that of the DNA mimics. We also present the structure determined by NMR spectroscopy of the pBP136 KlcA protein. The structure shows a novel protein fold and it is clearly not a DNA structural mimic.

Extensive DNA mimicry by the ArdA anti-restriction protein and its role in the spread of antibiotic resistance.

The ardA gene, found in many prokaryotes including important pathogenic species, allows associated mobile genetic elements to evade the ubiquitous Type I DNA restriction systems and thereby assist the spread of resistance genes in bacterial populations. As such, ardA contributes to a major healthcare problem. We have solved the structure of the ArdA protein from the conjugative transposon Tn916 and find that it has a novel extremely elongated curved cylindrical structure with defined helical grooves. The high density of aspartate and glutamate residues on the surface follow a helical pattern and the whole protein mimics a 42-base pair stretch of B-form DNA making ArdA by far the largest DNA mimic known. Each monomer of this dimeric structure comprises three alpha-beta domains, each with a different fold. These domains have the same fold as previously determined proteins possessing entirely different functions. This DNA mimicry explains how ArdA can bind and inhibit the Type I restriction enzymes and we demonstrate that 6 different ardA from pathogenic bacteria can function in Escherichia coli hosting a range of different Type I restriction systems.

[Antirestriction proteins ardA and Ocr as effective inhibitors of the type I restriction-modification enzymes].

Genes encoding antirestriction proteins (antirestrictases, inasmuch as the antirestriction proteins inhibit the activity of restriction-modification systems, but have no proper enzyme activity, the name antirestrictase is only tentative) are included in the composition of conjugative plasmids (genes ardABC) and some bacteriophages (genes ocr and darA). Antirestriction proteins inhibit of the type I restriction-modification enzymes and thus protect unmodified DNA of plasmids and bacteriophages from degradation. Antirestriction proteins belong to the "protein mimicry of DNA" family: the spatial structure is like the B-form of DNA, and therefore the antirestriction proteins operated on the principle of concurrent inhibition replacing DNA in the complex with the restriction-modification enzyme. Based on the prepared in vitro mutant forms of ArdA and Ocr, and also on natural proteins ArdA selectively inhibiting restriction activity of the type I enzymes, but not affecting their methylase activity, we have developed a model of complex formation between the antirestriction proteins and the restriction-modification enzymes R2M2S. Antirestriction proteins are capable of competing displacement of the DNA strand from two sites which are situated as follows: 1) in S-subunit (enzyme contact with the specific DNA site) and 2) in R-subunit (through this unit translocation of the DNA strand occurs followed by its degradation). Analysis of estriction and antimodification activities of proteins ArdA and Ocr depending on the expression level of genes ardA and ocr was performed (the cloning of the genes was done under strictly regulated promoter).

[Antirestriction and antimodification activities of the T7 Ocr protein: effect of mutations in interface].

Antirestriction protein Ocr (bacteriophage T7) is specific inhibitor of the type I restriction-modification enzymes. The bacteriophage T7 0.3 (ocr) gene is cloned in pUC18 vector. It was shown that T7 Ocr protein inhibits both restriction and modification activities of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. The mutation form of Ocr-Ocr F53D A57E, which inhibits only the restriction activity of EcoKI-enzyme, was constructed. The T7 0.3 (ocr) and the Photorhabdus luminescens luxCDABE genes were cloned in pZ-series vectors with the P(ltet0-1) promoter which is tightly repressible by the TetR repressor. Controlling the expression of the lux-genes encoding bacterial luciferase demonstrates that the P(ltet0-1) promoter can be regulated over and up to 5000 fold range by supplying anhydrotetracycline (aTc) to the E. coli MG1655Z1 tetR+ cells. It was determined the dependence of the effectiveness of the antirestriction activity of the Ocr and Ocr F53D A57E proteins on the intracellular concentration. It was shown that the values of the dissociation constants K(d) for Ocr and Ocr F53D A57E proteins with EcoKI enzyme differ in 1000 times: Kd (Ocr) = 10(-10) M, K(d) (Ocr F53D A57E) = 10(-7) M.

[Antirestriction and antimodification activities of the ArdA protein encoded by the IncI1 transmissive plasmids R-64 and ColIb-P9].

Proteins of the Ard family are specific inhibitors of type I restriction-modification enzymes. The ArdA of R64 is highly homologous to ColIb-P9 ArdA, differing only by four amino acid residues of the overall 166. However, unlike ColIb-P9 ArdA, which inhibits both the endonuclease and the methylase activities of EcoKI, the R64 ArdA protein inhibits only the endonuclease activity of this enzyme. The mutant forms of R64 ArdA--A29T, S43A, and Y75W, capable of partially reversing the protein to ColIb-P9 ArdA form--were produced by directed mutagenesis. It was demonstrated that only Y75W mutation of these three variants essentially influenced the functional activity of ArdA: the antimodification activity was restored to approximately 90-99%. It is assumed that R64 ArdA inhibits formation of the complex between unmodified DNA and the R subunit of the type I restriction-modification enzyme EcoKI (R2M2S), which translocates and cleaves DNA. ColIb-P9 ArdA protein is capable of forming the DNA complex not only with the R subunit, but also with the S subunit, which contacts sK site (containing modified adenine residues) in DNA. ArdA bound to the specific sK site inhibits concurrently the endonuclease and methylase activities of EcoKI (R2M2S), while ArdA bound to the nonspecific site in the R subunit blocks only its endonuclease activity.

[Antirestriction activity of the IncI1 transmissive plasmid R64 ArdA protein]. / Antirestriktsionnaia aktivnost' belka ArdA, kodiruemogo transmissivnoi plazmidoi R64.

The transmissive plasmid IncI1 R64 contains the ardA gene encoding the ArdA antirestriction protein. The R64 ardA gene locating in the leading region of plasmid R64 has been cloned and their sequence has been determined. Antirestriction proteins belonging to the Ard family are specific inhibitors of type I restriction-modification enzymes. The IncI1 ColIb-P9 and R64 are closely related plasmids, and the latter specifies an ArdA homologue that is predicted to be 97.6% (162 residues from 166) identical at the amino acid sequence level with the ColIb = P9 equivalent. However, the R64 ArdA selectively inhibits the restriction activity of EcoKi enzyme leaving significant levels of modification activity under conditions in which restriction was almost completely prevented. The ColIb-P9 ArdA inhibits restriction endonuclease and methyltransferase activities simultaneously. It is hypothesized that the ArdA protein forms two complexes with the type I restriction-modification enzyme (R2M2S): (1) with a specific region in the S subunit involved in contact with the sK site in DNA; and (2) with nonspecific region in the R subunit involved in DNA translocation and degradation by restriction endonuclease. The association of the ColIb-P9 ArdA with the specific region inhibits restriction endonuclease and methyltransferase activities simultaneously, whereas the association of the R64 ArdA with a nonspecific region inhibits only restriction endonuclease activity of the R2M2S enzyme.

Increasing DNA transfer efficiency by temporary inactivation of host restriction.

E. coli and Salmonella typhimurium are widely used bacterial hosts for genetic manipulation of DNA from prokaryotes and eukaryotes. Introduction of foreign DNA by electroporation or transduction into E. coli and Salmonella is limited by host restriction of incoming DNA by the recipient cells. Here, we describe a simple method that temporarily inactivates host restriction, allowing high-frequency DNA transfer. This technique might be readily applied to a wide range of bacteria to increase DNA transfer between strains and species.

IncI1 plasmid R64 encodes the ArsR protein that alleviates type I restriction.

The host-controlled EcoK restriction of unmodified phage lambda was five-fold alleviated in the wild-type Escherichia coli strain K12 carrying the R64 plasmid of the incompatibility group I1. The relevant gene was mapped between the origin of vegetative replication (rep, oriV) and the tet(r) gene about 60 kbp downstream from the origin of transfer, oriT. We cloned this gene inside the 613 bp long EcoRI-PstI fragment and sequenced it. Only one 351 bp long open reading frame (ORF) starting at 124 bp from the beginning of the insert was found in the sequence. Computer search in the current databases revealed that the putative protein is identical to the ArsR protein specified by the IncFI plasmid R773. ArsR is a repressor of the arsenical resistance (ars) operon, arsRDABC. There are no arsABC genes in the R64 plasmid since plasmid R64- (or pSR8)-mediated resistance of E. coli K12 cells to the arsenicals arsenate and arsenite was not detected. The gene arsR and the antirestriction genes ard (ardA and ardB) are non-homologous. However, comparison of the deduced amino acid sequence of ArsR with the ArdA and ArdB sequences revealed only one small region of similarity, a 9 amino acid motif found in different antirestriction proteins that is hypothesized to be an interaction site for antirestriction proteins with restriction endonucleases.

[Attenuation of type I restriction in Escherichia coli: effect of the ard gene in UV-irradiated cells]. / Oslablenie restriktsii I tipa u Escherichia coli: deistvie gena ard v UF-obluchennykh kletakh.

The effect of conjugative plasmids ColIb-P9 (incI1) and pKM 101 (incN), containing the active ard gene, on the efficiency of EcoK restriction of nonmodified phage lambda .0 in UV-irradiated Escherichia coli cells was studied. ard-Dependent antirestriction enzyme activity was shown to decrease in UV-irradiated cells. The efficiency of action of the ard plasmid gene on lambda .0 was also shown not to depend on cell helicases RecBCD and UvrD, in contrast to the UV-induced alleviation of EcoK restriction (SOS alleviation).

[The ard gene, coding for a type I restriction inhibitor, is present in plasmids of FII, B/O, and K-groups of incompatibility]. / Gen ard, kodiruiushchii ingibitor restriktsii I tipa, prisutstvuet v kon''iugativnykh plazmidakh FII, B/O i K-grupp nesovmestimosti.

The effect of conjugative plasmids of various incompatibility groups of the enterobacteria family on the activity of the cell restriction-modification system of type I (EcoK) was studied. Twenty-two conjugative plasmids of 15 incompatibility groups were tested. In addition to plasmids of the incI1 and incN groups studied earlier, conjugative plasmids of the incFII, incB/O, and incK groups were also shown to be able to weaken the action of type I restriction enzymes upon nonmodified DNA (Ard phenotype). A hybridization analysis of all the plasmid DNAs studied, using ard gene DNA sequences from the ColIb-P9 (incI1) plasmid as a probe, was performed. The ard locus of the R100 (incFII) plasmid was cloned in the pBR322 and pACYC184 vectors. The ard gene was located 2.5 kb from the oriT site in the leading region on the R100 conjugative plasmid.

Cycloheximide inhibits S-14 gene transcription and abolishes DNase I hypersensitive S-14 sites in the livers of euthyroid but not hypothyroid rats.

Earlier studies from our laboratory have demonstrated that cycloheximide administration to hypothyroid rats inhibited the induction of the hepatic mRNA-S14 by T3. These results suggested a role of short-lived proteins in the hormonal regulation of this gene. To define the possible mechanism of the cycloheximide effect, we examined the influence of cycloheximide on the in vitro transcription rate of the gene and its chromatin structure. Forty-five minutes after injection of cycloheximide to euthyroid rats, the in vitro transcriptional rate fell by 60% and this effect persisted for 4 h. In the same euthyroid rats, cycloheximide caused the disappearance of all four DNase I-hypersensitive sites situated in the 5'-flanking region of the gene. However, cycloheximide given to hypothyroid rats affected neither the basal transcription rate nor the chromatin structure. When cycloheximide was administered 30 min after an acute injection of T3 (200 micrograms/100 g BW) to hypothyroid animals, it completely blocked the hormone induction of the transcriptional rate. These results suggest that one or more labile proteins are required for maintenance of S14 chromatin structure in a configuration which permits hormonal regulation of gene expression. The ability of cycloheximide to block mRNA-S14 induction by T3 appears to be mediated at least in part by an inhibition of T3-stimulated transcription.