[Anti-Restriction Activity of ArdB Protein against EcoAI Endonuclease].

ArdB proteins are known to inhibit the activity of the type I restriction-modification (RM-I) system, in particular EcoKI (IA family). The mechanism of ArdB's activity still remains unknown; the spectrum of targets inhibited has been poorly studied. In this work, it was shown that the presence of the ardB gene from the R64 plasmid could suppress the activity of EcoAI endonuclease (IB family) in Escherichia coli TG1 cells. Due to the absence of specificity of ArdB to a certain RM-I system (it inhibits both the IA- and IB-family), it can be assumed that the mechanism of the anti-restriction activity of this protein does not depend on the sequence DNA at the recognition site nor the structure of the restriction enzyme of the RM-I systems.

Antibacterial, cytotoxicity and physical properties of laser--silver doped hydroxyapatite layers.

Hydroxyapatite layers with silver doping from 0.06 at.% to 14 at.% were prepared by laser deposition. The films' physical properties such as morphology, composition, crystallinity, Young's modulus and microhardness were measured. Films were amorphous or polycrystalline in dependence on deposition temperature (from RT to 600 °C). Antibacterial properties were tested using Escherichia coli and Bacillus subtilis cells. The antibacterial efficacy changed with silver doping from 4% to 100%. Cytotoxicity was studied by a direct contact test. Depending on doping and crystallinity the films were either non-toxic or mildly toxic.

Post-translational modification(s) and cell distribution of Streptomyces aureofaciens translation elongation factor Tu overproduced in Escherichia coli.

We cloned EF-Tu from Streptomyces aureofaciens on a pET plasmid and overproduced it using the T7 RNA polymerase system in Escherichia coli. Streptomyces EF-Tu represented more than 40% of the total cell protein and was stored mostly in inclusion bodies formed apically at both ends of E. coli cells. Analysis of the inclusion bodies by transmission and scanning electron microscopy did not reveal any internal or surface ultrastructures. We developed the method for purification of S. aureofaciens EF-Tu from isolated inclusion bodies based on the ability of the protein to aggregate spontaneously. EF-Tu present in inclusion bodies was not active in GDP binding. Purified protein showed a similar charge heterogeneity as EF-Tu isolated from the mycelium of S. aureofaciens and all of the isoforms reacted with EF-Tu antibodies. All isoforms also reacted with monoclonal antibodies against O-phosphoserine and O-phosphothreonine.

Cellular localization of Type I restriction-modification enzymes is family dependent.

Cellular localization of Type I restriction-modification enzymes EcoKI, EcoAI, and EcoR124I-the most frequently studied representatives of IA, IB, and IC families-was analyzed by immunoblotting of subcellular fractions isolated from Escherichia coli strains harboring the corresponding hsd genes. EcoR124I shows characteristics similar to those of EcoKI. The complex enzymes are associated with the cytoplasmic membrane via DNA interaction as documented by the release of the Hsd subunits from the membrane into the soluble fraction following benzonase treatment. HsdR subunits of the membrane-bound enzymes EcoKI and EcoR124I are accessible, though to a different extent, at the external surface of cytoplasmic membrane as shown by trypsinization of intact spheroplasts. EcoAI strongly differs from EcoKI and EcoR124I, since neither benzonase nor trypsin affects its association with the cytoplasmic membrane. Possible reasons for such a different organization are discussed in relation of the control of the restriction-modification activities in vivo.

Identification of the EcoKI and EcoR124I Type I restriction--modification enzyme subunits by non-equilibrium pH gradient two-dimensional gel electrophoresis.

Effectively optimized and reproducible procedure for monitoring the composition of type I restriction-modification endonucleases EcoKI and EcoR124I by non-equilibrium pH gradient two-dimensional (2-D) gel electrophoresis is described. Three subunits of the enzyme complex, which widely differ from one another in their isoelectric points and molar mass, were identified in crude cell extracts of E. coli. For the first time all three subunits of both EcoKI and EcoR124I were detected as distinct spots on a single 2-D gel. A sensitive immunoblotting procedure was suggested suitable for routine use in determining the identity of individual subunits. Potential application of this method for detailed studies of regulation of the function and stoichiometry of the enzyme complexes is discussed.

A novel mutant of the type I restriction-modification enzyme EcoR124I is altered at a key stage of the subunit assembly pathway.

The HsdS subunit of a type I restriction-modification (R-M) system plays an essential role in the activity of both the modification methylase and the restriction endonuclease. This subunit is responsible for DNA binding, but also contains conserved amino acid sequences responsible for protein-protein interactions. The most important protein-protein interactions are those between the HsdS subunit and the HsdM (methylation) subunit that result in assembly of an independent methylase (MTase) of stoichiometry M(2)S(1). Here, we analysed the impact on the restriction and modification activities of the change Trp(212)-->Arg in the distal border of the central conserved region of the EcoR124I HsdS subunit. We demonstrate that this point mutation significantly influences the ability of the mutant HsdS subunit to assemble with the HsdM subunit to produce a functional MTase. As a consequence of this, the mutant MTase has drastically reduced DNA binding, which is restored only when the HsdR (restriction) subunit binds with the MTase. Therefore, HsdR acts as a chaperon allowing not only binding of the enzyme to DNA, but also restoring the methylation activity and, at sufficiently high concentrations in vitro of HsdR, restoring restriction activity.

Localization of the type I restriction-modification enzyme EcoKI in the bacterial cell.

To localise the type I restriction-modification (R-M) enzyme EcoKI within the bacterial cell, the Hsd subunits present in subcellular fractions were analysed using immunoblotting techniques. The endonuclease (ENase) as well as the methylase (MTase) were found to be associated with the cytoplasmic membrane. HsdR and HsdM subunits produced individually were soluble, cytoplasmic polypeptides and only became membrane-associated when coproduced with the insoluble HsdS subunit. The release of enzyme from the membrane fraction following benzonase treatment indicated a role for DNA in this interaction. Trypsinization of spheroplasts revealed that the HsdR subunit in the assembled ENase was accessible to protease, while HsdM and HsdS, in both ENase and MTase complexes, were fully protected against digestion. We postulate that the R-M enzyme EcoKI is associated with the cytoplasmic membrane in a manner that allows access of HsdR to the periplasmic space, while the MTase components are localised on the inner side of the plasma membrane.

Two temperature-sensitive mutations in the DNA binding subunit of EcoKI with differing properties.

Two temperature-sensitive mutations in the hsdS gene, which encodes the DNA specificity subunit of the type IA restriction-modification system EcoKI, designated Sts1 (Ser(340)Phe) and Sts2 (Ala(204)Thr) had a different impact on restriction-modification functions in vitro and in vivo. The enzyme activities of the Sts1 mutant were temperature-sensitive in vitro and were reduced even at 30 degrees C (permissive temperature). Gel retardation assays revealed that the Sts1 mutant had significantly decreased DNA binding, which was temperature-sensitive. In contrast the Sts2 mutant did not show differences from the wild-type enzyme even at 42 degrees C. Unlike the HsdSts1 subunit, the HsdSts2 subunit was not able to compete with the wild-type subunit in assembly of the restriction enzyme in vivo, suggesting that the Sts2 mutation affects subunit assembly. Thus, it appears that these two mutations map two important regions in HsdS subunit responsible for DNA-protein and protein-protein interactions, respectively.

The effect of recA mutation on the expression of EcoKI and EcoR124I hsd genes cloned in a multicopy plasmid.

Type I restriction-modification (R-M) endonucleases are composed of three subunits--HsdR, required for restriction, and HsdM and HsdS which can produce a separate DNA methyltransferase. The HsdS subunit is required for DNA recognition. In this paper we describe the effect of cloned EcoKI and EcoR124I hsd genes on the resulting R-M phenotype. The variability in the expression of the wild type (wt) restriction phenotype after cloning of the wt hsd genes in a multicopy plasmid in Escherichia coli recA+ background suggests that the increased production of the restriction endonuclease from pBR322 is detrimental to the cell and this leads to the deletion of the cloned hsd genes from the hybrid plasmid and/or inactivation of the enzyme. The effect of a mutation in E. coli recA gene on the expression of R-M phenotype is described and discussed in relation to the role of the cell surface and the localization of the restriction endonuclease in the cell.

Isolation of a non-classical mutant of the DNA recognition subunit of the type I restriction endonuclease R.EcoR124I.

We have used deletion mutagenesis and PCR-based misincorporation mutagenesis to produce a collection of mutations in the central conserved region of the DNA binding subunit of the type IC restriction endonuclease EcoR124I. It has been proposed that this domain is involved in protein-protein interactions during the assembly of the endonuclease. While a large percentage of these mutations gave a classical Res- Mod- phenotype, one mutant was isolated with a nonclassical Res- Mod+ phenotype. The loss of restriction activity, but retention of the ability to modify indicates that this mutation cannot affect DNA binding and must alter the assembly of the endonuclease in such a way as to prevent DNA cleavage but allow methylation. This mutant resulted from a single amino acid change Trp212-->Arg. The location of the single amino acid change is at the border of the central conserved region and the second target recognition domain (TRD2) and suggests that this region is extremely important for the assembly of the methylase with the HsdR subunit into a functional restriction endonuclease.

Restriction endonucleases R.EcoKI and R.EcoR124I are probably located in different environments within the bacterial cell.

We describe the phenomenon of a transient state of R124I restriction deficiency after long-term storage of the E. coli[pCP1005] strain at 4 degrees C, or after growth of the culture in synthetic M9 medium with the nonmutagenic solvent dimethyl sulfoxide. The unusual high reversion from the R+ 124 to the R- 124 phenotype was observed only in E. coli strain transformed with the high-copy number plasmid pCP1005 carrying EcoR124I hsdR, M and S genes cloned, but not with strains carrying the natural conjugative plasmid R124. The effect of both treatments on the expression of EcoR124I phenotype in relation to the possible location of R.EcoR124I restriction endonuclease in E. coli is discussed.

Overproduction of the Hsd subunits leads to the loss of temperature-sensitive restriction and modification phenotype.

The genes hsdM and hsdS for M. EcoKI modification methyltransferase and the complete set of hsdR, hsdM and hsdS genes coding for R. EcoKI restriction endonuclease, both with and without a temperature-sensitive (ts) mutation in hsdS gene, were cloned in pBR322 plasmid and introduced into E. coli C (a strain without a natural restriction-modification (R-M) system). The strains producing only the methyltransferase, or together with the endonuclease, were thus obtained. The hsdSts-1 mutation, mapped previously in the distal variable region of the hsdS gene with C1 245-T transition has no effect on the R-M phenotype expressed from cloned genes in bacteria grown at 42 degrees C. In clones transformed with the whole hsd region an alleviation of R-M functions was observed immediately after the transformation, but after subculture the transformants expressed the wild-type R-M phenotype irrespective of whether the wild-type or the mutant hsdS allele was present in the hybrid plasmid. Simultaneous overproduction of HsdS and HsdM subunits impairs the ts effect of the hsdSts-1 mutation on restriction and modification.

Cloning, production and characterisation of wild type and mutant forms of the R.EcoK endonucleases.

The hsdR, hsdM and hsdS genes coding for R.EcoK restriction endonuclease, both with and without a temperature sensitive mutation (ts-1) in the hsdS gene, were cloned in pBR322 plasmid and introduced into E.coli C3-6. The presence of the hsdSts-1 mutation has no effect on the R-M phenotype of this construct in bacteria grown at 42 degrees C. However, DNA sequencing indicates that the mutation is still present on the pBR322-hsdts-1 operon. The putative temperature-sensitive endonuclease was purified from bacteria carrying this plasmid and the ability to cleave and methylate plasmid DNA was investigated. The mutant endonuclease was found to show temperature-sensitivity for restriction. Modification was dramatically reduced at both the permissive and non-permissive temperatures. The wild type enzyme was found to cleave circular DNA in a manner which strongly suggests that only one endonuclease molecule is required per cleavage event. Circular and linear DNA appear to be cleaved using different mechanisms, and cleavage of linear DNA may require a second endonuclease molecule. The subunit composition of the purified endonucleases was investigated and compared to the level of subunit production in minicells. There is no evidence that HsdR is prevented from assembling with HsdM and HsdSts-1 to produce the mutant endonuclease. The data also suggests that the level of HsdR subunit may be limiting within the cell. We suggest that an excess of HsdM and HsdS may produce the methylase in vivo and that assembly of the endonuclease may be dependent upon the prior production of this methylase.

Mutation in the specificity polypeptide of the type I restriction endonuclease R.EcoK that affects subunit assembly.

We describe the isolation and characterization of a temperature-sensitive mutation within the hsdS gene of the type I restriction and modification system EcoK. This mutation appears to affect the ability of the HsdR subunit to interact with the HsdS subunit when forming an active endonuclease. We discuss the possibility that this mutant, together with another mutation described previously, may define a discontinuous domain, involved in protein-protein interactions, within the HsdS polypeptide.

A mutation that converts serine340 of the HsdSK polypeptide to phenylalanine and its effects on restriction and modification in Escherichia coli K-12.

A hybrid hsdS gene, encoding the HsdSts + d polypeptide, was constructed by joining the proximal region of the wild-type (wt) hsdS sequence with the distal region of the hsdSts + d sequence, at the hsdS BglII site. The hybrid hsdS-Sts + d gene exerts a trans-dominant effect on restriction and modification, which points to the location of the temperature-sensitive (ts) trans-dominant (+ d) mutation in the gene hsdSts + d distal region. Sequencing of the region downstream from the HindIII target in the Escherichia coli K-12 hsdSts + d mutant was carried out. It is identical to the wt hsdS sequence (GenBank/EMBL accession number ECHSDK LV00288), except for a single base-pair transition C1245----T. The results obtained support the idea that the trans-dominant effect of the ts mutation described earlier is related to the single base-pair transition in the nonhomologous region of the hsdSts + d sequence.

The location of a temperature-sensitive trans-dominant mutation and its effect on restriction and modification in Escherichia coli K12.

An Escherichia coli K12 chromosomal EcoRI-BamHI fragment containing a mutant hsdS locus was cloned into plasmid pBR322. The mcrB gene, closely linked to hsdS, was used for selection of clones with the inserted fragment using T4 alpha gt57 beta gt14 and lambda vir. PvuII phages; the phage DNAs contain methylated cytosines and hence can be used to demonstrate McrB restriction. For the efficient expression of the hsdS gene, a BglII fragment of phage lambda carrying the pR promoter was inserted into the BamHI site of the hybrid plasmid. Under these conditions a trans-dominant effect of the hsdXts+d mutation on restriction and modification was detected. Inactivation of the hsdS gene by the insertion of the lambda phage BglII fragment into the BglII site within this gene resulted in the disappearance of the trans-dominant effect. When the cloned BamHI-EcoRI fragment was shortened by HpaI and EcoRI restriction enzymes, the trans-dominant effect was fully expressed. The results indicate that the Xts+d mutation is located in the hsdS gene. The effect of gene dosage of the HsdS subunit on the expression of Xts+d mutation was studied. The results of complementation experiments, using F'-merodiploids or plasmid pBR322 with an inserted Xts+d mutation, support the idea that the HsdSts+d product competes with the wild-type HsdS product, and has a quantitatively different effect on restriction and modification.

[Transfer of hybrid plasmids pRP1.2::MINI-Mu into Agrobacterium and Rhizobium cells]. / Perenos gibridnykh plazmid pRP1.2::MINI-Mu v kletki Agrobacterium i Rhizobium.

The study is devoted to determination of bacteriophage Mu genome regions responsible for transfer limitation and instability of the plasmids in cells of strains of practically important microorganisms. With this aim in view, we determined the frequency of transfer into Agrobacterium tumefaciens and Rhizobium meliloti cells of plasmids with mini-Mu phages carrying previously constructed deletions of various lengths. Sharp decrease has been noted in the frequency of transfer into A. tumefaciens strain PG2592 of all the plasmids used, as compared with the initial plasmid pRP1.2 with no dependence on the availability of mini-Mu killing functions. This gives evidence that deletions in the mini-Mu utilized do not include the sites affected by the recipient' restriction system. As regards R. meliloti L5-30-M27, it appeared that the transfer of pRM30 plasmid carrying mini-Mu 5 with conserved killing functions (the ability for autonomous transposition) is of the same frequency as the transfer of pRP1.2. In this mini-Mu, the region between the extreme HpAI sites in the right end is missing, this region being probably responsible for such low frequency of transfer into Rhizobium cells of Mu-containing plasmid.

Mini-Mu transposition of bacterial genes on the transmissible plasmid.

Using the pRM30 plasmid, an Aps deletion derivative of broad host range plasmid RP4 with integrated new miniMu 5 (11 kb), we followed the transfer of Escherichia coli chromosomal genes to the recipient strain. The miniMu 5-mediated transposition of chromosomal genes occurs onto the plasmid with integrated miniMu 5 rather than onto the "recipient" plasmid pNH602. The plasmid DNA in recipient cells was detected by electrophoresis. One of the acquired hybrid plasmids pTB2 was analyzed genetically and by restriction endodeoxyribonuclease digestion. A structure consisting of miniMu-chromosomal segment-miniMu as a product of Mu-mediated transposition was detected.

DNA restriction and modification in Escherichia coli: functional analysis of the role of the dnaC(D) gene product.

Escherichia coli strain PC-7 carries two independent temperature-sensitive mutations, one affecting the restriction and modification (R-M) phenotype and the other the DnaC(D) phenotype. The results of complementation and P1 transduction analysis of the mutation affecting the R-M phenotype implicate a fourth gene, designated hsdX, located close to the hsd three-gene complex. The properties of merodiploids constructed between appropriate recipients and F' elements with different mutations in hsdS, hsdR and hsdM genes might indicate that in strain PC-7 the temperature-sensitive products, determined by hsdR and hsdSK cistrons, are synthesized. The role of the temperature-sensitive dnaC(D) gene product in the formation of the restriction endonuclease was studied and no direct relation was found between the DnaC(D) and R-M phenotypes.