ABSTRACT
Conjugative plasmids and conjugative transposons contain the genes, which products specifically inhibit the type I restriction--modification systems. Here is shown that non-conjugative transposons Tn2l, Tn5053, Tn5045, Tn501, Tn402 partially inhibit the restriction activity of the type I restriction-modification endonuclease EcoKI (R2M2S1) in Escherichia coli cells K12 (the phenomenon of restriction alleviation, RA). Antirestriction activity of the transposons is determined by the MerR and ArdD proteins. Antirestriction activity of transposons is absent in mutants E. coli K12 clpX and clpP and is decreased in mutants E. coli K12 recA, recBC and dnaQ (mutD). Induction of the RA in response to the MerR and ArdD activities is consistent with the production of unmodified target sequences following DNA repair and DNA synthesis associated with recombination repair or replication errors. RA effect is determined by the ClpXP-dependent degradation of the endonuclease activity R subunit of EcoKI (R2M2S1).
Subject(s)
DNA Restriction Enzymes/biosynthesis , DNA Transposable Elements/physiology , DNA, Bacterial/biosynthesis , Escherichia coli K12/metabolism , Escherichia coli Proteins/biosynthesis , Proteolysis , Site-Specific DNA-Methyltransferase (Adenine-Specific)/biosynthesis , DNA Repair/physiology , DNA Replication/physiology , DNA Restriction Enzymes/genetics , DNA, Bacterial/genetics , Escherichia coli K12/genetics , Escherichia coli Proteins/genetics , Site-Specific DNA-Methyltransferase (Adenine-Specific)/geneticsABSTRACT
Antirestriction proteins ArdA and ArdB are specific inhibitors of the type I restriction-modification enzymes. The transmissible plasmid R64 ardA and yfeB (ardB) genes were cloned in pUC18 and pZE21 vectors. It was shown that the R64 ArdA and ArdB proteins inhibit only restriction activity of the type I restriction-modification enzyme (EcoKI) in Escherichia coli K12 cells. The dependence of the effectiveness of the antirestriction activity of the ArdA and ArdB proteins on the intracellular concentration was determined. Antirestriction activity of ArdB is independent from the ClpXP protease. Transcription of yfeB (ardB) gene in R64 plasmid is realized from the yfeA promoter.
Subject(s)
DNA Restriction-Modification Enzymes/metabolism , Escherichia coli K12/metabolism , Escherichia coli Proteins/biosynthesis , Plasmids/metabolism , Repressor Proteins/biosynthesis , DNA Restriction-Modification Enzymes/genetics , Endopeptidase Clp/genetics , Endopeptidase Clp/metabolism , Escherichia coli K12/genetics , Escherichia coli Proteins/genetics , Escherichia coli Proteins/metabolism , Plasmids/genetics , Promoter Regions, Genetic/physiology , Repressor Proteins/genetics , Transcription, Genetic/physiologyABSTRACT
Luminescence intensity of recombinant Escherichia coli and Bacillus subtilis strains with cloned luxCD(AB)E genes of the natural luminescent microorganism Photobacterium leiognathi was studied under the influence of 30 individual samples of human blood serum of different component composition. A relationship was found between the level of residual bioluminescence and degree of the bactericidal effect. Moreover, the inhibition of E. coli lux+ luminescence was shown to be related to activity of the complement-lysozyme system. The reaction of B. subtilis lux+ primarily depended on the presence of ß-lysin in the blood serum. These data provide an experimental substantiation of a new method of differential analysis of humoral factors of nonspecific innate immunity with recombinant luminescent bacteria.
