We have constructed hybrid plasmids pColD and pIbpA in which transcription of reporter genes luxCDABE from Photohabdus luminescens occurred from induced promoters: SOS-promoter of gene cdaA (pColD-CA23) and heat shock gene ibpA (E. coli MG1655 genome), respectively. Main parameters were measured: threshold concentration of an inducer and maximal response and minimal time of response for E. coli lux-biosensors bearing hybrid plasmids pColD and pRecA (SOS-response); pIbpA and pGrpe (heat shock) and pKatG and pSoxS (oxidative stress). Mitomicyn C, cis-diaminedichloroplatinum (SOS-response), ethanol and pentachlorophenol (heat shock), and hydrogen peroxide and methyl viologen (oxidative stress) were used as luminescence inducers. It was shown that the SOS-biosensor pColD is superior in main characteristics to the earlier constructed biosensor pRecA, and the heat biosensor pIbpA is more efficient than the earlier constructed pGrpE. Lux-biosensors (pKatG) and (pSoxS) are high sensitive to hydrogen peroxide and methyl viologen, respectively.

A broad spectrum of volatile organic compounds’ (VOCs’) biological activities has attracted significant scientific interest, but their mechanisms of action remain little understood. The mechanism of action of two VOCs—the cyclic monoterpenes (−)-limonene and (+)-α-pinene—on bacteria was studied in this work. We used genetically engineered Escherichia coli bioluminescent strains harboring stress-responsive promoters (responsive to oxidative stress, DNA damage, SOS response, protein damage, heatshock, membrane damage) fused to the luxCDABE genes of Photorhabdus luminescens. We showed that (−)-limonene induces the PkatG and PsoxS promoters due to the formation of reactive oxygen species and, as a result, causes damage to DNA (SOSresponse), proteins (heat shock), and membrane (increases its permeability). The experimental data indicate that the action of (−)-limonene at high concentrations and prolonged incubation time makes degrading processes in cells irreversible. The effect of (+)-α-pinene is much weaker: it induces only heat shock in the bacteria. Moreover, we showed for the first time that (−)-limonene completely inhibits the DnaKJE–ClpB bichaperone-dependent refolding of heat-inactivated bacterial luciferase in both E. coli wild type and mutant ΔibpB strains. (+)-α-Pinene partially inhibits refolding only in ΔibpB mutant strain.

It is shown for the first time for the Enterobacteriaceae family that a gene encoding L-methionine γ-lyase (MGL) is present in the genome of Citrobacter freundii. Homogeneous enzyme has been purified from C. freundii cells and its N-terminal sequence has been determined. The hybrid plasmid pUCmgl obtained from the C. freundii genomic library contains an EcoRI insert of about 3000 bp, which ensures the appearance of MGL activity when expressed in Escherichia coli TG1 cells. The nucleotide sequence of the EcoRI fragment contains two open reading frames. The first frame (the megL gene) encodes a protein of 398 amino acid residues that has sequence homology with MGLs from different sources. The second frame encodes a protein with sequence homology with proteins belonging to the family of permeases. To overexpress the megL gene it was cloned into pET-15b vector. Recombinant enzyme has been purified and its kinetic parameters have been determined. It is demonstrated that a presence of a hybrid plasmid pUCmgl, containing the megL gene in the E. coli K12 cells, leads to a decrease in efficiency of EcoKI-restriction. It seems likely that decomposition of L-methionine under the action of MGL leads to a decrease in the intracellular content of S-adenosylmethionine. Expression of the megL gene in the C. freundii genome occurs only upon induction by a significant amount of L-methionine.

Anti-restriction proteins ArdA and Ocr are specific inhibitors of type I restriction-modification enzymes. The IncI1 transmissible plasmid ColIb-P9 ardA and bacteriophage T7 0.3( ocr) genes were...