Induction of SOS Response by Autoregulatory Factors of Microorganisms

Among examined microbial growth regulators of alkyl hydroxybenzene group (hexylresorcinol, methylresorcinol, and hydroxyethylphenol), only hexylresorcinol induces cellular SOS response, demonstrating a dose-dependent increase of the induction factor in the SOS chromotest with the Escherichia coli PQ37 strain. At the highest of used concentrations (100 g/ml), hydroxyethylphenol and nonalkylated resorcinol were shown to exert a weak toxic effect, reducing the activity of constitutive alkaline phosphatase, but did not induce SOS response. Nontoxic methylresorcinol did not induce genome damage, which can trigger SOS functions. It is concluded that substitutions in phenolic ring affect genotoxic activity of alkylresorcinols.     

Effect of chemical chaperones on properties of lysozyme and the reaction center protein from Rhodobacter sphaeroides

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and the functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. Selected chemical chaperones are strongly different by the structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described within the frames of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic nuclus) moieties. This fact provides additional regulation of functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in denaturation temperature T(d), and an increase in the amplitude of equilibrium fluctuation, which allows him to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in the case of methylresorcinol. Therefore, methylresorcinol does not effect the functional properties of the RC protein. It was concluded that various chaperones at one and the same concentration and chaperones at different concentrations form diverse 3D structures of proteins, which differ by dynamic and functional characteristics.     

Influence of chemical chaperones on the properties of lysozyme and the reaction center protein from <Emphasis Type="Italic">Rhodobacter sphaeroides</Emphasis>

The influence of three chemical chaperones: glycerol, 4-hexylresorcinol, and 5-methylresorcinol on the structure, equilibrium fluctuations, and functional activity of the hydrophilic enzyme lysozyme and the transmembrane reaction center (RC) protein from Rb. sphaeroides in a broad range of concentrations has been studied. The chosen chemical chaperones differ strongly in their structure and action on hydrophilic and membrane proteins. The influence of the chemical chaperones (except methylresorcinol) on the structure, dynamics, and functional properties of lysozyme and RC protein are well described in the framework of extended models of preferential hydration and preferential interaction of protein with a chemical chaperone. A molecule of hexylresorcinol consists of a hydrophobic (alkyl radical) and a hydrophilic (aromatic core) moieties; this provides for additional regulation of the functional activity of lysozyme and RC by hexylresorcinol. The influence of methylresorcinol on proteins differs from that of glycerol and hexylresorcinol. Methylresorcinol interacts with the surface of lysozyme directly, not via water hydrogen bonds. This leads to a decrease in the denaturation temperature and an increase in the amplitude of equilibrium fluctuations, allowing it to be a powerful activator. Methylresorcinol interacts with the membrane RC protein only by the condensation of hydration water, which is negligible in this case. Therefore, methylresorcinol does not affect the functional properties of the RC protein. It is concluded that different chaperones at the same concentration as well as one and the same chaperone at different concentrations produce protein 3D structures differing in dynamic and functional characteristics.     

The effect of anabiosis autoinducers on the bacterial genome

The mutagenic activity of chemical analogues of microbial anabiosis autoinducers (the autoregulatory d1 factors of cell differentiation), which act to inhibit cell proliferation, to enhance cell tolerance, and to induce the transition of cells to anabiotic state, was studied using the Ames test. In the range of concentrations studied (0.1 to 100 micrograms/ml), alkyl-substituted hydroxybenzenes (AHBs) differing in hydrophobicity, i.e., methylresorcinol (C1-AHB) and hexylresorcinol (C6-AHB), as well as unsubstituted resorcinol, showed different growth-inhibiting and mutagenic effects. C6-AHB was found to inhibit the growth of Salmonella typhimurium TA100 and to induce its mutagenesis at a rate of 1.8 revertants/nmol. C1-AHB taken at low concentrations not only failed to inhibit bacterial growth but even stimulated it and exerted an antimutagenic effect. Unsubstituted resorcinol virtually did not influence bacterial growth and showed weak mutagenic activity. The growth-inhibiting effect of elevated C6-AHB concentrations correlated with the degree of the transition of the original phenotype producing S-type colonies to a phenotype producing R-type colonies. The role of AHB homologues, as microbial autoregulators with mutagenic activity, in the regulation and correlation of two processes (the phenotypic dissociation of microbial populations and the formation of resting microbial forms) is discussed. The accumulation of AHBs in senescent microbial cultures may induce adaptive mutations, change the expression of genes, and promote the development of minor cell subpopulations (phenotypes), thus providing for the adaptation of these cultures to varying environmental conditions.     

The Effect of Anabiosis Autoinducers on the Bacterial Genome

The mutagenic activity of chemical analogues of microbial anabiosis autoinducers (the autoregulatory d₁ factors of cell differentiation), which act to inhibit cell proliferation, to enhance cell tolerance, and to induce the transition of cells to an anabiotic state, was studied using the Ames test. In the range of concentrations studied (0.1 to 100 g/ml), alkyl-substituted hydroxybenzenes (AHBs) differing in hydrophobicity, i.e., methylresorcinol (C₁-AHB) and hexylresorcinol (C₆-AHB), as well as unsubstituted resorcinol, showed different growth-inhibiting and mutagenic effects. C₆-AHB was found to inhibit the growth of Salmonella typhimurium TA100 and to induce its mutagenesis at a rate of 1.8 revertants/nmol. C₁-AHB taken at low concentrations not only failed to inhibit bacterial growth but even stimulated it and exerted an antimutagenic effect. Unsubstituted resorcinol virtually did not influence bacterial growth and showed weak mutagenic activity. The growth-inhibiting effect of elevated C₆-AHB concentrations correlated with the degree of the transition of the original phenotype producing S-type colonies to a phenotype producing R-type colonies. The role of AHB homologues, as microbial autoregulators with mutagenic activity, in the regulation and correlation of two processes: the phenotypic dissociation of microbial populations and the formation of resting microbial forms, is discussed. The accumulation of AHBs in senescent microbial cultures may induce adaptive mutations, change the expression of genes, and promote the development of minor cell subpopulations (phenotypes), thus providing for the adaptation of these cultures to varying environmental conditions.     

Long-term preservation of DNA in aqueous solutions in the presence of the chemical analogues of microbial autoregulators

The fact of long-term preservation of the physicochemical properties of DNA molecules in aqueous solutions in complexes with methylresorcinol, hexylresorcinol, and tyrosol, the chemical analogues of microbial autoregulators (d1 factors) from the group of alkylhydroxybenzenes (AOB), was established. Compared to the control variants of storage of aqueous DNA solutions, the AOB influence consisted in the sum of correlating effects: the prevention of DNA degradation (according to spectrophotometric parameters) and the preservation of its viscous characteristics and electrophoretic mobility. The initial DNA properties were preserved to the greatest degree in the presence of hexylresorcinol, the compound with the longest alkyl radical. Possible mechanisms of the protective action of alkylhydroxybenzenes in relation to DNA are discussed, namely, the prevention of its hydrolysis due to isolation from the aqueous environment and maintaining DNA stability in the dormant forms of microorganisms.     

Long-term preservation of DNA in aqueous solutions in the presence of the chemical analogues of microbial autoregulators

he fact of long-term preservation of the physicochemical properties of DNA molecules in aqueous solutions in complexes with methylresorcinol, hexylresorcinol, and tyrosol, the chemical analogues of microbial autoregulators (d₁ factors) from the group of alkylhydroxybenzenes (AOB), was established. Compared to the control variants of storage of aqueous DNA solutions, the AOB influence consisted in the sum of correlating effects: the prevention of DNA degradation (according to spectrophotometric parameters) and the preservation of its viscous characteristics and electrophoretic mobility. The initial DNA properties were preserved to the greatest degree in the presence of hexylresorcinol, the compound with the longest alkyl radical. Possible mechanisms of the protective action of alkylhydroxybenzenes in relation to DNA are discussed, namely, the prevention of its hydrolysis due to isolation from the aqueous environment and maintaining DNA stability in the dormant forms of microorganisms.     

IR spectroscopic research on the impact of chemical analogues of autoregulatory d1 factors of microorganisms on structural changes in DNA

Using IR spectroscopy, we investigated the impact of chemical analogues of autoregulatory d1 factors of microorganisms (methylresorcinol, hexylresorcinol, and tyrosol) on the conformational changes in DNA in films upon altering (decreasing) the relative humidity. We analyzed the appearance/disappearance of characteristic absorption bands of A and B DNA forms and determined D1080/D1224, the ratio between the band intensities of symmetrical and asymmetrical oscillations in their phosphate groups. The data obtained suggest the slowing down of the B-->A structural transition in DNA in the presence of methylresorcinol and its speeding up in the presence of tyrosol. We discuss the mechanisms of this phenomenon in relation to the chemical composition of d1 factors and their biological function.     

The effect of alkylhydroxybenzenes on the antigen-binding capacity of antibodies

The effect of the chemical analogues of microbial extracellular autoregulators belonging to alkylhydroxybenzenes (AHB), hexylresorcinol (HR), and methylresorcinol (MR), on the interactions between specific antibodies and the corresponding antigens was studied. Nonlinear dependency of the inhibition of binding of AHB-modified antibodies on the AHB chemical structures and concentrations was revealed by enzyme immunoassay. Hexylresorcinol was shown to decrease the antibody affinity and avidity indices and simultaneously increase the indices of nonspecific binding of AHB-modified antibodies to antigens, thereby promoting the formation of “false” antigen-antibody complexes. The nonspecificity of the influence of AHB on the antigenbinding capacity of antibodies is an important characteristic of these effects, which allows us to consider AHB as unique “superhaptenes”.     

Genotoxicity and genotoxic enhancing effect of tetrandrine in Salmonella typhimurium

Tetrandrine has been used for the treatment of silicosis in China. The potential genotoxic and carcinogenic hazards of this drug were studied using the Salmonella/histidine reversion assay and the SOS/Umu test. The results show that tetrandrine was weakly mutagenic to Salmonella typhimurium TA98 with metabolic activation and did not induce SOS response. However, tetrandrine increased the mutagenic activity of benzo[alpha]pyrene, trinitrofluorenone (TNF), 2-aminoanthracene (2AA), diesel emission particles, airborne particles, and cigarette smoke condensate by more than 100%; the activity of aflatoxin B1 and fried beef was increased by over 75%. It also increased the 2AA and TNF-induced SOS response by more than 300%. These results indicated that tetrandrine was a weak promutagen inducing frameshift mutations and was a potent genotoxic enhancer. The mechanism for the genotoxic enhancement is not known. However, the fact that the increase in mutagenicity was noted only in TA98 and not in TA1538 suggested that the enhancement of genotoxicity by tetrandrine may result from an increase in error-prone DNA repair.     

IR spectroscopic research on the impact of chemical analogues of autoregulatory d<Subscript>1</Subscript> factors of microorganisms on structural changes in DNA

Using IR spectroscopy, we investigated the impact of chemical analogues of autoregulatory d₁ factors of microorganisms (methylresorcinol, hexylresorcinol, and tyrosol) on the conformational changes in DNA in films upon altering (decreasing) the relative humidity. We analyzed the appearance/disappearance of characteristic absorption bands of A and B DNA forms and determined D ₁₀₈₈/D ₁₂₂₄, the ratio between the band intensities of symmetrical and asymmetrical oscillations in their phosphate groups. The data obtained suggest the slowing down of the B → A structural transition in DNA in the presence of methylresorcinol and its speeding up in the presence of tyrosol. We discuss the mechanisms of this phenomenon in relation to the chemical composition of d 1 factors and their biological function.     

Escherichia coli DinB inhibits replication fork progression without significantly inducing the SOS response

The SOS response is readily triggered by replication fork stalling caused by DNA damage or a dysfunctional replicative apparatus in Escherichia coli cells. E. coli dinB encodes DinB DNA polymerase and its expression is upregulated during the SOS response. DinB catalyzes translesion DNA synthesis in place of a replicative DNA polymerase III that is stalled at a DNA lesion. We showed previously that DNA replication was suppressed without exogenous DNA damage in cells overproducing DinB. In this report, we confirm that this was due to a dose-dependent inhibition of ongoing replication forks by DinB. Interestingly, the DinB-overproducing cells did not significantly induce the SOS response even though DNA replication was perturbed. RecA protein is activated by forming a nucleoprotein filament with single-stranded DNA, which leads to the onset of the SOS response. In the DinB-overproducing cells, RecA was not activated to induce the SOS response. However, the SOS response was observed after heat-inducible activation in strain recA441 (encoding a temperature-sensitive RecA) and after replication blockage in strain dnaE486 (encoding a temperature-sensitive catalytic subunit of the replicative DNA polymerase III) at a non-permissive temperature when DinB was overproduced in these cells. Furthermore, since catalytically inactive DinB could avoid the SOS response to a DinB-promoted fork block, it is unlikely that overproduced DinB takes control of primer extension and thus limits single-stranded DNA. These observations suggest that DinB possesses a feature that suppresses DNA replication but does not abolish the cell's capacity to induce the SOS response. We conclude that DinB impedes replication fork progression in a way that does not activate RecA, in contrast to obstructive DNA lesions and dysfunctional replication machinery.     

Regulation of the manganese-containing superoxide dismutase is independent of the inducible DNA repair system in Escherichia coli

Studies on the induction of the manganese-containing superoxide dismutase in several strains of Escherichia coli with different mutations in recA and lexA revealed that the inductions of the Mn-isozyme and of the SOS system by oxygen free radicals are not coregulated. We also studied the synthesis of the manganese-superoxide dismutase in the temperature-dependent, protease-constitutive strain recA441(tif-1) that also contained a lac fusion in an SOS gene. A shift to the temperature at which recA441 has constitutive protease activity did not induce Mn-superoxide dismutase but did induce beta-galactosidase. The data clearly demonstrate that induction of the Mn-superoxide dismutase is independent of the SOS system.     

Effect of alkylhydroxybenzenes, microbial anabiosis inducers, on the structural organization of Pseudomonas aurantiaca DNA and on phenotypic dissociation induction

We revealed a relationship between alkylhydroxybenzene (AHB)-induced changes in the structural organization of supramolecular complexes (SC) of the DNA of Pseudomonas auraniaca and the phenotypic dissociation of this bacterium. The addition of 0.1-0.3 mM hexylresorcinol (C6-AHB), a chemical analogue of microbial anabiosis autoinducers, caused the formation of cystlike refractile cells (CRC) in these gram-negative, nonsporulating bacteria. Inoculating pseudomonad CRC on solid nutrient media resulted in phenotypic dissociation of the microbial population that yielded several variants with different colony structure and morphology. This manifested itself in the conversion of the original S-colony-forming phenotype into the R form and in the formation of less pigmented colonies. These transitions were possibly linked to AHB-induced structural changes in the DNA. In vitro studies revealed that AHB could interact with DNA SC, resulting in their structural modification that manifested itself in changes in their elastoviscosity. DNA supramolecular complexes isolated from proliferating, stationary-phase, and anabiotic P. aurantiaca cells differed in their elastoviscosity and capacity to interact with AHB homologues with different hydrophobicity, such as hexylresorcinol and methylresorcinol (C1-AHB). The DNA SC from actively proliferating cells were characterized by smaller elastoviscosity compared with those from stationary-phase and anabiotic cells, due to the difference in the DNA superspiralization degree and the physiological age of the bacteria involved. C6-AHB produced a pronounced relaxing effect on the DNA SC from exponential-phase P. aurantiaca cells. The less hydrophobic C1-AHB produced a similar effect on the DNA SC from stationary-phase cells. The curve of the dose-effect dependence of C6-AHB had a breaking point within the submillimolar (10(-4) M) concentration range. These concentrations induce the formation of cystlike anabiotic pseudomonad cells that are characterized by an unstable genotype and dissociate into distinct variants upon inoculation on solid media.     

SOS induction in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of complementary-strand DNA synthesis.

We report that the SOS response is induced in Escherichia coli by infection with mutant filamentous phage that are defective in initiation of the complementary (minus)-strand synthesis. One such mutant, R377, which lacks the entire region of the minus-strand origin, failed to synthesize any detectable amount of primer RNA for minus-strand synthesis. In addition, the rate of conversion of parental single-stranded DNA of the mutant to the double-stranded replicative form in infected cells was extremely slow. Upon infection, R377 induced the SOS response in the cell, whereas the wild-type phage did not. The SOS induction was monitored by (i) induction of beta-galactosidase in a strain carrying a dinD::lacZ fusion and (ii) increased levels of RecA protein. In addition, cells infected with R377 formed filaments. Another deletion mutant of the minus-strand origin, M13 delta E101 (M. H. Kim, J. C. Hines, and D. S. Ray, Proc. Natl. Acad. Sci. USA 78:6784-6788, 1981), also induced the SOS response in E. coli. M13Gori101 (D. S. Ray, J. C. Hines, M. H. Kim, R. Imber, and N. Nomura, Gene 18:231-238, 1982), which is a derivative of M13 delta E101 carrying the primase-dependent minus-strand origin of phage G4, did not induce the SOS response. These observations indicate that single-stranded DNA by itself induces the SOS response in vivo.     

Site-specific methylases induce the SOS DNA repair response in Escherichia coli.

Expression of the site-specific adenine methylase HhaII (GmeANTC, where me is methyl) or PstI (CTGCmeAG) induced the SOS DNA repair response in Escherichia coli. In contrast, expression of methylases indigenous to E. coli either did not induce SOS (EcoRI [GAmeATTC] or induced SOS to a lesser extent (dam [GmeATC]). Recognition of adenine-methylated DNA required the product of a previously undescribed gene, which we named mrr (methylated adenine recognition and restriction). We suggest that mrr encodes an endonuclease that cleaves DNA containing N6-methyladenine and that DNA double-strand breaks induce the SOS response. Cytosine methylases foreign to E. coli (MspI [meCCGG], HaeIII [GGmeCC], BamHI [GGATmeCC], HhaI [GmeCGC], BsuRI [GGmeCC], and M.Spr) also induced SOS, whereas one indigenous to E. coli (EcoRII [CmeCA/TGG]) did not. SOS induction by cytosine methylation required the rglB locus, which encodes an endonuclease that cleaves DNA containing 5-hydroxymethyl- or 5-methylcytosine (E. A. Raleigh and G. Wilson, Proc. Natl. Acad. Sci. USA 83:9070-9074, 1986).     

Genotoxic activity of some mycotoxins using the SOS chromotest

The genotoxic activity of 11 mycotoxins was investigated inEscherichia coli K 12. The induction of the SOS functionsfi A whose level of expression is monitored by means of asfi A:: lac Z operon fusion was assayed by measuring the-galactosidase activity in the PQ 37 strain. Most of these fungal metabolites did not induce SOS response in this bacterial test. Only aflatoxicol, a reduced metabolite of aflatoxin B₁ was well detected as an SOS inducer if metabolic activation was performed. Patulin, penicillic acid and viomellein are only weak inducing agents. The other fungal compounds tested failed to demonstrate a positive SOS inducing activity. Relationship between SOS chromotest, mutagenicity toSalmonella typhimurium andin vivo carcinogenicity was discussed.     

Increased superoxide radical production evokes inducible DNA repair in Escherichia coli

Paraquat induced the SOS response in Escherichia coli. This was measured in terms of acquired resistance towards UV lethality in a wild-type strain and in terms of appearance of beta-galactosidase activity in a din::Mu d(Ap lac) fusion strain. However measured, the induction of the SOS response by paraquat was entirely dioxygen-dependent; whereas induction of the SOS response by mitomycin C was independent of the presence of dioxygen. As expected, recA(Def) and lexA(Ind-) isogenic strains did not show the SOS response. It appears likely that O-2, whose intracellular production is increased by paraquat, leads to DNA damage which in turn induces the SOS response.     

Structural requirement for the induction of the adaptive response in Escherichia coli among N-(substituted alkyl)-N-nitrosoureas

Using E. coli CSH26 transformed with a plasmid carrying an alkA'-lacZ' fused gene, a series of N-(substituted alkyl)-N-nitrosoureas were subjected to a colorimetric assay to evaluate their capacity to induce the adaptive response, an inducible DNA-repair network in E. coli. Some of these derivatives induced the response in greater or lesser degrees, while others did not. Several structural requirements for the induction were disclosed. The capacity of these derivatives to induce the SOS response, which is another inducible DNA-repair network, was also evaluated using E. coli transformed with a plasmid carrying a umuC'-lacZ' fused gene. Since all the derivatives induced the SOS response, the structural requirements for the adaptive response disclosed in this study are substantially related to the molecular mechanism involved in the adaptive response.     

Drug-induced relaxation of supercoiled plasmid DNA in Bacillus subtilis and induction of the SOS response.

Whereas treatment with many different drugs led to induction of the SOS response in Bacillus subtilis, only inhibitors of DNA gyrase subunit B and, unexpectedly, polyether antibiotics (membrane ionophores) led to relaxation of supercoiled plasmid DNA. However, treatment with DNA gyrase subunit B inhibitors but not with polyethers led to SOS induction. Thus, the presence of underwound supercoiled DNA was not sufficient to induce the SOS response. Possible mechanisms by which polyethers induce relaxation of supercoiled DNA in vivo are discussed.