Effect of L-ethionine the expression of the SOS system in Escherichia coli

The effect of L-ethionine, the ethyl analog of the essential amino acid methionine the SOS system of Escherichia coli was studied. This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA⁺ Met⁺ RelA⁺ strain, nor in RecA⁺ Met⁻ RelA⁺ or RecA⁺ Met⁻ RelA⁻ mutants. Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42°C. Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift. Moreover, cultures of the recA441 mutant incubated at 42°C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid. On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA⁺ strain even if this compound is added to the cells several hours before irradiation.     

Expression of recA-gene dependent SOS functions in Salmonella typhimurium

Thymine starvation of RecA⁺ Thy^- strains of Salmonella typhimurium does not induce the inhibition of cellular respiration, one of the recA-gene dependent SOS functions. Nevertheless, thymine deprivation is able to produce a normal induction of prophage and thymineless death in these same strains. However, when these mutants are treated, in the presence of thymine, with UV-irradiation or bleomycin, they show a normal inhibition of cellular respiration and other SOS functions. Thus, one injurious treatment (thymine deprivation) may trigger prophage induction but not cessation of respiration, whereas another agent (UV-irradiation) may induce both. Together, these results suggest a possible discrimination in the pathways and conditions of expression of various SOS functions.     

Constitutive expression of SOS functions and modulation of mutagenesis resulting from resolution of genetic instability at or near the recA locus of Escherichia coli

Summary Cellular activities normally inducible by DNA damage (SOS functions) are expressed, without DNA damage, in recA441 (formerly tif-1) mutants of Escherichia coli at 42° C but not at 30° C. We describe a strain (SC30) that expresses SOS functions (including mutator activity, prophage induction and copious synthesis of recA protein) constitutively at both temperatures. SC30 is one of four stable subclones (SC strains) derived from an unstable recombinant obtained in a conjugation between a recA441 K12 donor and a recA ⁺ B/r-derived recipient. SC30 does not owe its SOS-constitutive phenotype to a mutation in the lexA gene (which codes the repressor of recA and other DNA damage-inducible genes), since it is lexA ⁺. Each of the SC strains expresses SOS functions in a distinctively anomalous way. We show that the genetic basis for the differences in SOS expression among the SC strains is located at or very near the recA locus. We propose that resolution of genetic instability in this region, in the original recombinant, has altered the pattern of expression of SOS functions in the SC strains.     

Effect of L-ethionine on the expression of the SOS system in Escherichia coli

The effect of L-ethionine, the ethyl analog of the essential amino acid methionine, on the SOS system of Escherichia coli was studied. This compound does not induce either inhibition of cell division nor cessation of cell respiration in a RecA+ Met+ RelA+ strain, nor in RecA+ Met- RelA+ or RecA+ Met- RelA- mutants. Nevertheless, L-ethionine blocks the expression of both cited SOS functions in a recA441 mutant when it is growing at the restrictive temperature of 42 degrees C. Furthermore, the inhibitory effect of the L-ethionine on the induction of the SOS system in this mutant is increased when the cells are preincubated for several hours in the presence of the analog, before the temperature shift. Moreover, cultures of the recA441 mutant incubated at 42 degrees C in the presence of both L-ethionine and L-methionine present the same behaviour as the cultures of this mutant growing at the same temperature but without either amino acid. On the other hand, L-ethionine does not have any effect on the expression of the two mentioned SOS functions when these are induced by UV-irradiation in a RecA+ strain even if this compound is added to the cells several hours before irradiation.     

Expression of the SOS response following simultaneous treatment with methyl-nitrosoguanidine and mitomycin C in Escherichia coli

Simultaneous treatment of Escherichia coli cultures with methyl-nitrosoguanidine and mitomycin C induces recA-dependent inhibition of respiration but not inhibition of cell division. This pattern of SOS functions expression is the same as that is found following treatment with methyl-nitrosoguanidine alone and contrary to the pattern induced after mitomycin C addition. The same result is obtained when a culture of E. coli RecA441 (formerly tif) is shifted to 42 degrees C and treated simultaneously with methyl-nitrosoguanidine. The suppressor effect of this compound over the pattern of SOS functions expression induced by mitomycin C or high temperature in recA441 mutants is directly related to the increase in its dose. Moreover, the division temperature-sensitive mutant ftsA treated with methyl-nitrosoguanidine and high temperature does not show any decrease in its normal filamentous growth when cultured at 42 degrees C. This indicates that the effect of methyl-nitrosoguanidine on the recA-independent inhibition of cell division is not due to any indiscriminate effect of this compound over the division process. These results suggest that the specific kind of lesion caused in DNA is very important in determining which SOS function is induced.     

Restoration of RecA protein activity by genetic complementation

Summary Bacteria carrying either recA430 or recA453-441 mutations are sensitive to UV-irradiation since they amplify the synthesis of RecA protein either poorly or not at all. We show here that, in a recA453-441 (recA430) heterodiploid, UV-resistance and amplification of RecA430 protein were restored, indicating that the cellular level of RecA-associated protease activity was high enough to inactivate LexA repressor. Prophage 434 repressor was also extensively inactivated, whereas RecA430 protein alone cannot cleave this substrate. On the other hand, during growth of the recA453-441(recA430) heterodiploid at 42° C in the presence of adenine, a treatment activating only RecA441 protein, RecA441 protease activity was as high as in a recA441 haploid. In contrast, following this inducing treatment, there was no complementation between RecA441 and RecA⁺ proteins in a recA453-441(recA ⁺) heterodiploid. These results indicate that multimerization of RecA protein molecules results in a functional interaction that, in some combination between RecA protein subunits, may enhance RecA-associated protease activity.Obra Social de la Caja de Ahorros de Valencia     

Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele

Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264→Glu) and recA665 (Tyr264→His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.     

Conditional lethality of the recA441 and recA730 mutants of Escherichia coli deficient in DNA polymerase I

E. coli strains bearing the recA441 mutation and various mutations in the polA gene resulting in enzymatically well-defined deficiencies of DNA polymerase I have been constructed. It was found that the recA441 strains bearing either the polA1 or polA12 mutation causing deficiency of the polymerase activity of pol I are unable to grow at 42 degrees C on minimal medium supplemented with adenine, i.e., when the SOS response is continuously induced in strains bearing the recA441 mutation. Under these conditions the inhibition of DNA synthesis is followed in recA441 polA12 by DNA degradation and loss of cell viability. A similar lethal effect is observed with the recA730 polA12 mutant. The recA441 strain bearing the polA107 mutation resulting in the deficiency of the 5'-3' exonuclease activity of pol I shows normal growth under conditions of continuous SOS response. We postulate that constitutive expression of the SOS response leads to an altered requirement for the polymerase activity of pol I.     

Nature of the SOS mutator activity: genetic characterization of untargeted mutagenesis in Escherichia coli

Summary In Escherichia coli, induction of the SOS functions by UV irradiation or by mutation in the recA gene promotes an SOS mutator activity which generates mutations in undamaged DNA. Activation of RecA protein by the recA730 mutation increases the level of spontaneous mutation in the bacterial DNA. The number of recA730-induced mutations is greatly increased in mismatch repair deficient strains in which replication errors are not corrected. This suggests that the majority of recA730-induced mutations (90%) arise through correctable, i.e. non-targeted, replication errors. This recA730 mutator effect is suppressed by a mutation in the umuC gene. We also found that dam recA730 double mutants are unstable, segregating clones that have lost the dam or the recA mutations or that have acquired a new mutation, probably in one of the genes involved in mismatch repair. We suggest that the genetic instability of the dam recA730 mutants is provoked by the high level of replication errors induced by the recA730 mutation, generating killing by coincident mismatch repair on the two unmethylated DNA strands. The recA730 mutation increases spontaneous mutagenesis of phage poorly. UV irradiation of recA730 host bacteria increases phage untargeted mutagenesis to the level observed in UV-irradiated recA ⁺ strains. This UV-induced mutator effect in recA730 mutants is not suppressed by a umuC mutation. Therefore UV and the recA730 mutation seem to induce different SOS mutator activities, both generating untargeted mutations.     

Restriction in vivo

Summary Degradation products of restricted T4 DNA induced filamentation, mutagenesis, and to a lesser extent, synthesis of recA protein in wild type cells but not in recA, lexA or recBC mutants of Escherichia coli. We conclude that the structural damage to the DNA caused by restriction cleavage and exonuclease V degradation can induce SOS functions. Degradation of restricted nonglucosylated T4 DNA by exonuclease V delayed cell division and induced filament formation and mutagenesis in lexA ⁺ but not in lexA ^- cells. Delay of cell division was also dependent upon recA and recBC funtions. Such degradation of DNA also dramatically increased mutagenesis in tif ^- Sfi^- cells at 42°C. The synthesis of recA protein continued in the restricting host after infection by the nonglucosylated T4 phage, but enhanced synthesis is not induced to the extent seen in SOS induced tif ^- cells grown at 42°. We also found that restriction of nonglucosylated T4 was alleviated in UV irradiated cells. The UV induced alleviation of rgl and r _K restriction depended upon post irradiation protein synthesis and was not observed in recA, lexA or recBC mutants.     

Influence of single-stranded DNA-binding protein on recA induction in Escherichia coli

Two ssb mutants of Escherichia coli, whic carry a lesion in the single-strand DNA-binding protein (SSB), are sensitive to UV-irradiation. We have investigated the influence of SSB on the “SOS” repair pathway by examining the levels of recA protein synthesis. These strains fail to induced normal levels of recA protein after treatment with nalidixic acid or ultraviolet light. The level of recA protein synthesis in wild-type cells is about three times greater than ssb cells. This deficiency in ssb mutants occurs in all strains and at all temperatures tested (30–41.5°). In contrast, the ssb-1 mutant has no effect on temperature-induced recA induction in a recA441 (tif-1) strain. Cells carrying ssb⁺ plasmids and overproducing normal DNA-binding protein surprisingly are moderated UV-sensitive and have reduced levels of recA protein synthesis. Together these results establish that single-strand DNA-binding protein is involved in the induction of recA, and accounts, at least in part, for the UV sensivitiy of ssb mutant. Three possible mechanisms to explain the role of SSB are discussed.     

Inducibility of the SOS response in a recA730 or recA441 strain is restored by transformation with a new recA allele

Escherichia coli RecA protein plays an essential role in both genetic recombination and SOS repair; in vitro RecA needs to bind ATP to promote both activities. Residue 264 is involved in this interaction; we have therefore created two new recA alleles, recA664 (Tyr264Glu) and recA665 (Tyr264His) bearing mutations at this site. As expected both mutations affected all RecA activities in vivo. Complementation experiments between these new alleles and wild-type recA or recA441 or recA730 alleles, both of which lead to constitutively activated RecA protein, were performed to further investigate the modulatory effects of these mutants on the regulation of SOS repair/recombination pathways. Our results provide further insight into the process of polymerization of RecA protein and its regulatory functions.     

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.     

Suppression of the UV-sensitive phenotype of Escherichia coli recF mutants by recA(Srf) and recA(Tif) mutations requires recJ+.

Mutations in recA, such as recA801(Srf) (suppressor of RecF) or recA441(Tif) (temperature-induced filamentation) partially suppress the deficiency in postreplication repair of UV damage conferred by recF mutations. We observed that spontaneous recA(Srf) mutants accumulated in cultures of recB recC sbcB sulA::Mu dX(Ap lac) lexA51 recF cells because they grew faster than the parental strain. We show that in a uvrA recB+ recC+ genetic background there are two prerequisites for the suppression by recA(Srf) of the UV-sensitive phenotype of recF mutants. (i) The recA(Srf) protein must be provided in increased amounts either by SOS derepression or by a recA operator-constitutive mutation in a lexA(Ind) (no induction of SOS functions) genetic background. (ii) The gene recJ, which has been shown previously to be involved in the recF pathway of recombination and repair, must be functional. The level of expression of recJ in a lexA(Ind) strain suffices for full suppression. Suppression by recA441 at 30 degrees C also depends on recJ+. The hampered induction by UV of the SOS gene uvrA seen in a recF mutant was improved by a recA(Srf) mutation. This improvement did not require recJ+. We suggest that recA(Srf) and recA(Tif) mutant proteins can operate in postreplication repair independent of recF by using the recJ+ function.     

Base pair substitution and frameshift mutagenesis induced by apurinic sites and two fluorene derivatives in a recA441 lexA (Def) strain

Summary One of the consequences of the induction of the Escherichia coli SOS system is the increased ability of the cells to perform mutagenesis. Induction of the SOS system is the result of derepression of a set of genes through a regulatory mechanism controlled by LexA and RecA. In response to an inducing signal, RecA is activated in a form that facilitates the proteolytic cleavage of LexA repressor. Previous works have shown that activated RecA plays a second role, i.e. it is required for the establishment of base pair substitution mutations promoted by UV irradiation. Using a forward mutatonal assay and recA441 lexA(Def) host bacteria, we show that the result can be extended not only to other mutagens promoting base pair substitution mutations (Apurinic sites, Ap sites and N-hydroxy-N-2-aminofluorene, N-OH-AF) but also mutagens promoting frameshift mutations (N-Acetoxy-N-2-acetylaminofluorene, N-AcO-AAF). In the recA441 lexA(Def) strain all the genes which are part of the lexA regulon, including recA itself, are expressed constitutively. The recA441 mutation allows RecA to acquire its activated form when the bacteria are grown at 42° C. We show that in such strains Ap sites or N-OH-AF induce a high level of mutations only when the bacteria are grown at 42° C. On the other hand, we show that N-AcO-AAF can promote mutations even at 30° C; the number of mutations being increased when the bacteria were grown at 42° C. Analysis of the mutants obtained at 30° C indicate that they belong to both type of mutations, UmuC-dependent or UmuC-independent. The much higher ability of N-AcO-AAF to induce RecA as compared to N-OH-AF strongly suggests that the former mutagen is able to induce at least partially the activated form of RexA441 even at 30°C in a strain which overproduces RecA, [lexA(Def)]. Furthermore, we show that the UmuC-independent type of mutagenesis induced by N-AcO-AAF depends on gene(s) that are part of the lexA regulon.     

The sfiA11 mutation prevents filamentation in a response to cell wall damage only in a recA+ genetic background

Summary N--palmitoyl-l-lysyl-l-lysine dihydrochloride ethyl ester (PLL) at sublethal doses causes filamentous growth of E. coli strains except sfiA mutants, which divide normally in its presence. PLL does not elicit the SOS responses as judged by prophage induction, an increase of RecA protein synthesis or induction of the sfiA operon in a sfiA::lacZ fusion strain. Thus, it appears that filamentation caused by PLL is not an SOS function and might be the result of membrane damage by PLL, which is an amphipathic compound and at higher doses causes cell lysis. This indicates that basal levels of the sfiA gene product are sufficient to inhibit cell division in the presence of PLL.We have found further that the phenotype of the sfiA mutation in the presence of PLL requires a recA ⁺ genetic background and does not occur in E. coli recA1 sfiA11, recA13 sfiA11, recA56 sfiA11 and recA441 sfiA11. All these strains, but rec441 sfiA11, however, regain the ability of sfiA11 mutants to divide in the presence of PLL after transformation with the RecA overproducing-plasmid pXO₂. This supports the conclusion that the RecA protein positively affects sfiA11-mediated cell division in the presence of the cell membrane damaging compound, PLL. The basal level of the RecA protein in the recA ⁺sfiA11 strain is sufficient for this process. An increased level due to overproduction from the multicopy plasmid pXO₂ exerts the same effect.     

A homozygous <Emphasis Type="Italic">recA</Emphasis> mutant of <Emphasis Type="Italic">Synechocystis</Emphasis> PCC6803: construction strategy and characteristics eliciting a novel RecA independent UVC resistance in dark

We report here the construction of a homozygous recA460::cam insertion mutant of Synechocystis sp. PCC 6803 that may be useful for plant molecular genetics by providing a plant like host free of interference from homologous recombination. The homozygous recA460::cam mutant is highly sensitive to UVC under both photoreactivating and nonphotoreactivating conditions compared to the wild type (WT). The liquid culture of the mutant growing in ~800 lx accumulates nonviable cells to the tune of 86% as estimated by colony counts on plates incubated at the same temperature and light intensity. The generation time of recA mutant in standard light intensity (2,500 lx) increases to 50 h compared to 28 h in lower light intensity (~800 lx) that was used for selection, thus explaining the earlier failures to obtain a homozygous recA mutant. The WT, in contrast, grows at faster rate (23 h generation time) in standard light intensity compared to that at ~800 lx (26 h). The Synechocystis RecA protein supports homologous recombination during conjugation in recA ^— mutant of Escherichia coli, but not the SOS response as measured by UV sensitivity. It is suggested that using this homozygous recA460::cam mutant, investigations can now be extended to dissect the network of DNA repair pathways involved in housekeeping activities that may be more active in cyanobacteria than in heterotrophs. Using this mutant for the first time we provide a genetic evidence of a mechanism independent of RecA that causes enhanced UVC resistance on light to dark transition.     

Physical mapping and characterization of the mitochondrial DNA and RNA sequences from mit- mutants defective in cytochrome oxidase peptide 1 (OXI 3).

Summary The striking similarity between the treatments that induce SOS functions and those that result in stable DNA replication (continuous DNA replication in the absence of protein synthesis) prompted us to examine the possibility of stable DNA replication being a recA ⁺ lexA ⁺-dependent SOS function. In addition to the treatments previously reported, ultraviolet (UV) irradiation or treatment with mitomycin C was also found to induce stable DNA replication.The thermal treatment of tif-1 strains did not result in detectable levels of stable DNA replication, but nalidixic acid readily induced the activity in these strains. The induction of stable DNA replication with nalidixic acid was severely suppressed in tif-1 lexA mutant strains. The inhibitory activity of lexA3 was negated by the presence of the spr-51 mutation, an intragenic suppressor of lexA3.Induced stable DNA replication was found to be considerably more resistant to UV irradiation than nromal replication both in a uvrA6 strain and a uvr ⁺ strain. The UV-resistant replication occurred mostly in the semiconservative manner. The possible roles of stable DNA replication in repair of damaged DNA are discussed.     

Genetic control of damage-inducible restriction alleviation in Escherichia coli K12: an SOS function not repressed by lexA

Summary The alleviation of K-specific DNA restriction after treatment of cells by UV or nalidixic acid has been studied in mutants with various alleles of recA and lexA and combinations of these alleles and with recB and recF mutations. The studies show that induction of restriction alleviation by UV or nalidixic acid is abolished in mutants in which the recA protein is defective (recA13, recA56), its protease activity is altered (recA430) or in which it cannot be efficiently activated (recA142). Thermoinduction of restriction alleviation was observed in tif mutant (recA441). In lexA amber mutants restriction alleviation is not constitutive but is still inducible. In a lexA3 mutant restriction alleviation is inducible by nalidixic acid provided that recA protein is overproduced as a result of a recA operator mutation. Induction by UV depends on the recF function and an unidentified function (Y) which is controlled by the lexA protein. The recBC enzyme is necessary for induction by UV or by nalidixic acid. Temperature shift experiments with a thermosensitive recB mutant indicate that the recBC enzyme functions in an early step during UV-induction. It is concluded that the damage-inducible function which alleviates restriction is similar to other damage inducible repair (SOS) functions in the dependence on activated recA protease for induction, but that it differs from these functions by the absence of a direct control through the lexA repressor.