Interplay of SOS induction,recombinant gene expression,and multimerization of plasmid vectors in Escherichia coli

Using pBR322- and pUC-derived plasmid vectors, a homologous (Escherichia coli native esterase) and three heterologous proteins (human interleukin-2, human interleukin-6, and Zymomonas levansucrase) were synthesized in E. coli IC2015(recA::lacZ) and GY4786 (sfiA::lacZ) strains. Via time-course measurement of beta-galactosidase activity in each recombinant culture, the SOS induction was estimated in detail and the results were systematically compared. In recombinant E. coli, the SOS response did not happen either with the recombinant insert-negative plasmid backbone alone or the expression vectors containing the homologous gene. Irrespective of gene expression level and toxic activity of synthesized foreign proteins, the SOS response was induced only when the heterologous genes were expressed using a particular plasmid vector, indicating strong dependence on the recombinant gene clone and the selection of a plasmid vector system. It is suggested that in recombinant E. coli the SOS response (i.e., activation of recA expression and initial sfiA expression) may be related neither to metabolic burden nor toxic cellular event(s) by synthesized heterologous protein, but may be provoked by foreign gene-specific interaction between a foreign gene and a plasmid vector. Unlike in E. coli XL1-blue(recA(-)) strains used, all expression vectors encoding each of the three heterologous proteins were multimerized in E. coli IC2015 strains in the course of cultivation, whereas the expression vectors containing the homologous gene never formed the plasmid multimers. The extent of multimerization was also dependent on a foreign gene insert in the expression vector. As a dominant effect of the SOS induction, recombinant plasmid vectors used for heterologous protein expression appear to significantly form various multimers in the recA(+) E. coli host.     

Protection of nonmodified phageλ againstEcoK restriction mediated byrecA protein

A study was conducted to establish whether the EcoK-specific restriction, which is alleviated in E. coli cells after UV induction of the SOS response (Day 1977), is also alleviated under the influence of an increased level of recA protein without induction of other SOS functions. The host cells used were E. coli K-12, strain AB2497, and its derivatives; the nonmodified phage lambda was a mutant b2b5(vir). An increase of the recA protein level was induced using the plasmid pX02, which is a recombinant of pBR322 carrying the recA gene of E. coli. AB2497(pX02) cells were found to exhibit a lower level of restriction than those without plasmid. The results indicate that the recA protein protects phage DNA during the process of restriction. A further factor affecting restriction is the growth phase of the culture of the restricting host: cells in the late stationary phase exhibit lower restriction than those in the exponential phase of growth. By a combination of these two factors (presence of plasmid pX02 and stationary growth phase) one can reduce the restriction of nonmodified phage about 300 times.     

Effect of induction of SOS response on expression of pBR322 genes and on plasmid copy number

Several lines of evidence are presented that indicate that the level of tetracycline resistance of Esherichia coli strains harboring plasmid pBR322 varies according to whether the SOS system of the host bacteria has been induced. These include use of strains in which the SOS system is expressed constitutively (lexA def.), is thermoinducible (recA441) or noninducible (lexA ind-), or is highly repressed (multiple copies of lexA+). Similar induction was observed with the product of another plasmid gene, beta-lactamase. The amounts of extractable plasmid DNA were also increased by SOS induction, and we propose that the SOS-induced increases in levels of tetracycline resistance and beta-lactamase activity are due to an increased plasmid copy number.     

Further studies on the lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

Our previous results on the genotoxic effect of 8-methoxypsoralen-induced lesions on pBR322 suggested an important involvement of an inducible error-free repair pathway in the repair of plasmid lesions. We present herein further results obtained in order to explore that possibility, together with a more general report on the subject. pBR322 treated with increasing concentrations of 8-MOP plus fixed UVA light irradiation was used to transform several E. coli strains differing in their repair capacities, and plasmid survival and mutagenesis were determined. Survival results suggested that crosslinks were completely lethal in pBR322 whereas monoadducts were partially removed from plasmid DNA mainly through an error-free excision pathway. A mutagenic repair pathway did not show a significant contribution to the total repair process. Cell preirradiation stimulated plasmid recovery in recA+ strains, including the umuC strain, thus confirming our previous results indicating that an inducible error-free repair had occurred. Globally, our results showed a strong requirement on the excision pathway for the repair of psoralen-damaged plasmid DNA. In contrast, the recA dependent pathway was needed only for SOS induction. After a theoretical correction of the data for estimating the effect only due to 8-MOP adducts, a different pattern of repair mechanisms appeared to be involved.     

Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant

We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease.     

The SOS-function-inducing activity of chemical mutagens in Escherichia coli

The SOS-function-inducing activities of 42 chemical mutagens were investigated in Escherichia coli K12. The induction of the SOS function was assayed by monitoring the beta-galactosidase activity in the sulA::lacZ fusion strain PQ37 . To correct for the inhibitory effects of test chemicals on mRNA or protein synthesis, the level of the constitutive alkaline phosphatase was assayed in parallel. Most of the mutagens reported to be mutagenic to the Ames' Salmonella tester strains showed the SOS-function-inducing activity. The inducible SOS repair may be responsible for not only base-change mutations but also frameshift mutations. However, 9-aminoacridine, ethidium bromide and 4-nitro-o-phenylenediamine did not induce the SOS function, suggesting that the mutagenesis induced by these mutagens may occur independently of SOS repair. Present results support the SOS mutagenesis model that error-prone SOS repair plays an important role in mutagenesis induced by most chemical mutagens.     

The induction of SOS function in Escherichia coli K-12/PQ37 by 4-nitroquinoline oxide (4-NQO) and fecapentaenes-12 and -14 is bile salt sensitive: implications for colon carcinogenesis

The response of Escherichia coli to genotoxic agents involves the triggering of a complex system of genes known as the SOS response. In E. coli PQ37, a test organism used for the assessment of genotoxicity, lacZ, the beta-galactosidase gene is placed under the control of sfiA, one of the SOS genes through an operon fusion. The induction of beta-galactosidase activity, when the organism is exposed to genotoxic agents, is an indirect measure of the genotoxic activity of the test compound. Incubation of E. coli PQ37 with either 4-nitroquinoline oxide (4-NQO) or one of the fecal mutagens, fecapentaene-12 or -14 (F-12 or F-14) in the presence of sodium taurocholate or sodium deoxycholate resulted in a significant enhancement of induction of beta-galactosidase activity. The molecular mechanisms of 4-NQO-induced mutagenesis in E. coli are similar to those of the effects of UV light in which both replication-dependent and repair-dependent pathways of mutagenesis exist. Since E. coli PQ37 is excision-repair-deficient, alternate pathways are involved in this system. Bile salts by themselves do not trigger the SOS response, and hence their role in enhancing the SOS-inducing potency of mutagens may involve the potentiation of the cleavage-inactivation of lexA (repressor of SOS) by the protein product of the SOS-controlled gene, recA. The potentiating effect of bile salts on the fecal mutagens, F-12 and F-14, has implications in their suspected role in colon carcinogenesis associated with high-fat, low-fiber diets.     

Relationships between structure of 5-nitro-2-furylethylenes and their SOS-function-inducing activities in Escherichia coli

The SOS-function-inducing activities of 36 furylethylenes were characterized in Escherichia coli K12. The induction of the SOS function was assayed by monitoring the beta-galactosidase activity in the sulA::lacZ fusion strain PQ 37. To correct for the inhibitory effects of test compounds on mRNA or protein synthesis, the level of the constitutive alkaline phosphatase was assayed in parallel. Tested furylethylenes included nine alkylesters and eleven N-alkylamides of 5-nitro-2-furylacrylic acid (NFAA) and fourteen derivatives differing not only in substituents at exocyclic double bond, but also in the position 5 of the furan ring. The induction of the SOS-function by the derivatives depends on the presence of 5-nitrofuran centre in their molecule; side chains in the position 2 modify the degree of SOS response. SOS-inducing potency of n-alkyl congeners decreases with increasing lipophilicity. Effect of derivatives with branched alkyl substituents is lower than expected from the behavior of the n-alkyl homologues. All derivatives with positive effect on SOS-function in E. coli show mutagenic activity on Salmonella typhimurium TA98 in Ames test.     

Effect of pKM101 plasmid on lethal and mutagenic damage in UV-irradiated E. coli strains

Introduction of the R-factor plasmid pKM101 increased resistance to UV-killing in uvr lexA(Ind-) recA+ strains of E. coli K12 as well as B, while their UV mutability was not affected. Similar effects were also observed in those strains when the 18-B plasmid (a pBR322 derivative carrying the region (about 5 kb) of the 35.4 kb pKM101 plasmid) was introduced. The muc genes which are considered to be involved in error-prone repair are contained in 18-B. These results suggest the possibility that the pKM101 effect requires the host recA gene and a common genetic region, including the muc genes, in both plasmids and is associated with some unmutable repair systems.     

Lethal and mutagenic effects of 8-methoxypsoralen-induced lesions on plasmid DNA

The genotoxic effect of 8-methoxypsoralen damages (monoadducts and crosslinks) on plasmid DNA was studied. pBR322 DNA was treated with several concentrations of 8-methoxypsoralen plus fixed UVA light irradiation. After transformation into E. coli cells with different repair capacities (uvrA, recA and wild-type), plasmid survival and mutagenesis in ampicillin- and tetracycline-resistant genes were analysed. Results showed that crosslinks were extremely lethal in all 3 strains; indeed, it seemed that they were not repaired even in proficient bacteria. Monoadducts were also found to be lethal although they were removed to some extent by the excision-repair pathway (uvrA-dependent). Damaged plasmid DNA appeared to induce mutagenic repair, but only in the wild-type strain. In order to study the influence of the SOS response on plasmid recovery, preirradiation of the host cells was also performed. Preirradiation of the uvrA or wild-type strains significantly increased plasmid recovery. Consistent with the expectations of SOS repair, no effect was observed in preirradiated recA cells. Plasmid recovery in the excision-deficient strain was mainly achieved by the mutagenic repair of some fraction of the lesions, probably monoadducts. The greatest increase in plasmid recovery was found in the wild-type strain. This likely involved the repair of monoadducts and some fraction of the crosslinks. We conclude that repair in preirradiated repair-proficient cells is carried out mainly by an error-free pathway, suggesting enhancement of the excision repair promoted by the induction of SOS functions.     

The plasmid carrying the temperature-sensitive mutation in the DNa-methylase gene of the PStI system: effect on host cells at nonpermissive temperature]

Temperature-sensitive (ts) derivatives of plasmid pRMP1, the derivative of PBR322 containing restriction and modification (RM) genes of the PstI system, were obtained using hydroxylamine mutagenesis. One of the isolated plasmids responsible for the inhibition of Escherichia coli cell growth at 42 degrees C, pRMPts, was analyzed in this work. Cells of Rec+ strains carrying this plasmid were unable to divide at 42 degrees C and formed long non-septated filaments that died upon prolonged cultivation. Cells of the RecA- strains carrying pRMPts did not form filaments at 42 degrees C and rapidly disappeared. On agar media with or without ampicillin, Rec+ and RecA- strains with this plasmid formed colonies of temperature-resistant (tr) derivatives with frequencies ranging from 1.5 x 10(-4) to 4 x 10(-6) in independent clones. The structure of plasmids from cells of tr-derivatives of Rec+ and RecA- strains carrying plasmid pRMPts was analyzed by the set of restriction enzymes. Reversions to the temperature-resistant phenotype were shown to result from the following events: (1) the insertional inactivation of the PstI restriction enzyme gene in pRMPts (the insertion of the IS1 element); (2) deletions in plasmid DNA fragments that partially or completely cover the restriction enzyme gene; (3) point mutations; and (4) others. The effect of the chromosomal sulA mutation on the maintenance of the ts-plasmid in bacterial cells was studied at 42 degrees C. High efficiency loss of the plasmid was detected in pRMPts-carrying Rec+ cells with the sulA::Tn5 mutation grown in liquid and solid nutrient media at this temperature. Under similar conditions, plasmid loss was not detected in SulA+ cells. On the basis of the data obtained, it is concluded that the ts-mutation is located in the DNA-methylase gene of plasmid pRMPts. Mutant DNA methylase was unable to methylate all sites in the chromosomal DNA at 42 degrees C. Some of the unmethylated sites can be digested with the PstI enzyme, which leads to the induction of SOS response in Rec+ cells or to total mortality in cells with the recA phenotype.     

Novel mechanism for UV sensitivity and apparent UV nonmutability of recA432 mutants: persistent LexA cleavage following SOS induction.

The recA432 mutant allele was isolated (T. Kato and Y. Shinoura, Mol. Gen. Genet. 156:121-131, 1977) by virtue of its defect in cellular mutagenesis (Mut-) and its hypersensitivity to damage by UV irradiation (UVs), which were phenotypes expected for a recA mutant. However, we found that in a different genetic background (lexA51 sulA211 uvrB+), recA432 mutants expressed certain mutant phenotypes but not the Mut- and UVs phenotypes (D.G. Ennis, N. Ossanna, and D.W. Mount, J. Bacteriol. 171:2533-2541, 1989). We present several lines of evidence that these differences resulted from the sulA genotype of the cell and that the apparent UVs and Mut- phenotypes of the sulA+ derivatives resulted from lethal filamentation of induced cells because of persistent derepression of sulA. First, transduction of sulA(Def) mutations into the recA432 strains restored cellular mutagenesis and resistance to UV. Second, recA432 sulA+ strains underwent filamentous death following SOS-inducing treatments. Third, cleavage of LexA repressor in a recA432 strain continued at a rapid rate long after UV induction, at a time when cleavage of the repressor in the recA+ parental strain had substantially declined. Fourth, we confirmed that a single mutation (recA432) conferring both the UVs and Mut- phenotypes mapped to the recA gene. These findings indicate that the RecA432 mutant protein is defective in making the transition back to the deactivated state following SOS induction; thus, the SOS-induced state of recA432 mutants is prolonged and can account for an excess of SulA protein, leading to filamentation. These results are discussed in the context of molecular models for RecA activation for LexA and UmuD cleavage and their roles in the control of mutagenesis and cell division in the SOS response.     

recA-genes of Erwinia chrysanthemi ENA49

The recA gene of Erwinia chrysanthemi ENA49 has been cloned in vivo in Escherichia coli K12, recA13 cells using the plasmid pULB113. On the basis or DNA repair and recombination deficiencies complementation, of restoration of the inducible "SOS"-response functions the functional identity of the cloned gene with the recA gene was concluded. The recA gene was localized in the 18th min region of the chromosomal genetical map of Erwinia chrysanthemi ENA49 between the genes proA and pheA.     

Convenient construction of strains useful for transducing recA mutations with bacteriophage P1

Abstract The generalized transducing phage P1 grew well on heterozygous Escherichia coli carrying recA srlC 300::Tn 10 on the chromosome and recA ⁺ on a pBR322-derived plasmid. Because of the close linkage of Tn 10 to recA mutations, including recA 1, recA 13, recA 56, recA deletion and recA allele of E. coli BNG30, the latter can be moved to other strains in transductional crosses for selective resistance to tetracycline.     

A stable derivative of pBR322 conferring increased tetracycline resistance and increased sensitivity to fusaric acid

A hybrid trp-tet promoter was formed on pBR322 by insertion of a segment containing part of the trp promoter at the ClaI site. The product plasmid, pDR42, conferred resistance to higher concentrations of tetracycline than pBR322. Cells bearing pDR42 were sensitive to lower concentrations of fusaric acid than were those bearing pBR322. Since the difference in growth on fusaric acid between the E. coli RR1 alone and the strain with pDR42 is greater than is the case with pBR322, an improved selection of tetracycline-sensitive (Tc^s) colonies out of a background of pDR42 specified tetracycline-resistant (Tc^r) colonies was observed.     

Role of the uvr gene in the SOS function inducing activity of two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2, in Escherichia coli K12

Two tryptophan pyrolysis products, Trp-P-1 and Trp-P-2 were assayed in the SOS-chromotest using PQ 37 (uvr A) and PQ 35 (uvr+) E. coli K12 strains, in the presence of S9 fraction from Aroclor-induced rats. Both compounds were able to induce the expression of SOS functions in uvr A bacteria, in the following order: Trp-P-1 less than Trp-P-2 less than aflatoxin B1, at low concentrations (less than 125 ng/assay). In this range, the induction of SOS functions was significantly decreased in the uvr+ strain. This implies that the uvr gene product plays an important role in the repair of genotoxic damage induced by Trp-P-1 and Trp-P-2. At higher concentrations (125-500 ng/assay), Trp-P-1 became more efficient in inducing SOS functions than Trp-P-2 and excision repair was less efficient than at low concentration.     

Structural and functional analysis of a cloned segment of Escherichia coli DNA that specifies proteins of a C4 pathway of serine biosynthesis.

The plasmid pDR121 is a pBR322 derivative that contains a 3.7-kilobase-pair EcoRI fragment of DNA from the 81.2-min region of the Escherichia coli chromosome. The genomic insert encodes threonine dehydrogenase and at least one other protein. Several physical and kinetic properties of threonine dehydrogenase, overproduced in cells harboring pDR121, are identical to those of pure threonine dehydrogenase from a haploid mutant of E. coli K-12 that produces this enzyme constitutively. Tester strains with serB or glyA mutations harboring pDR121 are prototrophs. The ability to confer prototrophy on such tester strains is associated with elevated levels of threonine dehydrogenase. The functional roles of various segments of the 3.7-kilobase-pair insert of pDR121 were analyzed by constructing specific deletions and insertions. Certain subclones retained the ability to specify threonine dehydrogenase without conferring prototrophy on tester strains. This suggests that at least one other protein encoded within pDR121 plays an essential role in the conversion of threonine to serine.     

Cloning of the recA gene of Bordetella pertussis and characterization of its product.

A recA gene of Bordetella pertussis was identified in a plasmid library by complementation of a recA mutation in E coli and subcloned as a 2.1-kb Sph I DNA fragment. Southern hybridization experiments showed no similarity to the E coli recA gene, but very strong similarity to other Bordetella species. E coli recA mutant cells containing the B pertussis recA gene at high gene dosage were resistant to DNA-damaging agents such as methyl methane sulfonate or 4-nitroquinoline-N-oxide, displayed induction of SOS functions, and were able to promote DNA recombination, but not induction of phage lambda. The latter phenotype distinguishes the B pertussis recA gene product from the corresponding proteins from most other Gram-negative organisms. Amino acid sequence comparisons revealed a high degree of structural conservation between prokaryotic RecA proteins.     

The pcsA gene is identical to dinD in Escherichia coli.

The pcsA68 mutant of Escherichia coli is a cold-sensitive mutant which forms long filaments with a large nucleoid in the central region at 20 degrees C. We here show that (i) the coding region for the pcsA gene is identical with orfY located upstream of pyrE and can be deleted without loss of viability; (ii) pcsA is also identical to dinD, a DNA damage-inducible gene, whose expression is regulated by the LexA-RecA system; (iii) the cold-sensitive phenotype of the pcsA68 mutation is suppressed by delta recA or lexA1 (Ind-) mutation, but not by sulA inactivation; (iv) overproduction of PcsA68 leads to inhibition of cell growth in recA+ and delta recA strains at 20 and 37 degrees C, but PcsA+ does not show such an effect at any temperature; (v) SOS response is induced in the pcsA68 mutant cells at 20 degrees C. We discuss the possible function of the pcsA gene, comparing it with the sulA or the dif-xerCD function. We also describe a new method for gene disruption with positive and negative selection.