Suppression of tif-mediated induction of SOS functions in Escherichia coli by an altered dnaB protein.

The tif-1 mutation in the Escherichia coli recA gene is known to cause induction of the various "SOS" functions at high temperature, including massive synthesis of the recA protein, lethal filamentation, elevated mutagenesis, and, in lambda lysogens, induction of prophage. It is shown here that the deoxyribonucleic acid initiation mutation dnaB252 suppresses all these manifestations of tif expression. Induction of lambda by ultraviolet irradiation, however, is not affected by the dnaB252 mutation. No similar suppression of tif is observed with other dnaB mutations affecting deoxyribonucleic acid elongation or with other deoxyribonucleic acid initiation mutations at the dnaA and dnaC loci. The fact that an alteration of the dnaB protein specifically suppresses tif-mediated SOS induction implies a role of the replication apparatus in this process, as has been suggested for ultraviolet induction. The induction of lambda is known to proceed via repressor cleavage, presumably promoted by an activated (protease) form of the recA protein. Since lambda induction is normal after ultraviolet irradiation of the tif-1 dnaB252(lambda) strain, tif-mediated induction in this strain may be blocked in a tif-specific step leading to activation of the recA (tif) protein. It is possible that the recA (tif) mutant protein may be directly involved in the replication complex in processes leading to this activation.     

Suppression of induction of SOS functions in an Escherichia coli tif-1 mutant by plasmid R100.1.

The tif-1 mutation in the recA gene of Escherichia coli caused, at 40 degrees C, lethal cell filamentation, induction of the recA protein, mutagenesis, and, in lambda lysogens, prophage induction. The presence of plasmid R100.1 in tif-1 strains suppressed tif-mediated cell filamentation and killing, recA protein induction, and prophage induction in lysogens. It also reduced mutagenesis in a tif-1 sfiA11(R100.1) strain. Plasmids F'lac, P1, and pMB9, in contrast, had little or no effect on tif-mediated induction of lambda. The presence of R100.1 did not inhibit the induction of the recA protein or of lambda by ultraviolet irradiation or mitomycin C treatment of tif-1(R100.1) or tif-1(lambda)(R100.1) strains.     

Dissociation of tsl-tif-Induced Filamentation and recA Protein Synthesis in Escherichia coli K-12

In Escherichia coli, expression of the tif-1 mutation (in the recA gene) induces the "SOS response" at 40 degrees C, including massive synthesis of the recA(tif) protein, cell filamentation, appearance of new repair and mutagenic activities, and prophage induction. Expression of the tsl-1 mutation (in the lexA gene) induces massive synthesis of the recA protein and cell filamentation at 42 degrees C, although other SOS functions are not induced. In this paper we show that the septation inhibition induced in tif and tsl strains at 42 degrees C is not due to the presence of a high concentration of recA protein since (i) no recA mutants (相似文献   

Effect of suppressors of SOS-mediated filamentation on sfiA operon expression in Escherichia coli

In Escherichia coli, the cell division block observed during the SOS response requires the product of the sfiA gene, whose expression is regulated negatively by the LexA repressor and positively by the RecA protease. We have studied the effect on sfiA expression of sfiA, sfiB, infA, and infB mutations, which are known to affect SOS-associated filamentation. To measure sfiA expression in the different strains, we first constructed a lambda transducing phage carrying an sfiA::lac operon fusion. Mutations at the sfiA locus (dominant and recessive) and the sfiB locus (recessive) had no effect on sfiA expression. The mutations tif (at the recA locus) and tsl (at the lexA locus) are known to induce filamentation and a high level of sfiA expression at 42 degrees C. The infB1 mutation, which suppresses filamentation in a tif tsl strain at 42 degrees C, reduced sfiA expression at 42 degrees C in tif tsl infB1 and tsl infB1 strains but not in a tif infB1 strain. The infA3 mutation, which suppresses tif-mediated filamentation, reduced induction of sfiA expression in a tif infA3 strain at 42 degrees C or after UV irradiation. The isolation and characterization of sfiA constitutive strains revealed only lexA-linked mutations in a sfiA-background, suggesting that LexA is the only readily eliminated repressor of the sfiA gene. Nevertheless, the infA and infB mutations could define elements involved in the regulation of sfiA expression.     

Constitutive expression of the SOS response in recA718 mutants of Escherichia coli requires amplification of RecA718 protein.

In recA718 lexA+ strains of Escherichia coli, induction of the SOS response requires DNA damage. This implies that RecA718 protein, like RecA+ protein, must be converted, by a process initiated by the damage, to an activated form (RecA) to promote cleavage of LexA, the cellular repressor of SOS genes. However, when LexA repressor activity was abolished by a lexA-defective mutation [lexA(Def)], strains carrying the recA718 gene (but not recA+) showed strong SOS mutator activity and were able to undergo stable DNA replication in the absence of DNA damage (two SOS functions known to require RecA activity even when cleavage of LexA is not necessary). lambda lysogens of recA718 lexA(Def) strains exhibited mass induction of prophage, indicative of constitutive ability to cleave lambda repressor. When the cloned recA718 allele was present in a lexA+ strain on a plasmid, SOS mutator activity and beta-galactosidase synthesis under LexA control were expressed in proportion to the plasmid copy number. We conclude that RecA718 is capable of becoming activated without DNA damage for cleavage of LexA and lambda repressor, but only if it is amplified above its base-line level in lexA+ strains. At amplified levels, RecA718 was also constitutively activated for its roles in SOS mutagenesis and stable DNA replication. The nucleotide sequence of recA718 reveals two base substitutions relative to the recA+ sequence. We propose that the first allows the protein to become activated constitutively, whereas the second partially suppresses this capability.     

Biochemical basis of the constitutive repressor cleavage activity of recA730 protein. A comparison to recA441 and recA803 proteins.

The recA730 mutation results in constitutive SOS and prophage induction. We examined biochemical properties of recA730 protein in an effort to explain the constitutive activity observed in recA730 strains. We find that recA730 protein is more proficient than the wild-type recA protein in the competition with single-stranded DNA binding protein (SSB protein) for single-stranded DNA (ssDNA) binding sites. Because an increased aptitude in the competition with SSB protein has been previously reported for recA441 protein and recA803 protein, we directly compared their in vitro activities with those of recA730 protein. At low magnesium ion concentration, both ATP hydrolysis and lexA protein cleavage experiments demonstrate that these recA proteins displace SSB protein from ssDNA in a manner consistent with their in vivo repressor cleavage activity, i.e. recA730 protein > recA441 protein > recA803 protein > recAwt protein. Additionally, a correlation exists between the proficiency of the recA proteins in SSB protein displacement and their rate of association with ssDNA. We propose that an increased rate of association with ssDNA allows recA730 protein to displace SSB protein from the ssDNA that occurs naturally in Escherichia coli and thereby to become activated for the repressor cleavage that leads to SOS induction. RecA441 protein is similarly activated for repressor cleavage; however, in this case, significant SSB protein displacement occurs only at elevated temperature. At physiological magnesium ion concentration, we argue that recA803 protein and wild-type recA protein do not displace sufficient SSB protein from ssDNA to constitutively induce the SOS response.     

Cloned truncated recA genes in E. coli II. Effects of truncated gene products on in vivo recA+ protein activity

The ability of plasmids carrying truncated recA genes to sensitize recA+ cells to UV-irradiation was dependent upon the size of the cloned recA gene fragment. Radiosensitization correlated with the inhibition of recombinational repair, and the in vivo reduction of recA protein recombinase activity, as measured by lambda bio 11 plating efficiency. W-reactivation was also abolished by the radiosensitizing plasmids, whilst DNA degradation control, naladixic acid induced filamentation and lambda induction were unaffected. UV-induced mutagenesis in excision proficient E. coli was unaffected, whilst excision deficient strains were hypermutable. It is suggested that these effects of plasmids bearing 22% or more of the recA gene are the result of the interaction of full-sized and truncated protein subunits to generate multimers unable to catalyze recombination.     

Role of the RecF gene product in UV mutagenesis of lambda phage

Summary E. coli recF mutants have a greatly reduced capacity for Weigle mutagenesis of ultraviolet light-irradiated lambda phage. A recF 332::Tn3 mutation was introduced into an E. coli recA441 lexA51 strain which constitutively expresses SOS functions. Weigle mutagenesis of phage lambda could occur in the resulting strain in the absence of host cell irradiation, and was increased when the recA441 (tif) allele was activated by increased temperature and excess adenine. The inability of recF strains to support Weigle mutagenesis can therefore be ascribed to a defect in expression of SOS functions after irradiation.     

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.     

5-Azacytidine: survival and induction of the SOS response in Escherichia coli K-12

Survival and induction of the SOS system by 5-azacytidine, an analog of cytidine, were studied in Escherichia coli K-12. This compound did not produce any effect on the viability of dcm and dam dcm mutants. Furthermore, recA430 and lexA1 strains (both mutations interfere with LexA repressor cleavage but not recombination proficiency) were more resistant than the wild-type strain of E. coli K-12. In contrast, recBC and recA13 mutants were more sensitive to 5-azacytidine than the wild type. Transient exposure of E. coli to 5-azacytidine for 60 min induced both recA-dependent inhibition of cell division and induction of lambda prophage in Dcm+ strains but not in Dcm- mutants. Expression of both functions was dependent on recBC exonuclease. On the other hand, 5-azacytidine was unable to trigger the induction of umuCD and mucB genes and no amplification of RecA protein synthesis in either Dcm+ or Dcm- strains was observed. These last results are in agreement with previously reported data suggesting that there is a discrimination in the expression of the several SOS functions and that some SOS genes may be induced without amplification of RecA protein synthesis.     

Prophage induction and cell division in E. coli. II. Linked (recA, zab) and unlinked (lex) suppressors of tif-1-mediated induction and filamentation

Summary The pleiotropy of tif-1, a mutation in E. coli K12 causing, among other effects, cellular filamentation at 42° and thermal induction of lysogenic derivatives, can be explained by the participation of the tif ⁺ gene product in more than one reaction pathway. Pathways that involve the tif ⁺ product may be analyzed by selection of secondary mutations that reverse both tif-1-mediated prophage induction and cell filamentation. Among revertants of a tif-1 lysogen among 20% are recombination deficient. These appear to carry a recA mutation. In addition to this class is a rarer (7%) phenotypically distinguishable class of revertants, called zab, first described here. Markers tif-1, recA and zab are closely linked. Mutations lex which are dominant and located near malB also appear (3%) among tif-1 revertants. The lex ⁺ function is needed for normal UV, X-ray and mitomycin C induction of prophage .The zab mutation resembles recA in causing (1) high sensitivity to UV, X-rays and mitomycin C, (2) drastic DNA degradation following UV irradiation but normal capacity to repair UV-damaged infecting phage (Hcr⁺), (3) failure to carry out UV reactivation and UV mutagenesis of UV-irradiated bacteriophage , (4) a markedly reduced level of spontaneous induction of . In contrast, other capacities, strikingly diminished by recA, are affected less, if at all by zab. Thus zab (1) permits 30–60% normal recombination proficiency, (2) shows real, although very low inducibility of by UV or mitomycin C, (3) permits 100% efficiency of plating of red gam, and (4) does not degrade DNA spontaneously.The hypothesis is proposed that the tif-1 mutation is a regulatory mutation controlling the activity, or more likely the synthesis of repair enzyme(s). The level of these repair enzyme(s), rather than DNA lesions, may govern the stability of the prophage repressor and the capacity of the bacteria to form septa.     

Derepression of colicin E1 synthesis in the constitutive tif mutant strain (spr tif sfi) and in a tif sfi mutant strain of Escherichia coli K-12.

We show here that expression of the colicin gene of the ColE1 plasmid is greatly derepressed in Escherichia coli K-12 strain DM1187 spr tif sfi, which is a constitutive tif mutant, altered in the lexA gene, and which shows constitutive expression of various pathways of the recA-dependent, lexA-blocked (SOS) repair system. In this strain colicin E1 synthesis is at least 100-fold greater than that observed in uninduced control strains (spr+ tif sfi and spr+ tif+ sfi). This result confirms the regulatory role of the lexA product in colicin E1 synthesis. Colicin yields by the uninduced strain DM1187 are as high as the maximum yields from mitomycin-induced control strains and often are several-fold higher. When the nonconstitutive tif sfi strain GC467 is raised to 43 degrees C to induce the SOS system, a low level of colicin synthesis is observed which is less than one-tenth of the yield obtained by induction with mitomycin C. Addition of adenine at the time of shift-up can increase the colicin yield of tif sfi to about one-third of the yield obtained with mitomycin C. We have also found that colicin overproduction can be detected by altered colony appearance in an overlay assay with colicin-sensitive bacteria. In addition, the lethality of the process of colicin synthesis is observed here without the use of bacteriostatic inducing agents.     

Efficiency of induction of prophage lambda mutants as a function of recA alleles.

Mutants of the cI gene of prophage lambda have been defined phenotypically in a recA+ host as noninducible (Ind-), inducible (Ind+), or induction sensitive (Inds). We showed that a phage lambda cI+ carrying operator mutations v2 and v3 displays an Inds phenotype, as does lambda cI inds-1. We characterized a fourth induction phenotype called induction resistant (Indr). Using these four prophage types, we tested the influence of bacterial recA mutations on prophage induction. Indr prophages were fully induced in recA441 bacteria whose RecA441 protein is activated constitutively. Indr prophages were not induced in a mutant overproducing RecA+ protein, confirming that RecA+ protein must be activated to promote prophage induction. Inds prophages were induced in recA142 and recA453-441 lysogens, previously described as deficient in prophage induction.     

Interaction of hybridoma antibodies with normal and mutant forms of Escherichia coli recA protein

We have prepared hybridomas which secrete antibodies against E. coli recA protein, by fusing spleen lymphocytes from immunized mice with P3X63-AG8 myeloma cells. This paper describes a preliminary survey of properties of antibody from a hybridoma population designated 156, which inhibits the strand pairing, strand assimilation, repressor cleavage, and DNA-dependent ATPase activities of recA protein. The 156 antibody consists of one or two species which have been tentatively identified as IgG2b. 156 Globulin reacts 4.6-fold more efficiently with denatured recA protein, and it reacts partially with the native tif-1, lexB30, recA44, and recA629 proteins, as well as with peptide fragments which are not common to all four proteins.     

Induction of prophage lambda by gamma-rays, mitomycin C and tif; repressor cleavage studied by immunoprecipitation.

Summary The fate of the repressor on induction of E. coli lysogens has been studied after -irradiation, mitomycin C treatment or by temperature step-up of a tif-1 mutant. The repressor and a fragment of the repressor were selectively precipitated from labelled cell lysates by anti-repressor serum and subjected to gel electrophoresis and autoradiography. Induction of lysogens by all of these treatments leads to cleavage of the repressor. Repressor cleavage closely parallels the inactivation of its DNA binding properties and seems to be the general fate of the repressor on induction, irrespective of the nature of the inducing treatment.The effect of chloramphenicol on the cleavage has been studied in order to assess the requirement for inducible protein synthesis in this process. In contrast to results obtained by mitomycin C treatment, tif mediated cleavage of the repressor does not require the induction of new protein synthesis. Although inactivation of the DNA binding properties of the repressor after induction by -irradiation does not appear to be inhibited by chloramphenicol (West, Powell and Emmerson, 1975), cleavage of the repressor is substantially inhibited by this compound. The possible implications of these findings are discussed in relation to inducible repair functions and the mechanism of repressor inactivation.     

New recA mutations that dissociate the various RecA protein activities in Escherichia coli provide evidence for an additional role for RecA protein in UV mutagenesis.

To isolate strains with new recA mutations that differentially affect RecA protein functions, we mutagenized in vitro the recA gene carried by plasmid mini-F and then introduced the mini-F-recA plasmid into a delta recA host that was lysogenic for prophage phi 80 and carried a lac duplication. By scoring prophage induction and recombination of the lac duplication, we isolated new recA mutations. A strain carrying mutation recA1734 (Arg-243 changed to Leu) was found to be deficient in phi 80 induction but proficient in recombination. The mutation rendered the host not mutable by UV, even in a lexA(Def) background. Yet, the recA1734 host became mutable upon introduction of a plasmid encoding UmuD*, the active carboxyl-terminal fragment of UmuD. Although the recA1734 mutation permits cleavage of lambda and LexA repressors, it renders the host deficient in the cleavage of phi 80 repressor and UmuD protein. Another strain carrying mutation recA1730 (Ser-117 changed to Phe) was found to be proficient in phi 80 induction but deficient in recombination. The recombination defect conferred by the mutation was partly alleviated in a cell devoid of LexA repressor, suggesting that, when amplified, RecA1730 protein is active in recombination. Since LexA protein was poorly cleaved in the recA1730 strain while phage lambda was induced, we conclude that RecA1730 protein cannot specifically mediate LexA protein cleavage. Our results show that the recA1734 and recA1730 mutations differentially affect cleavage of various substrates. The recA1730 mutation prevented UV mutagenesis, even upon introduction into the host of a plasmid encoding UmuD* and was dominant over recA+. With respect to other RecA functions, recA1730 was recessive to recA+. This demonstrates that RecA protein has an additional role in mutagenesis beside mediating the cleavage of LexA and UmuD proteins.     

Effects of overproduction of single-stranded DNA-binding protein on RecA protein-dependent processes in Escherichia coli

Overproduction of single-stranded DNA-binding protein (SSB) in Escherichia coli led to a decrease in the basal level of repressor LexA. Expression of the LexA-controlled genes was increased differentially, depending on the affinity of the LexA repressor for each promoter: expression of the recA and sfiA genes was increased 5-fold and 1.5-fold, respectively. Despite only a slight effect on expression of sfiA, which codes for an inhibitor of cell division, bacteria overproducing SSB produced elongated cells. In fact, the effect on cell shape appeared to be essentially independent of the expression of the sfiA and recA genes. Bacteria overproducing SSB were therefore phenotypically similar to bacteria partially starved of thymine, in which filamentation results from both sfiA-dependent and sfiA-recA-independent pathways. These data indicate that excess SSB acts primarily by perturbing DNA replication, thereby favoring gratuitous activation of RecA protein to promote cleavage of LexA protein. When bacteria overproducing SSB were exposed to a DNA-damaging agent such as ultraviolet light or mitomycin C, the recA and sfiA genes were fully induced. Induction of the sfiA gene occurred, however, at higher doses in bacteria overproducing SSB protein than in bacteria with normal levels of SSB. Whereas the efficiency of excision repair was apparently increased by excess SSB, the efficiency of post-replication recombinational repair was reduced as judged by a decrease in the recombination proficiency between a prophage and ultraviolet-irradiated heteroimmune infecting phage. Following induction of ssb+ bacteria with mitomycin C, the cellular content of SSB was slightly increased. These results provide evidence that SSB modulates RecA protein-dependent activities in vivo. It is proposed that SSB favors the formation of short complexes of RecA protein and single-stranded DNA that mediate cleavage of the LexA and lambda repressors, while it delays the formation of long nucleoprotein filaments, thereby slowing down RecA-promoted recombinational events in uninduced as well as in induced bacteria.     

Regulation of expression of the colicin gene of I1 group plasmid TP110.

The control of expression of the colicin Ib gene of the I1 group plasmid TP110 has been investigated. The colicin promoter was fused to the structural gene for beta-galactosidase, using the Mu d(Aprlac) phage, and the plasmid carrying this fusion was introduced into a variety of bacterial strains defective in genes involved in the "SOS" response. Colicin Ib belongs to that group of genes directly controlled by the repressor produced by the lexA gene, and expression was inducible by DNA-damaging agents. Mutations in uvrA, -B, and -C reduced the efficiency of induction by mitomycin C, as did mutations in recB. Mutations in recA and recF effectively prevented induction by mitomycin C, whereas mutations in lexA had contrasting effects, depending upon their effect on the properties of lexA protein. The spr-51 mutation (which inactivates lexA protein) led to constitutive expression, whereas the lexA3 mutation (which makes lexA protein refractory to cleavage by recA protein) completely inhibited inducible expression. In addition to lexA control, a TP110-coded function was identified which appeared able to inhibit colicin expression when the gene responsible was present in high copy number.     

Cell toxicity caused by products of the p(L) operon of bacteriophage lambda.

Induction of a lambda prophage causes the death of the host cell even in the absence of phage replication and lytic functions due to expression of functions from the lambda p(L) operon. We genetically modified the lambda prophage to determine which lambda p(L) operon functions were involved in cell killing. Viability assays and flow cytometry were used to monitor cell death and filamentation. The kil gene was shown to cause cell death and filamentation as described previously. Another killing activity was mapped within the p(L) operon to the gam gene. Inspection of the DNA sequence showed that there are two possible translation start points for both kil and gam. In both cases, the shorter of the two possible products could cause cell killing. The shorter products were also sufficient for the known filamentation and recombination activities of the respective Kil and Gam functions. The expression level of the p(L) operon is down-regulated by Cro repressor. In the absence of Cro, higher p(L) expression levels allow either Kil or Gam to be lethal or growth inhibitory, whereas at lowered expression in Cro-repressed conditions, only Kil is lethal. The filamentation function of Kil and recombination activity of Gam are unaffected at Cro-repressed levels of expression.