The Mechanism of Reca Pola Lethality: Suppression by Reca-Independent Recombination Repair Activated by the Lexa(def) Mutation in Escherichia Coli

The mechanism of recA polA lethality in Escherichia coli has been studied. Complementation tests have indicated that both the 5' -> 3' exonuclease and the polymerization activities of DNA polymerase I are essential for viability in the absence of RecA protein, whereas the viability and DNA replication of DNA polymerase I-defective cells depend on the recombinase activity of RecA. An alkaline sucrose gradient sedimentation analysis has indicated that RecA has only a minor role in Okazaki fragment processing. Double-strand break repair is proposed for the major role of RecA in the absence of DNA polymerase I. The lexA(Def)::Tn5 mutation has previously been shown to suppress the temperature-sensitive growth of recA200(Ts) polA25::spc mutants. The lexA(Def) mutation can alleviate impaired DNA synthesis in the recA200(Ts) polA25::spc mutant cells at the restrictive temperature. recF(+) is essential for this suppression pathway. recJ and recQ mutations have minor but significant adverse effects on the suppression. The recA200(Ts) allele in the recA200(Ts) polA25::spc lexA(Def) mutant can be replaced by δrecA, indicating that the lexA(Def)-induced suppression is RecA independent. lexA(Def) reduces the sensitivity of δrecA polA25::spc cells to UV damage by ~10(4)-fold. lexA(Def) also restores P1 transduction proficiency to the δrecA polA25::spc mutant to a level that is 7.3% of the recA(+) wild type. These results suggest that lexA(Def) activates a RecA-independent, RecF-dependent recombination repair pathway that suppresses the defect in DNA replication in recA polA double mutants.     

Measurement of in vivo expression of the recA gene of Escherichia coli by using lacZ gene fusions.

A recA-lacZ protein fusion was constructed in vivo by using bacteriophage Mu dII301(Ap lac). The fusion contained the promoter and first 47 codons of the recA mutant, as determined by DNA sequence analysis. The fusion was cloned and used to construct a recA-lacZ operon fusion at the same site within the recA gene. These fusions were introduced into the Escherichia coli chromosome at the lambda attachment site either as complete or cryptic lambda prophages. Synthesis of beta-galactosidase from these fusions was inducible by UV radiation. As the UV dose was increased, induction became slower and persisted for a longer period of time. At low doses of UV radiation, more beta-galactosidase was produced in a uvrA mutant than in a wild-type strain; however, at high doses, no induced synthesis of beta-galactosidase occurred in a uvrA mutant. recA+ strains carrying either the protein or operon fusion on a multicopy plasmid showed reduced survival after UV irradiation. This UV sensitivity was not exhibited by strains containing a single copy of either fusion, however; hence, the fusions provide a reliable measure of recA expression.     

Construction and characterization of two lexA mutants of Salmonella typhimurium with different UV sensitivities and UV mutabilities.

Salmonella typhimurium has a SOS regulon which resembles that of Escherichia coli. recA mutants of S. typhimurium have already been isolated, but no mutations in lexA have been described yet. In this work, two different lexA mutants of S. typhimurium LT2 have been constructed on a sulA background to prevent cell death and further characterized. The lexA552 and lexA11 alleles contain an insertion of the kanamycin resistance fragment into the carboxy- and amino-terminal regions of the lexA gene, respectively. SOS induction assays indicated that both lexA mutants exhibited a LexA(Def) phenotype, although SOS genes were apparently more derepressed in the lexA11 mutant than in the lexA552 mutant. Like lexA(Def) of E. coli, both lexA mutations only moderately increased the UV survival of S. typhimurium, and the lexA552 strain was as mutable as the lexA+ strain by UV in the presence of plasmids encoding MucAB or E. coli UmuDC (UmuDCEc). In contrast, a lexA11 strain carrying any of these plasmids was nonmutable by UV. This unexpected behavior was abolished when the lexA11 mutation was complemented in trans by the lexA gene of S. typhimurium. The results of UV mutagenesis correlated well with those of survival to UV irradiation, indicating that MucAB and UmuDCEc proteins participate in the error-prone repair of UV damage in lexA552 but not in lexA11. These intriguing differences between the mutagenic responses of lexA552 and lexA11 mutants to UV irradiation are discussed, taking into account the different degrees to which the SOS response is derepressed in these mutants.     

Roles of the sequence encoding tobacco etch virus capsid protein in genome amplification: requirements for the translation process and a cis-active element.

The roles of the capsid protein (CP) and the CP coding sequence of tobacco etch potyvirus (TEV) in genome amplification were analyzed. A series of frameshift-stop codon mutations that interrupted translation of the CP coding sequence at various positions were introduced into the TEV genome. A series of 3' deletion mutants that lacked the CP coding sequence beyond each of the frameshift-stop codon mutations were also produced. In addition, a series of 5' CP deletion mutants were generated. Amplification of genomes containing either frameshift-stop codon insertions after codons 1, 59, 103, and 138 or genomes containing the corresponding 3' deletions of the CP coding sequence was reduced by 100- to 1,000-fold relative to that of the parental genome in inoculated protoplasts. In contrast, a mutant containing a frameshift-stop codon after CP position 189 was amplified to 27% of the level of the parental virus, but the corresponding 3' deletion mutant lacking codons 190 to 261 was nonviable. Deletion mutants lacking CP codons 2 to 100, 2 to 150, 2 to 189, and 2 to 210 were amplified relatively efficiently in protoplasts, but a deletion mutant lacking codons 2 to 230 was nonviable. None of the amplification-defective frameshift-stop codon or deletion mutants was rescued in transgenic cells expressing TEV CP, although the transgenic CP was able to rescue intercellular movement defects of replication-competent CP mutants. Coupled with previous results, these data led to the conclusions that (i) TEV genome amplification requires translation to a position between CP codons 138 and 189 but does not require the CP product and (ii) the TEV CP coding sequence contains a cis-active RNA element between codons 211 and 246. The implications of these findings on mechanisms of RNA replication and genome evolution are discussed.     

Induction of mutations in bacteriophage T7 by gamma-rays: independence of host-repair mechanisms.

Amber mutants of bacteriophage T7 are reverted by gamma-rays to pseudo wild-type particles, i.e. particles able to propagate in a suppressorless host. The yield of revertants is much higher when the phage is irradiated in the presence of oxygen than when irradiated anoxically. Under particular gas conditions the efficiency of mutation induction differs by less than a factor of ten among six different amber codons in cistrons 1, 5, 6, 12, 17 and 19. The induction of mutations is not dependent on error-prone repair involving the recA or lexA genes of the host cell. It is estimated that of the damages that may be inflicted by gamma-rays upon an amber codon, fewer than 1 out of 85 results in reversion of the codon to pseudo wild-type.     

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.     

Isolation and characterization of noncleavable (Ind-) mutants of the LexA repressor of Escherichia coli K-12.

The LexA repressor of Escherichia coli represses a set of genes that are expressed in the response to DNA damage. After inducing treatments, the repressor is inactivated in vivo by a specific cleavage reaction which requires an activated form of RecA protein. In vitro, specific cleavage requires activated RecA at neutral pH and proceeds spontaneously at alkaline pH. We have isolated and characterized a set of lexA mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function. Forty-six independent mutants, generated by hydroxylamine and formic acid mutagenesis, were isolated by a screen involving the use of operon fusions. DNA sequence analysis identified 20 different mutations. In a recA mutant, all but four of the mutant proteins functioned as repressor as well as wild-type LexA. In a strain carrying a constitutively active recA allele, recA730, all the mutant proteins repressed a sulA::lacZ fusion more efficiently than the wild-type repressor, presumably because they were cleaved poorly or not at all by the activated RecA protein. These 20 mutations resulted in amino acid substitutions in 12 positions, most of which are conserved between LexA and four other cleavable proteins. All the mutations were located in the hinge region or C-terminal domain of the protein, portions of LexA previously implicated in the specific cleavage reactions. Furthermore, these mutations were clustered in three regions, around the cleavage site (Ala-84-Gly-85) and in blocks of conserved amino acids around two residues, Ser-119 and Lys-156, which are believed essential for the cleavage reactions. These three regions of the protein thus appear to play important roles in the cleavage reaction.     

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.     

Chromogenic method for rapid isolation of recA-like mutants of gram-negative bacteria.

We have devised a rapid and widely applicable color test for detecting recA-like mutants of gram-negative bacteria. The technique depends on decreased expression of an Escherichia coli recA-lacZ fusion in recA mutants and uses a broad-host-range plasmid to transfer the fusion gene into new species. We describe the isolation of a recA-like mutant of Pseudomonas syringae by this technique.     

Replication restart in UV-irradiated Escherichia coli involving pols II, III, V, PriA, RecA and RecFOR proteins

In Escherichia coli, UV-irradiated cells resume DNA synthesis after a transient inhibition by a process called replication restart. To elucidate the role of several key proteins involved in this process, we have analysed the time dependence of replication restart in strains carrying a combination of mutations in lexA, recA, polB (pol II), umuDC (pol V), priA, dnaC, recF, recO or recR. We find that both pol II and the origin-independent primosome-assembling function of PriA are essential for the immediate recovery of DNA synthesis after UV irradiation. In their absence, translesion replication or 'replication readthrough' occurs approximately 50 min after UV and is pol V-dependent. In a wild-type, lexA+ background, mutations in recF, recO or recR block both pathways. Similar results were obtained with a lexA(Def) recF strain. However, lexA(Def) recO or lexA(Def) recR strains, although unable to facilitate PriA-pol II-dependent restart, were able to perform pol V-dependent readthrough. The defects in restart attributed to mutations in recF, recO or recR were suppressed in a recA730 lexA(Def) strain expressing constitutively activated RecA (RecA*). Our data suggest that in a wild-type background, RecF, O and R are important for the induction of the SOS response and the formation of RecA*-dependent recombination intermediates necessary for PriA/Pol II-dependent replication restart. In con-trast, only RecF is required for the activation of RecA that leads to the formation of pol V (UmuD'2C) and facilitates replication readthrough.     

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.     

Yersinia enterocolitica type III secretion: mutational analysis of the yopQ secretion signal

Pathogenic Yersinia spp. secrete Yop proteins via the type III pathway. yopQ codons 1 to 15 were identified as a signal necessary and sufficient for the secretion of a fused reporter protein. Frameshift mutations that alter codons 2 to 15 with little alteration of yopQ mRNA sequence do not abolish type III transport, suggesting a model in which yopQ mRNA may provide a signal for secretion (D. M. Anderson and O. Schneewind, Mol. Microbiol. 31:1139-1148, 2001). In a recent study, the yopE signal was truncated to codons 1 to 12. All frameshift mutations introduced within the first 12 codons of yopE abolished secretion. Also, multiple synonymous mutations that changed the mRNA sequence of yopE codons 1 to 12 without altering the amino acid sequence did not affect secretion. These results favor a model whereby an N-terminal signal peptide initiates YopE into the type III pathway (S. A. Lloyd et al., Mol. Microbiol. 39:520-531, 2001). It is reported here that codons 1 to 10 of yopQ act as a minimal secretion signal. Further truncation of yopQ, either at codon 10 or at codon 2, abolished secretion. Replacement of yopQ AUG with either of two other start codons, UUG or GUG, did not affect secretion. However, replacement of AUG with CUG or AAA and initiating translation at the fusion site with npt did not permit Npt secretion, suggesting that the translation of yopQ codons 1 to 15 is a prerequisite for secretion. Frameshift mutations of yopQ codons 1 to 10, 1 to 11, and 1 to 12 abolished secretion signaling, whereas frameshift mutations of yopQ codons 1 to 13, 1 to 14, and 1 to 15 did not. Codon changes at yopQ positions 2 and 10 affected secretion signaling when placed within the first 10 codons but had no effect when positioned in the larger fusion of yopQ codons 1 to 15. An mRNA mutant of yopQ codons 1 to 10, generated by a combination of nine synonymous mutations, was defective in secretion signaling, suggesting that the YopQ secretion signal is not proteinaceous. A model is discussed whereby the initiation of YopQ polypeptide into the type III pathway is controlled by properties of yopQ mRNA.     

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.     

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.     

Isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12.

We describe the isolation and characterization of amber mutations in the lexA gene of Escherichia coli K-12. These mutations, designated spr(Am), were isolated and characterized in a lexA tif sfi genetic background. They abolished the sensitivity of the strain to UV light and resulted in high rates of synthesis of recA protein. Phage lambda+ failed to lysogenize the strains as observed with similar strains carrying non-amber spr mutations described previously, thereby indicating a constitutive expression of the phage induction pathway. Introduction of an amber suppressor mutation into a strain bearing the spr(Am) mutation restored expression of the LexA mutant phenotype. We conclude that spr mutations either inactivate or prevent synthesis of the lexA gene product and that loss of this product results in constitutive expression of the E. coli induction system in the tif sfi genetic background.     

DNA polymerase I and the bypassing of RecA dependence of constitutive stable DNA replication in Escherichia coli rnhA mutants.

In Escherichia coli rnhA mutants, several normally repressed origins (oriK sites) of DNA replication are activated. The type of DNA replication initiated from these origins, termed constitutive stable DNA replication, does not require DnaA protein or the oriC site, which are essential for normal DNA replication. It requires active RecA protein. We previously found that the lexA71(Def)::Tn5 mutation can suppress this RecA requirement and postulated that the derepression of a LexA regulon gene(s) leads to the activation of a bypass pathway, Rip (for RecA-independent process). In this study, we isolated a miniTn10spc insertion mutant that abolishes the ability of the lexA(Def) mutation to suppress the RecA requirement of constitutive stable DNA replication. Cloning and DNA sequencing analysis of the mutant revealed that the insertion occurs at the 3' end of the coding region of the polA gene, which encodes DNA polymerase I. The mutant allele, designated polA25::miniTn10spc, is expected to abolish the polymerization activity but not the 5'-->3' or 3'-->5' exonuclease activity. Thus, the Rip bypass pathway requires active DNA polymerase I. Since the lethal combination of recA(Def) and polA25::miniTn10spc could be suppressed by derepression of the LexA regulon only when DNA replication is driven by the oriC system, it was suggested that the bypass pathway has a specific requirement for DNA polymerase I at the initiation step in the absence of RecA. An accompanying paper (Y. Cao and T. Kogoma, J. Bacteriol. 175:7254-7259, 1993) describes experiments to determine which activities of DNA polymerase I are required at the initiation step and discusses possible roles for DNA polymerase in the Rip bypass pathway.