ATP hydrolysis during SOS induction in Escherichia coli.

Changes in cellular ATP concentration during SOS induction in strains of Escherichia coli with different levels of RecA and LexA proteins were studied. UV irradiation of RecA+ strains induced a twofold increase in the ATP concentration around the first 20 min, followed by a decrease to the values of nonirradiated cells. On the other hand, mutants defective in RecA protein or with either deficient RecA protease activity or cleavage-resistant LexA repressor did not show any decrease, suggesting that ATP consumption is related to LexA repressor hydrolysis. Furthermore, strains presenting a constitutive synthesis of RecA protein showed the same changes in ATP concentration as the wild-type strain. Likewise, the presence in a RecA+ strain of a LexA(Def) protein, which is defective in its capacity for binding specifically to SOS operators, did not disturb the changes in ATP when compared with the LexA+ RecA+ strain. Moreover, after UV irradiation, a LexA(Def) RecA- double mutant showed an important increase in ATP concentration, which remained elevated for at least 120 min after UV treatment.     

Inhibition of the SOS response of Escherichia coli by the Ada protein.

Induction of the adaptive response by N-methyl-N'-nitro-N-nitrosoguanidine (MNNG) caused a decrease in the UV-mediated expression of both recA and sfiA genes but not of the umuDC gene. On the other hand, the adaptive response did not affect the temperature-promoted induction of SOS response in a RecA441 mutant. The inhibitory effect on the UV-triggered expression of the recA and sfiA genes was not dependent on either the alkA gene or the basal level of RecA protein, but rather required the ada gene. Furthermore, an increase in the level of the Ada protein, caused by the runaway plasmid pYN3059 in which the ada gene is regulated by the lac promoter, inhibited UV-mediated recA gene expression even in cells to which the MNNG-adaptive treatment had not been applied. This inhibitory effect of the adaptive pretreatment was not observed either in RecBC- strains or in RecBC mutants lacking exonuclease V-related nuclease activity. However, RecF- mutants showed an adaptive response-mediated decrease in UV-promoted induction of the recA gene.     

Tandem cloning of bacteriophage T4 nrdA and nrdB genes and overproduction of ribonucleoside diphosphate reductase (alpha 2 beta 2) and a mutationally altered form (alpha 2 beta 2(93)).

To investigate the role of ribonucleoside diphosphate reductase in the deoxyribonucleoside triphosphate synthetase multienzyme complex induced by bacteriophage T4 infection and to study the expression of the T4 nrdA and nrdB genes, we have constructed separate plasmid expression strains overproducing their respective alpha 2 and beta 2 protein products. Because complementation of the two proteins to form an active alpha 2 beta 2 enzyme presented complications, nrdA and nrdB, each with its own tac promoter, were also cloned in tandem into a single expression vector. The resulting plasmid (pnrdAB) overproduces ribonucleoside diphosphate reductase. Phage T4 nrdB93, described by Wirak et al. (D. O. Wirak, K. S. Cook, and G. R. Greenberg, J. Biol. Chem. 263:6193-6201, 1988) contains a lesion in exon II of the gene. The mutation causes not only a temperature-sensitive inactivation of the catalytic structure of the beta 2(93) protein and of its ability to interact with alpha 2 protein to form the alpha 2 beta 2(93) enzyme but also a profound non-temperature-sensitive decrease in the formation of the beta 2(93) protein. An expression vector overproducing active alpha 2 beta 2(93) was constructed by site-directed mutagenesis of the nrdB gene.     

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.     

How Escherichia coli sets different basal levels in SOS operons

The recA and sfiA genes of Escherichia coli are SOS operons regulated negatively by the LexA repressor. The steady state level of expression of recA is 10-fold higher than that of sfiA, as measured by means of recA::lac and sfiA::lac operon fusions. To study the molecular basis of this difference, we have compared the expression of these two operons in strains in which the concentration of LexA repressor was normal (lexA+), zero (spr amber mutation) or higher than normal (plasmid pJL45, carrying the lexA gene linked to the lac promoter). The results indicate (i) that the recA promoter is about 4 times stronger than the sfiA promoter (as measured in the spr strains), (ii) that neither operon has a physiologically significant level of lexA-independent expression (pJL45 strains), and (iii) that the recA operator has about 2.5 times lower affinity than the sfiA operator for LexA repressor (comparison of lex+ and spr strains). Considering our previous results that the sfiA operon (high operator affinity of LexA) is derepressed very rapidly after inducing treatments and that the recA operon (low operator affinity) is repressed very rapidly when induction is stopped, we conclude that differences in operator affinity do not affect inducibility but serve only to set the basal levels of the different SOS functions.     

基因编辑技术对大肠杆菌yjjW基因点突变的两步法策略

【目的】为了实现对大肠杆菌靶基因的点突变,本研究将同源重组系统与CRISPR-Cas9技术相结合,探索一种高效、简捷的两步法策略。【方法】将靶基因的上下游同源臂和标记基因(amp)与pKOV质粒连接,获得pKOV-HR重组质粒。将pKOV-HR转化至大肠杆菌,借助其自身RecA重组系统,介导DNA发生同源重组,获得靶基因敲除菌株。随后,靶基因的点突变采用pSGKP-km和pCasKP-apr双质粒系统。首先,设计与amp结合具有定向引导作用的spacer序列,并利用overlap PCR获得带有靶基因点突变的同源臂,将spacer和同源臂与pSGKP-km质粒连接,获得重组质粒pSGKP-km-spacer-HR;接着,将pSGKP-km-spacer-HR和pCasKP-apr质粒转化至上述敲除菌株;最后,利用质粒表达的Cas9切割蛋白和λ-Red重组蛋白,发生DNA同源重组,获得靶基因点突变菌株。【结果】利用上述方法,既成功获得了大肠杆菌yjjW敲除菌株D7ΔyjjW::ampR,又实现了yjjW第24位碱基T到C的点突变,获得点突变菌株D7yjjW-24。【结论】本研究建立了一种高效、简捷的大肠杆菌靶基因点突变方法,amp基因的插入提供了有效的筛选标记,此外该方法建立的重组质粒pSGKP-km-spacer,可应用于同种菌株其他靶基因的点突变,能够缩短后续实验操作,为基因编辑技术的发展提供了科学理论以及实验操作基础。     

The Escherichia coli recA gene increases resistance of the yeast Saccharomyces cerevisiae to ionizing and ultraviolet radiation

Summary The Escherichia coli recA protein coding region was ligated into an extrachromosomally replicating yeast expression vector downstream of the yeast alcohol dehydrogenase promoter region to produce plasmid pADHrecA. Transformation of the wild-type yeast strains YNN-27 and 7799-4B, as well as the recombination-deficient rad52-t C5-6 mutant, with this shuttle plasmid resulted in the expression of the bacterial 38 kDa RecA protein in exponential phase cells. The wild-type YNN27 and 7799-4B transformants expressing the bacterial recA gene showed increased resistance to the toxic effects of both ionizing and ultraviolet radiation. RecA moderately stimulated the UV-induced mutagenic response of 7799-4B cells. Transformation of the rad52-t mutant with plasmid pADHrecA did not result in the complementation of sensitivity to ionizing radiation. Thus, the RecA protein endows the yeast cells with additional activities, which were shown to be error-prone and dependent on the RAD52 gene.     

A partially deficient mutant, recA1730, that fails to form normal nucleoprotein filaments.

Summary The phenotype of the recA1730 mutant is highly dependent on the level of expression of the RecA1730 protein. If the recA1730 gene was expressed from its own promoter, the cells were deficient in recombination and SOS induction. In contrast, when the recA1730 gene was expressed under the control of recAo98, a constitutive operator that increased the RecA1730 concentration 20-fold, cells became proficient in recombination and SOS induction. Likewise, in crude extracts, fivefold more RecA1730 than RecAwt was required to produce full cleavage of LexA protein. The requirement for a high RecA1730 concentration for recombination and LexA cleavage suggests that the recA1730 defect alters a common reaction step. In fact, in vitro data show that the impaired assembly of RecA1730 protein on single-stranded DNA (ssDNA) can account for the mutant phenotype. Purified RecA1730 protein was assayed in vitro for ssDNA binding and ATPase activities. RecA1730, like RecAwt, retained ssDNA equally well on nitrocellulose filters; this activity was specifically inhibited by a monoclonal anti-RecA antibody. However, RecA1730 protein did not form complete filaments on ssDNA, as shown by two observations: (i) most of the protein did not elute with ssDNA during gel filtration; and (ii) binding of RecA1730 to ssDNA did not protect it from being digested by DNaseI. RecA1730 hydrolysed ATP in high salt but was defective in ssDNA-dependent ATP hydrolysis. These results strongly suggest that RecA1730 binds to ATP and ssDNA but does not form normal nucleoprotein filaments.Abbreviations RecAwt RecA wind-type protein - ssDNA singlestranded DNA - dsDNA dmble-stranded DNA     

Heterogeneity in expression of the <Emphasis Type="Italic">Escherichia coli</Emphasis> colicin K activity gene <Emphasis Type="Italic">cka</Emphasis> is controlled by the SOS system and stochastic factors

Phenotypic diversity provides populations of prokaryotic and eukaryotic organisms with the flexibility required to adapt to and/or survive environmental perturbations. Consequently, there is much interest in unraveling the molecular mechanisms of heterogeneity. A classical example of heterogeneity in Escherichia coli is the subset (3%) of the population that expresses the colicin K activity gene (cka) upon nutrient starvation. Here, we report on the mechanism underlying this variable response. As colicin synthesis is regulated by the LexA protein, the central regulator of the SOS response, we focused on the role of LexA and the SOS system in the variable cka expression. Real-time RT-PCR showed that the SOS system, without exogenous DNA damage, induces moderate levels of cka expression. The use of cka-gfp fusions demonstrated that modification of the conserved LexA boxes in the cka promoter region affected LexA binding affinity and the percentage of cka-gfp expressing cells in the population. A lexA-gfp fusion showed that the lexA gene is highly expressed in a subset of bacteria. Furthermore, cka-gfp fusions cloned into higher copy plasmid vectors increased the percentage of cka-gfp positive bacteria. Together, these results indicate that the bistability in cka expression in the bacterial population is determined by (1) basal SOS activity, (2) stochastic factors and possibly (3) the interplay of LexA dimers at cka operator. Other LexA regulated processes could exhibit similar regulation.     

Interactions between epsilon, the proofreading subunit of DNA polymerase III, and proteins involved in the SOS response of Escherichia coli

Summary Epsilon, a fidelity subunit of Escherichia coli DNA Polymerase III, is encoded by dnaQ ⁺. dnaQ49 is a recessive allele that confers temperature-sensitive and saltsuppressible phenotypes for both replication fidelity and viability. SOS mutagenesis in E. coli is regulated by LexA and requires activated RecA (RecA^*) and the products of the umuDC operon. dnaQ49 strains with various recA, lexA and umuDC alleles were constructed to determine if activities induced as part of the SOS response influence epsilon activity. We found: (1) both UmuDC and RecA^* independently enhance the dnaQ49 mutator phenotype, and (2) expression of RecA^* activity in the absence of UmuDC function increases the temperature sensitivity for viability of dnaQ49. These results support the hypothesis that RecA and one or both of the UmuDC proteins interact with the replication complex during SOS mutagenesis.     

Molecular cloning, sequence and regulation of expression of the recA gene of the phototrophic bacterium Rhodobacter sphaeroides

The recA gene of Rhodobacter sphaeroides 2.4.1 has been isolated by complementation of a UV-sensitive RecA^– mutant of Pseudomonas aeruginosa. Its complete nucleotide sequence consists of 1032 bp, encoding a polypeptide of 343 amino acids. The deduced amino acid sequence displayed highest identity to the RecA proteins from Rhizobium mehloti, Rhizobium phaseoli, and Agrobacterium tumefaciens. An Escherichia coli-like SOS consensus region, which functions as a binding site for the LexA repressor molecule was not present in the 215 by upstream region of the R. sphaeroides recA gene. Nevertheless, by using a recA-lacZ fusion, we have shown that expression of the recA gene of R. sphaeroides is inducible by DNA damage. A recA-defective strain of R. sphaeroides was obtained by replacement of the active recA gene by a gene copy inactived in vitro. The resulting recA mutant exhibited increased sensitivity to UV irradiation, and was impaired in its ability to perform homologous recombination as well as to trigger DNA damage-mediated expression. This is the first recA gene from a Gram-negative bacterium that lacks an E. coli-like SOS box but whose expression has been shown to be DNA damage-inducible and auto-regulated.     

Increase in plasmid transformation efficiency in SOS-induced Escherichia coli cells

Summary UV irradiation of competent cells of Escherichia coli K12 produced an increase in the efficiency of transformation with plasmid DNA. This phenomenon has been called IPTE (increase in plasmid transformation efficiency) and is dependent on the activated state of the RecA protein. IPTE is independent of the lexA, recB recC, and recF genes. It is not related to the size or the replicon type of the plasmid. Furthermore, it is also induced in cells which have been previously treated with other SOS system-inducing agents such as bleomycin, mitomycin C, or nalidixic acid. IPTE is therefore similar to other repair (SOS) functions inducible by DNA damage since all of them are dependent upon activation of the RecA protein. IPTE differs from other SOS functions in the absence of a direct control by the LexA repressor.     

New phenotypes associated with mucAB: alteration of a MucA sequence homologous to the LexA cleavage site.

Most mutagenesis by UV and many chemicals in Escherichia coli requires the products of the umuDC operon or an analogous plasmid-derived operon mucAB. Activated RecA protein is also required for, or enhances, this process. MucA and UmuD proteins share homology with the LexA protein, suggesting that they might interact with the RecA protein as LexA does. We used oligonucleotide-directed mutagenesis to alter a site in MucA homologous to the Ala-Gly cleavage site of LexA. The mutation, termed mucA101(Glu26), results in a change of Gly26 of MucA to Glu26. A lexA(Def) recA441 umuC122::Tn5 strain carrying a mucA101(Glu26)B+ plasmid did not exhibit the greatly increased frequency of spontaneous mutagenesis in response to RecA activation that a strain carrying a mucA+B+ plasmid did but retained a basal recA-dependent ability to confer increased spontaneous mutagenesis that was independent of the state of RecA activation. These results are consistent with a model in which RecA plays two distinct roles in mutagenesis apart from its role in the cleavage of LexA. A pBR322-derived plasmid carrying mucA+B+, but not one carrying mucA101(Glu26)B+, inhibited the UV induction of SOS genes, suggesting that MucA+ and MucA(Glu26) proteins may have different abilities to compete with LexA for activated RecA protein. The spectrum of UV-induced mutagenesis was also altered in strains carrying the mucA101(Glu26) mutation. These results are consistent with the hypothesis that activated RecA protein interacts with wild-type MucA protein, possibly promoting proteolytic cleavage, and that this interaction is responsible for facilitating certain mutagenic processes.     

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.     

Purification of an SOS repressor from Bacillus subtilis.

We have identified in Bacillus subtilis a DNA-binding protein that is functionally analogous to the Escherichia coli LexA protein. We show that the 23-kDa B. subtilis protein binds specifically to the consensus sequence 5'-GAACN4GTTC-3' located within the putative promoter regions of four distinct B. subtilis DNA damage-inducible genes: dinA, dinB, dinC, and recA. In RecA+ strains, the protein's specific DNA binding activity was abolished following treatment with mitomycin C; the decrease in DNA binding activity after DNA damage had a half-life of about 5 min and was followed by an increase in SOS gene expression. There was no detectable decrease in DNA binding activity in B. subtilis strains deficient in RecA (recA1, recA4) or otherwise deficient in SOS induction (recM13) following mitomycin C treatment. The addition of purified B. subtilis RecA protein, activated by single-stranded DNA and dATP, abolished the specific DNA binding activity in crude extracts of RecA+ strains and strains deficient in SOS induction. We purified the B. subtilis DNA-binding protein more than 4,000-fold, using an affinity resin in which a 199-bp DNA fragment containing the dinC promoter region was coupled to cellulose. We show that B. subtilis RecA inactivates the DNA binding activity of the purified B. subtilis protein in a reaction that requires single-stranded DNA and nucleoside triphosphate. By analogy with E. coli, our results indicate that the DNA-binding protein is the repressor of the B. subtilis SOS DNA repair system.     

Measurement of SOS expression in individual Escherichia coli K-12 cells using fluorescence microscopy

Many recombination, DNA repair and DNA replication mutants have high basal levels of SOS expression as determined by a sulAp-lacZ reporter gene system on a population of cells. Two opposing models to explain how the SOS expression is distributed in these cells are: (i) the 'Uniform Expression Model (UEM)' where expression is evenly distributed in all cells or (ii) the 'Two Population Model (TPM)' where some cells are highly induced while others are not at all. To distinguish between these two models, a method to quantify SOS expression in individual bacterial cells was developed by fusing an SOS promoter (sulAp) to the green fluorescent protein (gfp) reporter gene and inserting it at attlambda on the Escherichia coli chromosome. It is shown that the fluorescence in sulAp-gfp cells is regulated by RecA and LexA. This system was then used to distinguish between the two models for several mutants. The patterns displayed by priA, dnaT, recG, uvrD, dam, ftsK, rnhA, polA and xerC mutants were explained best by the TPM while only lexA (def), lexA3 (ind-) and recA defective mutants were explained best by the UEM. These results are discussed in a context of how the processes of DNA replication and recombination may affect cells in a population differentially.     

Genetic and morphological characterization of ftsB and nrdB mutants of Escherichia coli.

The ftsB gene of Escherichia coli is believed to be involved in cell division. In this report, we show that plasmids containing the nrdB gene could complement the ftsB mutation, suggesting that ftsB is an allele of nrdB. We compared changes in the cell shape of isogenic nrdA, nrdB, ftsB, and pbpB strains at permissive and restrictive temperatures. Although in rich medium all strains produced filaments at the restrictive temperature, in minimal medium only a 50 to 100% increase in mean cell mass occurred in the nrdA, nrdB, and ftsB strains. The typical pbpB cell division mutant also formed long filaments at low growth rates. Visualization of nucleoid structure by fluorescence microscopy demonstrated that nucleoid segregation was affected by nrdA, nrdB, and ftsB mutations at the restrictive temperature. Measurements of beta-galactosidase activity in lambda p(sfiA::lac) lysogenic nrdA, nrdB, and ftsB mutants in rich medium at the restrictive temperature showed that filamentation in the nrdA mutant was caused by sfiA (sulA) induction, while filamentation in nrdB and ftsB mutants was sfiA independent, suggesting an SOS-independent inhibition of cell division.     

RecA protein of Escherichia coli has a third essential role in SOS mutator activity.

The DNA damage-inducible SOS response of Escherichia coli includes an error-prone translesion DNA replication activity responsible for SOS mutagenesis. In certain recA mutant strains, in which the SOS response is expressed constitutively, SOS mutagenesis is manifested as a mutator activity. Like UV mutagenesis, SOS mutator activity requires the products of the umuDC operon and depends on RecA protein for at least two essential activities: facilitating cleavage of LexA repressor to derepress SOS genes and processing UmuD protein to produce a fragment (UmuD') that is active in mutagenesis. To determine whether RecA has an additional role in SOS mutator activity, spontaneous mutability (tryptophan dependence to independence) was measured in a family of nine lexA-defective strains, each having a different recA allele, transformed or not with a plasmid that overproduces either UmuD' alone or both UmuD' and UmuC. The magnitude of SOS mutator activity in these strains, which require neither of the two known roles of RecA protein, was strongly dependent on the particular recA allele that was present. We conclude that UmuD'C does not determine the mutation rate independently of RecA and that RecA has a third essential role in SOS mutator activity.