Wild-type lexA Research Articles

SOS involvement in stress-inducible biofilm formation

Bacterial biofilm formation can be induced by antimicrobial and DNA damage agents. These agents trigger the SOS response, in which SOS sensor RecA stimulates auto-cleavage of repressor LexA. These observations lead to a hypothesis of a connection between stress-inducible biofilm formation and the RecA-LexA interplay. To test this hypothesis, three biofilm assays were conducted, viz. the standard 96-well assay, confocal laser scanning microscopy, and the newly developed biofilm-on-paper assay. It was found that biofilm stimulation by the DNA replication inhibitor hydroxyurea was dependent on RecA and appeared repressed by the non-cleavable LexA of Pseudomonas aeruginosa. Surprisingly, deletion of lexA led to reduction of both normal and stress-inducible biofilm formation, suggesting that the wild-type LexA contributes to biofilm formation. The decreases was not the result of poor growth of the mutants. These results suggest SOS involvement in hydroxyurea-inducible biofilm formation. In addition, with the paper biofilm assay, it was found that degradation of the biofilm matrix DNA by DNase I appeared to render the biofilms susceptible to the replication inhibitor. The puzzling questions concerning the roles of LexA in DNA release in the biofilm context are discussed.

4011 POSTER Results of a phase 3, randomized study of patients with advanced renal cell carcinoma (RCC) and poor prognostic features treated with temsirolimus, interferon-a or the combination of temsirolimus + interferon-a

This chapter describes a system for analyzing both homo- and heterodimerization of full-length proteins and protein fragments in an Escherichia coli background. The characterization of protein-protein interactions can reveal much about the structure and function of individual proteins and protein complexes. Many methods designed to identify these interactions have been reported, including yeast two hybrid and plasmid-based bacterial systems. This system has been successful in determining interactions between proteins involved in the biosynthesis of the polysialic acid capsule of E. coli K1. These interactions include those between full-length proteins as well as those between full-length proteins and protein fragments. However, it is not necessary to restrict the system's use to studying only Escherichia coli protein interactions. Another feature of this system is that the detection of interaction between fusions is not disrupted by the tandem expression of unfused subunits in the host strain. Chloramphenicol acetyltransferase (CAT) fused to the wild-type LexA DNA binding domain(DBD) efficiently repressed lacZ transcription in SU101, even though this strain carries a transposon that expresses subunits of the same type I CAT. This data also illustrates that some multimeric proteins can be studied using this system, since CAT is a well-characterized homotrimer.

Improved Model of a LexA Repressor Dimer Bound to recA Operator

A complete three dimensional model for the LexA repressor dimer bound to the recA operator site consistent with relevant biochemical and biophysical data for the repressor was proposed from our laboratory when no crystal structure of LexA was available. Subsequently, the crystal structures of four LexA mutants Δ1–67 S119A, S119A, G85D and Δ1-67 quadruple mutant in the absence of operator were reported. It is examined in this paper to what extent our previous model was correct and how, using the crystal structure of the operator-free LexA dimer we can predict an improved model of LexA dimer bound to recA operator. In our improved model, the C-domain dimerization observed repeatedly in the mutant operator-free crystals is retained but the relative orientation between the two domains within a LexA molecule changes. The crystal structure of wild type LexA with or without the recA operator cannot be solved as it autocleaves itself. We argue that the ‘cleavable’ cleavage site region found in the crystal structures is actually the more relevant form of the region in wildtype LexA since it agrees with the value of the pre-exponential Arrhenius factor for its auto- cleavage, absence of various types of trans-cleavages, difficulty in modifying the catalytic serine by diisopropyl flourophosphate and lack of cleavage at Arg 81 by trypsin; hence the concept of a ‘conformational switch’ inferred from the crystal structures is meaningless.

Use of LexA-based system to identify protein-protein interactions in vivo

Publisher Summary This chapter describes a system for analyzing both homo- and heterodimerization of full-length proteins and protein fragments in an Escherichia coli background. The characterization of protein-protein interactions can reveal much about the structure and function of individual proteins and protein complexes. Many methods designed to identify these interactions have been reported, including yeast two hybrid and plasmid-based bacterial systems. This system has been successful in determining interactions between proteins involved in the biosynthesis of the polysialic acid capsule of E. coli K1. These interactions include those between full-length proteins as well as those between full-length proteins and protein fragments. However, it is not necessary to restrict the system's use to studying only Escherichia coli protein interactions. Another feature of this system is that the detection of interaction between fusions is not disrupted by the tandem expression of unfused subunits in the host strain. Chloramphenicol acetyltransferase (CAT) fused to the wild-type LexA DNA binding domain(DBD) efficiently repressed lacZ transcription in SU101, even though this strain carries a transposon that expresses subunits of the same type I CAT. This data also illustrates that some multimeric proteins can be studied using this system, since CAT is a well-characterized homotrimer.

Analysis of the expression of the Rhodobacter sphaeroides lexA gene.

The regulation of the Rhodobacter sphaeroides lexA gene has been analyzed using both gel-mobility experiments and lacZ gene fusions. PCR-mediated mutagenesis demonstrated that the second GAAC motif in the sequence GAACN7GAACN7GAAC located upstream of the R. sphaeroides lexA gene is absolutely necessary for its DNA damage-mediated induction. Moreover, mutagenesis of either the first or the third GAAC motif in this sequence reduced, but did not abolish, the inducibility of the R. sphaeroides lexA gene. A R. sphaeroides lexA-defective (Def) mutant has also been constructed by replacing the active lexA gene with an inactivated gene copy constructed in vitro. Crude extracts of the R. sphaeroides lexA(Def) strain are unable to form any protein-DNA complex when added to the wild-type lexA promoter of R. sphaeroides. Likewise, the R. sphaeroides lexA(Def) cells constitutively express the recA and lexA genes. All these data clearly indicate that the lexA gene product is the negative regulator of the R. sphaeroides SOS response. Furthermore, the morphology, growth and viability of R. sphaeroides lexA(Def) cultures do not show any significant change relative to those of the wild-type strain. Hence, R. sphaeroides is so far the only bacterial species whose viability is known not to be affected by the presence of a lexA(Def) mutation.

Identification of additional genes belonging to the LexA regulon in Escherichia coli.

Exposure of Escherichia coli to a variety of DNA-damaging agents results in the induction of the global 'SOS response'. Expression of many of the genes in the SOS regulon are controlled by the LexA protein. LexA acts as a transcriptional repressor of these unlinked genes by binding to specific sequences (LexA boxes) located within the promoter region of each LexA-regulated gene. Alignment of 20 LexA binding sites found in the E. coli chromosome reveals a consensus of 5'-TACTG(TA)5CAGTA-3'. DNA sequences that exhibit a close match to the consensus are said to have a low heterology index and bind LexA tightly, whereas those that are more diverged have a high heterology index and are not expected to bind LexA. By using this heterology index, together with other search criteria, such as the location of the putative LexA box relative to a gene or to promoter elements, we have performed computational searches of the entire E. coli genome to identify novel LexA-regulated genes. These searches identified a total of 69 potential LexA-regulated genes/operons with a heterology index of <15 and included all previously characterized LexA-regulated genes. Probes were made to the remaining genes, and these were screened by Northern analysis for damage-inducible gene expression in a wild-type lexA+ cell, constitutive expression in a lexA(Def) cell and basal expression in a non-inducible lexA(Ind-) cell. These experiments have allowed us to identify seven new LexA-regulated genes, thus bringing the present number of genes in the E. coli LexA regulon to 31. The potential function of each newly identified LexA-regulated gene is discussed.

The mutA mistranslator tRNA-induced mutator phenotype requires recA and recB genes, but not the derepression of lexA-regulated functions.

The mapping of mutA and mutC mutator alleles to the glyV and glyW glycine tRNA genes, respectively, and the subsequent discovery that the mutA phenotype is abolished in a DeltarecA strain raise the possibility that asp --> gly misinsertion may induce a novel mutagenic pathway. The recA requirement suggests three possibilities: (i) the SOS mutagenesis pathway is activated in mutA cells; (ii) loss of recA function interferes with mutA-promoted asp --> gly misinsertion; or (iii) a hitherto unrecognized recA-dependent mutagenic pathway is activated by translational stress. By assaying the expression levels of a reporter plasmid bearing a umuC :lacZ fusion, we show that the SOS regulon is not in a derepressed state in mutA cells. Neither overexpression of the lexA gene through a multicopy plasmid nor replacement of the wild-type lexA allele with the lexA1[Ind-] allele interferes with the expression of the mutA phenotype. The mutA phenotype is unaffected in cells defective for dinB, as shown here, and is unaffected in cells defective for umuD and umuC genes, as shown previously. We show that mutA-promoted asp --> gly misinsertion occurs in recA- cells and, therefore, the requirement for recA is 'downstream' of mistranslation. Finally, we show that the mutA phenotype is abolished in cells deficient for recB, suggesting that cellular recombination functions may be required for the expression of the mutator phenotype. We propose that translational stress induces a previously unrecognized mutagenic pathway in Escherichia coli.

Mutant LexA proteins with specific defects in autodigestion.

In self-processing biochemical reactions, a protein or RNA molecule specifically modifies its own structure. Many such reactions are regulated in response to the needs of the cell by an interaction with another effector molecule. In the system we study here, specific cleavage of the Escherichia coli LexA repressor, LexA cleaves itself in vitro at a slow rate, but in vivo cleavage requires interaction with an activated form of RecA protein. RecA acts indirectly as a coprotease to stimulate LexA autodigestion. We describe here a new class of lexA mutants, lexA (Adg-; for autodigestion-defective) mutants, termed Adg- for brevity. Adg- mutants specifically interfered with the ability of LexA to autodigest but left intact its ability to undergo RecA-mediated cleavage. The data are consistent with a conformational model in which RecA favors a reactive conformation capable of undergoing cleavage. To our knowledge, this is the first example of a mutation in a regulated self-processing reaction that impairs the rate of self-processing without markedly affecting the stimulated reaction. Had wild-type lexA carried such a substitution, discovery of its self-processing would have been difficult; we suggest that, in other systems, a slow rate of self-processing has prevented recognition that a reaction is of this nature.

Interaction of Escherichia coli RecA Protein with LexA Repressor: II. INHIBITION OF DNA STRAND EXCHANGE BY THE UNCLEAVABLE LexA S119A REPRESSOR ARGUES THAT RECOMBINATION AND SOS INDUCTION ARE COMPETITIVE PROCESSES

The Escherichia coli RecA protein is involved in SOS induction, DNA repair, and homologous recombination. In vitro, RecA protein serves as a co-protease to cleave LexA repressor, the repressor of the SOS regulon; in addition, RecA protein promotes homologous pairing and DNA strand exchange, steps important to homologous recombination and DNA repair. To determine if these two functions of RecA protein are competing or parallel, the effect of uncleavable LexA S119A repressor on RecA protein-dependent activities was examined. LexA S119A repressor inhibits both the single-stranded DNA (ssDNA)-dependent ATP hydrolysis and DNA strand exchange activities of RecA protein. As for wild-type LexA repressor (Rehrauer, W. M., Lavery, P. E., Palmer, E. L., Singh, R. N., and Kowalczykowski, S. C. (1996) J. Biol. Chem. 271, 23865-23873), inhibition of ATP hydrolysis is dependent upon the presence of E. coli single-stranded DNA binding (SSB) protein, arguing that LexA repressor affects the competition between RecA protein and SSB protein for ssDNA binding sites. In contrast, inhibition of DNA strand exchange activity is SSB protein-independent, suggesting that LexA S119A repressor blocks a site required for DNA strand exchange. These results imply that there is a common site on the RecA protein filament for secondary DNA and LexA repressor binding and raise the possibility that the recombination and co-protease activities of the RecA protein filament are competitive.

Relationship between the functional regions of the RecA protein and ATP hydrolysis in UV-irradiated Escherichia coli cells

The time course of the intracellular ATP concentration in several UV-irradiated RecA protease constitutuve (Cptc) mutants of E. coli has been studied. All Cptc mutants harboring a mutation in region 3 of the RecA protein (including amino acid residues 298–301) increased ATP after UV damage but without any subsequent decrease. Nevertheless, these mutants induced the SOS response after UV irradiation. Likewise, truncated RecA proteins lacking region 3 are also unable to carry out massive ATP hydrolysis in UV-irradiated cells. On the other hand, mutants in region 1 (including amino acids 25–39) or 2 (amino acids 157–184) of the RecA protein showed increase in ATP concentration during the first 20 min following UV irradiation, which dropped afterwards to the basal level. All these data indicate that region 3 of the RecA protein must be involved in the ATP hydrolysis process. Futhermore, a relationship between the quantity of the UV-mediated ATP produced and the strength of the different RecA Cptc mutants has also been found. Accordingly, both lexA71::Tn5 and null lexA mutants of E. coli only show a cellular ATP increase after UV irradiation when containing a multicopy plasmid carrying either a wild-type lexA or a lexA(Ind−) gene.

In vitro analysis of mutant LexA proteins with an increased rate of specific cleavage

Specific cleavage of LexA repressor plays a crucial role in the SOS response of Escherichia coli. In vivo, cleavage requires an activated form of RecA protein. However, previous work has shown that the mechanism of cleavage is unusual, in that the chemistry of cleavage is probably carried out by residues in the repressor, and not those in RecA; RecA appears to facilitate this reaction, acting as a coprotease. We recently described a new type of lexA mutation, a class termed lexA (Ind S) and here called Ind S, that confers an increased rate of in vivo cleavage. Here, we have characterized the in vitro cleavage of these Ind S mutant proteins, and of several double mutant proteins containing an Ind S mutation and one of several mutations, termed Ind −, that decrease the rate of cleavage. We found, first, that the autodigestion reaction for the Ind S mutant proteins had a higher maximum rate and a lower apparent p K a than wild-type LexA. Second, the Ind S mutations had little or no effect on the rate of RecA-mediated cleavage, measured at low protein concentrations, implying that the value of k cat K m was unaffected. Third, the rate of autodigestion for the double-mutant proteins, relative to wild-type, was about that rate predicted from the product of the effects of the two single mutations. Finally, by contrast, these proteins displayed the same rate of RecA-mediated cleavage as did the single Ind − mutant protein. We interpret these data to mean that the Ind S mutations mimic to some extent the effect of RecA on cleavage, perhaps by favoring a conformational change in LexA. We present and analyze a model that embodies these conclusions.

Mutant LexA proteins with an increased rate of in vivo cleavage.

LexA repressor of Escherichia coli is inactivated by a specific cleavage reaction that requires activated RecA protein in vivo. This cleavage reaction can proceed in vitro in the presence of activated RecA or as an intramolecular RecA-independent reaction, termed autodigestion, that is stimulated by alkaline pH. Here we describe a set of LexA mutant proteins that undergo a greatly increased rate of specific cleavage in vivo, compared with wild-type LexA. Efficient in vivo cleavage of these mutant proteins also took place without RecA. Several lines of evidence suggest that cleavage occurred via a mechanism similar to autodigestion. These mutations changed Gln-92, which lies near the cleavage site, to tyrosine, phenylalanine, or tryptophan. The latter mutation increased the rate of cleavage approximately 500-fold. These findings imply that the rate of wild-type LexA cleavage has been optimized during evolution to make the SOS system properly responsive to DNA-damaging treatments. Availability of these mutants will aid in the understanding of rate-limiting steps in intramolecular reactions.

Repression of the E coli recA gene requires at least two LexA protein monomers

To analyze the DNA binding domain of E coli LexA reprressor and to test whether the repressor binds as a dimer to DNA, negative dominant lexA mutations affecting the binding domain have been isolated. A large number of amino acid substitutions between amino acid positions 39 and 46 were introduced using cassette mutagenesis. Mutants defective in DNA binding were identified and then examined for dominance to lexA +. A number of substitutions weakened repressor function partially, whereas other substitutions led to a repressor with no demonstrable activity and a defective dominant phenotype. Since the LexA binding site has dyad symmetry, we infer that this dominance results from interaction of monomers of wild-type LexA protein with mutant monomers and that an oligomeric form of repressor binds to operator. The binding of LexA protein to operator DNA was investigated further using a mutant protein, LexA408, which recognizes a symmetrically altered operator mutant but not wild-type operator. A mixture of mutant LexA408 and LexA + proteins, but neither individual protein, bound to a hybrid recA operator consisting of mutant and wild-type operator half sites. These results suggest that at least 1 LexA protein monomer interacts with each operator half site. We discuss the role of LexA oligomer formation in binding of LexA to operator DNA.

Autodigestion and RecA-dependent cleavage of Ind− mutant LexA proteins

The LexA repressor of Escherichia coli undergoes a specific cleavage reaction in vivo, an event that leads to derepression of the SOS regulon and requires an activated form of RecA protein. In vitro, cleavage requires RecA at neutral pH; at alkaline pH, a spontaneous cleavage reaction termed autodigestion takes place. Both autodigestion and RecA-mediated cleavage cut the same bond, and are observed for the same set of substrates, suggesting that RecA acts indirectly to stimulate LexA self-cleavage at neutral pH, perhaps binding to LexA and acting as an allosteric effector. We previously isolated a set of lexA(Ind−) mutants that are deficient in in vivo RecA-mediated cleavage but retain significant repressor function. Here, we describe the in vitro cleavage of purified mutant proteins. All of those tested were deficient in both cleavage reactions. Although most of them were equally deficient in both reactions, some were more deficient in one reaction than the other. Several mutant proteins appeared to have defects in binding to RecA. Autodigestion of all but one of the poorly cleavable mutant proteins reached a maximum rate at pH around 10, as does wild-type LexA. The exception was KR156, which changed Lys156, a residue previously implicated in the mechanism of cleavage, to Arg, another basic residue; for this protein, the rate of autodigestion increased with pH at values above 11. RecA-mediated cleavage of KR156 was 1% the wild-type rate at pH 7, but increased with increasing pH to a plateau at pH 9.5, where the rate was 40% the wild-type rate. In contrast, an essentially constant rate was observed for wild-type LexA over the pH range 6 to 11. We suggest, first, that deprotonation of Arg156 and, by inference, Lys156 in the wild-type protein, is required for both autodigestion and RecA-mediated cleavage; and second, that RecA acts to reduce the pKa of Lys156, allowing efficient cleavage of wild-type repressor under physiological conditions.

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.

Effect of a lexA41(Ts) mutation on DNA repair in recA(Def) derivatives of Escherichia coli K-12.

Derivatives of Escherichia coli K-12 carrying a deletion of the recA gene survive exposure to UV (254 nm) better if they also contain the lexA41 mutation which codes for a labile LexA protein. This effect of the lexA41 mutation is not observed in comparable strains carrying a uvr A6 mutation. Using two independent methods to detect pyrimidine dimers we found that UV irradiated RecA deficient cells removed dimers from their DNA more rapidly if they contained the lexA41 mutation than if they contained the wild-type lexA gene. Our results are consistent with the idea that a relatively high level of UvrABC incision nuclease resulting from inefficient repression of the corresponding genes by the labile LexA41 protein facilitates excision of pyrimidine dimers from the DNA of UV irradiated cells.

Cleavage of the Escherichia coli lexA protein by the recA protease.

The recA and lexA proteins of EScherichia coli are involved in a complex regulatory circuit that allows the expression of a diverse set of functions after DNA damage or inhibition of DNA replication. Exponentially growing cells contain a low level of recA protein, and genetic evidence suggests that lexA protein is involved in its regulation, perhaps as a simple repressor. Recent models for recA derepression after DNA damage have suggested that an early event in this process is the proteolytic cleavage of lexA protein, leading to high-level expression of recA. We present several lines of evidence that the specific protease activity of the recA protein, previously described with the lambda repressor as substrate, is capable of cleaving the wild-type lexA+ protein. First, lexA protein can be cleaved in vitro under the same conditions as prevously described for lambda repressor cleavage in a reaction requring both recA protease and ATP or an analogue, adenosine 5'-[lambda-thio]-triphosphate. Second, lexA protein can be observed in vivo as a physical entity after infection with lambda lexA+ transducing phage of host strains containing ittle or no active protease, but not in strains containing high levels of active protease. Finally, infection of host cells containing active protease with a lambda lexA+ transducing phage does not lead to repression of recA, but does so in cells lacking active protease. In all of these conditions the mutant lexA3 protein is largely resistant to inactivation or cleavage; this resistance can explain the dominant phenotype of lexA3 over lexA+. We discuss models for recA derepression and re-establishment of repression which propose that modulation of the protease activity of recA protein regulates both of these transitions.

The lexA gene product represses its own promoter.

The products of the lexA and recA genes of Escherichia coli regulate the cellular response to DNA damage (the SOS response). Here we describe the cloning of the wild-type lexA gene and the identification of its 24,000-dalton protein product. We also describe construction, by recombination in vitro, of a phage that bears the lexA promoter fused to the lacZ gene. Experiments with this fusion phage and with multicopy plasmids that carry the lexA gene showed that the lexA gene product represses of its own promoter. This repression occurs even if the cell has no recA gene, showing that the lexA protein need not be complexed to the recA protein for activity. Moreover, the presence of multicopy plasmids that carry the lexA gene blocks expression of all SOS responses tested. This presumably results from two effects: (i) repression of the recA gene, the product of which is required to activate many of these responses; and (ii) direct repression of other functions involved in the SOS response.

Identification of the lexA gene product of Escherichia coli K-12.

The Escherichia coli lexA gene encodes a product important in induction of the recA gene and the expression of various cellular functions, including mutagenesis and prophage induction. As a start in a biochemical analysis of the lexA function, a family of lambda transducing phages carrying lexA+, lexA3, lexA3 spr-54, and lexA3 spr-55 alleles of the lexA gene was isolated and characterized. Polypeptides synthesized by these phages were examined. lambdalexA+ made a distinctive protein 24 kilodaltons (kd) in size. Lambda lexA3, which encodes an active mutant form of the protein dominant to wild-type function, made a slightly larger protein 25 kd in size. The latter protein was shown to be the mutant lexA3 gene product by the fact that lambda lexA3 spr-55, which carries an amber mutation in lexA3, made the 25-kd protein in hosts with an amber suppressor but not in a suppressor-free host. In hosts carrying a multicopy lexA3 plasmid, neither the 25-kd nor the 24-kd protein was made. This result suggests that lexA is autoregulated and that expression of the 24-kd protein made by lambda lexA+ is subject to the same controls. This and other evidence argues that the 24-kd protein is the product of the wild-type lexA+ gene.