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PubMed1988

Increased expression of the Escherichia coli umuDC operon restores SOS mutagenesis in lexA41 cells.

The lexA41 allele of Escherichia coli encodes a semidefective mutant repressor that is also resistant to RecA facilitated cleavage. Cells harboring the lexA41 allele were found previously to repress only a subset of operons in the SOS regulon. lexA41 cells cannot promote SOS mutagenesis, presumably because one or more operons required for mutagenesis are repressed by this mutant repressor. Using the lac regulatory system to increase the expression of the umuDC operon, we were able to restore mutagenesis in the lexA41 mutant. We conclude that the products of the umuDC operon appear to be uniquely limiting in this mutant.

Alleles↗
PubMed1999

Toxic mutations in the recA gene of E. coli prevent proper chromosome segregation.

The recA gene of Escherichia coli is the prototype of the recA/RAD51/DMC1/uvsX gene family of strand transferases involved in genetic recombination. In order to find mutations in the recA gene important in catalytic turnover, a genetic screen was conducted for dominant lethal mutants. Eight single amino acid substitution mutants were found to prevent proper chromosome segregation and to kill cells in the presence or absence of an inducible SOS system. All mutants catalyzed some level of recombination and constitutively stimulated LexA cleavage. The mutations occur at the monomer-monomer interface of the RecA polymer or at residues important in ATP hydrolysis, implicating these residues in catalytic turnover. Based on an analysis of the E96D mutant, a model is presented in which slow RecA-DNA dissociation prevents chromosome segregation, engendering lexA-independent, lethal filamentation of cells.

Adenosine Triphosphate↗
PubMed2002

Mistranslation induced by streptomycin provokes a RecABC/RuvABC-dependent mutator phenotype in Escherichia coli cells.

Translational stress-induced mutagenesis (TSM) refers to the mutator phenotype observed in Escherichia coli cells expressing a mutant allele (mutA or mutC) of the glycine tRNA gene glyV (or glyW). Because of an anticodon mutation, expression of the mutA allele results in low levels of Asp-->Gly mistranslation. The mutA phenotype does not require lexA-regulated SOS mutagenesis functions, and appears to be suppressed in cells defective for RecABC-dependent homologous recombination functions. To test the hypothesis that the TSM response is mediated by non-specific mistranslation rather than specific Asp-->Gly misreading, we asked if streptomycin (Str), an aminoglycoside antibiotic known to promote mistranslation, can provoke a mutator phenotype. We report that Str induces a strong mutator phenotype in cells bearing certain alleles of rpsL, the gene encoding S12, an essential component of the ribosomal 30 S subunit. The phenotype is strikingly similar to that observed in mutA cells in its mutational specificity, as well as in its requirement for RecABC-mediated homologous recombination functions. Expression of Str-inducible mutator phenotype correlates with mistranslation efficiency in response to Str. Thus, mistranslation in general is able to induce the TSM response. The Str-inducible mutator phenotype described here defines a new functional class of rpsL alleles, and raises interesting questions on the mechanism of action of Str, and on bacterial response to antibiotic stress.

Anti-Bacterial Agents↗
PubMed2002

Characterization of Xanthomonas axonopodis pv. citri LexA: recognition of the LexA binding site.

Levels of l exA transcripts are markedly increased upon exposure of Xanthomonas axonopodis pathovar citri ( X. a. pv. citri) to the DNA-damaging agent mitomycin C. Preliminary electrophoretic mobility-shift data led us to propose that binding of LexA protein to the sequence upstream of the lexA coding region is responsible for low promoter activity in the uniduced state. We determined that the LexA protein binds to the region located between the transcription start site and the translation initiation codon of the lexA gene of X. a. pv. citri. Using a DNase I footprinting technique, we identified a 19-bp palindromic sequence, TTAGTAGTAATACTACTAA (TTAGN(11)CTAA), located in this region as the binding sequence for the LexA protein of X. a. pv. citri, and showed that the two halves of the palindrome have to be in the inverted repeat orientation to permit binding of LexA. We also showed that almost any mutation in this sequence, including changes in the length of the spacer region of the palindrome, destroyed its ability to bind LexA both in vitro and in vivo.

Bacterial Proteins↗
PubMed1993

UmuC product contributes to the inhibition of dimer excision produced by thymine-less-amino acid-less pretreatment in UV-irradiated Escherichia coli.

In UV-irradiated Escherichia coli, predamaged by thymine-amino acid starvation or a UV predose, a large amount of dimers may remain unexcised and may be tolerated by an error-free mechanism, which requires the function of uvr, recA and lexA genes. A possible role of the umuC gene in both the inhibition of dimer excision and the toleration of unexcised dimers is investigated. Data suggest that the UmuC gene product is not absolutely necessary for the inhibition of dimer excision in UV-irradiated thymine-less-amino acid-less pretreated cells, but that it contributes to it. However, the UmuC product does not seem to be involved in the toleration of unexcised dimers which is dependent on uvr, recA and lexA.

Bacterial Proteins↗
PubMed1999

An Arabidopsis 14-3-3 protein can act as a transcriptional activator in yeast.

The 14-3-3 proteins are a group of highly conserved and widely distributed eukaryotic proteins with diverse functions. One 14-3-3 protein, AFT1 from Arabidopsis thaliana, was found to be able to activate transcription in yeast. When fused to the DNA-binding domain of a bacterial protein LexA, AFT1 can activate transcription of reporter genes that contain LexA operator sequences in their promoters. Although the in vivo function of AFT1 is not completely known, its similarity to previously identified proteins found in transcription complexes of Arabidopsis and maize suggests that AFT1 and some other 14-3-3 proteins may activate gene expression in other systems as well.

14-3-3 Proteins↗
PubMed2000

Interaction of the PA2G4 (EBP1) protein with ErbB-3 and regulation of this binding by heregulin.

The processes by which ErbB-3, an inactive tyrosine kinase, exerts its biological effects are poorly understood. Using the yeast two-hybrid system, we have isolated an ErbB-3 binding protein (Ebp1) that interacts with the juxtamembrane domain of ErbB-3. This protein is identical to that predicted to be encoded for by the human PA2G4 gene. Ebp1 is the human homologue of a previously identified cell cycle-regulated mouse protein p38-2G4. Two transcripts of ebp1 mRNA (1.7 and 2.2 kb) were detected in several normal human organs. The interaction of Ebp1 with ErbB-3 was examined in vitro and in vivo. The first 15 amino acids of the juxtamembrane domain of ErbB-3 were essential for Ebp1 binding in vitro. Treatment of AU565 cells with the ErbB-3 ligand heregulin resulted in dissociation of Ebp1 from ErbB-3. Ebp1 translocated from the cytoplasm into the nucleus following heregulin stimulation. These findings suggest that Ebp1 may be a downstream member of an ErbB-3-regulated signal transduction pathway.

Animals↗
PubMed2006

Identification of the Acidobacterium capsulatum LexA box reveals a lateral acquisition of the Alphaproteobacteria lexA gene.

Acidobacterium capsulatum is the most thoroughly studied species of a new bacterial phylogenetic group designated the phylum Acidobacteria. Through a tblastn search, the A. capsulatum lexA gene has been identified, and its product purified. Electrophoretic mobility shift assays have shown that A. capsulatum LexA protein binds specifically to the direct repeat GTTCN(7)GTTC motif. Strikingly, this is also the LexA box of the Alphaproteobacteria, but had not previously been described outside this subclass of the Proteobacteria. In addition, a phylogenetic analysis of the LexA protein clusters together Acidobacterium and the Alphaproteobacteria, moving the latter away from their established phylogenetic position as a subclass of the Proteobacteria, and pointing to a lateral gene transfer of the lexA gene from the phylum Acidobacteria, or an immediate ancestor, to the Alphaproteobacteria. Lastly, in vivo experiments demonstrate that the A. capsulatum recA gene is DNA-damage inducible, despite the fact that a LexA-binding sequence is not present in its promoter region.

Alphaproteobacteria↗
PubMed2005

Characterization of functional domains of the Vibrio cholerae virulence regulator ToxT.

The toxT gene encodes an AraC family transcriptional activator that is responsible for regulating virulence gene expression in Vibrio cholerae. Analysis of ToxT by dominant/negative assays and a LexA-based reporter system demonstrated that the N-terminus of the protein contains dimerization determinants, indicating that ToxT likely functions as a dimer. Additionally, a natural variant of ToxT with only 60% identity in the N-terminus, as well as a mutant form of ToxT with an altered amino acid in the N-terminus (L107F), exhibited altered transcriptional responses to bile, suggesting that the N-terminus is involved in environmental sensing. The C-terminus of ToxT functions to bind DNA and requires dimerization for stable binding in vitro, as demonstrated by gel shift analysis. Interestingly, a dimerized form of the ToxT C-terminus is able to bind DNA in vitro but is transcriptionally inactive in vivo, indicating that the N-terminus contains determinants that are required for transcriptional activation. These results provide a model for a two-domain structure for ToxT, with an N-terminal dimerization and environmental sensing domain and a C-terminal DNA-binding domain; unlike other well-studied AraC family proteins, both domains of ToxT appear to be required for transcriptional activation.

Amino Acid Substitution↗
PubMed2006

Analysis of the interaction between hPFTAIRE1 and PLZF in a yeast two-hybrid system.

hPFTAIRE1 is a Cdc2-related kinase family member. To search its substrates and regulatory proteins, hPFTAIRE1 was fused to LexA and used as a bait to screen a human brain LexA two-hybrid library. In this screening, seven hPFTAIRE1 interacting proteins, including promyelocytic leukemia zinc finger (PLZF), were obtained. The interaction between PLZF and hPFTAIRE1 was confirmed by beta-galactosidase assay and Leu growth activity. PLZF encodes a transcription factor belonging to the POZ/BTB domain and Krüpel zinc finger (POK) family. The highly conserved POZ/BTB domain plays a critical role in protein-protein interaction. We deleted the POZ/BTB and Krüpel zinc finger domains, respectively, and observed the interaction between hPFTAIRE1 and truncated PLZFs by liquid beta-galactosidase activity assay. A weak interaction was detected between hPFTAIRE1 and PLZF. We also observed the interaction between PLZF and another Cdc2-related kinase, PCTAIRE1. A similar result was observed. The interaction between PLZF and hPFTAIRE1 or PCTAIRE1 was confirmed by co-immunoprecipitation assay in a yeast system. PLZF is a phosphoprotein and plays multiple roles during cell growth. Our results suggest that hPFTAIRE1 and PCTAIRE1 may play important roles in the functional regulation of PLZF.

Animals↗
PubMed1983

The Salmonella typhimurium LT2 uvrD gene is regulated by the lexA gene product.

The uvrD gene product apparently plays a role in the repair of UV damage, in mismatch repair, and in genetic recombination. A lower level of expression of the Salmonella typhimurium LT2 uvrD gene was observed in maxicells prepared from an Escherichia coli strain that contained a lexA+ plasmid than in maxicells prepared from an E. coli strain that lacked functional LexA protein. These results suggest that the uvrD+ gene is repressed by the LexA protein and is thus a member of the set of genes whose expression is increased by "SOS"-inducing treatments.

Bacterial Proteins↗
PubMed2005

Error-prone DNA polymerase IV is regulated by the heat shock chaperone GroE in Escherichia coli.

An insertion in the promoter of the operon that encodes the molecular chaperone GroE was isolated as an antimutator for stationary-phase or adaptive mutation. The groE operon consists of two genes, groES and groEL; point mutations in either gene conferred the same phenotype, reducing Lac+ adaptive mutation 10- to 20-fold. groE mutant strains had 1/10 the amount of error-prone DNA polymerase IV (Pol IV). In recG+ strains, the reduction in Pol IV was sufficient to account for their low rate of adaptive mutation, but in recG mutant strains, a deficiency of GroE had some additional effect on adaptive mutation. Pol IV is induced as part of the SOS response, but the effect of GroE on Pol IV was independent of LexA. We were unable to show that GroE interacts directly with Pol IV, suggesting that GroE may act indirectly. Together with previous results, these findings indicate that Pol IV is a component of several cellular stress responses.

Adaptation, Biological↗
PubMed1996

Vectors encoding alternative antibiotic resistance for use in the yeast two-hybrid system.

We have altered the antibiotic resistance of the reporter plasmids and the pJG4-5 activation-domain and pEG202 DNA binding-domain plasmids used in the Brent interaction trap/two-hybrid system. These plasmids were each previously ampicillin-resistant, resulting in an inefficient purification of any one plasmid from a yeast strain containing all three plasmids that constitute the complete interaction trap. By creating derivatives of each of these plasmids expressing either kanamycin or chloramphenicol resistance, along with the parent plasmids, we now have the option to use the interaction trap in yeast with three E. coli differentially selectable vectors. This will allow isolation of any one plasmid by purifying all of the interaction trap plasmids from yeast simultaneously and plating E. coli transformed with the plasmids onto the appropriate antibiotic plate to select the particular plasmid of interest.

Ampicillin Resistance↗
PubMed2001

Identification of some DNA damage-inducible genes of Mycobacterium tuberculosis: apparent lack of correlation with LexA binding.

The repair of DNA damage is expected to be particularly important to intracellular pathogens such as Mycobacterium tuberculosis, and so it is of interest to examine the response of M. tuberculosis to DNA damage. The expression of recA, a key component in DNA repair and recombination, is induced by DNA damage in M. tuberculosis. In this study, we have analyzed the expression following DNA damage in M. tuberculosis of a number of other genes which are DNA damage inducible in Escherichia coli. While many of these genes were also induced by DNA damage in M. tuberculosis, some were not. In addition, one gene (ruvC) which is not induced by DNA damage in E. coli was induced in M. tuberculosis, a result likely linked to its different transcriptional arrangement in M. tuberculosis. We also searched the sequences upstream of the genes being studied for the mycobacterial SOS box (the binding site for LexA) and assessed LexA binding to potential sites identified. LexA is the repressor protein responsible for regulating expression of these SOS genes in E. coli. However, two of the genes which were DNA damage inducible in M. tuberculosis did not have identifiable sites to which LexA bound. The absence of binding sites for LexA upstream of these genes was confirmed by analysis of LexA binding to overlapping DNA fragments covering a region from 500 bp upstream of the coding sequence to 100 bp within it. Therefore, it appears most likely that an alternative mechanism of gene regulation in response to DNA damage exists in M. tuberculosis.

Bacterial Proteins↗
PubMed1991

Induction of the SOS response in Escherichia coli inhibits Tn5 and IS50 transposition.

In response to DNA damage or the inhibition of normal DNA replication in Escherichia coli, a set of some 20 unlinked operons is induced through the RecA-mediated cleavage of the LexA repressor. We examined the effect of this SOS response on the transposition of Tn5 and determined that the frequency of transposition is reduced 5- to 10-fold in cells that constitutively express SOS functions, e.g., lexA(Def) strains. Furthermore, this inhibition is independent of recA function, is fully reversed by a wild-type copy of lexA, and is not caused by an alteration in the levels of the Tn5 transposase or inhibitor proteins. We isolated insertion mutations in a lexA(Def) background that reverse this transposition defect; all of these mapped to a new locus near 23 min on the E. coli chromosome.

Alleles↗
PubMed1994

Beta subunit of DNA polymerase III holoenzyme is induced upon ultraviolet irradiation or nalidixic acid treatment of Escherichia coli.

Exposure of Escherichia coli to UV irradiation or nalidixic acid, which induce both the SOS and heat shock responses, led to a 3-4-fold increase in the amount of the beta subunit of DNA polymerase III holoenzyme, as assayed by Western blot analysis using anti-beta antibodies. Such an induction was observed also in a delta rpoH mutant lacking the heat shock-specific sigma 32 subunit of RNA polymerase, but it was not observed in recA13 or lexA3 mutants, in which the SOS response cannot be induced. Mapping of transcription initiation sites of the dnaN gene, encoding the beta subunit, using the S1 nuclease protection assay showed essentially no induction of transcription upon UV irradiation, indicating that induction is regulated primarily at the post-transcriptional level. Analysis of translational gene fusions of the dnaN gene, encoding the beta subunit, to the lacZ reporter gene showed induction of beta-galactosidase activity upon UV irradiation of cells harboring the fusion plasmids. Elimination of a 5' flanking DNA sequence in which the dnaN promoters P1 and P2 were located, did not affect the UV inducibility of the gene fusions. Thus, element(s) present from P3 downstream were sufficient for the UV induction. The induction of the dnaN-lacZ gene fusions was dependent on the recA and lexA gene products, but not on the rpoH gene product, in agreement with the immunoblot analysis. The dependence of dnaN induction on the SOS regulators was not mediated via classical repression by the LexA repressor, since the dnaN promoter does not contain a sequence homologous to the LexA binding site, and dnaN mRNA was not inducible by UV light. This suggests that SOS control may be imposed indirectly, by a post-transcriptional mechanism. The increased amount of the beta subunit is needed, most likely, for increased replication and repair activities in cells which have been exposed to UV radiation.

Bacterial Proteins↗
PubMed2002

Assigning numbers to the arrows: parameterizing a gene regulation network by using accurate expression kinetics.

A basic challenge in systems biology is to understand the dynamical behavior of gene regulation networks. Current approaches aim at determining the network structure based on genomic-scale data. However, the network connectivity alone is not sufficient to define its dynamics; one needs to also specify the kinetic parameters for the regulation reactions. Here, we ask whether effective kinetic parameters can be assigned to a transcriptional network based on expression data. We present a combined experimental and theoretical approach based on accurate high temporal-resolution measurement of promoter activities from living cells by using green fluorescent protein (GFP) reporter plasmids. We present algorithms that use these data to assign effective kinetic parameters within a mathematical model of the network. To demonstrate this, we employ a well defined network, the SOS DNA repair system of Escherichia coli. We find a strikingly detailed temporal program of expression that correlates with the functional role of the SOS genes and is driven by a hierarchy of effective kinetic parameter strengths for the various promoters. The calculated parameters can be used to determine the kinetics of all SOS genes given the expression profile of just one representative, allowing a significant reduction in complexity. The concentration profile of the master SOS transcriptional repressor can be calculated, demonstrating that relative protein levels may be determined from purely transcriptional data. This finding opens the possibility of assigning kinetic parameters to transcriptional networks on a genomic scale.

Algorithms↗