Operator-constitutive Mutations Research Articles

Effect of SOS-induced levels of imuABC on spontaneous and damage-induced mutagenesis in Caulobacter crescentus

imuABC (imuAB dnaE2) genes are responsible for SOS-mutagenesis in Caulobacter crescentus and other bacterial species devoid of umuDC. In this work, we have constructed operator-constitutive mutants of the imuABC operon. We used this genetic tool to investigate the effect of SOS-induced levels of these genes upon both spontaneous and damage-induced mutagenesis. We showed that constitutive expression of imuABC does not increase spontaneous or damage-induced mutagenesis, nor increases cellular resistance to DNA-damaging agents. Nevertheless, the presence of the operator-constitutive mutation rescues mutagenesis in a recA background, indicating that imuABC are the only genes required at SOS-induced levels for translesion synthesis (TLS) in C. crescentus. Furthermore, these data also show that TLS mediated by ImuABC does not require RecA, unlike umuDC-dependent mutagenesis in E. coli.

Operator-constitutive mutation in the recA gene enhances radiation resistance of Escherichia coli

Plasmid pUC19-recAoc carrying a mutant allele of the recA gene, which plays the key role in the control of the SOS repair system and homologous recombinational repair, causes a 1.5-fold increase in radiation resistance of Escherichia coli DeltarecA cells, as compared to the wild-type recA+ cells. The protective effect of this plasmid is drastically reduced in mutant lexA3 recADelta21 deficient in the LexA protein and in induction of the SOS regulon. Plasmid pUC19-recAoc effectively suppresses UV sensitivity of the DeltarecA mutant. Mutation recAo20 allows constitutive high-level synthesis of the RecA protein. This mutation impairs the SOS box in the operator site of the recA gene and enhances heterology of the dimer LexA binding site. These data confirm that high level of the RecA protein synthesis per se is not sufficient for the expression of gamma-inducible functions and that the derepression of lexA-dependent genes, other than recA gene, is necessary for the complete induction of the SOS repair system.

Genetic Suppressors and Recovery of Repressed Biochemical Memory

Many of the Reflections articles in this journal have focused on particular biological problems that the authors have devoted their careers to or on particular techniques that have facilitated their biological studies. Although I began my career as a biochemist, one of the most common threads to my research has been the exploitation of a genetic approach, the analysis of suppressor mutations. Particularly in the last 20 years, the success of this approach has led me into biological problems that required a remembering or relearning of my early training in biochemistry. This article will describe the voyage from chemistry and biochemistry to genetics and then to a recovery of remnants of my biochemical memory. As a graduate student working under Lowell Hager at Harvard in the late 1950s, my thesis research was chemical and biochemical, as I performed the chemical synthesis and studied the biosynthesis of the chlorinated mold product caldariomycin (2,2-dichloro-1,3-cyclopentanediol) (1–3). With his postdoctoral fellow Paul Shaw, Lowell had discovered an enzyme, chloroperoxidase, that was capable of utilizing chloride ion to chlorinate organic compounds (4). Part of my work was to determine how this enzyme might be involved in the biosynthesis of caldariomycin. Apparently, the work became somewhat well known. This I learned when, toward the end of my Ph.D. work, I attended a FASEB meeting in Atlantic City to give a short presentation. As I walked into the conference center on the first day, a scientist passing by me stopped to look at my badge, hesitated a moment, and then commented “Beckwith... hmmm, oh yes, I remember... chlorination!” Despite this really quite limited chemical/biochemical notoriety, I became more and more fascinated by bacterial genetics in graduate school. I proceeded to do postdoctoral work in several bacterial genetics laboratories.

Induction of only one SOS operon, umuDC, is required for SOS mutagenesis in Escherichia coli.

The actions of UmuDC and RecA proteins, respectively in SOS mutagenesis are studied here with the following experimental strategy. We used lexAl (Ind-) bacteria to maintain all SOS proteins at their basal concentrations and then selectively increased the concentration of either UmuDC or RecA protein. For this purpose, we isolated operator-constitutive mutations oc in the umuDC and umuD'C operons and also used the oc98-recA mutation. The oc1-umuDC mutation prevents LexA repressor from binding to the operator and improves the Pribnow box consensus sequence. As a result, 5000 UmuD and 500 UmuC molecules per cell were produced in lexAl bacteria. This concentration is sufficient to restore SOS mutagenesis. The level of RecA protein present in the repressed state promoted full UmuD cleavage. Overproduction of RecA alone did not promote SOS mutagenesis. Increasing the level of RecA in the presence of high concentrations of UmuDC proteins has no further effect on SOS mutagenesis. We conclude that, after DNA damage, umuDC is the only SOS operon that must be induced in Escherichia coli to promote SOS mutagenesis.

Cairnsian mutagenesis inEscherichia coli: Genetic evidence for two pathways regulated bymutS andmutL genes

The phenomenon of Cairnsian mutagenesis was studied inEscherichia coli mutants bearing mutations inmutS,mutL,recA andlexA genes. It is shown that development of resistance to exogenous valine could be used as an example of Cairnsian response. Strains defective inmutS andmutL show a high frequency of Cairnsian mutagenesis to valine resistance. The response inmutS mutants is dependent upon cleavability of the LexA protein whereas that inmutL is not. The latter is independent ofrecA also. The need for LexA protein cleavage inmutS mutants can be bypassed by over-production of the RecA protein due to arecA operator constitutive mutation. Genetic evidence is presented to show that the products ofmutS andmutL genes negativelycontrol two pathways of Cairnsian mutagenesis. Cairnsian response is also elicited whenmutS ormutL strains are grown under conditions wherein a required nutrient is present in sub-optimal concentrations. Random, unselected mutagenic events are likely to occur during or after Cairnsian mutagenesis provided the cells are SOS inducible.

Salmonella typhimurium metC operator-constitutive mutations

We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (lambda Clac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying lambda Clac mutant phage exhibit high beta-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.

Salmonella typhimurium metCoperator-constitutive mutations

We used an Escherichia coli lac deletion strain lysogenized with a metC-lacZ fusion phage (λClac) to select operator-constitutive mutations in the Salmonella typhimurium metC gene control region. The mutations were located in a region containing 2 tandemly repeated 8 bp palindromes previously proposed to be the MetJ repressor binding site. Lysogens carrying λClac mutant phage exhibit high β-galactosidase levels that are only partially repressible by methionine. The results suggest that the mutations disrupt the methionine control system mediated by the metJ gene product.

Salmonella typhimurium metE operator-constitutive mutations

We used a metE-lacZ fusion phage (λElac) to select for mutants with operator-constitutive mutations in the Salmonella typhimurium metE control region. All of the mutations identified were found to lie within a region containing tandemly-repeating 8-bp palindromes with the consensus sequence 5′-AGACGTCT-3′, previously proposed to be the binding region for the metJ-encoded represser. Lysogens carrying mutant λElac phage exhibit high β-galactosidase levels that are only partially repressible by methionine. Although repression of metE expression by vitamin B 12 is not disrupted in metJ + lysogens, vitamin B 12 repression is disrupted in lysogens lacking an active MetJ repressor. These results suggest that there is an interaction between the metJ-encoded repressor and the vitamin B 12 repression system mediated by the metH gene product.

CarP, a novel gene regulating the transcription of the carbamoylphosphate synthetase operon of Escherichia coli

The carAB operon, encoding carbamoylphosphate synthetase (CPSase; EC 6.3.5.5) is transcribed from two tandem promoters. The upstream promoter (P1) is controlled by pyrimidines and the downstream promoter (P2) is controlled by arginine. We have isolated a new type of constitutive mutation ( carP) that specifically affects the control of the pyrimidine-sensitive promoter but does not appear to influence other genes of the pyrimidine pathway. The carP mutation acts in trans and is dominant, which suggests that the carP product is an activator of car transcription. The downstream promoter P2, which is repressed by arginine, overlaps two operator modules characteristic of the arginine regulon. We have isolated two operator-constitutive mutations that specifically affect P2; both map in the upstream ARG box at a strongly conserved position.

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.

Operator constitutive mutation of 3-hydroxy-3-methylglutaryl coenzyme A reductase promoter abolishes protein binding to sterol regulatory element.

Through substitution mutagenesis we identified the promoter elements responsible for basal expression and sterol-mediated repression of transcription of the gene for 3-hydroxy-3-methylglutaryl coenzyme A reductase, a rate-controlling enzyme of cholesterol biosynthesis. Mutant promoters containing 277 base pairs (bp) of reductase 5' flanking sequence were inserted into recombinant plasmids upstream of the coding region for bacterial chloramphenicol acetyltransferase. The plasmids were transfected into hamster fibroblasts, and transcription was measured in the presence and absence of sterols. Mutations in three regions that are known to bind nuclear proteins markedly reduced transcription. Mutation of another protein-binding region of 20 bp in length did not reduce transcription, but it did abolish sterol-mediated repression, producing an operator constitutive phenotype. This mutation also abolished protein binding to the corresponding 20-bp region of DNA as determined by footprinting assays. When a DNA fragment containing these 20 bp was inserted into the herpes simplex virus thymidine kinase promoter, sterol-mediated repression was observed. This sequence contains an octanucleotide that shows a 7/8-bp match with a previously identified regulatory sequence in repeat 2 of the low density lipoprotein receptor promoter, another sterol-repressible gene. We hypothesize that this octanucleotide, GTGGCGGTG, is the core binding site for a sterol-dependent protein that represses transcription.

Isolation and genetic analysis of operator-constitutive mutants of the H1 operon in Salmonella typhimurium.

In phase-2 cells of diphasic Salmonella strains, expression of H1 is repressor, coded for by the rh1 gene. A procedure for the isolation of operator-constitutive (H1-Oc) mutants of the H1 operon is described. Using three-factor crosses between an H1-Oc H1 strain and H1-O+ H1 strains, where motility recovery via H1-phase (or phase 1) flagellation was used as the selected marker and the H1-O character was the unselected marker, the relative position of the H1-Oc site to the H1 gene was determined. A diphasic H1-Oc strain produced, in phase 2, copolymer filaments composed of H1 and H2 flagellin.

Transcriptional control in the EcoRI-F immunity region of Bacillus subtilis phage φ105 : Identification and unusual structure of the operator

We present the first evidence, in Bacillus subtilis, for gene regulation through the classical mechanism of repressor-operator interaction. The EcoRI-F immunity region ( immF) of lysogenic phage φ105 contains two promoters, P M and P R, in divergent orientations. P M initiates transcription of the φ105 repressor ( c φ105 ) gene, whereas P R most probably signals the onset of the lytic pathway. Fusions between each of these promoters and the cat-86 gene were constructed, and in-vivo promoter activities were determined, in both the presence and absence of the functional c φ105 product, using S 1 nuclease analysis and chloramphenicol acetyl transferase assays. The results showed that transcription from P M is stimulated, whereas P R activity is negatively controlled by the repressor. This differential regulation appears to be mediated by recognition of a 14 base-pair (bp) sequence, 5′ GACGGAAATACAAG 3′, three identical copies of which are present as direct repeats. Two copies, O R1 and O R2, are located closely together in the non-transcribed region between P M and P R, but do not overlap with the −35 and −10 regions of these promoters. The third copy, O R3, is located some 250 bp downstream from P R, within the coding region ( ORF3) of the proximal gene of the P R transcription unit. When a 231 bp restriction fragment containing only O R3 was inserted between a strong constitutive promoter (P 138) and the cat-86 gene, the in-vivo expression of chloramphenicol resistance was considerably reduced in the presence, but not in the absence, of φ105 repressor. This hybrid P 138-O R- cat-86 construct was subsequently used to select in vivo for operator-constitutive (O c) mutations. Of 25 O c mutants analyzed, all showed base alterations or deletions affecting the 14 bp sequence. We show further that insertion of a chemically synthesized oligonucleotide, containing the 14 bp O R sequence, at a site more than 100 bp downstream from the constitutive P 138 is sufficient for transcription to become negatively controlled by φ105 repressor. In comparison with previously identified Gram-negative bacterial and phage operators, the most unusual aspect of the φ105 O R sequence appears to be its complete lack of 2-fold rotational symmetry.

Multiple repressor binding sites in the genome of bacteriophage P1.

After digestion of bacteriophage P1 DNA with EcoRI in the presence of P1 repressor, 6 repressor binding sites were identified in 5 of 26 EcoRI fragments. Binding sites were localized by the decreased mobility of DNA fragment-repressor complexes during electrophoresis and by DNase protection ("footprinting") analysis. The repressor binding sites, or operators, comprise a 17-base-pair-long consensus sequence lacking symmetrical elements. Three operators can be related to known genes, whereas the function of the others is still unknown. The mutant P1 bac, rendering ban expression constitutive, is identified as an operator-constitutive mutation of the ban operon.

Transcriptional and translational signals of the uidA gene in Escherichia coli K12.

The expression of uidA is negatively controlled by the products of the uidR and uxuR genes and is sensitive to catabolite repression. The locations of the transcriptional and translational signals of uidA were determined using lac gene fusions and S1 mapping experiments. The promoter structure of uidA resembles that of a promoter activated by cAMP receptor protein (CRP); putative control regions are located at positions -10 and -35 (relative to the transcription start site), are separated by more than 17 bp and exhibit poor homology with the normally recognized consensus sequences. Moreover, 80 bp separate the promoter from the translational signals. No CRP binding site was detected in the promoter region of uidA. Two operator sites, 01 and 02, were identified: 01 has a greater affinity for the UidR repressor, whereas 02 has a greater affinity for the UxuR repressor, but the two repressor molecules are able to bind at both the 01 and 02 sites. Analysis of two operator constitutive mutations allowed the location of one of the two UidR repressor binding sites; it contains palindromic units spanning the TaqI site of the uidA control region.

Regulatory role of recF in the SOS response of Escherichia coli: impaired induction of SOS genes by UV irradiation and nalidixic acid in a recF mutant.

We isolated a new recF mutant of Escherichia coli K-12 by insertion of transposon Tn5 into the recF gene. This recF400::Tn5 allele displayed the same phenotypic characteristics as the classic recF143 mutation. By using Mu d(Ap lac) fusions, the induction of nine SOS genes, including recA, umuC, dinA, dinB, dinD, dinF, recN, and sulA, by UV irradiation and nalidixic acid was examined. Induction of eight genes by the two agents was impaired by recF400::Tn5 to different extents. The ninth fused SOS gene, dinF, was no longer inducible by UV when combined with recF400::Tn5. The generally impaired SOS response in recF strains did not result from weak induction of recA protein synthesis, since a recA operator-constitutive mutation did not alleviate the inhibitory effect of the recF mutation. The results suggest that recF plays a regulatory role in the SOS response. It is proposed that this role is to optimize the signal usage by recA protein to become a protease.

Operator-constitutive mutations of the Escherichia coli metF gene.

The Escherichia coli metF gene codes for 5,10-methylene-tetrahydrofolate reductase, the enzyme that leads to the formation of N-methyltetrahydrofolate, supplying the methyl group of methionine. Transcription of metF, as well as most of the methionine genes, is repressed by the metJ gene product complexed with S-adenosylmethionine. A metF'-'lacZ gene fusion was used to isolate mutants that have altered expression from the metF promoter. The nucleotide sequences of the metF regulatory region from five such mutants were determined. The mutations were located in the region previously defined as the potential target of the methionine repressor by its similarity to other binding sites. The mutationally defined metF operator thus consists of a 40-base-pair-long region, with five 8-base-pair imperfect palindromes spanning the metF transcription start. The altered operators do not recognize the purified repressor in an in vitro transcription-translation system, although the repressor binds efficiently to the metF wild-type operator.

Transcription control of the aroP gene in Escherichia coli K-12: analysis of operator mutants.

The nucleotide sequence of the region containing the promoter-operator for the aroP gene was determined. The start site of aroP transcription was identified by using S1 nuclease mapping and primer extension techniques. Examination of the nucleotide sequence revealed the presence of two "TYR R" boxes which are similar to those identified in the regulatory regions of other genes in the tyrR regulon. Bisulfite-induced aroP operator-constitutive mutants were analyzed, and the base-pair changes responsible for alterations in aroP regulation were located within these boxes.

Mutational and in vivo methylation analysis of F-factor PifC protein binding to the pif operator and the region containing the primary origin of mini-F replication.

We have used in vivo methods to identify multiple DNA-binding sites for the negatively autoregulated mini-F replication factor PifC. Sequence analysis of pif operator constitutive mutants, isolated as insensitive to repression by PifC, establishes the structure of pifO. This site contains a 17-base-pair (bp) region of dyad symmetry with 7-bp perfect inverted repeats separated by 3 bp. In vivo DNA methylation studies with dimethyl sulfate show that the reactivity of five of six guanine residues in the pifO region is altered in the presence of PifC protein. In addition, there are several sites of PifC-dependent methylation enhancement and protection upstream of pifO within repeated sequences bearing homology to pifO. The significance of the repeated PifC binding sequences and their relationship to the primary origin of mini-F replication (oriV1) are discussed.

Evolutionary divergence of genes for ornithine and aspartate carbamoyl-trasferases-complete sequence and mode of regulatio of theEscherichia coli argFgene; comparison ofargFwithargIandpyrB

The complete nucleotide sequence of argF is presented, together with that of an operator-constitutive mutant. ArgF is compared with the other gene coding for ornithine carbamoyltransferase (OTCase) in E. coli K-12, argI, and with pyrB, encoding the catalytic monomer of aspartate carbamoyltransferase (ATCase). ArgF and argI appear very closely related having emerged from a relatively recent ancestor gene. The relationship between OTCase and ATCase appears more distant. Nevertheless, the homology observed between the two proteins (mainly in the polar domain) suggests a common origin.