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PubMed2003

Error-prone DNA polymerase IV is controlled by the stress-response sigma factor, RpoS, in Escherichia coli.

An insertion in rpoS, which encodes the general stress response sigma factor sigma 38, was isolated as an antimutator for 'stationary-phase' or 'adaptive' mutation. In the rpoS mutant strain the levels of error-prone DNA polymerase Pol IV were reduced. Pol IV is encoded by the dinB gene, and the amount of its transcript was also reduced in rpoS mutant cells. In wild-type cells, the levels of Pol IV increased in late stationary phase and stayed elevated for several days of continuous incubation, whereas in rpoS defective cells Pol IV was not induced and declined during prolonged incubation. Even in cells missing LexA, the repressor of dinB, maximum Pol IV expression required RpoS. These results suggest that induction of Pol IV is part of a cellular response to starvation and other stresses.

Bacterial Proteins↗
PubMed1997

Expression of the Pseudomonas aeruginosa uvrA gene is constitutive.

By complementation of an Escherichia coli uvrA-defective mutant, the uvrA gene of Pseudomonas aeruginosa has been isolated from a genomic library. The nucleotide sequence of the promoter and the 5'-coding region of this gene have been determined. A P. aeruginosa SOS consensus region, which functions as a binding site for the LexA repressor molecule, was not present in the 629-bp upstream region of the cloned uvrA gene. Analysis of transcriptional fusions with the lacZ gene demonstrates that, contrary to what happens with its homologous gene of E. coli, the P. aeruginosa uvrA gene is not DNA damage-inducible. However, the UvrA protein must be functional in P. aeruginosa cells because an uvrA-defective mutant is extremely sensitive to UV radiation.

Adenosine Triphosphatases↗
PubMed1986

Functional dissection of a eukaryotic transcriptional activator protein, GCN4 of yeast.

Yeast GCN4 protein binds specifically to the promoters of amino acid biosynthetic genes and coordinately induces their transcription. Serially deleted GCN4 and hybrid LexA-GCN4 proteins were assayed for specific DNA binding activity in vitro, and for stimulation of transcription in vivo. The specific DNA binding activity resides in the 60 C-terminal amino acids, a basic region of GCN4. However, certain deletions containing the entire DNA binding region are unable to activate transcription and instead act as repressors in vivo. The activation function appears to critically involve just 19 amino acids that are centrally located in an acidic region of GCN4. In addition to their functional separation, the DNA binding and transcriptional activation regions of the protein can be separated physically by elastase cleavage. The implications of these results for the mechanisms of DNA sequence recognition and transcription activation are discussed.

Binding Sites↗
PubMed1982

Homologies between different procaryotic DNA-binding regulatory proteins and between their sites of action.

Comparison of the amino acid sequences of 13 procaryotic regulatory proteins, including the products of genes crp (catabolite activator protein; CAP), lacI, galR , lexA, lysR, araC, trpR, and tnpR of Escherichia coli, of genes cI, cII and cro of phage lambda, cro of phage 434, and c2 of phage P22, has revealed two regions of homology. The sites of action of these proteins also share common features in their DNA sequence. Taking into account the models proposed for the lambda repressors, cro and cI, and for CAP, a general type of DNA-protein interaction is suggested.

Amino Acid Sequence↗
PubMed1983

Novel mechanism of cell division inhibition associated with the SOS response in Escherichia coli.

Certain Escherichia coli strains were shown to possess a novel system of cell division inhibition, called the SfiC+ phenotype. SfiC+ filamentation had a number of properties similar to those of sfiA-dependent division inhibition previously described: (i) both are associated with the SOS response induced by expression of the recA(Tif) mutation, (ii) both are associated with cell death, (iii) both are amplified in mutants lacking the Lon protease, and (iv) both are suppressed by sfiB mutations. SfiC+ filamentation and sfiA-dependent division inhibition differed in (i) the physiological conditions under which loss of viability is observed, (ii) the extent of amplification in lon mutants, (iii) their genetic regulation (SfiC+ filamentation is not under direct negative control of the LexA repressor), and (iv) their genetic determinants (SfiC+ filamentation depends on a locus, sfiC+, near 28 min on the E. coli map and distinct from sfiA).

ATP-Dependent Proteases↗
PubMed1983

Regulation of expression of the colicin gene of I1 group plasmid TP110.

The control of expression of the colicin Ib gene of the I1 group plasmid TP110 has been investigated. The colicin promoter was fused to the structural gene for beta-galactosidase, using the Mu d(Aprlac) phage, and the plasmid carrying this fusion was introduced into a variety of bacterial strains defective in genes involved in the "SOS" response. Colicin Ib belongs to that group of genes directly controlled by the repressor produced by the lexA gene, and expression was inducible by DNA-damaging agents. Mutations in uvrA, -B, and -C reduced the efficiency of induction by mitomycin C, as did mutations in recB. Mutations in recA and recF effectively prevented induction by mitomycin C, whereas mutations in lexA had contrasting effects, depending upon their effect on the properties of lexA protein. The spr-51 mutation (which inactivates lexA protein) led to constitutive expression, whereas the lexA3 mutation (which makes lexA protein refractory to cleavage by recA protein) completely inhibited inducible expression. In addition to lexA control, a TP110-coded function was identified which appeared able to inhibit colicin expression when the gene responsible was present in high copy number.

Cloning, Molecular↗
PubMed1986

Capacity of RecA protein to bind preferentially to UV lesions and inhibit the editing subunit (epsilon) of DNA polymerase III: a possible mechanism for SOS-induced targeted mutagenesis.

The RecA protein of Escherichia coli is required for SOS-induced mutagenesis in addition to its recombinational and regulatory roles. Most SOS-induced mutations probably occur during replication across a DNA lesion (targeted mutagenesis). We have suggested previously that RecA might participate in targeted mutagenesis by binding preferentially to the site of the DNA damage (e.g., pyrimidine dimer) because of its partially unwound character; DNA polymerase III (polIII) will then encounter RecA-coated DNA at the lesion and might replicate across the damaged site with reduced fidelity. In this report, we analyze at a biochemical level two major predictions of this model. With respect to lesion recognition, we show that purified RecA protein binds more efficiently to UV-irradiated double-stranded DNA than to nonirradiated DNA, as judged by filter-binding and gel electrophoresis assays. With respect to replication fidelity, Fersht and Knill-Jones [Fersht, A. R. & Knill-Jones, J. W. (1983) J. Mol. Biol. 165, 669-682] have found that RecA inhibits the 3'----5' exonuclease (editing function) of polIII holoenzyme. We extend this observation by demonstrating that RecA inhibits the exonuclease of the purified editing subunit of polIII, epsilon protein. Thus, we suggest that the activities of RecA required for targeted mutagenesis are lesion-recognition, followed by localized inhibition of the editing capacity of the epsilon subunit of polIII holoenzme. In this proposed mechanism, one activation signal for RecA for mutagenesis is the lesion itself. Because UV-irradiated, double-stranded DNA efficiently activates RecA for cleavage of the LexA repressor, the lesion itself may also often serve as an activation signal for induction of SOS-controlled genes.

Antibody Diversity↗
PubMed2006

Genetic mosaic with dual binary transcriptional systems in Drosophila.

MARCM (mosaic analysis with a repressible cell marker) involves specific labeling of GAL80-minus and GAL4-positive homozygous cells in otherwise heterozygous tissues. Here we demonstrate how the concurrent use of two independent binary transcriptional systems may facilitate complex MARCM studies in the Drosophila nervous system. By fusing LexA with the VP16 acidic activation domain (VP16) or the GAL4 activation domain (GAD), we obtained both GAL80-insensitive and GAL80-suppressible transcriptional factors. LexA::VP16 can mediate MARCM-independent binary transgene induction in mosaic organisms. The incorporation of LexA::GAD into MARCM, which we call dual-expression-control MARCM, permits the induction of distinct transgenes in different patterns among GAL80-minus cells in mosaic tissues. Lineage analysis with dual-expression-control MARCM suggested the presence of neuroglioblasts in the developing optic lobes but did not indicate the production of glia by postembryonic mushroom body neuronal precursors. In addition, dual-expression-control MARCM with a ubiquitous LexA::GAD driver revealed many unidentified cells in the GAL4-GH146-positive projection neuron lineages.

Animals↗
PubMed2005

Phage regulatory circuits and virulence gene expression.

In many pathogenic bacteria, genes that encode virulence factors are located in the genomes of prophages. Clearly bacteriophages are important vectors for disseminating virulence genes, but, in addition, do phage regulatory circuits contribute to expression of these genes? Phages of the lambda family that have genes encoding Shiga toxin are found in certain pathogenic Escherichia coli (known as Shiga toxin producing E. coli) and the filamentous phage CTXphi, that carries genes encoding cholera toxin (CTX), is found in Vibrio cholerae. Both the lambda and CTXphi phages have repressor systems that maintain their respective prophages in a quiescent state, and in both types of prophages this repressed state is abolished when the host cell SOS response is activated. In the lambda type of prophages, only binding of the phage-encoded repressor is involved in repression and this repressor ultimately controls Shiga toxin production and/or release. In the CTXphi prophage, binding of LexA, the bacterial regulator of SOS, in addition to binding of the repressor is involved in repression; the repressor has only limited control over CTX production and has no influence on its release.

Bacteriophages↗
PubMed1996

Syntactic recognition of regulatory regions in Escherichia coli.

MOTIVATION: One of the most common methodologies to identify cis-regulatory sites in regulatory regions in the DNA is that of weight matrices, as testified by several articles in this issue. An alternative to strengthen the computational predictions in regulatory regions is to develop methods that incorporate more biological properties present in such DNA regions. The grammatical implementation presented in this paper provides a concrete example in this direction. RESULTS: On the basis of the analysis of an exhaustive collection of regulatory regions in Escherichia coli, a grammatical model for the regulatory regions of sigma 70 promoters has been developed. The terminal symbols of the grammar represent individual sites for the binding of activator and repressor proteins, and include the precise position of sites in relation to transcription initiation. Combining these symbols, the grammar generates a large number of different sentences, each of which can be searched for matching against a collection of regulatory regions by means of weight matrices specific for each set of sites for individual proteins. On the basis of this grammatical model, a Prolog syntactic recognizer is presented here. Specific subgrammars for ArgR, LexA and TyrR were implemented. When parsing a collection of 128 sigma 70 promoter regions, the syntactic recognizer produces a much lower number of false-positive sites than the standard search using weight matrices.

Algorithms↗
PubMedDate not supplied

[The SOS and catabolytic repression systems can play a key role in the regulation of infection of Escherichia coli cells with F-specific filamentous phages M13, f1 and fd].

Theoretical analysis of DNA sequences revealed recognition sites for two global E. coli cellular regulons in M13, fd and fl phage's genomes. Both Px and Pv promoters have SOS operator sequences and therefore must be repressed by the lexA protein. PIII and PIV contains CRP-cAMP recognition sequences in activating positions and hence will be activated by the cAMP receptor protein. The model is proposed for the phage life cycle's control in the persistent infection of E. coli cells by F-specific filamentous phages.

Coliphages↗
PubMed2004

The pKO2 linear plasmid prophage of Klebsiella oxytoca.

Temperate bacteriophages with plasmid prophages are uncommon in nature, and of these only phages N15 and PY54 are known to have a linear plasmid prophage with closed hairpin telomeres. We report here the complete nucleotide sequence of the 51,601-bp Klebsiella oxytoca linear plasmid pKO2, and we demonstrate experimentally that it is also a prophage. We call this bacteriophage phiKO2. An analysis of the 64 predicted phiKO2 genes indicate that it is a fairly close relative of phage N15; they share a mosaic relationship that is typical of different members of double-stranded DNA tailed-phage groups. Although the head, tail shaft, and lysis genes are not recognizably homologous between these phages, other genes such as the plasmid partitioning, replicase, prophage repressor, and protelomerase genes (and their putative targets) are so similar that we predict that they must have nearly identical DNA binding specificities. The phiKO2 virion is unusual in that its phage lambda-like tails have an exceptionally long (3,433 amino acids) central tip tail fiber protein. The phiKO2 genome also carries putative homologues of bacterial dinI and umuD genes, both of which are involved in the host SOS response. We show that these divergently transcribed genes are regulated by LexA protein binding to a single target site that overlaps both promoters.

Amino Acid Sequence↗
PubMed2000

Escherichia coli cells defective for the recN gene display constitutive elevation of mutagenesis at 3,N(4)-ethenocytosine via an SOS-induced mechanism.

The Escherichia coli UVM (UV Modulation of mutagenesis) response is a DNA damage-inducible mutagenic pathway detected as significantly increased mutagenesis at 3,N4-ethenocytosine (epsilon C) lesions borne on transfected single-stranded M13 vector DNA. All major classes of DNA-damaging agents can induce UVM, and the phenomenon is independent of previously characterized mutagenic responses in E. coli. To understand this phenomenon further, we set out to identify and characterize mutants in the UVM response. Screening a mutant bank of cells defective for 1-methyl-3-nitro-1-nitrosoguanidine-inducible genes revealed that defects in the recN gene cause a constitutive elevation of mutagenesis at epsilon C residues. In contrast to normal cells that show approximately 6% mutagenesis at epsilon C lesions, but approximately 60% upon UVM induction, recN-defective strains display approximately 50% mutagenesis at epsilon C lesion sites in untreated cells. However, the recN-mediated mutagenesis response was found to require the recA gene and the umuDC genes, and could be suppressed in the presence of a plasmid harbouring the SOS transcriptional repressor LexA. These results imply that recN cells are constitutively active for SOS mutagenesis functions. The observation that epsilonC mutagenesis is enhanced in recN cells confirms previous findings that mutagenesis at epsilonC can also be independently elevated by the SOS pathway.

Bacterial Proteins↗
PubMed1992

Detecting interactions between eukaryotic proteins in bacteria.

Few convenient genetic assays are available to study protein-protein interactions. This report describes a genetic scheme in E. coli to detect protein-protein interactions based on the concept of cooperative DNA binding of two interacting proteins. The yeast regulatory proteins GAL4 and GAL80, which are known to interact with each other, were used to test the scheme. A fusion protein, LexA-GAL80, was found to exert a cooperative effect on the DNA-binding activity of GAL4 as monitored by a bacterial repression assay.

Base Sequence↗
PubMed1991

Escherichia coli DNA polymerase II is homologous to alpha-like DNA polymerases.

The Escherichia coli polB gene encodes DNA polymerase II and is regulated by the SOS system. We sequenced a 4081 nucleotide segment of the E. coli chromosome that contains the polB gene and its flanking regions. DNA polymerase II, as deduced from the DNA sequence, consists of 782 amino acids, has a molecular weight of 89,917, and is structurally homologous to alpha-like DNA polymerases, which include eukaryotic replicative DNA polymerases. Comparison of the sequences of the alpha-like DNA polymerases including E. coli DNA polymerase II showed that there were nine highly conserved regions, and we constructed an unrooted phylogenetic tree of the DNA polymerases based on the differences in these conserved regions. The DNA polymerases of herpes groups viruses and the DNA polymerases that use protein priming for the initiation of replication form two separate subfamilies that occupy opposite locations in the tree. Other DNA polymerases, including E. coli DNA polymerase II, human DNA polymerase alpha, and yeast DNA polymerase I, occupy the central regions between the two subfamilies and they are rather distantly related to each other. The transcription initiation site of polB was identified by analysis of in vivo transcripts, and the promoter was assigned upstream of the polB coding region. The recognition sequence of the LexA repressor (SOS box) was identified by a footprinting experiment. It overlaps the -35 sequence of the polB promoter.

Amino Acid Sequence↗
PubMed1996

Structure/functional properties of the yeast dual regulator protein NGG1 that are required for glucose repression.

NGG1p/ADA3p is a yeast dual function regulator required for the complete glucose repression of GAL4p-activated genes (Brandl, C. J., Furlanetto, A. M., Martens, J. A., and Hamilton, K. S. (1993) EMBO J. 12, 5255-5265). Evidence for a direct role for NGG1p in regulating activator function is supported by the finding that NGG1p is also required for transcriptional activation by GAL4p-VPl6 and LexA-GCN4p (Pina, B., Berger, S. L., Marcus, G. A., Silverman, N., Agapite, J., and Guarente, L. (1993) Mol. Cell. Biol. 13, 5981-5989). By analyzing deletion derivatives of the 702-amino acid protein, we identified a region essential for glucose repression within residues 274-373. Essential sequences were further localized to a segment rich in Phe residues that is predicted to be an amphipathic alpha helix. As well as finding mutations within this region that reduced glucose repression, we identified mutations that made NGG1p a better repressor. In addition, NGG1p probably represses GAL4p activity as part of a complex containing ADA2p because single and double disruptions of ngg1 and ada2 had comparable effects on glucose repression. We also localized a transcriptional activation domain within the amino-terminal amino acids of NGG1p that is proximal or overlapping the region required for glucose repression. Activation by GAL4p-NGG1p(1-373) requires ADA2p; however, activation by GAL4p-NGG1p(1-308), is ADA2p-independent. This suggests that a site required for ADA2p interaction lies between amino acids 308 and 373 and that ADA2p has a regulatory role in activation by GAL4p-NGG1p(1-373).

Alleles↗
PubMed1983

Proteins required for ultraviolet light and chemical mutagenesis. Identification of the products of the umuC locus of Escherichia coli.

umuC is the genetic locus showing the greatest specificity for the "error-prone repair" process that is responsible for u.v. and chemical mutagenesis in Escherichia coli. By generating a probe specific to umuC DNA, we have been able to clone the umuC locus. Through a combination of subcloning and Tn1000 mutagenesis, we have identified a region of 2.2 X 10(3) bases which contains the information necessary to complement umuC mutations. This region of DNA codes for two polypeptides with molecular weights of 16,000 and 45,000. The genes for these proteins are organized in an operon that is repressed by the lexA protein. Complementation of previously isolated umuC mutations revealed that these proteins correspond to two complementation groups, umuC, which codes for the 45,000 Mr protein, and umuD, which codes for the 16,000 Mr protein, and that therefore both proteins are essential for "error-prone repair" in E. coli.

Bacterial Proteins↗
PubMed1990

Nucleotide sequence and further characterization of the Synechococcus sp. strain PCC 7002 recA gene: complementation of a cyanobacterial recA mutation by the Escherichia coli recA gene.

The nucleotide sequence and transcript initiation site of the Synechococcus sp. strain PCC 7002 recA gene have been determined. The deduced amino acid sequence of the RecA protein of this cyanobacterium is 56% identical and 73% similar to the Escherichia coli RecA protein. Northern (RNA) blot analysis indicates that the Synechococcus strain PCC 7002 recA gene is transcribed as a monocistronic transcript 1,200 bases in length. The 5' endpoint of the recA mRNA was mapped by primer extension by using synthetic oligonucleotides of 17 and 27 nucleotides as primers. The nucleotide sequence 5' to the mapped endpoint contained sequence motifs bearing a striking resemblance to the heat shock (sigma 32-specific) promoters of E. coli but did not contain sequences similar to the E. coli SOS operator recognized by the LexA repressor. An insertion mutation introduced into the recA locus of Synechococcus strain PCC 7002 via homologous recombination resulted in the formation of diploids carrying both mutant and wild-type recA alleles. A variety of growth regimens and transformation procedures failed to produce a recA Synechococcus strain PCC 7002 mutant. However, introduction into these diploid cells of the E. coli recA gene in trans on a biphasic shuttle vector resulted in segregation of the cyanobacterial recA alleles, indicating that the E. coli recA gene was able to provide a function required for growth of recA Synechococcus strain PCC 7002 cells. This interpretation is supported by the observation that the E. coli recA gene is maintained in these cells when antibiotic selection for the shuttle vector is removed.

Amino Acid Sequence↗