RESUMO
A multi-step screening procedure was devised to identify new operators for the LexA repressor in the sequenced portions of the genomes of Escherichia coli and its plasmids and bacteriophages. Sequence analysis methods were employed initially to distinguish true LexA operators from "operator-like" sequences stored within the GenBank and EMBL databases. The affinity of purified LexA protein for cloned DNA fragments containing several of the prospective new sites was then assessed using quantitative electrophoretic mobility shift assays and site-directed mutagenesis. Calculated binding affinities were compared directly with values determined for known and mutant LexA operators in concurrent experiments. Three E. coli chromosomal segments (near pyrC, hsdS and ntrla) and two bacteriophage sequences (near the P1 cre and lambda oop genes) bound LexA protein specifically. These sites and most others identified in the screening are located immediately upstream of known genes and/or large open reading frames. These results and additional transcription data demonstrate that several of the sequences define new DNA damage-inducible (din) genes and include the previously uncharacterized dinD locus. Furthermore, the search identified an SOS gene within the genome of P1 which encodes a protein that is homologous to UmuD', the RecA-promoted cleavage product of the umuD gene. The success of the combinatorial approach described here suggests that analogous searches for new regulatory sequences within the E. coli genome and the genomes of other organisms will also yield favorable results.
Assuntos
Proteínas de Bactérias/metabolismo , Proteínas de Ligação a DNA/metabolismo , Escherichia coli/genética , Genes Bacterianos , Regiões Operadoras Genéticas/genética , Proteínas Repressoras/metabolismo , Resposta SOS em Genética/genética , Serina Endopeptidases , Sequência de Aminoácidos , Sequência de Bases , Sítios de Ligação , Southern Blotting , Cromossomos Bacterianos , Colífagos/genética , Eletroforese em Gel de Poliacrilamida , Modelos Genéticos , Dados de Sequência Molecular , Mutagênese Sítio-DirigidaRESUMO
Exchange of nucleotide (nt) sequences between the catIJF and pcalJF regions of the Acinetobacter calcoaceticus chromosome appears to contribute to frequent repair of mutations, including removal of a Tn5 insertion from pcaJ. Repaired nt sequences are the products of nonreciprocal genetic exchange. The length of donor catIJF nt tracts recovered in repaired pcaIJF DNA ranged from less than 315 nt to more than 881 nt. This evidence does not distinguish gene conversion from natural transformation as a cause of repair, but natural transformation does not appear to contribute significantly, because it, unlike the repair process, is inactive in the presence of DNase. High-frequency recombination between catIJF and pcaIJF raises the question of why DNA between these chromosomal regions is stable. It is possible that some of the recombinational processes associated with gene conversion are unlike those underlying natural transformation.
Assuntos
Acinetobacter calcoaceticus/genética , Reparo do DNA , DNA Bacteriano/genética , Conversão Gênica , Sequência de Bases , Evolução Biológica , Cromossomos Bacterianos/fisiologia , Elementos de DNA Transponíveis , Dados de Sequência Molecular , Recombinação Genética , Homologia de Sequência do Ácido Nucleico , Transformação BacterianaRESUMO
The Acinetobacter calcoaceticus pcaJ and catJ genes, nearly identical in DNA sequence, differ in transcriptional control and are separated by more than 20 kb of chromosomal DNA. The pcaJ3125 mutation is repaired frequently in organisms containing the wild-type catJ gene. This high-frequency repair is eliminated in strains lacking the catJ gene, which suggests that recombination between the homologous catJ and pcaJ genes contributes to the high-frequency repair of the pcaJ3125 mutation. We report here that the high-frequency repair also requires a functional recA gene. The A. calcoaceticus recA gene was cloned in Escherichia coli by complementation of a recA mutation in the host strain. The nucleotide sequence of a 1506 bp DNA fragment containing A. calcoaceticus recA was determined. The amino acid sequences of RecA from E. coli and A. calcoaceticus shared 71% identity. The DNA sequences differed in that a consensus binding site for binding of LexA repressor, represented upstream from recA in E. coli, is not evident in the corresponding region of the A. calcoaceticus DNA sequence. A Tn5 insertion was introduced into the A. calcoaceticus recA gene. Selection for Tn5-encoded kanamycin resistance allowed the inactivated recA gene to be recombined by natural transformation into the A. calcoaceticus chromosome. Strains that had acquired the mutant gene were sensitive to both MMS and u.v. light, were deficient in natural transformation, and failed to carry out catJ-dependent high-frequency repair of the pcaJ3125 mutation.
Assuntos
Acinetobacter calcoaceticus/genética , Proteínas de Bactérias , Proteínas de Ligação a DNA , Conversão Gênica/fisiologia , Genes Bacterianos/genética , Recombinases Rec A/genética , Acinetobacter calcoaceticus/metabolismo , Sequência de Aminoácidos , Sequência de Bases , Clonagem Molecular , Elementos de DNA Transponíveis , Escherichia coli/genética , Teste de Complementação Genética , Biblioteca Genômica , Dados de Sequência Molecular , Mutação/fisiologia , Recombinases Rec A/química , Recombinases Rec A/metabolismo , Mapeamento por Restrição , Análise de Sequência de DNA , Homologia de Sequência de Aminoácidos , Transformação BacterianaRESUMO
A strain of Acinetobacter calcoaceticus that demonstrates unusually high competence for natural transformation by linear DNA has proven valuable for analysis of genes and gene clusters associated with aromatic catabolism. The transformation system allowed gap repair to be used to recover mutant chromosomal DNA within recombinant plasmids. The sizes of the recovered fragments, 5 and 7 kilobase pairs in length, indicate that gap repair will be a useful procedure for isolation of wild-type and modified gene clusters from the A. calcoaceticus chromosome.