RESUMEN
The transposase (Tnp) of the bacterial transposon Tn5 acts 50- to 100-fold more efficiently on elements located cis to the site of its synthesis compared with those located in trans. In an effort to understand the basis for this cis preference, we have screened for Tnp mutants that exhibit increased transposition activity in a trans assay. Two mutations in the carboxyl terminus were isolated repeatedly. The EK345 mutation characterized previously increases Tnp activity eightfold both in cis and in trans. The novel LP372 mutation, however, increases Tnp activity 10-fold specifically in trans. Combining both mutations increases Tnp activity 80-fold. Interestingly, the LP372 mutation maps to a region shown previously to be critical for interaction with Inh, an inhibitor of Tn5 transposition, and results in reduced inhibition activity by both Tnp and Inh. Tnp also inhibits Tn5 transposition in trans, and this has been suggested to occur by the formation of inactive Tnp multimers. Because Inh and (presumably) Tnp inhibit Tn5 transposition by forming defective multimers with Tnp, the inhibition defect of the trans-active LP372 mutant suggests that the cis preference of Tnp may also be attributable to nonproductive Tnp-Tnp multimerization. In addition, we show that increasing the synthesis of EK345/LP372 Tnp, but not wild-type Tnp, leads to very high levels of transposition, presumably because this altered Tnp is defective in the inhibitory activity of the wild type protein.
Asunto(s)
Escherichia coli/enzimología , Nucleotidiltransferasas/metabolismo , Secuencia de Bases , Elementos Transponibles de ADN , ADN Bacteriano/genética , Escherichia coli/genética , Datos de Secuencia Molecular , Mutación , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Reacción en Cadena de la Polimerasa , Conformación Proteica , Activación Transcripcional , TransposasasRESUMEN
Overexpression of the Tn5 transposase (Tnp) was found to be lethal to Escherichia coli. This killing was not caused by transposition or dependent on the transpositional or DNA binding competence of Tnp. Instead, it was strictly correlated with the presence of a wild-type N terminus. Deletions removing just two N-terminal amino acids of Tnp resulted in partial suppression of this effect, and deletions of Tnp removing 3 or 11 N-terminal amino acids abolished the killing effect. This cytotoxic effect of Tnp overexpression is accompanied by extensive filament formation (i.e., a defect in cell division) and aberrant nucleoid segregation. Four E. coli mutants were isolated which allow survival upon Tnp overexpression, and the mutations are located at four discrete loci. These suppressor mutations map near essential genes involved in cell division and DNA segregation. One of these mutations maps to a 4.5-kb HindIII region containing the ftsYEX (cell division) locus at 76 min. A simple proposition which accounts for all of these observations is that Tnp interacts with an essential E. coli factor affecting cell division and/or chromosome segregation and that overexpression of Tnp titrates this factor below a level required for viability of the cell. Furthermore, the N terminus of Tnp is necessary for this interaction. The possible significance of this phenomenon for the transposition process is discussed.
Asunto(s)
Elementos Transponibles de ADN , Escherichia coli/enzimología , Escherichia coli/crecimiento & desarrollo , Genes Bacterianos , Mutagénesis , Nucleotidiltransferasas/biosíntesis , Supresión Genética , División Celular/genética , Mapeo Cromosómico , Cromosomas Bacterianos , ADN Bacteriano/metabolismo , Desoxirribonucleasa HindIII , Escherichia coli/genética , Expresión Génica , Genes Letales , Plásmidos , Mapeo Restrictivo , Eliminación de Secuencia , TransposasasRESUMEN
A series of deletions were constructed in the 476 amino acid Tn5 transposase in order to assemble an initial domain structure for this protein. The first four amino acids were found to be important for transposition activity but not for DNA binding to the Tn5 outside end (OE). Larger amino-terminal deletions result in the complete loss of transposition in vivo and the concomitant loss of specific DNA binding. Four point mutants and a six base-pair deletion in the amino terminus between residues 20 and 36 were also found to impair DNA binding to the OE. Analysis of a series of carboxy-terminal deletions has revealed that the carboxy terminus may actually mask the DNA binding domain, since deletions to residues 388 and 370 result in a large increase in DNA binding activity. In addition, the carboxy-terminal deletion to residue 370 results in a significant increase in the mobility of the Tnp-OE complex indicative of a change in the oligomeric state of this complex. Further carboxy-terminal deletions beyond residue 370 also abolished DNA binding activity. These results indicate that the first four amino acids of Tnp are important for transposition but not DNA binding, a region between residues 5 and 36 is critical for DNA binding, the wild-type carboxy terminus acts to inhibit DNA binding, and that a region towards the carboxy terminus, defined by residues 370 to 387, is critical for Tnp multimeric interactions.
Asunto(s)
Elementos Transponibles de ADN , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/metabolismo , Nucleotidiltransferasas/metabolismo , Secuencia de Aminoácidos , Composición de Base , Secuencia de Bases , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Leucina Zippers , Datos de Secuencia Molecular , Nucleotidiltransferasas/química , Nucleotidiltransferasas/genética , Plásmidos , Mutación Puntual , Polímeros , Eliminación de Secuencia , TransposasasRESUMEN
Tn5 is a composite transposon consisting of two IS50 sequences in inverted orientation with respect to a unique, central region encoding several antibiotic resistances. The IS50R element encodes two proteins in the same reading frame which regulate the transposition reaction: the transposase (Tnp), which is required for transposition, and an inhibitor of transposition (Inh). The inhibitor is a naturally occurring deletion variant of Tnp which lacks the N-terminal 55 amino acids. In this report, we present the purification of both the Tnp and Inh proteins and an analysis of their DNA binding properties. Purified Tnp, but not Inh, was found to bind specifically to the outside end of Tn5. Inh, however, stimulated the binding activity of Tnp to outside-end DNA and was shown to be present with Tnp in these bound complexes. Inh was also found to exist as a dimer in solution. These results indicate that the N-terminal 55 amino acids of Tnp are required for sequence-specific binding. They also suggest that Inh inhibits transposition by forming mixed oligomers with Tnp which still bind to the ends of the transposon but are defective for later stages of the transposition reaction.
Asunto(s)
Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , Proteínas de Unión al ADN/metabolismo , Escherichia coli/enzimología , Nucleotidiltransferasas/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/aislamiento & purificación , Cromatografía en Gel , Clonación Molecular , ADN Bacteriano/metabolismo , Proteínas de Unión al ADN/biosíntesis , Proteínas de Unión al ADN/aislamiento & purificación , Electroforesis en Gel de Poliacrilamida , Escherichia coli/genética , Immunoblotting , Nucleotidiltransferasas/biosíntesis , Nucleotidiltransferasas/aislamiento & purificación , Plásmidos , Proteínas Recombinantes/biosíntesis , Proteínas Recombinantes/aislamiento & purificación , Proteínas Recombinantes/metabolismo , TransposasasRESUMEN
The Fis (factor for inversion stimulation) protein of Escherichia coli was found to influence the frequency of transposon Tn5 and insertion sequence IS50 transposition. Fis stimulated both Tn5 and IS50 transposition events and also inhibited IS50 transposition in Dam-bacteria. This influence was not due to regulation by Fis of the expression of the Tn5 transposition proteins. We localized, by DNase I footprinting, one Fis site overlapping the inside end of IS50 and give evidence to strongly suggest that when Fis binds to this site, IS50 transposition is inhibited. The Fis site at the inside end overlaps three Dam GATC sites, and Fis bound efficiently only to the unmethylated substrate. Using a mobility shift assay, we also identified another potential Fis site within IS50. Given the growth phase-dependent expression of Fis and its differential effect on Tn5 versus IS50 transposition in Dam-bacteria, we propose that the high levels of Fis present during exponential growth stimulate transposition events and might bias those events toward Tn5 and away from IS50 transposition.
Asunto(s)
Proteínas Portadoras/metabolismo , Elementos Transponibles de ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Escherichia coli , Escherichia coli/genética , Adenina/análogos & derivados , Adenina/metabolismo , Secuencia de Bases , Sitios de Unión , Proteínas Portadoras/farmacología , Ciclo Celular/fisiología , ADN Bacteriano/metabolismo , Factor Proteico para Inverción de Estimulación , Factores de Integración del Huésped , Datos de Secuencia Molecular , Metiltransferasa de ADN de Sitio Específico (Adenina Especifica)/metabolismoRESUMEN
Three genes from the Bacillus subtilis major che-fla operon have been cloned and sequenced. Two of the genes encode proteins that are homologous to the Escherichia coli and Salmonella typhimurium flagellar biosynthetic proteins FliP and FliQ. The third gene, designated fliZ, encodes a 219-amino-acid protein with a predicted molecular mass of 24,872 Da. FliZ is not significantly homologous to any known proteins. Null mutants in fliP and fliZ do not have flagella; however, motility can be restored to the fliZ null mutant by expression of fliZ from a plasmid. FliZ has a conventional N-terminal signal sequence that does not direct secretion of the protein but appears to target the protein to the membrane. Two possible models of insertion of FliZ into the membrane are described.
Asunto(s)
Bacillus subtilis/genética , Proteínas Bacterianas/genética , Proteínas de Escherichia coli , Flagelos/metabolismo , Proteínas de la Membrana , Secuencia de Aminoácidos , Proteínas Bacterianas/química , Secuencia de Bases , Southern Blotting , Genes Bacterianos , Prueba de Complementación Genética , Datos de Secuencia Molecular , Mutación/genética , Operón/genética , Plásmidos/genética , Señales de Clasificación de Proteína/genética , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismoRESUMEN
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.