RESUMEN
The amount of tryptophanase was estimated in Escherichia coli deltadnaJ and deltadnaKdnaJ mutants. Densitometric analysis of polyacrylamide gels demonstrated that the amount of tryptophanase was diminished in both mutants. DnaK and DnaJ molecular chaperones apparently influence the amount of tryptophanase, the expression of which is regulated at all transcription steps, including transcription elongation. The half-life of GreA and GreB proteins (being activators of transcription elongation of the tna operon) are diminished in both mutants suggesting the involvement of DnaK and DnaJ in the stability of these proteins.
Asunto(s)
Proteínas de Escherichia coli/metabolismo , Escherichia coli/metabolismo , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Factores de Transcripción/metabolismo , Triptofanasa/metabolismo , Escherichia coli/genética , Proteínas de Escherichia coli/genética , Expresión Génica , Genes Bacterianos , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/genética , Semivida , Proteínas de Choque Térmico/genética , Mutación , Factores de Elongación Transcripcional , Triptofanasa/genéticaRESUMEN
The effect of mutations in dnaK and dnaJ genes on the expression of two operons that are part of cysteine regulon was determined using Escherichia coli strains harboring cysPTWA::lacZ and cysJIH::lacZ fusions. Null dnaJ and dnaKdnaJ mutants were impaired in beta-galactosidase expression from both fusions. Efficient complementation of this defect by wild-type alleles present on a low-copy number plasmid was achieved. The presence of the pMH224 plasmid coding for CysB* protein defective in DNA binding lowered beta-galactosidase expression from cysPTWA::lacZ fusion strain harboring wild-type dnaKdnaJ alleles but did not diminish enzyme expression in delta dnaJ and delta dnaKdnaJ strains.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/genética , Genes Bacterianos , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Alelos , Fusión Artificial Génica , Cisteína/genética , Escherichia coli/enzimología , Expresión Génica , Prueba de Complementación Genética , Proteínas del Choque Térmico HSP40 , Operón Lac , Mutación , Operón , Plásmidos/genética , beta-Galactosidasa/genéticaAsunto(s)
Bacterias/genética , Proteínas de Escherichia coli/metabolismo , Extensión de la Cadena Peptídica de Translación/fisiología , Factores de Transcripción/metabolismo , ARN Polimerasas Dirigidas por ADN/metabolismo , Células Eucariotas/metabolismo , Transcripción Genética/fisiología , Factores de Elongación TranscripcionalRESUMEN
The role of two chaperone proteins, DnaK and the cooperating factor DnaJ, in Escherichia coli antibiotic susceptibility to three antibiotics (a beta-lactam, chloramphenicol, tetracycline) has been studied. It was found that null dnaJ and dnaKdnaJ mutants are impaired in the functions leading to antibiotic susceptibility. The secretion of beta-lactamase to the periplasmic space is diminished in both mutants, and the additive effect of the two mutations was observed. The activity of chloramphenicol acetyltransferase is also impaired in an additive manner in both mutant strains. Tetracycline uptake is changed only in the double deletion mutant. These defects were observed only during incubation at high temperature (42 degrees C). Efficient complementation of some of these defects by the wild-type alleles introduced on low-copy number plasmid was achieved. Minimal inhibitory concentrations and the titer of the wild-type strains, delta dnaJ and delta dnaKdnaJ mutants treated with ampicillin, chloramphenicol, and tetracycline were also determined. Higher susceptibility of both mutants to chloramphenicol and tetracycline, as compared to their wild-type parent, was observed only after 1 h preincubation of cultures at 42 degrees C. On the contrary, both mutants were less susceptible to ampicillin than their parent strain.
Asunto(s)
Antibacterianos/farmacología , Proteínas de Escherichia coli , Escherichia coli/efectos de los fármacos , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Mutación , Ampicilina/farmacología , Cloranfenicol/farmacología , Cloranfenicol O-Acetiltransferasa/metabolismo , Escherichia coli/enzimología , Escherichia coli/genética , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/metabolismo , Proteínas de Choque Térmico/metabolismo , Pruebas de Sensibilidad Microbiana/métodos , Tetraciclina/metabolismo , beta-Lactamasas/metabolismoRESUMEN
Escherichia coli null dnaJ and dnaKdnaJ mutants were defective in the biosynthesis and secretion of several enzymes. The synthesis of beta-galactosidase induced in delta dnaJ and delta dnaKdnaJ mutants was abolished at 42 degrees C and significantly decreased at 30 and 37 degrees C. The activity of alkaline phosphatase in the periplasm in both mutant strains at high temperature was lower than in the wild-type strain. The synthesis of b-type cytochromes was defective in two deletion mutants while the synthesis of nitrate reductase-A at 42 degrees C was influenced by dnaK mutation only. The lack of DnaK and DnaJ does not impair the activity of catechol 2,3-dioxygenase irrespective of growth temperature.
Asunto(s)
Proteínas Bacterianas/biosíntesis , Dioxigenasas , Proteínas de Escherichia coli , Escherichia coli/enzimología , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Chaperonas Moleculares/genética , Fosfatasa Alcalina/metabolismo , Proteínas Bacterianas/metabolismo , Catecol 2,3-Dioxigenasa , Grupo Citocromo b/biosíntesis , Grupo Citocromo b/metabolismo , Escherichia coli/genética , Eliminación de Gen , Proteínas del Choque Térmico HSP40 , Calefacción , Nitrato-Reductasa , Nitrato Reductasas/biosíntesis , Nitrato Reductasas/metabolismo , Oxigenasas/metabolismo , beta-Galactosidasa/biosíntesis , beta-Galactosidasa/metabolismoRESUMEN
E. coli defects in response to nutritional starvation caused by DnaK and DnaJ proteins deprivation are examined. The ability of delta dnaKdnaJ mutant to survive carbon, nitrogen and phosphorus starvation is highly impaired while delta dnaJ mutant is characterized by the diminished survival of phosphorus starvation only. delta dnaKdnaJ mutant grows slowly utilizing maltose and glycerol and delta dnaJ mutant utilizes glycerol inefficiently. The growth on alternate nitrogen sources is comparable to wild-type strain.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/fisiología , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas de Choque Térmico/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/fisiología , Carbono/metabolismo , Medios de Cultivo/química , Escherichia coli/química , Glicerol , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Maltosa , Mutación , Nitrógeno/metabolismo , Fósforo/metabolismoRESUMEN
Mutations in the heat shock genes, dnaK and dnaJ, cause severe defects of several cellular functions. Null dnaJ and dnaKdnaJ mutations can be transduced in a restricted range of temperature. The efficiency of transformation with three unrelated plasmids, viz pACYC184, pBR322 and pSC101, is two times lower in dnaK mutants while the dnaJ mutant is characterized by slightly impaired transformation with pSC101 only. The lack of DnaJ function negatively influences the stability of pSC101 at 42 degrees C, and this plasmid cannot be stably maintained at 30 degrees C in the delta dnaKdnaJ mutant. The double deletion mutant, delta dbaKdnaJ, is characterized by impaired osmoadaptation. The galactokinase content is lower in both mutants tested compared with wild-type strains even at 30 degrees C. The efficient complementation of some of these defects by the wild-type alleles present on low-copy number plasmid was achieved.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/fisiología , Genes Bacterianos , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Adaptación Fisiológica , Alelos , Escherichia coli/genética , Escherichia coli/metabolismo , Galactoquinasa/metabolismo , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas de Choque Térmico/fisiología , Mutagénesis , Presión OsmóticaAsunto(s)
Bacterias/genética , Bacteriófagos/genética , Transcripción Genética/genética , Secuencia de Bases , Mapeo Cromosómico , ARN Polimerasas Dirigidas por ADN/metabolismo , Escherichia coli/genética , Datos de Secuencia Molecular , Estructura Secundaria de Proteína , ARN de Transferencia/metabolismo , Operón de ARNr/genéticaRESUMEN
Escherichia coli defects caused by the overproduction of DnaK and DnaJ proteins are presented. Induced overproduction of DnaK and DnaJ was determined by SDS-PAGE electrophoresis and immunoblots analysis. Elevated levels of both proteins are not deleterious for such cellular functions as growth, survival and cell morphology. However, instability of pJW14 plasmid (derivative of pACYC184) was observed as the result of dnaKdnaJ operon overexpression.
Asunto(s)
Proteínas de Escherichia coli , Escherichia coli/crecimiento & desarrollo , Escherichia coli/genética , Proteínas HSP70 de Choque Térmico/biosíntesis , Proteínas de Choque Térmico/biosíntesis , Recuento de Colonia Microbiana , Electroforesis en Gel de Poliacrilamida , Escherichia coli/metabolismo , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , Immunoblotting , Isopropil Tiogalactósido/farmacología , Operón , Plásmidos/genética , EspectrofotometríaRESUMEN
Escherichia coli dnaJ and dnaKdnaJ mutants are defective in cell growth and survival at high temperature. Lack of DnaK and DnaJ proteins results in cell filamentation and leads to the defect in motility. Synthesis of DNA and protein is inhibited at 42 degrees C, especially in double-deletion mutant. Degradation of protein was observed in both mutants at high temperature. Complementation of these defects can be achieved by the expression of the wild-type alleles from low-copy number plasmid.
Asunto(s)
Proteínas Bacterianas/fisiología , Proteínas de Escherichia coli , Escherichia coli/crecimiento & desarrollo , Proteínas HSP70 de Choque Térmico/fisiología , Proteínas de Choque Térmico/fisiología , Mutación , Proteínas Bacterianas/biosíntesis , Proteínas Bacterianas/genética , ADN Bacteriano/biosíntesis , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos/fisiología , Proteínas del Choque Térmico HSP40 , Proteínas HSP70 de Choque Térmico/genética , Proteínas de Choque Térmico/genética , TemperaturaAsunto(s)
Bacterias/genética , Proteínas de Escherichia coli , Terminación de la Cadena Péptídica Traduccional , Factor Rho/metabolismo , Adenosina Trifosfato/metabolismo , Proteínas Bacterianas/metabolismo , Secuencia de Bases , Datos de Secuencia Molecular , Factores de Elongación de Péptidos/metabolismo , Factores de Terminación de Péptidos/metabolismo , ARN Bacteriano/metabolismo , Factor Rho/química , Factores de Transcripción/metabolismo , Transcripción Genética , Factores de Elongación TranscripcionalRESUMEN
Amino acid analysis of pure murein isolated from cells of T. neapolitanus revealed the typical constituents of most mureins form Gram-negative bacteria. i.e. glutamic acid, alanine and diaminopimelic acid, but the molecular ratio ot these was unusual, being approximately 1: 1: 1. The reduced amount of alanine was explained by the absence of monomers containing tetrapeptide side chains, as revealed by h. p. 1. c. analysis, [(3)H]glutamic acid, [(3)H]diaminopimelic acid and [(3)H]N-acetylglucosamine were incorporated into the murein and allowed to determine the degree of its crosslinkage (28%) and the occurrence of turnover.
Asunto(s)
Aminoácidos/análisis , Halothiobacillus/química , Halothiobacillus/citología , Peptidoglicano/química , Amino Azúcares/análisis , Marcaje Isotópico , Peptidoglicano/aislamiento & purificación , Peptidoglicano/metabolismo , Tritio/químicaRESUMEN
A restriction map of Thiobacillus versutus plasmid pTAV1 was constructed using EcoRI, BamHI and SalI restriction enzymes. Knowledge of the restriction map is an obligatory starting point for genetic and molecular studies of this, so far cryptic, plasmid.
Asunto(s)
Plásmidos/genética , Thiobacillus/genética , Desoxirribonucleasa BamHI/metabolismo , Desoxirribonucleasa EcoRI/metabolismo , Desoxirribonucleasas de Localización Especificada Tipo II/metabolismo , Plásmidos/fisiología , Mapeo RestrictivoRESUMEN
RNA-dependent ATPase activity of Rho + and two mutant proteins Rho15 and Rho301 was studied. It was shown that monomeric Rho forms oligomers in the presence of ATP. This ATP-induced structural change of Rho allows protection of the protein from heat inactivation. Poly(C), which highly activates Rho ATPase, was found to potentiate heat inactivation of Rho301, but no Rho + and Rho15, only under optimal conditions of ATP hydrolysis. It was also shown that Rho301 is defective in interaction with RNA. The molecular model postulating that Rho-catalysed ATP hydrolysis with free RNA involves the cyclic process of protein dissociation and reassembly is postulated.