RESUMEN
The sipM gene of Bacillus megaterium encoding a type I signal peptidase (SPase) was isolated and structurally characterized. RNA analysis revealed a transcript size in accordance with a bicistronic operon comprising sipM and an adjacent open reading frame. Inactivation of sipM by targeted gene disruption could not be achieved, indicating its essential role for cell viability since there might be no other type I SPase of major importance present in B. megaterium. Plasmid-assisted amplification of the gene resulted in an increase in activity of the heterologous glucanase used as an extracellular reporter, suggesting a potential bottleneck for protein secretion within this species.
Asunto(s)
Bacillus megaterium/enzimología , Bacillus megaterium/genética , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Serina Endopeptidasas/genética , Serina Endopeptidasas/metabolismo , Secuencia de Aminoácidos , Proteínas Bacterianas/genética , Secuencia de Bases , Celulasa/genética , Celulasa/metabolismo , ADN Bacteriano/química , ADN Bacteriano/aislamiento & purificación , Eliminación de Gen , Dosificación de Gen , Expresión Génica , Orden Génico , Genes Bacterianos , Genes Esenciales , Genes Reporteros , Datos de Secuencia Molecular , Mutagénesis Insercional , Operón , Alineación de Secuencia , Análisis de Secuencia de ADN , Homología de Secuencia de Aminoácido , Transcripción GenéticaRESUMEN
The bamM gene from Bacillus megaterium DSM319 encoding an extracellular beta-amylase was isolated and completely sequenced. Chromosomal inactivation by deletion mutagenesis resulted in total loss of amylolytic activity, indicative of a single starch-degrading enzyme. Functional characterization of the expressed protein revealed a maltogenic enzyme exhibiting optimal activities at pH 7.5 and 50 degrees C. Amylase expression is subject to catabolite repression by glucose. A putative cis-acting catabolite-responsive element (CRE) was identified; it is located within the bamM coding region, matching the position of the predicted signal peptide processing site. Base substitutions introduced by site-directed mutagenesis within the bamM-CRE--retaining unchanged the amino acid sequence--provoked a remarkable relief from carbon catabolite repression (CCR), thereby proving functionality of the CRE for CCR.
Asunto(s)
Bacillus megaterium/enzimología , Carbono/metabolismo , Represión Enzimática , Elementos de Respuesta , beta-Amilasa/genética , Secuencia de Aminoácidos , Bacillus megaterium/genética , Secuencia de Bases , Clonación Molecular , Datos de Secuencia Molecular , Mutagénesis Sitio-DirigidaRESUMEN
The beta-galactosidase-encoding bgaM gene of Bacillus megaterium DSM319 and the divergently orientated bgaR operon were isolated and sequenced. Both traits are subject to catabolite repression. A set of single-gene replacement mutants was generated and used to analyze gene function. BgaR was found to be a XylS/AraC-type positive transcriptional regulator of bgaM; a potential regulator binding site overlaps the bgaM promoter. A mechanism for regulation of beta-galactosidase expression in B. megaterium is proposed.
Asunto(s)
Bacillus megaterium/genética , Regulación Bacteriana de la Expresión Génica , Genes Bacterianos/genética , Transactivadores/metabolismo , beta-Galactosidasa/genética , Bacillus megaterium/enzimología , Proteínas Bacterianas , Secuencia de Bases , Sitios de Unión , Clonación Molecular , Proteínas de Unión al ADN , Eliminación de Gen , Expresión Génica , Regulación Bacteriana de la Expresión Génica/efectos de los fármacos , Regulación Enzimológica de la Expresión Génica/efectos de los fármacos , Genes Bacterianos/fisiología , Glucosa/metabolismo , Glucosa/farmacología , Lactosa/metabolismo , Lactosa/farmacología , Modelos Genéticos , Datos de Secuencia Molecular , Mutagénesis Insercional , Nitrofenilgalactósidos/metabolismo , Sistemas de Lectura Abierta/genética , Operón/genética , Regiones Promotoras Genéticas/genética , Análisis de Secuencia de ADN , Transactivadores/genética , Transcripción Genética/efectos de los fármacos , beta-Galactosidasa/metabolismoRESUMEN
An efficient method for gene replacement in Bacillus megaterium was developed and used to inactivate the chromosomal neutral protease gene (nprM) from strain DSM319. A temperature-dependant suicide vector was constructed to allow replacement of the normal chromosomal copy with an altered version of the nprM gene. One mutant B. megaterium MS941 was selected for further characterization. Measurement of extracellular protease activity from strain MS941 indicated the existence of an additional minor extracellular protease in B. megaterium. Inhibitor studies revealed that this minor protease, comprising only 1.4% of the wild-type total extracellular protease activities, is a serine-type enzyme.
Asunto(s)
Bacillus megaterium/enzimología , Bacillus megaterium/genética , Proteínas Bacterianas , Endopeptidasas/genética , Secuencia de Bases , Mapeo Cromosómico , Clonación Molecular , ADN Bacteriano/genética , Endopeptidasas/metabolismo , Escherichia coli/genética , Eliminación de Gen , Genes Bacterianos , Técnicas Genéticas , Vectores Genéticos , Datos de Secuencia Molecular , Mutación , Plásmidos/genéticaRESUMEN
The leuC gene, encoding 3-isopropylmalate dehydrogenase, the nprM gene (neutral protease) and a sporulation gene coding for a putative spoIV protein (spoIV) from Bacillus megaterium DSM 319 were cloned and the nucleotide sequences were determined. The leuC gene is 1101 bp in length, preceded by a ribosome binding site; no promoter consensus sequence could be found. The nucleotide sequence from nprM when compared to the recently published gene from B. megaterium ATCC 14581 exhibited only a 17-base pair deviation. From a sporulation mutant isolated after transposon-mutagenesis with transposon Tn917 the insertion site of the transposon was cloned and adjacent chromosomal fragments were characterized. An open reading frame that encodes for a putative spo protein of 247 amino-acid residues was identified.
