RESUMEN
The Bacillus megaterium genes uvrB and uvrA, encoding two subunits of the (A)BC excinuclease, which is responsible for nucleotide excision repair, were isolated and functionally characterized. RNA analyses revealed co-transcription of both genes probably forming a bicistronic operon. Expression of uvrB and uvrA was inducible by the DNA-damaging agent mitomycin C. This finding agrees with the presence of a potential DinR box within the uvrBA promoter. Single inactivation of uvrB or uvrA as well as the parallel knockout of both genes resulted in mutants highly sensitive to UV irradiation. Thus, this locus represents an attractive target for generating biologically safe containment strains of B. megaterium.
Asunto(s)
Bacillus megaterium/efectos de la radiación , Proteínas Bacterianas/fisiología , ADN Helicasas/genética , ADN Bacteriano/efectos de la radiación , Rayos Ultravioleta , Bacillus megaterium/enzimología , Bacillus megaterium/genética , Proteínas Bacterianas/genética , Daño del ADN , ADN Helicasas/fisiología , Reparación del ADN , Mitomicina/farmacología , Mutación , Operón/genética , Transcripción GenéticaRESUMEN
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
Monoamine oxidase (MAO) was characterized in tissue homogenates from pancreatic islets, exocrine pancreas, and liver from rats. Phenylethylamine was preferentially deaminated by pancreatic islet MAO while 5-hydroxytryptamine was preferentially deaminated by MAO from exocrine pancreas, and tyramine was a good substrate for both tissues. All three substrates were well deaminated by liver tissue. Clorgyline, a selective inhibitor of MAO-A, preferentially inhibited deamination of 5-hydroxytryptamine by all three tissue homogenates, while deprenyl, a selective inhibitor of MAO-B, preferentially inhibited deamination of phenylethylamine. In the case of pargyline, a less selective MAO-B inhibitor, the preference in favour of phenylethylamine was less pronounced. According to these results, MAO in pancreatic islets can be classified as predominantly type B enzyme species and MAO in exocrine pancreas as predominantly type A enzyme species while both types of the enzyme are present in the liver. Using the same three MAO substrates and compared with the effects of the selective enzyme inhibitors, clorgyline and deprenyl, tranylcypromine can be classified as a potent nonselective inhibitor of MAO in homogenates of all three tissues investigated with a slight preference in favour of the inhibition of the B-form of the enzyme, while in contrast amezinium can be classified as a weak nonselective inhibitor of MAO with a slight preference in favour of the inhibition of the A-form of the enzyme. All MAO inhibitors tested also inhibited insulin secretion by isolated incubated rat pancreatic islets, however only at IC50 which were two to three decimal powers higher than those necessary for the inhibition of the MAO activity, thus indicating that inhibition of MAO activity and inhibition of insulin secretion are apparently not closely related.