RESUMEN
In addition to the known response regulator ErbR (former AgmR) and the two-component regulatory system EraSR (former ExaDE), three additional regulatory proteins have been identified as being involved in controlling transcription of the aerobic ethanol oxidation system in Pseudomonas aeruginosa. Two putative sensor kinases, ErcS and ErcS', and a response regulator, ErdR, were found, all of which show significant similarity to the two-component flhSR system that controls methanol and formaldehyde metabolism in Paracoccus denitrificans. All three identified response regulators, EraR (formerly ExaE), ErbR (formerly AgmR) and ErdR, are members of the luxR family. The three sensor kinases EraS (formerly ExaD), ErcS and ErcS' do not contain a membrane domain. Apparently, they are localized in the cytoplasm and recognize cytoplasmic signals. Inactivation of gene ercS caused an extended lag phase on ethanol. Inactivation of both genes, ercS and ercS', resulted in no growth at all on ethanol, as did inactivation of erdR. Of the three sensor kinases and three response regulators identified thus far, only the EraSR (formerly ExaDE) system forms a corresponding kinase/regulator pair. Using reporter gene constructs of all identified regulatory genes in different mutants allowed the hierarchy of a hypothetical complex regulatory network to be established. Probably, two additional sensor kinases and two additional response regulators, which are hidden among the numerous regulatory genes annotated in the genome of P. aeruginosa, remain to be identified.
Asunto(s)
Proteínas Bacterianas/metabolismo , Etanol/metabolismo , Proteínas Quinasas/metabolismo , Pseudomonas aeruginosa/metabolismo , Factores de Transcripción/metabolismo , Regulación Bacteriana de la Expresión Génica , Redes Reguladoras de Genes , Genes Bacterianos , Mutagénesis Sitio-Dirigida , Operón , Oxidación-Reducción , Paracoccus denitrificans/genética , Paracoccus denitrificans/metabolismo , Proteínas Quinasas/genética , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/genética , Transducción de Señal , Factores de Transcripción/genética , Transcripción GenéticaRESUMEN
Gene PA1990 of Pseudomonas aeruginosa, located downstream of pqqE and encoding a putative peptidase, was shown to be involved in excretion of PQQ into the culture supernatant. This gene is cotranscribed with the pqqABCDE cluster and was named pqqH. A PA1990::Km(r) mutant (VK3) did not show any effect in growth behaviour; however, in contrast to the wild-type, no excretion of PQQ into the culture supernatant was observed. The putative pqqF gene of P. aeruginosa was shown to be essential for PQQ biosynthesis. A pqqF::Km(r) mutant did not grow aerobically on ethanol, because of its inability to produce PQQ. Transcription of the pqqABCDEH operon was induced upon aerobic growth on ethanol, 1-propanol, 1,2-propanediol and 1-butanol, while on glycerol, succinate and acetate, transcription was low. Transcription of the pqqABCDEH operon was also found upon anoxic growth on ethanol with nitrate as electron acceptor, but no PQQ was produced. Expression of the pqqABCDEH operon is regulated at the transcriptional level. In contrast, the pqqF operon appeared to be transcribed constitutively at a very low level under all growth conditions studied.
Asunto(s)
Regulación Bacteriana de la Expresión Génica , Operón , Cofactor PQQ/biosíntesis , Pseudomonas aeruginosa/genética , Transcripción Genética , 1-Butanol/metabolismo , 1-Propanol/metabolismo , Proteínas de la Membrana Bacteriana Externa/genética , Proteínas de la Membrana Bacteriana Externa/metabolismo , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Elementos Transponibles de ADN , ADN Bacteriano/genética , ADN Bacteriano/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Genes Bacterianos , Prueba de Complementación Genética , Familia de Multigenes , Mutación , Regiones Promotoras Genéticas , Propilenglicol/metabolismo , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/metabolismo , Quinona Reductasas/genética , Quinona Reductasas/metabolismo , ARN Bacteriano/genética , ARN Bacteriano/metabolismoRESUMEN
The response regulator AgmR was identified to be involved in the regulation of the quinoprotein ethanol oxidation system of Pseudomonas aeruginosa ATCC 17933. Interruption of the agmR gene by insertion of a kanamycin-resistance cassette resulted in mutant NG3, unable to grow on ethanol. After complementation with the intact agmR gene, growth on ethanol was restored. Transcriptional lacZ fusions were used to identify four operons which are regulated by the AgmR protein: the exaA operon encodes the pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase, the exaBC operon encodes a soluble cytochrome c(550) and an aldehyde dehydrogenase, the pqqABCDE operon carries the PQQ biosynthetic genes, and operon exaDE encodes a two-component regulatory system which controls transcription of the exaA operon. Transcription of exaA was restored by transformation of NG3 with a pUCP20T derivative carrying the exaDE genes under lac-promoter control. These data indicate that the AgmR response regulator and the exaDE two-component regulatory system are organized in a hierarchical manner. Gene PA1977, which appears to form an operon with the agmR gene, was found to be non-essential for growth on ethanol.