RESUMEN
In this work, we have characterized the soluble lytic transglycosylase (SltF) from Rhodobacter sphaeroides that interacts with the scaffolding protein FlgJ in the periplasm to open space at the cell wall peptidoglycan heteropolymer for the emerging rod. The characterization of the genetic context of flgJ and sltF in alphaproteobacteria shows that these two separate genes coexist frequently in a flagellar gene cluster. Two domains of unknown function in SltF were studied, and the results show that the deletion of a 17-amino-acid segment near the N terminus does not show a recognizable phenotype, whereas the deletion of 47 and 95 amino acids of the C terminus of SltF disrupts the interaction with FlgJ without affecting the transglycosylase catalytic activity of SltF. These mutant proteins are unable to support swimming, indicating that the physical interaction between SltF and FlgJ is central for flagellar formation. In a maximum likelihood tree of representative lytic transglycosylases, all of the flagellar SltF proteins cluster in subfamily 1F. From this analysis, it was also revealed that the lytic transglycosylases related to the type III secretion systems present in pathogens cluster with the closely related flagellar transglycosylases.IMPORTANCE Flagellar biogenesis is a highly orchestrated event where the flagellar structure spans the bacterial cell envelope. The rod diameter of approximately 4 nm is larger than the estimated pore size of the peptidoglycan layer; hence, its insertion requires the localized and controlled lysis of the cell wall. We found that a 47-residue domain of the C terminus of the lytic transglycosylase (LT) SltF of R. sphaeroides is involved in the recognition of the rod chaperone FlgJ. We also found that in many alphaproteobacteria, the flagellar cluster includes a homolog of SltF and FlgJ, indicating that association of an LT with the flagellar machinery is ancestral. A maximum likelihood tree shows that family 1 of LTs segregates into seven subfamilies.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/enzimología , Glicosiltransferasas/metabolismo , Filogenia , Rhodobacter sphaeroides/enzimología , Proteínas Bacterianas/genética , Flagelos/genética , Glicosiltransferasas/genética , Funciones de Verosimilitud , Mutación , Peptidoglicano/metabolismo , Rhodobacter sphaeroides/genética , Eliminación de Secuencia , Sistemas de Secreción Tipo III/genéticaRESUMEN
Rhodobacter sphaeroides has two different sets of flagellar genes. Under the growth conditions commonly used in the laboratory, the expression of the fla1 set is constitutive, whereas the fla2 genes are not expressed. Phylogenetic analyses have previously shown that the fla1 genes were acquired by horizontal transfer from a gammaproteobacterium and that the fla2 genes are endogenous genes of this alphaproteobacterium. In this work, we characterized a set of mutants that were selected for swimming using the Fla2 flagella in the absence of the Fla1 flagellum (Fla2(+) strains). We determined that these strains have a single missense mutation in the histidine kinase domain of CckA. The expression of these mutant alleles in a Fla1(-) strain allowed fla2-dependent motility without selection. Motility of the Fla2(+) strains is also dependent on ChpT and CtrA. The mutant versions of CckA showed an increased autophosphorylation activity in vitro. Interestingly, we found that cckA is transcriptionally repressed by the presence of organic acids, suggesting that the availability of carbon sources could be a part of the signal that turns on this flagellar set. Evidence is presented showing that reactivation of fla1 gene expression in the Fla2(+) background strongly reduces the number of cells with Fla2 flagella.
Asunto(s)
Proteínas Bacterianas/metabolismo , Flagelos/metabolismo , Regulación Bacteriana de la Expresión Génica , Proteínas Quinasas/metabolismo , Rhodobacter sphaeroides/enzimología , Proteínas Bacterianas/genética , Flagelos/genética , Histidina Quinasa , Proteínas Quinasas/genética , Rhodobacter sphaeroides/genética , Rhodobacter sphaeroides/metabolismoRESUMEN
Cytochrome c oxidase (COX) employs electrons obtained from cytochrome c to bring about the reduction of oxygen to water. It is known that the electrons originate from the haem edge of cytochrome c and enters bovine COX at Trp-104. It is also known that Tyr-105, Glu-198 and Asp-158 of COX subunit II play roles in the enzyme's catalysis but how these roles are linked to electron transfer remain unclear. Recently, we proposed that electrons travel from the haem edge of cytochrome c to CuA, the first metal redox centre of COX, by a hydrogen/hydride ion relay using six residues. Now using a similar computer assisted approach, we investigate the extent to which this hydride/hydrogen ion mechanism is common amongst oxidases. The crystal structures of COX from P denitrificans, R sphaeroides and T thermophilus and quinol oxidase from E coli were downloaded and their binding domains analysed. As with bovine, all four oxidases had only nine amino acid residues in that region and both the sequences and three-dimensional structures were highly conserved. We propose that these residues function as a hydrogen/hydride ion relay, participating directly in electron transfer to CuA. We further suggest that this electron transfer mechanism might be a common feature in oxidases.
Asunto(s)
Citocromos c , Complejo IV de Transporte de Electrones , Hemo , Hidrógeno , Secuencia de Aminoácidos , Animales , Bovinos , Citocromos c/química , Citocromos c/metabolismo , Transporte de Electrón , Complejo IV de Transporte de Electrones/química , Complejo IV de Transporte de Electrones/metabolismo , Escherichia coli/enzimología , Hemo/química , Hemo/metabolismo , Hidrógeno/química , Hidrógeno/metabolismo , Oxidación-Reducción , Paracoccus denitrificans/enzimología , Protones , Rhodobacter sphaeroides/enzimología , Thermus thermophilus/enzimologíaRESUMEN
Cytochrome c oxidase (COX) employs electrons obtained from cytochrome c to bring about the reduction of oxygen to water. It is known that the electrons originate from the haem edge of cytochrome c and enters bovine COX at Trp-104. It is also known that Tyr-105, Glu-198 and Asp-158 of COX subunit II play roles in the enzyme's catalysis but how these roles are linked to electron transfer remain unclear. Recently, we proposed that electrons travel from the haem edge of cytochrome c to CuA, the first metal redox centre of COX, by a hydrogen/hydride ion relay using six residues. Now using a similar computer assisted approach, we investigate the extent to which this hydride/hydrogen ion mechanism is common amongst oxidases. The crystal structures of COX from P denitrificans, R sphaeroides and T thermophilus and quinol oxidase from E coli were downloaded and their binding domains analysed. As with bovine, all four oxidases had only nine amino acid residues in that region and both the sequences and three-dimensional structures were highly conserved. We propose that these residues function as a hydrogen/hydride ion relay, participating directly in electron transfer to CuA. We further suggest that this electron transfer mechanism might be a common feature in oxidases.
La citocromo c oxidasa (COX) emplea electrones obtenidos del citocromo c para producir la reducción del oxígeno a agua. Se sabe que los electrones originan a partir del hemo del citocromo c, y entran en la COX bovina en Trp-104. También se conoce que Tyr-105, Glu-198 y Asp-158 de la subunidad II de COX, desempeñan papeles en la catálisis de la enzima, pero no hay todavía claridad en cuanto a cómo estos papeles se hallan vinculados con la transferencia de electrones. Recientemente, sugerimos que los electrones viajan del borde del hemo del citocromo c al CuA, el primer centro metálico de reacción redox de la COX, por un relé iónico hidrógeno-hidruro, usando seis residuos. Ahora, usando un enfoque similar computarizado, investigamos hasta que punto este mecanismo de iones hidrógeno/hidruro es común entre las oxidasas. Se bajaron y analizaron los dominios de unión de las estructuras cristalinas de la COX de P denitrificans, R sphaeroides, y T thermophilus, y de la quinol oxidasa de la E coli. Como en el caso de la bovina, las cuatro oxidasas tenían sólo nueve residuos de aminoácido en esa región, y tanto las secuencias como las estructuras tridimensionales presentaban un alto grado de conservación. Proponemos que estos residuos funcionan como un relé iónico hidrógeno-hidruro, participando directamente en una transferencia de electrones al CuA. Asimismo, sugerimos que este mecanismo de transferencia de electrones podría ser un rasgo común de las oxidasas.
Asunto(s)
Animales , Bovinos , Complejo IV de Transporte de Electrones/metabolismo , Citocromos c/metabolismo , Hemo/química , Hidrógeno/metabolismo , Oxidación-Reducción , Paracoccus denitrificans/enzimología , Protones , Rhodobacter sphaeroides/enzimología , Secuencia de Aminoácidos , Thermus thermophilus/enzimología , Escherichia coli/enzimologíaRESUMEN
The F(1)F(O) and F(1)-ATPase complexes of Paracoccus denitrificans were isolated for the first time by ion exchange, gel filtration, and density gradient centrifugation into functional native preparations. The liposome-reconstituted holoenzyme preserves its tight coupling between F(1) and F(O) sectors, as evidenced by its high sensitivity to the F(O) inhibitors venturicidin and diciclohexylcarbodiimide. Comparison and N-terminal sequencing of the band profile in SDS-PAGE of the F(1) and F(1)F(O) preparations showed a novel 11-kDa protein in addition to the 5 canonical alpha, beta, gamma, delta, and epsilon subunits present in all known F(1)-ATPase complexes. BN-PAGE followed by 2D-SDS-PAGE confirmed the presence of this 11-kDa protein bound to the native F(1)F(O)-ATP synthase of P. denitrificans, as it was observed after being isolated. The recombinant 11 kDa and epsilon subunits of P. denitrificans were cloned, overexpressed, isolated, and reconstituted in particulate F(1)F(O) and soluble F(1)-ATPase complexes. The 11-kDa protein, but not the epsilon subunit, inhibited the F(1)F(O) and F(1)-ATPase activities of P. denitrificans. The 11-kDa protein was also found in Rhodobacter sphaeroides associated to its native F(1)F(O)-ATPase. Taken together, the data unveil a novel inhibitory mechanism exerted by this 11-kDa protein on the F(1)F(O)-ATPase nanomotor of P. denitrificans and closely related alpha-proteobacteria.
Asunto(s)
Inhibidores Enzimáticos/aislamiento & purificación , Subunidades de Proteína/metabolismo , ATPasas de Translocación de Protón/química , Inhibidores Enzimáticos/metabolismo , Peso Molecular , Paracoccus denitrificans/enzimología , ATPasas de Translocación de Protón/antagonistas & inhibidores , ATPasas de Translocación de Protón/aislamiento & purificación , Rhodobacter sphaeroides/enzimologíaRESUMEN
Three halotolerant bacterial strains; Rhodobacter sphaeroides ES16 (the wild type) and the two mutant strains of R. sphaeroides ES16, namely N20 and U7, were cultivated in glutamate-malate (GM) medium and screened for production of polyhydroxybutyrate (PHB). The mutant strains N20 and U7 were found to accumulate PHB (53.9 and 42.0 percent of DCW, respectively) 3.6 and 2.8 times higher than the wild type strain (19.5 percent of DCW), respectively. R. sphaeroides N20 were selected for studies on the effects of nutrient and environmental conditions on PHB accumulation. The optimal condition was 4 g/l acetate, 0.02 g/l (NH4)2SO4, C/N ratio of 6:1, 1.0 g/l K2HPO4, 1.0 g/l KH2PO4 and 3 percent NaCl with initial pH at 7.0. Under this optimal condition, the maximum PHB accumulation increased from 53.9 percent to 88 percent of DCW and 9.11 ± 0.08 g/l biomass, 8.02 +/- 0.10 g/l PHB concentration were achieved after 60 hrs cultivation at 37ºC. These results are the highest values ever obtained from photosynthetic bacteria reported so far.
Asunto(s)
Bacterias Gramnegativas Fotosintéticas Oxigénicas , Hidroxibutiratos , Rhodobacter sphaeroides/enzimología , Rhodobacter sphaeroides/metabolismo , Cromatografía de Gases , Fermentación , Microscopía Electrónica de Transmisión , Nitrógeno/administración & dosificaciónRESUMEN
BACKGROUND AND AIMS: Trypanosoma cruzi is the causative agent of Chagas disease or American trypanosomiasis. The parasite manifests a nutritional requirement for heme compounds because of its biosynthesis deficiency. The aim of this study has been to investigate the presence of metabolites and enzymes of porphyrin pathway, as well as ALA formation in epimastigotes of T. cruzi, Tulahuén strain, Tul 2 stock. METHODS: Succinyl CoA synthetase, 5-aminolevulinic acid (ALA) synthetase, 4,5-dioxovaleric (DOVA) transaminase, ALA dehydratase and porphobilinogenase activities, as well as ALA, porphobilinogen (PBG), free porphyrins and heme content were measured in a parasite cells-free extract. Extracellular content of these metabolites was also determined. RESULTS: DOVA, PBG, porphyrins and heme were not detected in acellular extracts of T. cruzi. However ALA was detected both intra- and extracellularly This is the first time that the presence of ALA (98% of intracellularly formed ALA) is demonstrated in the extracellular medium of a parasite culture. Regarding the ALA synthesizing enzymes, DOVA transaminase levels found were low (7.13+/-0.49EU/mg protein), whilst ALA synthetase (ALA-S) activity was undetectable. A compound of non-protein nature, low molecular weight, heat unstable, inhibiting bacterial ALA-S activity was detected in an acellular extract of T. cruzi. This inhibitor could not be identified with either ALA, DOVA or heme. CONCLUSIONS: ALA synthesis is functional in the parasite and it would be regulated by the heme levels, both directly and through the inhibitor factor detected. ALA formed can not be metabolized further, because the necessary enzymes are not active, therefore it should be excreted to avoid intracellular cytotoxicity.
Asunto(s)
5-Aminolevulinato Sintetasa/biosíntesis , Ácido Aminolevulínico/metabolismo , Trypanosoma cruzi/enzimología , 5-Aminolevulinato Sintetasa/antagonistas & inhibidores , Amoníaco-Liasas/metabolismo , Animales , Hemo/metabolismo , Porfobilinógeno/metabolismo , Rhodobacter sphaeroides/enzimología , Succinato-CoA Ligasas/metabolismo , Transaminasas/metabolismoRESUMEN
Rhodobacter sphaeroides expresses a bb3-type quinol oxidase, and two cytochrome c oxidases: cytochrome aa3 and cytochrome cbb3. We report here the characterization of the genes encoding this latter oxidase. The ccoNOQP gene cluster of R. sphaeroides contains four open reading frames with high similarity to all ccoNOQP/fixNOQP gene clusters reported so far. CcoN has the six highly conserved histidines proposed to be involved in binding the low spin heme, and the binuclear center metals. ccoO and ccoP code for membrane bound mono- and diheme cytochromes c. ccoQ codes for a small hydrophobic protein of unknown function. Upstream from the cluster there is a conserved Fnr/FixK-like box which may regulate its expression. Analysis of a R. sphaeroides mutant in which the ccoNOQP gene cluster was inactivated confirms that this cluster encodes the cbb3-type oxidase previously purified. Analysis of proton translocation in several strains shows that cytochrome cbb3 is a proton pump. We also conclude that cytochromes cbb3 and aa3 are the only cytochrome c oxidases in the respiratory chain of R. sphaeroides.
Asunto(s)
Complejo IV de Transporte de Electrones/genética , Oxidorreductasas/genética , Bombas de Protones/metabolismo , Rhodobacter sphaeroides/genética , Secuencia de Aminoácidos , Secuencia de Bases , Membrana Celular/enzimología , Clonación Molecular , Cobre , Complejo IV de Transporte de Electrones/química , Genes Bacterianos/genética , Prueba de Complementación Genética , Hemo , Histidina/química , Datos de Secuencia Molecular , Familia de Multigenes/genética , Mutación , Sistemas de Lectura Abierta/genética , Oxidorreductasas/química , Bombas de Protones/química , Bombas de Protones/genética , Mapeo Restrictivo , Rhodobacter sphaeroides/enzimología , Análisis de Secuencia de ADNRESUMEN
Incubation of porphobilinogen (PBG) with PBG deaminase from Rhodopseudomonas sphaeroides in carbonate buffer (pH 9.2) to total PBG consumption resulted in low yields of uroporphyrinogen I (uro'gen I). In the reaction mixture a pyrrylmethane accumulated, which at longer incubation periods was transformed into uro'gen I. The accumulated pyrrylmethane gave an Ehrlich reaction which was different from that of a 2-(aminomethyl)dipyrrylmethane or 2-(aminomethyl)tripyrrane. It resembled that of a bilane (tetrapyrrylmethane) but was different from that of a 2-(hydroxymethyl)bilane. The 13C NMR spectra of incubations carried out with [11-13C]PBG indicated that the pyrrylmethane was a tetrapyrrole with methylene resonances at 22.35-22.50 ppm. It was loosely bound to the deaminase, and when separated from the enzyme by gel filtration or gel electrophoresis, it immediately cyclized to uro'gen I. No enzyme-bound methylene could be detected by its chemical shift, suggesting that its line width must be very broad. When uro'gen III-cosynthase was added to the deaminase-tetrapyrrole complex, uro'gen III was formed at the expense of the latter in about 75% yield. The tetrapyrrole could only be partially displaced from the enzyme by ammonium ions, although a small amount of 2-(aminomethyl)bilane was always formed together with the tetrapyrrole intermediate. A protonated uro'gen I structure for this intermediate was ruled out by incubations using [2,11-13C]PBG. Uro'gen III formation from 2-(hydroxymethyl)bilane (HMB) and from the deaminase-tetrapyrrole intermediate was compared by using deaminase-cosynthase and cosynthase from several sources. It was found that while the HMB inhibited uro'gen III formation at higher concentrations and longer incubation times, uro'gen III formation from the complex did not decrease with time.