RESUMEN
Initial analysis has shown that the transcription of the Pseudomonas alcaligenes lipA gene, which encodes an extracellular lipase, is governed by the LipQR two-component system consisting of sensor kinase LipQ and DNA-binding regulator LipR. This study further analyzes lipA gene expression and demonstrates that the RNA polymerase σ54 is involved in the transcription. Purified LipR has an ATPase activity that is stimulated by the presence of lipA promoter DNA. Surface plasmon resonance measurements with purified and in vitro phosphorylated LipR reveal that phosphorylation of LipR is required for specific binding to the upstream activating sequence of the lipA promoter. Furthermore, mass spectrometric analysis combined with mutagenesis demonstrates that Asp52 is the phosphorylated aspartate. This analysis exposes LipR as a prominent member of the growing family of bacterial enhancer-binding proteins.
Asunto(s)
Proteínas Bacterianas/genética , Proteínas de Unión al ADN/genética , Lipasa/genética , Pseudomonas alcaligenes/genética , ARN Polimerasa Sigma 54/genética , Transcripción Genética/genética , Adenosina Trifosfatasas/metabolismo , Ácido Aspártico/metabolismo , Proteínas Bacterianas/biosíntesis , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/metabolismo , Proteínas Fúngicas/química , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Regulación Bacteriana de la Expresión Génica , Lipasa/biosíntesis , Mutación , Fosforilación , Pseudomonas alcaligenes/enzimología , Pseudomonas alcaligenes/metabolismo , ARN Polimerasa Sigma 54/química , ARN Polimerasa Sigma 54/metabolismo , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
PvdQ, an acylase from Pseudomonas aeruginosa PAO1, has been shown to have at least two functions. It can act as a quorum quencher due to its ability to degrade long-chain N-acylhomoserine lactones (AHLs), e.g. 3-oxo-C12-HSL, leading to a decrease in virulence factors. In addition, PvdQ is involved in iron homeostasis by playing a role in the biosynthesis of pyoverdine, the major siderophore of P. aeruginosa. In accordance with earlier studies on RNA level, we could show at the protein level that PvdQ is only expressed when iron is present at very low concentrations. We therefore set out to investigate the two functions of PvdQ under iron-limiting conditions. Gene deletion of pvdQ does not affect growth of P. aeruginosa but abrogates pyoverdine production, and results in an accumulation of 3-oxo-C12-HSL. Phenotypic analyses of our DeltapvdQ mutant at low iron concentrations revealed that this mutant is impaired in swarming motility and biofilm formation. Additionally, a plant and a Caenorhabditis elegans infection model demonstrated that the deletion of pvdQ resulted in reduced virulence. None of the phenotypes in the present study could be linked to the presence or absence of AHLs. These results clearly indicate that under iron-limiting conditions PvdQ plays a major role in swarming motility, in biofilm development and in infection that is more likely to be linked to the pyoverdine pathway rather than the LasI/LasR/3-oxo-C12-HSL quorum-sensing circuit.
Asunto(s)
Amidohidrolasas/metabolismo , Proteínas Bacterianas/metabolismo , Hierro/metabolismo , Pseudomonas aeruginosa/genética , 4-Butirolactona/análogos & derivados , 4-Butirolactona/biosíntesis , Amidohidrolasas/genética , Animales , Proteínas Bacterianas/genética , Biopelículas/crecimiento & desarrollo , Caenorhabditis elegans/microbiología , Eliminación de Gen , Regulación Bacteriana de la Expresión Génica , Homoserina/análogos & derivados , Homoserina/biosíntesis , Oligopéptidos/biosíntesis , Pseudomonas aeruginosa/enzimología , Pseudomonas aeruginosa/crecimiento & desarrollo , Pseudomonas aeruginosa/patogenicidad , Solanum tuberosum/microbiología , VirulenciaRESUMEN
Penicillin G acylase (PGA) is a widely studied bacterial enzyme of great industrial importance. Since its overproduction in the original organisms is mostly limited to the intracellular bacterial spaces which may lead to aggregation and cell toxicity, we have set out to explore the host organism Pseudomonas aeruginosa that possesses the Xcp machinery for secretion of folded proteins to the extracellular medium. We have made fusion proteins, consisting of Pseudomonas Sec- or Tat-specific signal peptides, the elastase propeptide and the mature penicillin G acylase. With all constructs we obtained production of PGA in P. aeruginosa, but we observed that processing of the PGA was temperature dependent and that the active enzyme could only be found after growth at 25 degrees C or lower temperatures. Remarkably, the mature protein, expressed from a TatProPGA hybrid, was not only found in the extracellular medium and the periplasm, but also in the cytoplasm as assessed by comparison to the reporter beta-lactamase protein. The unusual cytoplasmic localization of the mature protein strongly suggests that processing of PGA can also occur in the cytoplasm of P. aeruginosa. The extracellular localization of the TatProPGA hybrid was found not to be dependent on the tatABC-genes. The elastase signal sequence/propeptide combination appeared to be an inadequate carrier for transporting penicillin G acylase across the outer membrane of P. aeruginosa.
Asunto(s)
Proteínas de Escherichia coli/biosíntesis , Penicilina Amidasa/biosíntesis , Pseudomonas aeruginosa/enzimología , Proteínas Recombinantes de Fusión/biosíntesis , Proteínas Bacterianas , Clonación Molecular , Citoplasma/química , Proteínas de Escherichia coli/genética , Proteínas de Transporte de Membrana , Elastasa Pancreática/genética , Penicilina Amidasa/genética , Periplasma/química , Señales de Clasificación de Proteína/genética , Transporte de Proteínas , Pseudomonas aeruginosa/genética , Proteínas Recombinantes de Fusión/genéticaRESUMEN
Preliminary observations in a large-scale fermentation process suggested that the lipase expression of Pseudomonas alcaligenes can be switched on by the addition of certain medium components, such as soybean oil. In an attempt to elucidate the mechanism of induction of lipase expression, we have set up a search method for genes controlling lipase expression by use of a cosmid library containing fragments of P. alcaligenes genomic DNA. A screen for lipase hyperproduction resulted in the selection of multiple transformants, of which the best-producing strains comprised cosmids that shared an overlapping genomic fragment. Within this fragment, two previously unidentified genes were found and named lipQ and lipR. Their encoded proteins belong to the NtrBC family of regulators that regulate gene expression via binding to a specific upstream activator sequence (UAS). Such an NtrC-like UAS was identified in a previous study in the P. alcaligenes lipase promoter, strongly suggesting that LipR acts as a positive regulator of lipase expression. The regulating role could be confirmed by down-regulated lipase expression in a strain with an inactivated lipR gene and a threefold increase in lipase yield in a large-scale fermentation when expressing the lipQR operon from the multicopy plasmid pLAFR3. Finally, cell extracts of a LipR-overexpressing strain caused a retardation of the lipase promoter fragment in a band shift assay. Our results indicate that lipase expression in Pseudomonas alcaligenes is under the control of the LipQR two-component system.