03 Fakultät Chemie

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Author: search.filters.author.Billard, PatrickSubject: search.filters.subject.500

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    The PedS2/PedR2 two-component system is crucial for the rare earth element switch in Pseudomonas putida KT2440
    (2018) Wehrmann, Matthias; Berthelot, Charlotte; Billard, Patrick; Klebensberger, Janosch
    ABSTRACT In Pseudomonas putida KT2440, two pyrroloquinoline quinone-dependent ethanol dehydrogenases (PQQ-EDHs) are responsible for the periplasmic oxidation of a broad variety of volatile organic compounds (VOCs). Depending on the availability of rare earth elements (REEs) of the lanthanide series (Ln3+), we have recently reported that the transcription of the genes encoding the Ca2+-utilizing enzyme PedE and the Ln3+-utilizing enzyme PedH are inversely regulated. With adaptive evolution experiments, site-specific mutations, transcriptional reporter fusions, and complementation approaches, we now demonstrate that the PedS2/PedR2 (PP_2671/PP_2672) two-component system (TCS) plays a central role in the observed REE-mediated switch of PQQ-EDHs in P. putida. We provide evidence that in the absence of lanthanum (La3+), the sensor histidine kinase PedS2 phosphorylates its cognate LuxR-type response regulator PedR2, which in turn not only activates pedE gene transcription but is also involved in repression of pedH. Our data further suggest that the presence of La3+ lowers kinase activity of PedS2, either by the direct binding of the metal ions to the periplasmic region of PedS2 or by an uncharacterized indirect interaction, leading to reduced levels of phosphorylated PedR2. Consequently, the decreasing pedE expression and concomitant alleviation of pedH repression causes - in conjunction with the transcriptional activation of the pedH gene by a yet unknown regulatory module - the Ln3+-dependent transition from PedE- to PedH-catalyzed oxidation of alcoholic VOCs. IMPORTANCE The function of lanthanides for methanotrophic and methylotrophic bacteria is gaining increasing attention, while knowledge about the role of rare earth elements (REEs) in nonmethylotrophic bacteria is still limited. The present study investigates the recently described differential expression of the two PQQ-EDHs of P. putida in response to lanthanides. We demonstrate that a specific TCS is crucial for their inverse regulation and provide evidence for a dual regulatory function of the LuxR-type response regulator involved. Thus, our study represents the first detailed characterization of the molecular mechanism underlying the REE switch of PQQ-EDHs in a nonmethylotrophic bacterium and stimulates subsequent investigations for the identification of additional genes or phenotypic traits that might be coregulated during REE-dependent niche adaptation.
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    Rare earth element (REE)-dependent growth of Pseudomonas putida KT2440 relies on the ABC-transporter PedA1A2BC and is influenced by iron availability
    (2019) Wehrmann, Matthias; Berthelot, Charlotte; Billard, Patrick; Klebensberger, Janosch
    In the soil-dwelling organism Pseudomonas putida KT2440, the rare earth element (REE)-utilizing, and pyrroloquinoline quinone (PQQ)-dependent ethanol dehydrogenase PedH is part of a periplasmic oxidation system that is vital for growth on various alcoholic volatiles. Production of PedH and its Ca2+-dependent counterpart PedE is inversely regulated in response to lanthanide (Ln3+) bioavailability, a mechanism termed the REE-switch. In the present study, we demonstrate that copper, zinc, and in particular, iron availability influences this regulation in a pyoverdine-independent manner by increasing the minimal Ln3+ concentration required for the REE-switch to occur by several orders of magnitude. A combined genetic and physiological approach reveals that an ABC-type transporter system encoded by the gene cluster pedA1A2BC is essential for efficient growth on 2-phenylethanol with low (nanomolar) Ln3+ concentrations. In the absence of pedA1A2BC, a ∼100-fold higher La3+-concentration is needed for PedH-dependent growth but not for the ability to repress growth based on PedE activity. From these results, we conclude that cytoplasmic uptake of lanthanides through PedA1A2BC is essential to facilitate REE-dependent growth on 2-phenylethanol under environmental conditions with poor REE bioavailability. Our data further suggest that the La3+/Fe2+/3+ ratio impacts the REE-switch through the mismetallation of putative La3+-binding proteins, such as the sensor histidine kinase PedS2, in the presence of high iron concentrations. As such, this study provides an example for the complexity of bacteria-metal interactions and highlights the importance of medium compositions when studying physiological traits in vitro in particular in regard to REE-dependent phenomena.