Biology subjects

Klebensberger, J.

Publications and source records attributed to Klebensberger, J..

5 recordsLinked to original sources

bioRxiv2019

SiaABCD - A threonine phosphorylation pathway that controls biofilm formation in Pseudomonas aeruginosa

The critical role of bacterial biofilms in chronic human infections calls for novel anti-biofilm strategies targeting the regulation of biofilm development. However, the regulation of biofilm development is very complex and can include multiple, highly interconnected signal transduction/response pathways, which are incompletely understood. We demonstrated previously that in the opportunistic, human pathogen P. aeruginosa, the PP2C-like protein phosphatase SiaA and the di-guanylate cyclase SiaD control the formation of macroscopic cellular aggregates, a type of suspended biofilms, in response to surfactant stress. In this study, we demonstrate that the SiaABC proteins represent a signal response pathway that functions through a partner switch mechanism to control biofilm formation. We also demonstrate that SiaABCD functionality is dependent on carbon substrate availability for a variety of substrates, and that upon carbon starvation, SiaB mutants show impaired dispersal, in particular with the primary fermentation product ethanol. This suggests that carbon availability is at least one of the key environmental cues integrated by the SiaABCD system. Further, our biochemical, physiological and crystallographic data reveals that the phosphatase SiaA and its kinase counterpart SiaB balance the phosphorylation status of their target protein SiaC at threonine 68 (T68). Crystallographic analysis of the SiaA-PP2C domain shows that SiaA is present as a dimer. Dynamic modelling of SiaA with SiaC suggested that SiaA interacts strongly with phosphorylated SiaC and dissociates rapidly upon dephosphorylation of SiaC. Further, we show that the known phosphatase inhibitor fumonisin inhibits SiaA mediated phosphatase activity in vitro. In conclusion, the present work improves our understanding of how P. aeuruginosa integrates specific environmental conditions, such as carbon availability and surfactant stress, to regulate cellular aggregation and biofilm formation. With the biochemical and structural characterization of SiaA, initial data on the catalytic inhibition of SiaA, and the interaction between SiaA and SiaC, our study identifies promising targets for the development of biofilm-interference drugs to combat infections of this aggressive opportunistic pathogen. Author SummaryPseudomonas aeruginosa is a Gram-negative bacterium that is feared within clinical environments due to its potential to cause life-threatening acute and chronic infections. One cornerstone of its success is the ability to form and disperse from biofilms, which are self-made, multicellular structures that protect the individual cell from the human immune system and antibiotic treatment. As such, therapies that combine a biofilm-interference strategy and the use of antimicrobial drugs represent one of the promising strategies to tackle infections of this organism. With the current study, we gain a deeper understanding of the SiaABCD mediated biofilm formation in response to clinically relevant environmental conditions. Further, our structural and biochemical characterization of the PP2C-type protein-phosphatase SiaA and the partner switch protein SiaC suggest that both represent promising novel targets for the development of future anti-biofilms drugs based on a signal interference strategy.

microbiology↗

bioRxiv2019

Rare earth element (REE)-dependent growth of Pseudomonas putida KT2440 depends on the ABC-transporter PedA1A2BC and is influenced by iron availability

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. Expression 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 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 under environmental conditions with poor REE bioavailability. Our data further suggest that the La3+/Fe3+ 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 regards to REE-dependent phenomena.

microbiology↗

bioRxiv2019

The cellular response towards lanthanum is substrate specific and reveals a novel route for glycerol metabolism in Pseudomonas putida KT2440

Ever since the discovery of the first rare earth element (REE)-dependent enzyme, the physiological role of lanthanides has become an emerging field of research due to the potential environmental implications and biotechnological opportunities. In Pseudomonas putida KT2440, the two pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) PedE and PedH are inversely produced in response to La3+-availability. This REE-switch is orchestrated by a complex regulatory network including the PedR2/PedS2 two-component system and is important for efficient growth on several alcoholic volatiles. As P. putida is exposed to a broad variety of organic compounds in its natural soil habitat, the cellular responses towards La3+ during growth on various carbon and energy sources were investigated with a differential proteomic approach. Apart from the Ca2+-dependent enzyme PedE, the differential abundance of most other identified proteins was conditional and revealed a substrate specificity. Concomitant with the proteomic changes, La3+ had a beneficial effect on lag-phases while causing reduced growth rates and lower optical densities in stationary phase during growth on glycerol. When these growth phenotypes were evaluated with mutant strains, a novel metabolic route for glycerol utilization was identified that seems to be functional in parallel with the main degradation pathway encoded by the glpFKRD operon. The newly discovered route is initiated by PedE and/or PedH, which most likely convert glycerol to glyceraldehyde. In the presence of lanthanum, glyceraldehyde seems to be further oxidized to glycerate, which, upon phosphorylation to glycerate-2-phosphate by the glycerate kinase GarK, is finally channelled into the central metabolism. ImportanceThe biological role of rare earth elements has long been underestimated and research has mainly focused on methanotrophic bacteria. We have recently demonstrated that P. putida, a plant growth promoting bacterium that thrives in the rhizosphere of various feed crops, possesses a REE-dependent alcohol dehydrogenase (PedH), but knowledge about lanthanide-dependent effects on physiological traits in non-methylotrophic bacteria is still scarce. This study demonstrates that the cellular response of P. putida KT2440 towards La3+ is mostly substrate specific and that during growth on glycerol, La3+ has a severe effect on several growth parameters. We provide compelling evidence that the observed physiological changes are linked to the catalytic activity of PedH and thereby identify a novel route for glycerol metabolism in this biotechnological relevant organism. Overall, these findings demonstrate that lanthanides can alter important physiological traits of non-methylotrophic bacteria, which might consequently influence their competitiveness during colonization of various environmental niches.

microbiology↗

bioRxiv2018

The PedS2/PedR2 two-component system is crucial for the rare earth element switch in Pseudomonas putida KT2440

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 described 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 herein 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 suggests 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 fading 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 catalysed oxidation of alcoholic VOCs.\n\nIMPORTANCEThe function of lanthanides for methano- and methylotrophic bacteria is gaining increasing attention, while knowledge about the role of rare earth elements (REEs) in non-methylotrophic 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 non-methylotrophic bacterium and stimulates subsequent investigations for the identification of additional genes or phenotypic traits that might be co-regulated during REE-dependent niche adaptation.

microbiology↗

bioRxiv2017

Functional Role Of Lanthanides In Enzymatic Activity And Transcriptional Regulation Of PQQ-Dependent Alcohol Dehydrogenases In Pseudomonas putida KT2440

The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems, based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs), which are often functionally redundant. Using purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440, the present study reports the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a non-methylotrophic bacterium. PedH exhibits enzyme activity on a similar substrate range as its Ca2+-dependent counterpart PedE, including linear and aromatic primary and secondary alcohols as well as aldehydes, however, only in the presence of lanthanide ions including La3+, Ce3+, Pr3+, Sm3+ or Nd3+. Reporter assays revealed that PedH not only has a catalytic function, but is also involved in the transcriptional regulation of pedE and pedH, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 - 10 nM of lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results we conclude that functional redundancy and inverse regulation of PedE and PedH represents an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to different lanthanide availability.\n\nIMPORTANCEDue to their low bioavailability, lanthanides have long been considered as biologically inert. In recent years however, the identification of lanthanides as a cofactor in methylotrophic bacteria has attracted tremendous interest among various biological fields. The present study reveals that one of the two PQQ-ADHs produced by the model organism P. putida KT2440 also utilizes lanthanides as a cofactor, thus expanding the scope of lanthanide employing bacteria beyond the methylotrophs. Similar to methyloptrophic bacteria, a complex regulatory network is involved in the lanthanide-responsive switch between the two PQQ-ADHs encoded by P. putida KT2440. We further show that functional production of at least one of the enzymes is crucial for efficient growth with several volatile alcohols. Overall, our study provides a novel understanding for the redundancy of PQQ-ADHs observed in many organisms and further highlights the importance of lanthanides for bacterial metabolism, particularly in soil environments.

microbiology↗