Janosch Klebensberger

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.

The biological role of REEs has long been underestimated, and research has mainly focused on methanotrophic and methylotrophic bacteria. We have recently demonstrated that P. putida , a plant growth-promoting bacterium that thrives in the rhizosphere of various food crops, possesses a REE-dependent alcohol dehydrogenase (PedH), but knowledge about REE-specific effects on physiological traits in nonmethylotrophic bacteria is still scarce. This study demonstrates that the cellular response of P. putida to lanthanum (La ³⁺ ) is mostly substrate specific and that La ³⁺ availability highly affects the growth of cells on glycerol. Further, a novel route for glycerol metabolism is identified, which is initiated by PedE and/or PedH activity and provides a growth advantage to this biotechnologically relevant organism by allowing a faster onset of growth. Overall, these findings demonstrate that lanthanides can affect physiological traits in nonmethylotrophic bacteria and might influence their competitiveness in various environmental niches.

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⁺/Fe^2+/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.

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 Summary: Pseudomonas 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.

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 Ca ²⁺ -dependent counterpart PedE is inversely regulated in response to lanthanide (Ln ³⁺ ) 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 Ln ³⁺ 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) Ln ³⁺ concentrations. In the absence of pedA1A2BC , a ~100-fold higher La ³⁺ 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 La ³⁺ /Fe ³⁺ ratio impacts the REE-switch through the mismetallation of putative La ³⁺ -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.

Summary: Pollution from ethylene glycol, and plastics containing this monomer, represent a significant environmental problem. The investigation of its microbial metabolism therefore provides insights into the environmental fate of this pollutant and also enables its utilization as a carbon source for microbial biotechnology. Here, we reveal the genomic and metabolic basis of ethylene glycol metabolism in Pseudomonas putida KT2440. Although this strain cannot grow on ethylene glycol as sole carbon source, it can be used to generate growth‐enhancing reducing equivalents upon co‐feeding with acetate. Mutants that utilize ethylene glycol as sole carbon source were isolated through adaptive laboratory evolution. Genomic analysis of these mutants revealed a central role of the transcriptional regulator GclR, which represses the glyoxylate carboligase pathway as part of a larger metabolic context of purine and allantoin metabolism. Secondary mutations in a transcriptional regulator encoded by PP_2046 and a porin encoded by PP_2662 further improved growth on ethylene glycol in evolved strains, likely by balancing fluxes through the initial oxidations of ethylene glycol to glyoxylate. With this knowledge, we reverse engineered an ethylene glycol utilizing strain and thus revealed the metabolic and regulatory basis that are essential for efficient ethylene glycol metabolism in P. 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 La ³⁺ -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 La ³⁺ during growth on various carbon and energy sources were investigated with a differential proteomic approach. Apart from the Ca ²⁺ -dependent enzyme PedE, the differential abundance of most other identified proteins was conditional and revealed a substrate specificity. Concomitant with the proteomic changes, La ³⁺ 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. Importance: The 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 La ³⁺ is mostly substrate specific and that during growth on glycerol, La ³⁺ 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.

Mixed-species biofilms display a number of emergent properties, including enhanced antimicrobial tolerance and communal metabolism. These properties may depend on interspecies relationships and the structure of the biofilm. However, the contribution of specific matrix components to emergent properties of mixed-species biofilms remains poorly understood. Using a dual-species biofilm community formed by the opportunistic pathogens Pseudomonas aeruginosa and Staphylococcus aureus, we found that whilst neither Pel nor Psl polysaccharides, produced by P. aeruginosa, affect relative species abundance in mature P. aeruginosa and S. aureus biofilms, Psl production is associated with increased P. aeruginosa abundance and reduced S. aureus aggregation in the early stages of biofilm formation. Our data suggest that the competitive effect of Psl is not associated with its structural role in cross-linking the matrix and adhering to P. aeruginosa cells but is instead mediated through the activation of the diguanylate cyclase SiaD. This regulatory control was also found to be independent of the siderophore pyoverdine and Pseudomonas quinolone signal, which have previously been proposed to reduce S. aureus viability by inducing lactic acid fermentation-based growth. In contrast to the effect mediated by Psl, Pel reduced the effective crosslinking of the biofilm matrix and facilitated superdiffusivity in microcolony regions. These changes in matrix cross-linking enhance biofilm surface spreading and expansion of microcolonies in the later stages of biofilm development, improving overall dual-species biofilm growth and increasing biovolume severalfold. Thus, the biofilm matrix and regulators associated with matrix production play essential roles in mixed-species biofilm interactions. IMPORTANCE Bacteria in natural and engineered environments form biofilms that include many different species. Microorganisms rely on a number of different strategies to manage social interactions with other species and to access resources, build biofilm consortia, and optimize growth. For example, Pseudomonas aeruginosa and Staphylococcus aureus are biofilm-forming bacteria that coinfect the lungs of cystic fibrosis patients and diabetic and chronic wounds. P. aeruginosa is known to antagonize S. aureus growth. However, many of the factors responsible for mixed-species interactions and outcomes such as infections are poorly understood. Biofilm bacteria are encased in a self-produced extracellular matrix that facilitates interspecies behavior and biofilm development. In this study, we examined the poorly understood roles of the major matrix biopolymers and their regulators in mixed-species biofilm interactions and development.

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.