Unraveling the c-di-GMP modulation network in the opportunistic pathogen Pseudomonas aeruginosa

While in many bacteria the entirety of c-di-GMP modulating enzymes drive a global switch to a biofilm mode of growth phenotype, the various players and sub-sets of c-di-GMP modulating enzymes are responsible for smaller and in parts non-overlapping bacterial phenotypes.

In this project we will exploit our extensive expertise in RNA-seq in order to evaluate how intracellular c-di-GMP levels that are produced locally by the individual c-di-GMP cyclases impact the transcriptional profile in P. aeruginosa and how this facilitates the establishment of c-di-GMP dependent phenotypes that are mediated by the individual c-di-GMP pathways

Oxygen-dependent control of a diguanylate cyclase lacking canonical sensory and regulatory domains

The production of alginate by Pseudomonas aeruginosa is induced under anaerobic conditions that are for example occurring in the lung of cystic fibrosis patients. Increased levels of alginate are detected after 1 h exposure to 0% oxygen. The membrane bound diguanylate cyclase SadC is involved in stimulating alginate biosynthesis and shows increased c-di-GMP production under oxygen limitation; however, it does not contain canonical oxygen-binding domains.

The aim of this study is to gain insight into the mechanism underlying the oxygen-dependent regulation of SadC and alginate production.

Spatiotemporal control of c-di-GMP signaling in Shewanella via phosphodiesterase PdeB

Many bacterial species harbor a plethora of proteins potentially involved in synthesis and turnover of the second messenger c-di-GMP. Among more than 50 of such proteins, only a single one, PdeB, is a key regulator of flagella-mediated motility of our model species, Shewanella putrefaciens, in response to nutrient conditions.

In this project we aim at understanding how PdeB is capable of eliciting such a specific response, to identify corresponding input signals and output mechanisms and to characterize the role of the PdeB domains in this process.

A light-triggered behavioural switch in cyanobacterial motility

Cyanobacteria are phototrophic prokaryotes performing plant-like photosynthesis. Like for all phototrophs light sensing is of crucial importance for cyanobacteria to acclimate to the light environment. They have to choose the optimal light conditions for photosynthesis and at the same time they avoid high- and UV-light stress. The concentration of the second messenger molecule c-di-GMP controls the decision of cyanobacteria to move towards a light source or to induce biofilm formation.

In this project we investigate how light signals sensed by photoreceptors are transmitted to downstream components of a so far unknown c-di-GMP related signal transduction chain that controls cyanobacterial motility.

Regulation of development in Myxococcus xanthus by the second messenger c-di-GMP

Myxococcus xanthus initiates a developmental program in response to starvation that culminates in the formation of multicellular, spore-filled fruiting bodies. Development crucially depends on the coordination of temporally regulated changes in gene expression, motility, intercellular signaling, and signaling by nucleotide-based second messengers including (p)ppGpp and, as we have recently shown, c-di-GMP. c-di-GMP accumulates at elevated levels during development and this increase is essential for development.

The ultimate goal of this project is to understand how c-di-GMP accumulation is regulated during development and to identify the effector proteins that bind c-di-GMP and implement the effects of changing c-di-GMP levels.

Dynamic basis of the molecular mechanism of nucleotide secondary messenger-sensing riboswitches by NMR spectroscopy

Riboswitches are cis-acting regulatory RNA elements involved into the modulation of translational or transcriptional efficiency, binding small-molecule ligands.

In this project second-messenger binding bacterial riboswitches are investigated by NMR methods. NMR offers unmatched precision for such scientific studies, enabling investigations in atomic resolution. Applied methods range from the characterization of the conformational space of riboswitches over structural profiling to kinetic investigation involving a multitude of NMR methods such as proton 1D, 2D-NOESY, HSQC and application of modern isotope labeling methods.

Sensory mechanisms and local signaling in
c-di-GMP-mediated signal transduction in E. coli biofilm formation

Bacterial biofilms are large cellular aggregates in which bacteria are encased in a protective extracellular selfproduced polymeric matrix. In Escherichia coli macrocolony biofilms this matrix consists of amyloid protein fibres (curli) and cellulose arranged in a complex architecture that is under the control of multiple enzymes that make and break c-di-GMP.

The project aims at clarifying molecular signaling mechanisms in this c-di-GMP control network, in particular how environmental and cellular signals are sensed via N-terminal sensory domain of these enzymes and how c-di-GMP as a second messenger is sensed by novel effector mechanisms that control matrix production and other biofilm functions.

Gene regulation in Pseudomonas aeruginosa by
c-di-GMP-dependent alterations in mRNA stability

Next to proteins also RNAs can bind c-di-GMP and function as c-di-GMP effectors. Conserved RNA domains located in the 5┬┤UTR of mRNAs have been demonstrated to be c-di-GMP targets and to affect gene expression via the formation of riboswitches. Those c-di GMP-dependent riboswitches are present in various bacterial species but they have not been identified in the opportunistic pathogen Pseudomonas aeruginosa.

In this project we will apply extensive RNA profiling and systematically monitor cellular translation processes by ribosome profiling in order to specifically analyze the influence of c-di-GMP as posttranscriptional modulators of gene expression in P. aeruginosa.

The role of the c-di-GMP specific phosphodiesterase NbdA in NO-induced biofilm dispersal in Pseudomonas aeruginosa

Dispersion is a process by which bacteria transit from a biofilm to a motile growth state to colonize new sites. Alterations in c-di-GMP levels were shown to be associated with biofilm dispersal in a number of different bacteria including the opportunistic pathogen Pseudomonas aeruginosa. The signaling molecule nitric oxide (NO) is one factor known to induce biofilm dispersion through stimulation of c-di-GMP degrading phosphodiesterase (PDE) activity.

The project aims at understanding the role of the membrane-anchored PDE NbdA in this process, its transcriptional regulation and interaction with other players in the dispersion network.