Ralf Paul

Allosteric control of cyclic di-GMP signaling

Cyclic di-guanosine monophosphate is a bacterial second messenger that has been implicated in biofilm formation, antibiotic resistance, and persistence of pathogenic bacteria in their animal host. Although the enzymes responsible for the regulation of cellular levels of c-di-GMP, diguanylate cyclases (DGC) and phosphodiesterases, have been identified recently, little information is available on the molecular mechanisms involved in controlling the activity of these key enzymes or on the specific interactions of c-di-GMP with effector proteins. By using a combination of genetic, biochemical, and modeling techniques we demonstrate that an allosteric binding site for c-di-GMP (I-site) is responsible for non-competitive product inhibition of DGCs. The I-site was mapped in both multi- and single domain DGC proteins and is fully contained within the GGDEF domain itself. In vivo selection experiments and kinetic analysis of the evolved I-site mutants led to the definition of an RXXD motif as the core c-di-GMP binding site. Based on these results and based on the observation that the I-site is conserved in a majority of known and potential DGC proteins, we propose that product inhibition of DGCs is of fundamental importance for c-di-GMP signaling and cellular homeostasis. The definition of the I-site binding pocket provides an entry point into unraveling the molecular mechanisms of ligand-protein interactions involved in c-di-GMP signaling and makes DGCs a valuable target for drug design to develop new strategies against biofilm-related diseases.

Role of the GGDEF regulator PleD in polar development of Caulobacter crescentus

Several members of the two‐component signal transduction family have been implicated in the control of  polar development in Caulobacter crescentus: PleC and DivJ, two polarly localized histidine sensor kinases; and the response regulators DivK and PleD. The PleD protein was shown previously to be required during the swarmer‐to‐stalked cell transition for flagellar ejection and efficient stalk biogenesis. Here, we present data indicating that PleD also controls the onset of motility and a cell density switch immediately preceding cell division. Constitutively active alleles of pleD or wspR, an orthologue from Pseudomonas fluorescens, almost completely suppressed C. crescentus motility and inhibited the increase in swarmer cell density during cell differentiation. The observation that these alleles also had a dominant‐negative effect on motility in a pleC divJ and a pleC divK mutant background indicated that PleD is located downstream of the other components in the signal transduction cascade, which controls the activity of the flagellar motor. In addition, the presence of a constitutive pleD or wspR allele resulted in a doubling of the average stalk length. Together, this is consistent with a model in which the active form of PleD, PleD∼P, negatively controls aspects of differentiation in the late predivisional cell, whereas it acts positively on polar development during the swarmer‐to‐stalked cell transition. In agreement with such a model, we found that DivJ, which localizes to the stalked pole during cell differentiation, positively controlled the in vivo phosphorylation status of PleD, and the swarmer pole‐specific PleC kinase modulated this status in a negative manner. Furthermore, domain switch experiments demonstrated that the WspR GGDEF output domain from P. fluorescens is active in C. crescentus, favouring a more general function for this novel signalling domain over a specific role such as DNA or protein interaction. Possible roles for PleD and its C‐terminal output domain in modulating the polar cell surface of C. crescentus are discussed.

Activation of the diguanylate cyclase PleD by phosphorylation-mediated dimerization

Diguanylate cyclases (DGCs) are key enzymes of second messenger signaling in bacteria. Their activity is responsible for the condensation of two GTP molecules into the signaling compound cyclic di-GMP. Despite their importance and abundance in bacteria, catalytic and regulatory mechanisms of this class of enzymes are poorly understood. In particular, it is not clear if oligomerization is required for catalysis and if it represents a level for activity control. To address this question we perform in vitro and in vivo analysis of the Caulobacter crescentus diguanylate cyclase PleD. PleD is a member of the response regulator family with two N-terminal receiver domains and a C-terminal diguanylate cyclase output domain. PleD is activated by phosphorylation but the structural changes inflicted upon activation of PleD are unknown. We show that PleD can be specifically activated by beryllium fluoride in vitro, resulting in dimerization and c-di-GMP synthesis. Cross-linking and fractionation experiments demonstrated that the DGC activity of PleD is contained entirely within the dimer fraction, confirming that the dimer represents the enzymatically active state of PleD. In contrast to the catalytic activity, allosteric feedback regulation of PleD is not affected by the activation status of the protein, indicating that activation by dimerization and product inhibition represent independent layers of DGC control. Finally, we present evidence that dimerization also serves to sequester activated PleD to the differentiating Caulobacter cell pole, implicating protein oligomerization in spatial control and providing a molecular explanation for the coupling of PleD activation and subcellular localization.

Crystal structure of oxidized flavodoxin, an essential protein in Helicobacter pylori

The redox protein flavodoxin has been shown earlier to be reduced by the pyruvate‐oxidoreductase (POR) enzyme complex of Helicobacter pylori, and also was proposed to be involved in the pathogenesis of gastric mucosa‐associated lymphoid‐tissue lymphoma (MALToma). Here, we report its X‐ray structure, which is similar to flavodoxins of other bacteria and cyanobacteria. However, H. pylori flavodoxin has an alanine residue near the isoalloxazine ring of its cofactor flavin mononucleotide (FMN), while the other previously crystallized flavodoxins have a larger hydrophobic residue at this position. This creates a solute filled hole near the FMN cofactor of H. pylori flavodoxin. We also show that flavodoxin is essential for the survival of H. pylori, and conclude that its structure can be used as a starting point for the modeling of an inhibitor for the interaction between the POR‐enzyme complex and flavodoxin.

Mutations of the Helicobacter pylori genes rdxA and pbp1 cause resistance against metronidazole and amoxicillin.

ABSTRACT To investigate amoxicillin and metronidazole resistance ofHelicobacter pylori, we compared putative resistance genes between resistant strains obtained in vitro and their sensitive parent strain. All metronidazole-resistant strains hadrdxA mutations, and an amoxicillin-resistant strain hadpbp1 and pbp2 mutations. By transforming PCR products of these mutated genes into antibiotic-sensitive strains, we showed that rdxA null mutations were sufficient for metronidazole resistance, while pbp1mutations contributed to amoxicillin resistance of H. pylori.