Biosynthesis and regulation of cyclic lipopeptides in Pseudomonas fluorescens

Cyclic lipopeptides (CLPs) are surfactant and antibiotic metabolites produced by a variety of bacterial genera. For the genus Pseudomonas, many structurally different CLPs have been identified. CLPs play an important role in surface motility of Pseudomonas strains, but also in virulence and attachment/detachment to and from surfaces. In this Ph.D. thesis project, two new CLP biosynthesis clusters were identified in Pseudomonas fluorescens and fully sequenced. In P. fluorescens strain SBW25, the viscosin biosynthesis cluster was identified by bioinformatic analyses of the genome followed by genetic and chemical analyses. For P. fluorescens strain SS101, the genes for massetolide biosynthesis were identified via random mutagenesis followed by cloning, sequencing and chemical analyses. Biosynthesis of viscosin and massetolide is governed by three nonribosomal peptide synthetase (NRPS) genes, designated viscABC and massABC, respectively. The viscosin and massetolide biosynthesis gene clusters are very similar, but different from CLP gene clusters described for other Pseudomonas as the viscA and massA genes are physically disconnected from viscBC and massBC, respectively. Viscosin differs from massetolide A only at position number four in the peptide moiety, which is a valine in viscosin and an isoleucine in massetolide A. Because of the modular structure of the NRPSs and the co-linearity of the assembly process, transfer of the mass genes of strain SS101 into strain SBW25 resulted in the production of both massetolide A and viscosin, demonstrating that the assembly line for CLP biosynthesis in Pseudomonas can be altered leading to the production of non-native products. Compared to the understanding of CLP biosynthesis, not so much is known about the regulation. This thesis shows that the GacA/GacS two-component system regulates massetolide and viscosin biosynthesis in strains SS101 and SBW25, respectively. No indications were found that massetolide or viscosin biosynthesis is regulated by quorum sensing via N-acylhomoserine lactones. Site-directed mutagenesis of the LuxR-type regulator genes luxR-vA and luxR-vBC flanking the viscosin biosynthesis cluster resulted in a loss of viscosin production, indicating that both LuxR-type transcriptional regulators are important for viscosin biosynthesis in strain SBW25. Phylogenetic analyses further suggested that these LuxR-type transcriptional regulators do not contain the autoinducerbinding domain found for the quorum sensing-associated LuxR regulator in Vibrio fischeri. Instead, the LuxR-type regulator genes flanking the massetolide and viscosin biosynthesis genes are closely related to the LuxR-type regulators identified for syringomycin/ syringopeptin biosynthesis and appear to belong to a separate LuxR-type regulator subfamily, different from the autonomous effector domain protein GerE. Via random mutagenesis and subsequent screening for massetolide-deficient mutants, also other regulator genes were identified including clpP. ClpP is a serine protease that plays a crucial role in intracellular refolding and degradation of proteins, which is an essential process for the viability of cells. ClpP was shown to affect transcription of luxR-mA, thereby regulating transcription of the massetolide biosynthesis genes. Results further suggested that, at the transcriptional level, ClpPmediated regulation of massetolide biosynthesis operates independently from regulation by the GacA/GacS two-component system. In conclusion, the results of this thesis led to the identification of several genes and previously unknown pathways involved in regulation of CLP biosynthesis and highlighted the complexity of the signaling cascades underlying CLP biosynthesis in Pseudomonas. CLPs have diverse functions for the producing bacterial strains, including a role in motility, biofilm formation, antimicrobial activity and virulence. Also in establishment and persistence in plant environments, CLPs were shown to confer a competitive advantage. A new function of CLPs, identified in a collaboration with Mark Mazzola (USDA) and presented in this thesis, is their protective effects against predation by protozoa. In vitro assays showed that both massetolide and viscosin can lyse the trophozoites of Naeglaria americana and that wild type strains SS101 and SBW25 were substantially less sensitive to protozoan grazing than their CLP-deficient mutants. Also in soil containing N. americana, population densities of wild type strains SS101 and SBW25 were significantly higher compared to the massetolide and viscosin-deficient mutants, showing that CLP production confers a competitive advantage in survival in complex environments. Moreover, transcription of the CLP-biosynthesis genes increased significantly upon protozoan grazing, indicating that the Pseudomonas strains sense the protozoa and react by producing CLPs as defense compounds. Which signal triggers the induction of the CLP biosynthesis genes is not known yet and currently under investigation. Based on these results, we postulate that CLPs are an important component of the preingestional defense mechanisms of bacteria against protozoan predation, not only due to their lytic effects on protozoa, but also because CLPs contribute to evasion of protozoan grazing via altered cell surface properties, swimming and swarming, and microcolony and biofilm formation.