Genetic and functional characterization of the gene cluster directing the biosynthesis of putisolvin I and II in Pseudomonas putida strain PCL1445

Pseudomonas putida PCL1445 secretes two cyclic lipopeptides, putisolvin I and putisolvin II, which possess a surface-tension-reducing ability, and are able to inhibit biofilm formation and to break down biofilms of Pseudomonas species including Pseudomonas aeruginosa. The putisolvin synthetase gene cluster (pso) and its surrounding region were isolated, sequenced and characterized. Three genes, termed psoA, psoB and psoC, were identified and shown to be involved in putisolvin biosynthesis. The gene products encode the 12 modules responsible for the binding of the 12 amino acids of the putisolvin peptide moiety. Sequence data indicate that the adenylation domain of the 11th module prioritizes the recognition of Val instead of Leu or Ile and consequently favours putisolvin I production over putisolvin II. Detailed analysis of the thiolation domains suggests that the first nine modules recognize the D form of the amino acid residues while the two following modules recognize the L form and the last module the L or D form, indifferently. The psoR gene, which is located upstream of psoA, shows high similarity to luxR-type regulatory genes and is required for the expression of the pso cluster. In addition, two genes, macA and macB, located downstream of psoC were identified and shown to be involved in putisolvin production or export

Regulation of the biosynthesis of cyclic lipopeptides from Pseudomonas putida PCL1445

Pseudomonas putida strain PCL1445 produces two cyclic lipopeptides, named putisolvins I and II, which represent a Novel class of biosurfactants. Putisolvins reduce the surface tension between liquid and air, and disrupt already existing biofilms of several Pseudomonas sp., including those of the opportunistic human pathogen P. aeruginosa, and therefore offers opportunities for interesting application in the medical and industrial field. In this Ph.D Thesis, I describe the regulation of cyclic lipopeptide biosynthesis in P. putida. A dnaK gene and a GacA/GacS two-component signalling system were discovered and were shown to be involved in biosurfactants production. Given that DnaK is a heat-chock protein, we investigated the role of temperature on putisolvin production. The results showed that biosurfactant production in P. putida is up-regulated at low temperatures and that DnaK is required for putisolvin production. Bacteria co-ordinate their activities by producing and detecting small diffusible signal molecules (N-acylhomoserine lactones) which enable a population of organized bacteria to act as a community by forming a biofilm. Such a co-operative behaviour is named “quorum sensing” and plays a central role in the lifestyle of bacteria. P. putida PCL1445 was shown to produce N-acylhomoserine lactones which control the production of putisolvins and regulate biofilm formation. We hypothesise that the bacterial community co-ordinates the biosynthesis of putisolvin when nutrients become limiting resulting in a detachment of part of the bacterial cell population. In this Ph.D Thesis, Novel mechanisms for the regulation of cyclic lipopeptide biosynthesis are described, which contribute to the understanding of their role for bacterial proliferation in the rhizosphere.This work was financed by the grant number 700.50.015 from the Council for Chemical Sciences of the Netherlands Foundation for Scientific Research (NOW/CW)UBL - phd migration 201

Distinctive features of the Gac‐Rsm

Productive plant–bacteria interactions, either beneficial or pathogenic, require that bacteria successfully sense, integrate and respond to continuously changing environmental and plant stimuli. They use complex signal transduction systems that control a vast array of genes and functions. The Gac-Rsm global regulatory pathway plays a key role in controlling fundamental aspects of the apparently different lifestyles of plant beneficial and phytopathogenic Pseudomonas as it coordinates adaptation and survival while either promoting plant health (biocontrol strains) or causing disease (pathogenic strains). Plant-interacting Pseudomonas stand out for possessing multiple Rsm proteins and Rsm RNAs, but the physiological significance of this redundancy is not yet clear. Strikingly, the components of the Gac-Rsm pathway and the controlled genes/pathways are similar, but the outcome of its regulation may be opposite. Therefore, identifying the target mRNAs bound by the Rsm proteins and their mode of action (repression or activation) is essential to explain the resulting phenotype. Some technical considerations to approach the study of this system are also given. Overall, several important features of the Gac-Rsm cascade are now understood in molecular detail, particularly in Pseudomonas protegens CHA0, but further questions remain to be solved in other plant-interacting Pseudomonas.This research was supported by grants BIO2014-55075-P and BIO2017-83533-P from the ERDF/Spanish Ministry of Science, Innovation and Universities - State Research Agency. M.D.F. was supported by a FPU contract from the Spanish MECD/MEFP (ECD/1619/2013)