TheppuI-rsaL-ppuRQuorum-Sensing System Regulates Biofilm Formation ofPseudomonas putidaPCL1445 by Controlling Biosynthesis of the Cyclic Lipopeptides Putisolvins I and II

Abstract

Pseudomonas putida strain PCL1445 produces two cyclic lipopeptides, putisolvin I and putisolvin II, which possess surface tension-reducing abilities and are able to inhibit biofilm formation and to break down existing biofilms of several Pseudomonas spp., including P. aeruginosa. Putisolvins are secreted in the culture medium during growth at late exponential phase, indicating that production is possibly regulated by quorum sensing. In the present study, we identified a quorum-sensing system in PCL1445 that is composed of ppuI, rsaL, and ppuR and shows very high similarity with gene clusters of P. putida strains IsoF and WCS358. Strains with mutations in ppuI and ppuR showed a severe reduction of putisolvin production. Expression analysis of the putisolvin biosynthetic gene in a ppuI background showed decreased expression, which could be complemented by the addition of synthetic 3-oxo-C 10 -N-acyl homoserine lactone (3-oxo-C 10 -AHL) or 3-oxo-C 12 -AHL to the medium. An rsaL mutant overproduces AHLs, and production of putisolvins is induced early during growth. Analysis of biofilm formation on polyvinylchloride showed that ppuI and ppuR mutants produce a denser biofilm than PCL1445, which correlates with decreased production of putisolvins, whereas an rsaL mutant shows a delay in biofilm production, which correlates with early production of putisolvins. The results demonstrate that quorum-sensing signals induce the production of cyclic lipopeptides putisolvin I and II and consequently control biofilm formation by Pseudomonas putida.Bacteria can form multicellular aggregates generally referred to as biofilms on biotic and abiotic surfaces. Such communities are ubiquitous in natural environments but can also be found in industrial and clinical settings, for example, on artificial surfaces of medical devices, thereby greatly contributing to infections (34).Pseudomonas putida strain PCL1445 is capable of forming biofilms on roots and on polyvinylchloride (PVC) in a commonly used biofilm assay (21). We have shown that P. putida PCL1445 produces two novel lipodepsipeptides, putisolvin I and II, consisting of a C 6 lipid moiety and a 12-amino-acid peptide, which are produced by a putisolvin synthetase gene designated as psoA (21). A mutant with impaired putisolvin biosynthesis was shown to form a thicker biofilm than the wild-type strain. In addition, purified putisolvins I and II inhibit biofilm formation and break down existing biofilms of various Pseudomonas spp., including the opportunistic human pathogen P. aeruginosa (21). The production of putisolvins occurs at the end of the exponential growth phase (21), which may indicate that the production is mediated through a quorum-sensing mechanism. The term quorum sensing describes an environmental sensing system which allows bacteria to monitor their own population density. Quorum sensing in gramnegative bacteria relies on the interaction of small diffusible signal molecules belonging to the class of N-acyl homoserine lactones (AHLs). They are synthetized via the LuxI...

Potential Emergence of Multi-quorum Sensing Inhibitor Resistant (MQSIR) Bacteria

“…QSS in Pseudomonas corrugata, P. mediterranea, P. putida, and P. syringae are mediated by AHLs like C6HSL, C8HSL and 3OC6HSL and 3OC12HSL (Table 3) [70,[109][110][111]. QS regulated plasmid transfer in Rhizobium etli is under the control of 3OC8HSL and OHC8HSL signals ( Table 3).…”

Core Principles of Bacterial Autoinducer Systems

“…In several Pseudomonas putida strains, an AI system (called Ppu) triggers the release of the cell-associated surfactant putisolvin (79). Putisolvin promotes the removal of cells from the colony/biofilm.…”

Massetolide A Biosynthesis inPseudomonas fluorescens

“…6A). To investigate the relationship between massetolide A production and expression of each of the three mass genes, RNA was isolated from samples taken at specific time points (8,12,16, and 24 h) in the growth curve and cDNA was subjected to Q-PCR with different primers specific for each of the three mass genes (positions of the primers are indicated in Fig. 1B).…”