Lipopeptides (LP) are produced by diverse bacterial genera and are composed of a lipid tail linked to a short linear or cyclic oligopeptide (Raaijmakers et al., 2010). LP are surface-active compounds, exhibit broad-spectrum antibiotic activities, and have diverse natural functions in motility, biofilm formation, and virulence (Raaijmakers et al., 2006; Raaijmakers & Mazzola, 2012). The LP massetolide A was identified in Pseudomonas fluorescens SS101, a biocontrol strain isolated from the wheat rhizosphere (de Souza et al., 2003; de Bruijn et al., 2008), and has potent surfactant and broad-spectrum antimicrobial activities (Gerard et al., 1997; van de Mortel et al., 2009). LP biosynthesis is governed by large multimodular nonribosomal peptide synthetases (NRPS) and is well studied in Pseudomonas and Bacillus (Finking & Marahiel, 2004; Raaijmakers et al., 2006). In contrast, relatively little is known about the regulatory networks, and the signal transduction pathways involved in LP biosynthesis. Among the global regulatory systems, two-component regulators play an important role in the regulation of LP biosynthesis (Raaijmakers et al., 2010). For example, the GacS/GacA two-component system in Pseudomonas functions as a master switch, and a mutation in either one of the two genes results in loss of LP production (Kitten et al., 1998; Koch et al., 2002; Dubern et al., 2005; de Bruijn et al., 2007). Also in Bacillus, LP biosynthesis is regulated by a two-component system as was shown for ComA/ComP in surfactin biosynthesis (Sullivan, 1998). Also quorum sensing plays a role in the regulation of LP biosynthesis in some species and strains. For example, in Bacillus, the cell-density-dependent pheromone ComX and the phosphatase RapC are involved in surfactin biosynthesis (Duitman et al., 2007). For Pseudomonas fluorescens 5064 and Pseudomonas putida PCL1445, N-acyl homoserine lactones (N-AHLs) regulate viscosin and putisolvin biosynthesis, respectively (Cui et al., 2005; Dubern et al., 2006). However, for various other Pseudomonas strains belonging to the same species, including P. fluorescens strain SS101, subject of this study, no evidence was found for a role of N-AHL-mediated regulation of LP biosynthesis (Dumenyo et al., 1998; Kinscherf & Willis, 1999; Andersen et al., 2003; de Bruijn et al., 2007, 2008). This indicates that cell-density-dependent regulation of LP biosynthesis can differ between species and between strains of the same species. In addition to these two global regulatory systems, LuxR-type transcriptional regulators that flank the LP biosynthesis genes have been shown to regulate syringomycin, syringopeptin, syringofactin, putisolvin, entolysin, viscosin, and massetolide biosynthesis (Lu et al., 2002; Wang et al., 2006; Berti et al., 2007; Dubern et al., 2008; de Bruijn & Raaijmakers, 2009b; Vallet-Gely et al., 2010). In P. putida, also DnaK was identified as an additional regulator of putisolvin biosynthesis (Dubern et al., 2005).
The study presented here focuses on identification of regulatory genes of massetolide biosynthesis in the beneficial rhizobacterium P. fluorescens SS101. Massetolides consist of a 9-amino acid cyclic peptide moiety linked to a 3-hydroxydecanoic acid tail and belong to the viscosin LP group (Raaijmakers et al., 2006). Massetolide biosynthesis is governed by three NRPS genes, designated massA, massB, and massC, flanked by two LuxR-type regulatory genes massAR and massBCR (de Bruijn et al., 2008; de Bruijn & Raaijmakers, 2009b). We previously identified the two-component system GacS/GacA and the serine protease ClpP as regulators of massetolide biosynthesis in strain SS101 (de Bruijn & Raaijmakers, 2009a). At the transcriptional level, ClpP-mediated regulation of massetolide biosynthesis appears to operate independently from the regulation by GacS/GacA (de Bruijn & Raaijmakers, 2009a). Based on these previous findings, a tentative regulation model was proposed where ClpP regulates, alone or together with a yet unknown chaperone other than ClpX, massetolide biosynthesis via degradation of putative transcriptional repressors of massAR, and/or via modulation of the citric acid cycle and amino acid metabolism (de Bruijn & Raaijmakers, 2009b). The aims of this study were to (1) perform a genome-wide search for new regulatory genes of massetolide biosynthesis in P. fluorescens SS101, (2) determine the role of these genes in the regulation of massetolide production, and (3) investigate the putative role of these regulatory genes in other phenotypic traits of P. fluorescens SS101. To this end, we screened two independent random plasposon mutant libraries (c. 8000 mutants total) for a reduced or loss of massetolide production. Thirteen putative regulatory mutants were found. Genetic, phenotypic, chemical, and transcriptional analyses were performed to elucidate the functions of three regulatory genes in massetolide biosynthesis and in other phenotypic traits, including swarming motility, siderophore production, and extracellular protease activity.