bs_bs_banner Environmental Microbiology Reports (2013) 5(4), 608–619 doi:10.1111/1758-2229.12061 The Gac regulon of Pseudomonas fluorescens SBW25 Xu Cheng,1† Irene de Bruijn,1† Introduction Menno van der Voort,1† Joyce E. Loper2 and Comparative genome analyses of different plant- Jos M. Raaijmakers1* associated Pseudomonas species and strains revealed 1Laboratory of Phytopathology, Wageningen University, substantial heterogeneity within the Pseudomonas fluo- Droevendaalsesteeg 1, 6708 PB, Wageningen, rescens group, with a pangenome of close to 14 000 the Netherlands. genes (Silby et al., 2009; 2011; Loper et al., 2012). A core 2USDA-ARS Horticultural Crops Research Laboratory genome of only 1491 genes was identified for all of the and Department of Botany and Plant Pathology, Oregon sequenced Pseudomonas species, representing 24–36% State University, Corvallis, OR, USA. of any individual genome (Loper et al., 2012). In each of the genomes, orphan genes were discovered for traits Summary that were not known previously, including genes for insect toxins, novel type II, III and VI secretion systems as well Transcriptome analysis of Pseudomonas fluorescens as gene clusters with unknown functions. Interestingly, SBW25 showed that 702 genes were differentially almost all of the traits associated with biological control of regulated in a gacS::Tn5 mutant, with 300 and 402 plant pathogens or other multitrophic interactions mapped genes up- and downregulated respectively. Similar outside of the core genome to variable genomic regions to the Gac regulon of other Pseudomonas species, present in only individual strains or a limited subset of genes involved in motility, biofilm formation, strains. The study by Loper and colleagues (2012) as well siderophore biosynthesis and oxidative stress were as previous genome analyses of other bacterial genera differentially regulated in the gacS mutant of SBW25. (Gross et al., 2007; Zerikly and Challis, 2009) further Our analysis also revealed, for the first time, that indicated that the orphan biosynthetic pathways outnum- transcription of 19 rhizosphere-induced genes and of ber by far the number of bioactive compounds currently genes involved in type II secretion, (exo)polysaccha- identified. ride and pectate lyase biosynthesis, twitching motility Approaches to explore and exploit microbial (meta)ge- and an orphan non-ribosomal peptide synthetase nomes for orphan biosynthetic pathways and novel (NRPS) were significantly affected in the gacS mutant. bioactive compounds are numerous and include: (i) het- Furthermore, the gacS mutant inhibited growth of erologous expression of genomic fragments obtained oomycete, fungal and bacterial pathogens signifi- from culturable or unculturable microorganisms (i.e. cantly more than wild type SBW25. Since RP-HPLC metagenomics) followed by activity assays and metabolic analysis did not reveal any potential candidate profiling, (ii) in silico mining of microbial genomes for metabolites, we focused on the Gac-regulated orphan consensus sequence motifs or modules followed by struc- NRPS gene cluster that was predicted to encode an ture prediction, gene inactivation studies and/or geno- eight-amino-acid ornicorrugatin-like peptide. Site- misotopic approaches, or (iii) inactivation of orphan bio- directed mutagenesis indicated that the encoded synthetic pathways by manipulation of regulatory genes peptide is not involved in the enhanced antimicrobial or by challenging microorganisms with specific culture activity of the gacS mutant but may function as a conditions or other external cues (Gross et al., 2007; siderophore. Collectively, this genome-wide analysis McAlpine, 2009; Scherlach and Hertweck, 2009; Li et al., revealed that a mutation in the GacS/A two- 2012). Following a combination of the two latter component regulatory system causes major tran- approaches, Hassan and colleagues (2010) presented scriptional changes in SBW25 and significantly the global-regulator-based genome mining strategy to enhances its antimicrobial activities by yet unknown decipher the metabolome of Pseudomonas protegens mechanisms. (formerly P. fluorescens) strain Pf-5 and to identify orphan gene clusters for novel secondary metabolites. In Pseudomonas, the GacS/GacA two-component Received 5 December, 2012; accepted 1 April, 2013. *For corre- system is a highly conserved global regulatory system spondence. E-mail [email protected]; Tel. (+31) 317 48 94 27; Fax (+31) (0)317 48 34 12. †These authors contributed equally to the and comprises the membrane-bound sensor kinase GacS manuscript. that, upon recognition of a yet unknown environmental © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology 609 X. Cheng et al. signal, is activated and phosphorylates the response with that of P. protegens strain Pf-5 and harbours multiple regulator GacA. The GacS/GacA two-component system gene clusters for which the functions are yet unknown controls the biosynthesis of numerous secondary metabo- (Loper et al., 2012). Here, we conducted a genome-wide lites in Pseudomonas and mutations (spontaneous or transcriptome comparison between wild type strain site-directed) in the gacS or gacA genes generally lead to SBW25 and a Tn5 mutant disrupted in the gacS gene. a substantial reduction in antimicrobial activity of Pseu- The functions of several of the differentially expressed domonas strains (Haas and Defago, 2005). In P. prote- genes were studied. The in vitro activity assays unexpect- gens Pf-5, it also has broad effects on iron homeostasis, edly showed an enhanced antimicrobial activity of the enhancing transcript levels of genes functioning in gacS mutant against several economically important plant siderophore biosynthesis and various mechanisms for and fish pathogens. The role of a specific orphan NRPS iron uptake (Hassan et al., 2010). The GacS/GacA two- gene cluster in this enhanced antimicrobial activity was component system also regulates the expression of studied in more detail by site-directed mutagenesis. several genes involved in virulence, biofilm formation, motility, quorum sensing, stress responses and survival (Kinscherf and Willis, 1999; Haas and Keel, 2003; Results and discussion Raaijmakers et al., 2010; Yamazaki et al., 2012). The Gac regulon of P. fluorescens SBW25 Here we investigated the Gac regulon of P. fluorescens SBW25, a well-known model strain for studying bacterial For the transcriptome analysis, we isolated RNA from evolution and adaptation (Rainey, 1999; Gal et al., 2003; cells grown for 48 h at 25°C on one-fifth PDA agar plates Kassen et al., 2004; Silby et al., 2009; Scanlan and to match the conditions used to assess the antimicrobial Buckling, 2012). Strain SBW25 also promotes plant activity of SBW25 and mutants (see Supporting informa- growth and controls plant diseases caused by fungal tion for detailed description of the materials and methods and oomycete pathogens (Rainey, 1999; Naseby et al., used). The results showed that Tn5-based inactivation of 2001; de Bruijn et al., 2007). To date, however, little is gacS significantly affected the transcriptome of strain known about the genes and metabolites involved in plant SBW25: a total of 1807 genes were differentially regu- growth promotion and antimicrobial activity of SBW25: lated (fold change > 2.0, P < 0.0001) in the gacS mutant, it does not produce the typical antibiotic compounds with a total of 935 and 872 genes up- or downregulated 2,4-diacetylphloroglucinol, phenazines, pyrrolonitrin or respectively (Fig. 1). For our analyses, we focused pyoluteorin (Loper et al., 2012), but produces mostly on genes with a fold change > 4.0 and a siderophores (Timms-Wilson et al., 2000; Moon et al., P-value < 0.0001. Using these more stringent criteria, 702 2008) and the cyclic lipopeptide viscosin (de Bruijn et al., genes were differentially expressed in the gacS mutant of 2007). Strain SBW25 shares only two-thirds of its genome SBW25, with 300 and 402 genes significantly up- and Fig. 1. Genome-wide representation of the genes most differentially expressed in the gacS mutant of Pseudomonas fluorescens SBW25. Each point is one of the annotated genes in the SBW25 genome, with the x-axis showing gene order (the origin of replication at 0 and 6009), and the y-axis showing the fold change of transcript abundance of each gene in the gacS mutant relative to the wild type. Panels (A) and (B) represent different scales of the y-axis. Annotated functions of boxed genes further discussed in the text are: (a) viscosin biosynthesis cluster (PFLU2552, PFLU2553, PFLU4007), (b) metalloprotease aprA (PFLU3146), (c) sod gene cluster (PFLU0873–0877), (d) sigma factor rpoS (PFLU1302), (e) siderophore biosynthesis genes (PFLU2534–2550), (f) type VI secretion system (PFLU6014-6026), (g) flagellar biosynthesis genes (PFLU4437–4456), (h) the putative ornicorrugatin NRPS gene cluster (PFLU3220–3225). © 2013 John Wiley & Sons Ltd and Society for Applied Microbiology, Environmental Microbiology Reports, 5, 608–619 The Gac regulon of SBW25 610 Table 1. Grouping of differentially regulated genes (fold change > 4.0, P < 0.0001) of the gacS mutant of P. fluorescens SBW25 into role categories. Transcript level in gacS mutant versus wild type Upregulated Downregulated Number %of Role categories of CDSs CDSs n % n % A Amino acid synthesis 167 2.8 4 2.4 10 6.0 B Biosynthesis of cofactors, prosthetic groups and carriers 203 3.4 4 2.0 6 3.0 C Cell envelope 499 8.3 31 6.2 50 10.0 D Cellular processes 502 8.4 31 6.2 77 15.3 E Central intermediary metabolism 297 4.9