Materials and methods.

Fig. S1. Location of the Tn5 transposon insertion (A) and the 19 microarray 60-mer probes in the gacS gene of P. fluorescens SBW25 (B). Probes are in grey and the overlap between probes is depicted in dark grey.

Fig. S2. Organization of the sod gene cluster in P. fluorescens SBW25, including sodA, fumC1, a transport related membrane protein (PFLU0877), and two hypothetical proteins (PFLU0873, PFLU0875). For each of the genes, fold changes in transcription ± standard error of the mean were determined for the gacS mutant by Q-PCR analysis; these are indicated below each of the genes.

Fig S3. Phenotypic characterization of wild type strain SBW25, gacS mutant, complemented gacS mutant (gacS compl.) and gacS+ empty vector (gacS ev). For complementation, a 3763 bp fragment containing the gacS gene with putative promoter and terminator was obtained by PCR with specific primers (Table S1). The PCR fragment was first subcloned in pGEM-T Easy (Promega) and subsequently cloned into the shuttle vector pME6031 (Heeb et al., 2000). The obtained pME6031-gacS construct and pME6031 empty vector control were electroporated into the gacS::Tn5 mutant of P. fluorescens SBW25 and the presence of the vectors was verified by PCR with pME6031-specific primers (Table S2, Supporting information).

A. Siderophore detection on CAS media: top, observation under normal light; bottom, under UV light; the size of the orange halos is a measure for the amount of siderophores produced.

B. Extracellular protease activity on skimmed milk agar plates; a halo around the bacterial colony represents protease activity.

C. Biofilm formation in KB liquid media in a 96-well PVC plate: the cells in the biofilm were stained with crystal violet (left); biofilm formation was quantified spectrophotometrically (right).

D. Swimming motility (top) and swarming motility (bottom) on soft SSM agar medium [0.3% and 0.6% agar (w/v) respectively].

Fig. S4. RP-HPLC analysis of culture extracts of P. fluorescens SBW25, the gacS mutant and the viscA mutant grown on one-fifth strength PDA agar medium for 48 h. RP-HPLC chromatograms at 210 nm are shown for wild type SBW25, the gacS mutant and the viscA mutant.

Fig. S5. Activity of wild type SBW25, the gacS mutant and the viscA mutant against Phytophthora infestans on agar plates supplemented with 0, 50 or 100 μM FeSO4.


Table S1. Plasmids and oligonucleotide primers used in this study.

Table S2. Specific functions, corresponding genes and their relative expression levels in the gacS mutant of P. fluorescens SBW25 that are highlighted in the manuscript.

Table S3. Genes (#702) differentially expressed (fold change > 4.0, P < 0.0001) in the gacS mutant of P. fluorescens SBW25. Certain genes fall in more than one role category and therefore are presented more than once in the table.

Table S4. Comparison of genes significantly affected in expression in the gacS mutant of P. fluorescens SBW25 with orthologous genes significantly affected in expression in gacS/A mutants of P. protegens Pf-5 and P. aeruginosa PAO1. For Pf-5 and PA01, data were obtained from Hassan and colleagues (2010) and Brencic and colleagues (2009) respectively.

Table S5. TonB-dependent receptors and linked extracytoplasmic function (ECF) sigma factors and anti-sigma factor regulators that were transcriptionally modulated in the gacS mutant of P. fluorescens SBW25.

Table S6. Quantification of the antimicrobial activity of wild-type P. fluorescens SBW25 and the gacS mutant against different oomycetes, fungi and bacteria.

emi412061-sup-0003-si.zip154KSupporting Information

Please note: Wiley Blackwell is not responsible for the content or functionality of any supporting information supplied by the authors. Any queries (other than missing content) should be directed to the corresponding author for the article.