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emi12050-sup-0001-suppl.doc68K

Supplementary information for Experimental procedures.

emi12050-sup-0002-FigS1.doc1205K

Fig. S1. Schematic representation of the PCR approach to study the expression and transcriptional organization of the Fit toxin locus in P. fluorescens CHA0. The locations of amplicons generated by RT-PCR and RT-qPCR are indicated, respectively, above and below the large arrows representing the eight genes of the locus. Numbered lines with goal posts indicate the fragments amplified with primer pairs internal to each gene or spanning contiguous genes to reveal expression and transcriptional links (see Tables S1 and S2 for more details). Suggested major transcripts are indicated by thick dashed arrows. image, putative promoter.

emi12050-sup-0003-FigS2.doc837K

Fig. S2. Evidence for fitABCDE RNA by Northern blot analysis. Total RNA was extracted from P. fluorescens wild-type CHA0 and the ΔfitH mutant CHA1158 at the end of the exponential growth phase and loaded onto a 12% polyacrylamide gel. A band corresponding to the expected 16.5 kb fitABCDE transcript (marked with an arrow) was detected with a digoxigenin-labelled single-stranded probe directed against fitB RNA.

emi12050-sup-0004-FigS3.doc3424K

Fig. S3. SDS-PAGE and Western blot analysis of FitD expression in P. fluorescens CHA0 and derivatives expressing a FitD–mCherry fusion protein, using polyclonal antibodies directed against FitD (A) or mCherry (B). Lanes: 1, E. coli BL21/pCOLDI::fitD; 2, wild-type CHA0; 3, CHA0-mche (CHA0::attTn7-mcherry); 4, CHA1163 (CHA0::fitD-mcherry); 5, CHA1158 (ΔfitH mutant); 6, CHA1175 (CHA1158::fitD-mcherry); 7, CHA1185 (FitG overexpressing derivative CHA0::attTn7-Ptac/lacIq-fitG; IPTG-induced); 8, CHA5010 (CHA1185::fitD-mcherry; IPTG-induced); M, molecular mass standards. BL21/pCOLDI::fitD served as a positive control for FitD detection. Bacteria were grown in LB medium to the early stationary growth phase. The inclined up arrows indicate the FitD protein on the SDS-9% PAGE gel which was stained with Coomassie brilliant blue.

emi12050-sup-0005-FigS4.doc1048K

Fig. S4. Complementation of the fitH deletion in P. fluorescens CHA1175. Panels DsRed micrographs (from left to right) show Fit insect toxin expression in single cells of the wild type (CHA1163), the ΔfitH deletion mutant (CHA1175), the complemented ΔfitH mutant CHA5031 (i.e. CHA1175:: attTn7-Ptac/lacIq-fitH; without and with IPTG induction respectively), all equipped for expression of a chromosomally encoded FitD–mCherry fusion protein. Cells were harvested from late exponential growth-phase cultures in LB medium and analysed by fluorescence microscopy.

emi12050-sup-0006-FigS5.doc1247K

Fig. S5. Toxicity of P. fluorescens CHA0 and derived mutants to Galleria mellonella as assessed by following (A) the percentage of surviving larvae and (B) the progress of melanization of the larvae over time. Aliquots of 50 cells of wild-type CHA0 (grey circles), the ΔfitH mutant CHA1158 (open triangles), the fitG overexpressing mutant CHA1185 (Ptac/lacIq-fitG; IPTG induced; dark squares), and the ΔfitD mutant CHA1151 (open circles) were injected per larva in 5 μl of sterile 0.9% NaCl solution. Controls (crosses) received 5 μl of sterile NaCl solution. Eighteen larvae per treatment were tested. The experiment was repeated twice with similar results.

emi12050-sup-0007-TableS1.doc52K

Table S1. Transcriptional analysis of the fit locus (see also Fig. S1).

emi12050-sup-0008-TableS2.doc48K

Table S2. Primers used for RT-PCR analyses.

emi12050-sup-0009-TableS3.doc52K

Table S3. Primer pairs used for mutant and reporter construction.

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