Supplementary Figure Legends
Figure S1 Phylogenetic position of NZI7. The phylogenetic position of NZI7 was analysed using
concatenated sequence data from the highly conserved housekeeping genes gyrB and rpoD
(Yamamoto et al 2000) obtained from the NCBI database and generated for P. fluorescens NZI7
and F113, and P. chlororaphis PCL1391 using primers described in (Hwang et al 2005).
Concatenated sequences were aligned using ClustalW (Thompson et al 1994) and neighbourjoining phylogenetic analysis and bootstrapping (n= 2000) performed using PAUP (Sinauer
Associates Inc, US). Very similar results were obtained using other tree construction methods.
Trees were visualised in Dendroscope (Huson et al 2007). (T) indicates type strains. Genomesequenced strains in which EDB cluster-like genes were detected are highlighted in red, genome
sequenced strains in which EDB cluster-like genes were not detected are highlighted in blue.
Figure S2. Genomic distribution of transposon mutations. We used BLASTN to map the
positions of each of the NZI7 transposon insertions onto the completely sequenced chromosome of
Pseudomonas protegens Pf-5 (RefSeq: NC_004129). The outermost circular track indicates the
insertions found in mutants having an edible phenotype but wild-type in all other phenotypic
screens (“edible specific”). The second track shows insertions found in mutants that were edible
but also showed additional non-wild-type phenotypes (“edible non-specific”). The third track
indicates the positions of insertions that did not give an edible phenotype (“non-edible”). The next
track, in green, shows the alignment of the NZI7 genome assembly (GenBank: AJXF00000000.1)
with the genome of Pf-5 (RefSeq: NC_004129) to indicate regions of conserved sequence in the
genomes of strains NZI7 and Pf-5. The innermost track indicates positions of predicted genes in
the genome of Pf-5. The image was generated using CGView software (Stothard and Wishart
2005).
Figure S3. Biochemical complementation with thiamine or anthranilate restores nematode
repellence to NZI7 mutants with insertions in thiamine and anthranilate biosynthesis genes.
Lawns of mutants 11G5 (thiG, thiamine synthase), 50C11 (anthranilate synthase) and 91G1
(chorismate synthase) were grown on NGM or NGM supplemented with 1 mM thiamine or
anthranilate for 24 hours and inoculated with 15-20 nematodes. Plates were photographed four
days after transfer of nematodes. Scale bars= 2 mm.
Figure S4 NZI7 mutants that lack the ability to produce a tolaasin-like toxin are unable to
cause disease symptoms on mushrooms. A. bisporus button mushroom cubes were inoculated
with KB growth medium (control), NZI7 wild-type and NZI7 mutants 5e3 (TOL) and 79g1 (EDB).
Brown blotch symptoms were produced by wild-type bacteria and the EDB cluster mutant 79g1 but
not by 5e3, which contains an insertion in the non-ribosomal peptide synthase cluster responsible
for synthesis of the tolaasin-like toxin. Images taken 48 hours post inoculation; scale bar= 1 cm.
Figure S5 Gene clusters similar to the EDB cluster in other genome-sequenced
pseudomonads. A. ACT alignment of the NZI7 EDB cluster against the Pf-5 genome. B.
Schematic of EDB-like clusters identified in other pseudomonads. Values represent % amino acid
identity compared to the NZI7 EDB cluster ORFs. ORFs marked in orange are incomplete in the
draft genome assemblies. *Percent amino acid identity is based on partial alignment using
available sequence data.
Figure S6 Factors influencing the expression of EDB genes. The lux reporter cartridge in the
mini-Tn5::gfp::lux transposon inserted into EDB mutants 79G1 (ORF1) and 54G10 (ORF8) was
used to assess the expression of the EDB gene cluster in response to different factors. For each
assay, the photon count integrated over 1 second (relative light units, RLU) corrected for OD600 is
plotted versus time (hourly readings).
A
pH. Mutant 54G10 was grown at 28ºC in LB broth buffered to different pH values with 50
mM MES (2-(N-morpholino)ethanesulfonic acid). Error bars = s.e.m; n= 4.
B.
Standard laboratory media. Mutant 79G1 was grown at 28ºC in liquid media with the
same nutritional composition as the solid media used for nematode assays (NGM, nematode
growth medium; BHI, brain heart infusion) and in commonly used laboratory media (M9 (minimal
medium); Medium 1A (minimal medium); LB (rich medium); KB (rich medium)). Error bars = s.e.m;
n= 6.
C.
Iron availability. Mutant 54G10 was grown at 28ºC in KB broth supplemented with FeCl3 or
the iron chelator 2,2-dipyridyl at different concentrations. There was a noticeable reduction in
growth with 5 x 10-4 M 2,2- dipyridyl (only half the final OD600 reached in the other treatments) and
the strain did not grow at 5 x 10-3 M 2,2- dipyridyl. Error bars = s.e.m; n= 8.
D
Carbon:nitrogen balance. EDB mutant 54G10 was grown at 28ºC in M9 minimal medium
containing Difco casamino acids at different concentrations. Error bars = s.e.m; n= 8.
E
Carbon source. Mutant 79G1 was grown at 28ºC in KB (minus glycerol) supplemented
with alternative carbon sources at 0.11M. Error bars = s.e.m; n= 8.
F.
Autoregulation. Mutant 79G1 and the complemented strain 79G1 (pLAF4EDB) were used
to assess the presence of the cloned EDB cluster affected the expression of the lux reporter
cartridge in 79G1. Strains were grown in LB (NZI7; 79G1) or LB with 20 µM tetracycline (79G1
(pLAF4EDB) and 79G1 (pLAFR6)) at 28ºC. Error bars = s.e.m; n=8.
Figure S7. Heterologous expression of the NZI7 EDB cluster. Pseudomonads transformed with
pLAFR6 or pLAF4EDB were grown as lawns on BHI plates for 24 hours at 28ºC prior to extraction
of total RNA. The top image shows RT-PCR results for ORF 8 expression using primers RT54g10f
and RT54g10r. The bottom image shows RT-PCR results for the housekeeping gene rpoD using
primers rpoD_RT2f and rpoD_RT2r. Labels indicate strains transformed with the vector control
pLAFR6 (-) or pLAF4EDB (+). 1= P. entomophila, 2= P. fluorescens NZ43, 3= P. fluorescens
NZ047, 4= P. tolaasii NCPPB 2192, 5= P. fluorescens NZ092, 6= P. fluorescens NZ096. L
indicates 100 bp ladder.
Figure S8. NZI7 hcnB/EDB double mutants lose lethality and nematode repellence on BHI
but retain some ability to inhibit nematode growth relative to NZI7 gacS mutants. Images are
taken 3 days after ~20 adult C. elegans were placed on bacterial lawns. No nematodes could be
seen on lawns of wild-type NZI7 (not shown) or the hcnB mutant 64d2 (A). Nematodes and a few
eggs were visible on lawns of the hcnB/EDB double mutant 64d2-e2 (B), but substantially more
were present on the ΔgacS mutant (C). Scale bar= 2 mm.
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