Figure 1.
The hybrid sensor kinase FitF is essential for Fit toxin expression.
(A) Domain topology of FitF and FitH and putative signal transduction pathways (blue arrays) and phosphotransfer reactions (black arrows) between domains and proteins predicted by NCBI Conserved Domain Search [42] and SMART [43]. The conserved amino acid residues predicted by NCBI Conserved Domain Search to be phosphorylated or to be important for signal recognition are indicated with their respective amino acid positions. Hpt, phosphotransfer domain; PASc, cytoplasmic Per-Arnt-Sim (PAS) domain; PASp, periplasmic PAS domain; REC, receiver domain; TM, transmembrane region. (B) Epifluorescence microscopy of hemolymph extracts from larvae of G. mellonella infected with FitD-mCherry reporter strains with the wild-type (CHA1176) and ΔfitF mutant (CHA1174-gfp2) background for 24 h. The injected strains harbor a constitutive GFP cell tag for identification, expression of FitD-mCherry can be seen in the DsRed channel. Strain CHA0-gfp2 was used as a negative control. Bars represent 10 µm, micrographs are false-colored. The experiment was repeated twice with similar results. (C) Systemic virulence assay with injection of wild-type (in black, CHA0) and isogenic mutants (ΔfitF in red, CHA1154; ΔfitD in blue, CHA1151) of P. protegens CHA0 into last instar larvae of G. mellonella. Saline solution served as a negative control (in gray). Significant differences between the different treatments are indicated with *** (p-value<0.0001; Log-rank test). The experiment was repeated twice with similar results.
Figure 2.
Expression of the Fit insect toxin can be induced in an insect hemolymph-mimicking medium (GIM).
(A) The FitD-mCherry reporter strain of P. protegens CHA0 (CHA1163) was grown in different media and red fluorescence intensities of single cells were quantified by epifluorescence microscopy in the exponential (8 h post inoculation) and stationary (24 h post inoculation) growth phase. Results are the mean and standard deviation of population averages of single cell fluorescence intensities from three independent cultures (n = on average approx. 3200 cells per treatment and time point). Treatments labeled with a different letter are significantly different (p-values<0.0001; two-way ANOVA with Tukey's HSD test for post-hoc comparisons). The experiment was performed three times with similar results. (B) Quantification of the expression of FitD-mCherry in the wild-type background of CHA0 (CHA1163) in GIM and M9 L-malate with or without root extracts from field-grown wheat (n = on average approx. 2600 cells per treatment and time point). Characters indicate significant differences between the treatments (p-values<0.05; two-way ANOVA with Tukey's HSD test for post-hoc comparisons). The experiment was repeated twice with similar results. (C) Quantification of the expression of FitD-mCherry in the wild-type (CHA1163) and ΔfitF deletion mutant (CHA1174) background of strain CHA0 grown in GIM for 24 h at 25°C (n = 2768–3239 cells per strain). Re-introducing a single copy of fitF from CHA0 (CHA5066) or PCL1391 (CHA5073) in the bacterial chromosome rescued the expression of FitD-mCherry. Means labeled with a different letter are significantly different (p-value<0.05; one-way ANOVA with Tukey's HSD test for post-hoc comparisons). The experiment was performed three times with similar results.
Figure 3.
FitFp is homologous to the periplasmic DctB-like sensor domain.
(A) Multiple sequence alignment of the periplasmic region of FitF and DctB homologs (selection). Amino acid residues that are identical to FitF are highlighted in yellow. Secondary structures of DctB were deduced from the corresponding crystal structures and are displayed on top (H, alpha helix; E, beta sheet; -, coil). Pa, P. aeruginosa PAO1; Pp, P. protegens CHA0; Pc, P. chlororaphis PCL1391; Sm, S. meliloti; Vc, V. cholerae. (B) Phylogenetic tree with sequences obtained from BLASTp searches using the periplasmic sequence of FitF of P. protegens CHA0 and of homologs of DctBp. MAFFT was used for sequence alignment and the Minimum Evolution method in MEGA [44] for inferring the evolutionary history of the proteins. The percentage of replicate trees in which the associated proteins clustered together in the bootstrap test (500 replicates) is shown next to the branches. Evolutionary distances, which were computed using the Poisson correction method, are drawn to scale and are in the units of the number of amino acid substitutions per site. The corresponding protein sequences can be found in File S1. The predicted domain topology of the entire proteins is depicted for groups of interest. Domains that are displayed in half do not exist in all proteins of the respective group. PhoQ was used as out group. (C) Tertiary structure prediction for P. protegens FitFp by Phyre2 in comparison with crystal structures of DctBp of V. cholerae (PDB code 3BY9) and S. meliloti (PDB code 3E4O). Other modeling programs predicted highly similar structures (data not shown). (D) Site-directed mutagenesis of the native fitF gene in the FitD-mCherry reporter strain CHA1163. The sites of the mutated residues are depicted in panel A and Figure 1C. Microscopic quantification of the expression of FitD-mCherry in the wild-type and individual mutant backgrounds of CHA0 grown for 24 h in GIM. Results are the mean and standard deviation of population averages of single cell fluorescence intensities from three independent cultures (n = on average approx. 2900 cells per strain). Characters indicate significant differences between the means (p-values<0.01; one-way ANOVA with Tukey's HSD test for post-hoc comparisons). The experiment was performed three times with similar results.
Figure 4.
Site-directed mutagenesis of fitF and fitH.
Site-directed mutagenesis of the native fitF and fitH genes in the FitD-mCherry reporter strain CHA1163. Quantification of the expression of FitD-mCherry in the wild-type (CHA1163) and individual mutant backgrounds of CHA0 (CHA5056, CHA5075, CHA1174, CHA5084, and CHA1175) grown for 24 h in GIM. Results are the mean and standard deviation of population averages of single cell fluorescence intensities from three independent cultures (n = on average approx. 2900 cells per strain). Characters indicate significant differences between the means (p-values<0.001; one-way ANOVA with Tukey's HSD test for post-hoc comparisons). The experiment was repeated twice with similar results.
Figure 5.
A DctBp-FitFc chimera regulates toxin expression similarly to wild-type FitF.
(A) A chimeric protein of the cytoplasmic portion of FitF and the N-terminal part of DctB including its double-PASp sensor domain and the transmembrane regions was constructed by fusing the respective P. protegens CHA0 genes using the conserved DNA sequence coding for the second transmembrane region as a linker. A CitAp-FitFc chimera was constructed analogously using E. coli citA. (B) Expression of FitD-mCherry in the ΔfitF reporter strain CHA1174 complemented with either wild-type fitF (CHA5066), the dctB‘-’fitF chimeric gene (CHA5093) or the citA‘-’fitF chimeric gene (CHA5151) in different media for 24 h. Results are the mean and standard deviation of population averages of single cell fluorescence intensities from three independent cultures (n = on average approx. 3590 cells per treatment). Characters indicate significant differences between the means (p-values<0.05; one-way ANOVA with Tukey's HSD test for post-hoc comparisons). The experiment was performed three times with similar results. (C) Quantification by epifluorescence microscopy of FitD-mCherry expression in reporter strains CHA5066, CHA5093, CHA5151, and CHA1175 (ΔfitH, positive control), all harboring the plasmid pPROBE-TT for GFP-tagging of the cells, grown for five days on roots of cucumber. Shown are means and standard deviations of population averages of single cell fluorescence intensities of bacteria isolated from six independent plants (n = on average approx. 1170 cells per strain). Characters indicate significant differences between the means (p-values<0.05; one-way ANOVA with Tukey's HSD test for post-hoc comparisons). The experiment was repeated twice with similar results. (D) Galleria injection assay with wild-type (in black, CHA0) and isogenic mutants (ΔfitF in red, CHA1154; ΔfitD in blue, CHA1151; ΔfitF dctB‘-’fitF in green, CHA5150) of P. protegens CHA0 into last instar larvae of G. mellonella. Saline solution served as a negative control (in gray). Significant differences between the different treatments are indicated with *** (p-value<0.0001; Log-rank test). The experiment was repeated twice with similar results.
Figure 6.
Fit toxin expression is controlled in a host-specific manner.
The insectidical toxin is expressed by P. protegens CHA0 only in certain insect species and not on plant roots. (A) Epifluorescence microscopy of hemolymph isolated from S. littoralis, T. molitor and A. pisum infected with FitD-mCherry reporter strains with the wild-type (CHA1176) and ΔfitH mutant (CHA1178, positive control) background. The bacteria harbor a constitutive GFP cell tag for identification, expression of FitD-mCherry can be seen in the DsRed channel. Strain CHA0-gfp2 was used as a negative control. Bars represent 10 µm, micrographs are false-colored. The experiments were performed at least twice with similar results. (B) Epifluorescence microscopy of plant roots (or root washes) three to five days after the inoculation with the same reporter strains as in panel A, with or without co-inoculation with the phytopathogen Fusarium oxysporum f. sp. radicis-lycopersici. The experiments were performed twice with similar results.
Figure 7.
Model for evolution of FitF via a domain shuffling event involving a DctB ancestor.
The ancestor of the gene coding for the sensor kinase DctB was duplicated several times in various proteobacterial species. One dctB gene copy underwent a fusion with a gene encoding a histidine kinase-response regulator hybrid protein, possibly by homologous recombination via a conserved region coding for the second transmembrane region of the sensor proteins. This domain shuffling event resulted in the expression of a hybrid histidine kinase with a dual PASp domain architecture in the periplasmic portion. Selective pressure then led to adaptive modifications in the protein sequence and domain topology (i.e. insertion of a second PASc domain in P. chlororaphis). Domain shuffling and subsequent modifications during the evolution of FitF significantly contributed to the ability of P. protegens CHA0 to produce its insecticidal toxin in a host-specific manner and as a result to the evolution of insect pathogenicity in this biocontrol bacterium. Inhibition of FitF by plant-derived molecules may be a mechanism helping the bacterium to distinguish between the plant and insect host. The evolution of FitF may have taken place in bacterial species other than P. protegens, implying horizontal gene transfer.