Antimicrobial and Insecticidal: Cyclic Lipopeptides and Hydrogen Cyanide Produced by Plant-Beneficial Pseudomonas Strains CHA0, CMR12a, and PCL1391 Contribute to Insect Killing.

Particular groups of plant-beneficial fluorescent pseudomonads are not only root colonizers that provide plant disease suppression, but in addition are able to infect and kill insect larvae. The mechanisms by which the bacteria manage to infest this alternative host, to overcome its immune system, and to ultimately kill the insect are still largely unknown. However, the investigation of the few virulence factors discovered so far, points to a highly multifactorial nature of insecticidal activity. Antimicrobial compounds produced by fluorescent pseudomonads are effective weapons against a vast diversity of organisms such as fungi, oomycetes, nematodes, and protozoa. Here, we investigated whether these compounds also contribute to insecticidal activity. We tested mutants of the highly insecticidal strains Pseudomonas protegens CHA0, Pseudomonas chlororaphis PCL1391, and Pseudomonas sp. CMR12a, defective for individual or multiple antimicrobial compounds, for injectable and oral activity against lepidopteran insect larvae. Moreover, we studied expression of biosynthesis genes for these antimicrobial compounds for the first time in insects. Our survey revealed that hydrogen cyanide and different types of cyclic lipopeptides contribute to insecticidal activity. Hydrogen cyanide was essential to full virulence of CHA0 and PCL1391 directly injected into the hemolymph. The cyclic lipopeptide orfamide produced by CHA0 and CMR12a was mainly important in oral infections. Mutants of CMR12a and PCL1391 impaired in the production of the cyclic lipopeptides sessilin and clp1391, respectively, showed reduced virulence in injection and feeding experiments. Although virulence of mutants lacking one or several of the other antimicrobial compounds, i.e., 2,4-diacetylphloroglucinol, phenazines, pyrrolnitrin, or pyoluteorin, was not reduced, these metabolites might still play a role in an insect background since all investigated biosynthetic genes for antimicrobial compounds of strain CHA0 were expressed at some point during insect infection. In summary, our study identified new factors contributing to insecticidal activity and extends the diverse functions of antimicrobial compounds produced by fluorescent pseudomonads from the plant environment to the insect host

Role of Gluconic Acid Production in the Regulation of Biocontrol Traits of Pseudomonas fluorescens CHA0▿

The rhizobacterium Pseudomonas fluorescens CHA0 promotes the growth of various crop plants and protects them against root diseases caused by pathogenic fungi. The main mechanism of disease suppression by this strain is the production of the antifungal compounds 2,4-diacetylphloroglucinol (DAPG) and pyoluteorin (PLT). Direct plant growth promotion can be achieved through solubilization of inorganic phosphates by the production of organic acids, mainly gluconic acid, which is one of the principal acids produced by Pseudomonas spp. The aim of this study was to elucidate the role of gluconic acid production in CHA0. Therefore, mutants were created with deletions in the genes encoding glucose dehydrogenase (gcd) and gluconate dehydrogenase (gad), required for the conversion of glucose to gluconic acid and gluconic acid to 2-ketogluconate, respectively. These enzymes should be of predominant importance for rhizosphere-colonizing biocontrol bacteria, as major carbon sources provided by plant root exudates are made up of glucose. Our results show that the ability of strain CHA0 to acidify its environment and to solubilize mineral phosphate is strongly dependent on its ability to produce gluconic acid. Moreover, we provide evidence that the formation of gluconic acid by CHA0 completely inhibits the production of PLT and partially inhibits that of DAPG. In the Δgcd mutant, which does not produce gluconic acid, the enhanced production of antifungal compounds was associated with improved biocontrol activity against take-all disease of wheat, caused by Gaeumannomyces graminis var. tritici. This study provides new evidence for a close association of gluconic acid metabolism with antifungal compound production and biocontrol activity in P. fluorescens CHA0

FitFp is homologous to the periplasmic DctB-like sensor domain.

<p>(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, <i>P. aeruginosa</i> PAO1; Pp, <i>P. protegens</i> CHA0; Pc, <i>P. chlororaphis</i> PCL1391; Sm, <i>S. meliloti</i>; Vc, <i>V. cholerae</i>. (B) Phylogenetic tree with sequences obtained from BLASTp searches using the periplasmic sequence of FitF of <i>P. protegens</i> CHA0 and of homologs of DctBp. MAFFT was used for sequence alignment and the Minimum Evolution method in MEGA <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003964#ppat.1003964-Tamura1" target="_blank">[44]</a> 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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003964#ppat.1003964.s006" target="_blank">File S1</a>. 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 <i>P. protegens</i> FitFp by Phyre2 in comparison with crystal structures of DctBp of <i>V. cholerae</i> (PDB code 3BY9) and <i>S. meliloti</i> (PDB code 3E4O). Other modeling programs predicted highly similar structures (data not shown). (D) Site-directed mutagenesis of the native <i>fitF</i> gene in the FitD-mCherry reporter strain CHA1163. The sites of the mutated residues are depicted in panel A and <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003964#ppat-1003964-g001" target="_blank">Figure 1C</a>. 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.</p

Expression of the Fit insect toxin can be induced in an insect hemolymph-mimicking medium (GIM).

<p>(A) The FitD-mCherry reporter strain of <i>P. protegens</i> 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 Δ<i>fitF</i> 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 <i>fitF</i> 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.</p

Fit toxin expression is controlled in a host-specific manner.

<p>The insectidical toxin is expressed by <i>P. protegens</i> CHA0 only in certain insect species and not on plant roots. (A) Epifluorescence microscopy of hemolymph isolated from <i>S. littoralis</i>, <i>T. molitor</i> and <i>A. pisum</i> infected with FitD-mCherry reporter strains with the wild-type (CHA1176) and Δ<i>fitH</i> 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-<i>gfp2</i> 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 <i>Fusarium oxysporum</i> f. sp. <i>radicis-lycopersici</i>. The experiments were performed twice with similar results.</p

Model for evolution of FitF via a domain shuffling event involving a DctB ancestor.

<p>The ancestor of the gene coding for the sensor kinase DctB was duplicated several times in various proteobacterial species. One <i>dctB</i> 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 <i>P. chlororaphis</i>). Domain shuffling and subsequent modifications during the evolution of FitF significantly contributed to the ability of <i>P. protegens</i> 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 <i>P. protegens</i>, implying horizontal gene transfer.</p

Site-directed mutagenesis of <i>fitF</i> and <i>fitH</i>.

<p>Site-directed mutagenesis of the native <i>fitF</i> and <i>fitH</i> 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.</p