Relationships Between Root Pathogen Resistance, Abundance and Expression of \u3ci\u3ePseudomonas\u3c/i\u3e Antimicrobial Genes, and Soil Properties in Representative Swiss Agricultural Soils

Strains of Pseudomonas that produce antimicrobial metabolites and control soilborne plant diseases have often been isolated from soils defined as disease-suppressive, i.e., soils, in which specific plant pathogens are present, but plants show no or reduced disease symptoms. Moreover, it is assumed that pseudomonads producing antimicrobial compounds such as 2,4-diacetylphloroglucinol (DAPG) or phenazines (PHZ) contribute to the specific disease resistance of suppressive soils. However, pseudomonads producing antimicrobial metabolites are also present in soils that are conducive to disease. Currently, it is still unknown whether and to which extent the abundance of antimicrobials-producing pseudomonads is related to the general disease resistance of common agricultural soils. Moreover, virtually nothing is known about the conditions under which pseudomonads express antimicrobial genes in agricultural field soils. We present here results of the first side-by-side comparison of 10 representative Swiss agricultural soils with a cereal-oriented cropping history for (i) the resistance against two soilborne pathogens, (ii) the abundance of Pseudomonas bacteria harboring genes involved in the biosynthesis of the antimicrobials DAPG, PHZ, and pyrrolnitrin on roots of wheat, and (iii) the ability to support the expression of these genes on the roots. Our study revealed that the level of soil disease resistance strongly depends on the type of pathogen, e.g., soils that are highly resistant to Gaeumannomyces tritici often are highly susceptible to Pythium ultimum and vice versa. There was no significant correlation between the disease resistance of the soils, the abundance of Pseudomonas bacteria carrying DAPG, PHZ, and pyrrolnitrin biosynthetic genes, and the ability of the soils to support the expression of the antimicrobial genes. Correlation analyses indicated that certain soil factors such as silt, clay, and some macro- and micronutrients influence both the abundance and the expression of the antimicrobial genes. Taken together, the results of this study suggests that pseudomonads producing DAPG, PHZ, or pyrrolnitrin are present and abundant in Swiss agricultural soils and that the soils support the expression of the respective biosynthetic genes in these bacteria to various degrees. The precise role that these pseudomonads play in the general disease resistance of the investigated agricultural soils remains elusive

Defining early steps in <i>Bacillus subtilis</i> biofilm biosynthesis

ABSTRACT The Bacillus subtilis extracellular biofilm matrix includes an exopolysaccharide (EPS) that is critical for the architecture and function of the community. To date, our understanding of the biosynthetic machinery and the molecular composition of the EPS of B. subtilis remains unclear and incomplete. This report presents synergistic biochemical and genetic studies built from a foundation of comparative sequence analyses targeted at elucidating the activities of the first two membrane-committed steps in the EPS biosynthetic pathway. By taking this approach, we determined the nucleotide sugar donor and lipid-linked acceptor substrates for the first two enzymes in the B. subtilis biofilm EPS biosynthetic pathway. EpsL catalyzes the first phosphoglycosyl transferase step using uridine diphosphate (UDP)-di-N-acetyl bacillosamine as phospho-sugar donor. EpsD is a predicted GT-B fold (GT4 family) retaining glycosyl transferase that catalyzes the second step in the pathway that utilizes the product of EpsL as an acceptor substrate and UDP-N-acetyl glucosamine as the sugar donor. Thus, the study defines the first two monosaccharides at the reducing end of the growing EPS unit. In doing so, we provide the first evidence of the presence of bacillosamine in an EPS synthesized by a Gram-positive bacterium. IMPORTANCE Biofilms are the communal way of life that microbes adopt to increase survival. Key to our ability to systematically promote or ablate biofilm formation is a detailed understanding of the biofilm matrix macromolecules. Here, we identify the first two essential steps in the Bacillus subtilis biofilm matrix exopolysaccharide (EPS) synthesis pathway. Together, our studies and approaches provide the foundation for the sequential characterization of the steps in EPS biosynthesis, using prior steps to enable chemoenzymatic synthesis of the undecaprenyl diphosphate-linked glycan substrates

Protecting maize from rootworm damage with the combined application of arbuscular mycorrhizal fungi, Pseudomonas bacteria and entomopathogenic nematodes.

Diabrotica virgifera virgifera LeConte, the western corn rootworm (WCR), is the most destructive pest of maize in North America, and has recently spread across central Europe. Its subterranean larval stages are hard to reach with pesticides and it has evolved resistance to conventional management practices. The application of beneficial soil organisms is being considered as a sustainable and environmental friendly alternative. In a previous study, the combined application in wheat fields of arbuscular mycorrhizal fungi, entomopathogenic Pseudomonas bacteria, and entomopathogenic nematodes was found to promote growth and protection against a natural pest infestation, without negative cross effects. Because of the insect-killing capacity of the bacteria and nematodes, we hypothesized that the application of these organisms would have similar or even greater beneficial effects in WCR-infested maize fields. During three consecutive years (2015-2017), we conducted trials in Missouri (USA) in which we applied the three organisms, alone or in combinations, in plots that were artificially infested with WCR and in non-infested control plots. For two of the three trials, we found that in plots treated with entomopathogenic nematodes and/or entomopathogenic Pseudomonas bacteria, roots were less damaged than the roots of plants in control plots. During one year, WCR survival was significantly lower in plots treated with Pseudomonas than in control plots, and the surviving larvae that were recovered from these plots were lighter. The bacterial and nematodes treatments also enhanced yield, assessed as total grain weight, in one of the trials. The effects of the treatments varied considerable among the three years, but they were always positive for the plants

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

A DctBp-FitFc chimera regulates toxin expression similarly to wild-type FitF.

<p>(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 <i>P. protegens</i> CHA0 genes using the conserved DNA sequence coding for the second transmembrane region as a linker. A CitAp-FitFc chimera was constructed analogously using <i>E. coli citA</i>. (B) Expression of FitD-mCherry in the Δ<i>fitF</i> reporter strain CHA1174 complemented with either wild-type <i>fitF</i> (CHA5066), the <i>dctB</i>‘-’<i>fitF</i> chimeric gene (CHA5093) or the <i>citA‘-’fitF</i> 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 (Δ<i>fitH</i>, 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) <i>Galleria</i> injection assay with wild-type (in black, CHA0) and isogenic mutants (Δ<i>fitF</i> in red, CHA1154; Δ<i>fitD</i> in blue, CHA1151; Δ<i>fitF dctB‘-’fitF</i> in green, CHA5150) of <i>P. protegens</i> CHA0 into last instar larvae of <i>G. mellonella</i>. 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.</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

The hybrid sensor kinase FitF is essential for Fit toxin expression.

<p>(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 <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003964#ppat.1003964-MarchlerBauer1" target="_blank">[42]</a> and SMART <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1003964#ppat.1003964-Schultz1" target="_blank">[43]</a>. 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 <i>G. mellonella</i> infected with FitD-mCherry reporter strains with the wild-type (CHA1176) and Δ<i>fitF</i> mutant (CHA1174-<i>gfp2</i>) 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-<i>gfp2</i> 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 (Δ<i>fitF</i> in red, CHA1154; Δ<i>fitD</i> in blue, CHA1151) of <i>P. protegens</i> CHA0 into last instar larvae of <i>G. mellonella</i>. 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.</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