Figure 1.
Growth inhibition of Xanthomonas species by P.putida BW11M1.
Spotted BW11M1 cells (2 µl of 107 CFU/ml) were overlayed with Xanthomonas indicator cells. (A) X. alfalfae subsp. alfalfae LMG 497; (B) X. axonopodis pv. manihotis LMG 784; (C) X. hortorum pv. hederae LMG 7411; (D) X. sacchari LMG 471; (E) X. translucens pv. cerealis LMG 679; (F) X. translucens pv. graminis LMG 726; (G) X. translucens pv. hordei LMG 737; (H) X. vasicola pv. holcicola LMG 736; (I) X. vasicola pv. musacearum LMG 785.
Figure 2.
Xantholysin biosynthetic gene clusters of P.putida BW11M1.
The organization of the genomic regions with the xantholysin synthetases genes (xtlA, xtlB and xtlC), the associated regulatory gene (xtlR) and putative export genes (xtlD, xtlE and xtlF) is shown (GenBank accession numbers: KC297505 (xtlFRA); KC297506 (xtlBCDE), together with the position of the sequenced fosmid-cloned genomic fragments. Plasposon insertion sites generating mutants without antagonistic activity X. alfalfae subsp. alfalfae LMG 497 are indicated (solid lines). Mutants selected for further phenotypic characterization are highlighted in blue font. For the encoded NRPS enzymes, the modular composition and domain architecture is visualized. The predicted amino acid sequence is based on the substrate specificity of the 14 modules, as inferred by phylogenetic analysis of Pseudomonas A-domain sequences (Fig. 3) and assuming consecutive co-linear biosynthesis by XtlA, XtlB and XtlC. The experimentally determined structure of the main biosynthetic product, xantholysin A, is shown.
Figure 3.
Phylogeny-based substrate specificity prediction of xantholysin synthetases.
Cladogram of maximum-likelihood tree inferred from amino acid sequence alignment of adenylation (A) domains extracted from functionally characterized Pseudomonas NRPSs. Lipopeptide-specific codes used for NRPS enzymes: Arf (arthrofactin, P. fluorescens MIS38); Etl (entolysin, P. entomophila L48); Mass (massetolide, P. fluorescens SS101); Ofa (orfamide, P. fluorescens Pf-5); Pso (putisolvin, P. putida PCL1445); Syf (syringafactin, P. syringae DC3000); Syp (syringopeptin, P. syringae pv. syringae B301D); Syr (syringomycin, P. syringae pv. syringae strain B301D); Visc (viscosin, P. fluorescens SBW25); Wip (WLIP, P. fluorescens LMG 5329); Wlp (WLIP, P. putida RW10S2); Xtl (xantholysin, P. putida BW11M1; highlighted in larger bold font). For each domain the substrate specificity is indicated in parentheses using the standard amino acid three-letter code (for xantholysin, as determined in this work). Non-protein amino acids are annotated as follows: allo-threonine (aTHR); 2,3-dehydro-2-aminobutyric acid (DHB); 2,4-diaminobutyric acid (DAB); 3-hydroxyaspartate (OH-ASP); unidentified residue (Leu or Ile) in putisolvin II (XLE). Clusters comprising xantholysin domains are highlighted in different colors. The tree was rooted with the divergent SyrB1 domain.
Figure 4.
Phenotypes of P.putida BW11M1 and xantholysin-deficient mutants.
(A) Antagonistic activity against X. axonopodis pv. manihotis LMG 784. (B) Antagonistic activity against X. translucens pv. cerealis LMG 679. (C) Formation of brown blotch on sliced Agaricus bisporus tissue. (D) Hemolysis on horse blood TSB agar plate. (E) Swarming on 0.8% TSB agar plate. (F) Biofilm formation on pegs visualized by staining of adherent cells. WT: BW11M1 wild type; xtlA, xtlB, xtlC, and xtlR: mutants CMPG2183, CMPG2187, CMPG2198, and CMPG2201, respectively; xltR+: mutant CMPG2201 with pCMPG6204 containing xtlR of P. putida BW11M1. The phenotypes shown for the selected xtlA, xtlB and xtlC mutants are representative for the other xtl NRPS mutants (Table S1). The quantitative data for biofilm formation are shown in Fig. 5.
Figure 5.
Biofilm formation by P.putida BW11M1 and xantholysin-deficient mutants.
Abbreviations as in Fig. 4. Error bars indicate standard deviations. The analysis of variance (ANOVA) test was used to evaluate significant differences (p<0.001; indicated with different letters above the bars) between the wild type (set to 100%; blue), mutants (red), and complemented regulatory mutant (green).
Figure 6.
Modular architecture of the xantholysin synthetases and other Pseudomonas enzymes with similar A domains.
The respective starter NRPS genes are located distantly from the gene pairs encoding the middle and terminating NRPSs, except for the putisolvin operon psoABC [16]. Synthesis of the peptide moiety by the consecutive action of the modules (with only A domains shown) proceeds in a co-linear fashion, with incorporation of the respective amino acid at the positions corresponding to the numbered boxes. If a minor lipopeptide variant with a different amino acid at a certain position has been identified, this residue is shown in parentheses with smaller font. Xle indicates that the residue’s identity (either Leu or Ile) was not resolved in putisolvin II. The connected residues form a depsi bond. Similarity between the different systems is visualized according to Rokni-Zadeh et al. [18]: two domains with a patristic distance <0.45 (summed branch lengths in a maximum-likelihood tree constructed from aligned A-domain sequences; see Fig. 3) are represented in the same color.
Table 1.
Bacterial strains and plasmids.