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. 2012 Jul;78(14):4826-34.
doi: 10.1128/AEM.00335-12. Epub 2012 Apr 27.

Genetic and functional characterization of cyclic lipopeptide white-line-inducing principle (WLIP) production by rice rhizosphere isolate Pseudomonas putida RW10S2

Hassan Rokni-Zadeh  1 , Wen Li, Aminael Sanchez-Rodriguez, Davy Sinnaeve, Jef Rozenski, José C Martins, René De Mot
Affiliations

Genetic and functional characterization of cyclic lipopeptide white-line-inducing principle (WLIP) production by rice rhizosphere isolate Pseudomonas putida RW10S2

Hassan Rokni-Zadeh et al. Appl Environ Microbiol. 2012 Jul.

Abstract

The secondary metabolite mediating the GacS-dependent growth-inhibitory effect exerted by the rice rhizosphere isolate Pseudomonas putida RW10S2 on phytopathogenic Xanthomonas species was identified as white-line-inducing principle (WLIP), a member of the viscosin group of cyclic lipononadepsipeptides. WLIP producers are commonly referred to by the taxonomically invalid name "Pseudomonas reactans," based on their capacity to reveal the presence of a nearby colony of Pseudomonas tolaasii by inducing the formation of a visible precipitate ("white line") in agar medium between both colonies. This phenomenon is attributed to the interaction of WLIP with a cyclic lipopeptide of a distinct structural group, the fungitoxic tolaasin, and has found application as a diagnostic tool to identify tolaasin-producing bacteria pathogenic to mushrooms. The genes encoding the WLIP nonribosomal peptide synthetases WlpA, WlpB, and WlpC were identified in two separate genomic clusters (wlpR-wlpA and wlpBC) with an operon organization similar to that of the viscosin, massetolide, and entolysin biosynthetic systems. Expression of wlpR is dependent on gacS, and the encoded regulator of the LuxR family (WlpR) activates transcription of the biosynthetic genes and the linked export genes, which is not controlled by the RW10S2 quorum-sensing system PmrR/PmrI. In addition to linking the known phenotypes of white line production and hemolytic activity of a WLIP producer with WLIP biosynthesis, additional properties of ecological relevance conferred by WLIP production were identified, namely, antagonism against Xanthomonas and involvement in swarming and biofilm formation.

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Figures

Fig 1
Fig 1
Growth inhibition of Xanthomonas species by P. putida RW10S2. Spotted RW10S2 cells (2 μl; 107 CFU/ml) were overlaid with Xanthomonas indicator cells. For each indicator, the growth inhibition halo (radius in mm; mean ± standard deviations [SD] of three repeats) is indicated in parentheses: (A) X. citri pv. malvacearum LMG 761 (8.3 ± 0.6); (B) X. axonopodis pv. manihotis LMG 784 (7.6 ± 0.3); (C) X. translucens pv. cerealis LMG 679 (4.6 ± 0.6); (D) X. albilineans LMG 494 (4.5 ± 0.5); (E) X. hortorum pv. hederae LMG 7411 (4.3 ± 0.3); (F) Xanthomonas sp. pv. zinnia LMG 8692 (2.6 ± 0.6); (G) X. translucens pv. graminis LMG 726 (2.8 ± 0.3); (H) X. campestris pv. pelargonii LMG 10342 (3.1 ± 0.6); (I) X. alfalfae pv. alfalfae LMG 497 (2.6 ± 0.3).
Fig 2
Fig 2
Organization of the wlp gene cluster in P. putida RW10S2 (A) and its transcriptional analysis (B). (A) The labeled circles show predicted NRPS domains encoded by wlpA (NRPS1), wlpB (NRPS2), and wlpC (NRPS3): C, condensation; A, adenylation; T, thiolation; TE, thioesterase. The condensation domains with putative dual condensation/epimerization function are underlined. The condensation domain with predicted lipo-initiation activity, attaching a fatty acid (FA), is labeled Ci. For the nine modules, amino acid specificity based on in silico analysis of the respective A domains is indicated. The vertical bold lines mark the plasposon insertion sites leading to WLIP production deficiency. The mutants in bold were verified by Southern blot analysis (see Fig. S1 in the supplemental material) and phenotypically characterized. (B) The composite panel shows the amplicons obtained by RT-PCR transcript analysis using primer couples for the specified pairs of adjacent convergent genes and for 16S rRNA (internal control). RNA was extracted from the wild type (WT) and from mutants CMPG2173 (wlpR) and CMPG2134 (gacS).
Fig 3
Fig 3
Phenotypic characterization of P. putida RW10S2 and representative mutants lacking WLIP production. (A) Antagonism against X. citri pv. malvacearum LMG 761. (B) White-line formation obtained by confronting P. putida RW10S2 with P. tolaasii CH36 (lower bacterial streak) (C) Hemolysis on a horse blood TSA plate. (D) Swarming on 0.8% TSA. (E) Biofilm formation on polystyrene pegs visualized by staining of adherent cells. WT, RW10S2 wild type; wlpA through wlpR, mutants CMPG2170, CMPG2169, CMPG2120, and CMPG2173, respectively; wlpR+, mutant CMPG2173 with pCMPG6125 containing wlpR from P. putida RW10S2; wlpR*, mutant CMPG2173 with pCMPG6116 containing xtlR from P. putida BW11M1; gacS, mutant CPMG2134; gacS+, mutant CMPG2134 with pCMPG6203 containing gacS from P. putida RW10S2; gacS*, mutant CMPG2134 with pCMPG6113 containing gacS from P. putida RW10S1. The phenotypes shown for the selected wlpA, wlpB, wlpC, and gacS mutants are representative for the other wlp NRPS and gacS mutants (Fig. 2; see Table S1 in the supplemental material). The corresponding quantitative data are shown in Fig. 4 (biofilm formation) and in Table S3 in the supplemental material (antagonism, hemolysis, and swarming).
Fig 4
Fig 4
Biofilm formation by P. putida RW10S2 and selected mutants affected in WLIP production (abbreviations are as in Fig. 3). Error bars indicate standard deviations. Analysis of variance (ANOVA) was used to evaluate significant differences (P < 0.001) between the wild type (set to 100%), mutants, and complemented mutants (indicated with different letters above the bars).
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