Characterization of the biocontrol activity of pseudomonas fluorescens strain X reveals novel genes regulated by glucose.

Pseudomonas fluorescens strain X, a bacterial isolate from the rhizosphere of bean seedlings, has the ability to suppress damping-off caused by the oomycete Pythium ultimum. To determine the genes controlling the biocontrol activity of strain X, transposon mutagenesis, sequencing and complementation was performed. Results indicate that, biocontrol ability of this isolate is attributed to gcd gene encoding glucose dehydrogenase, genes encoding its co-enzyme pyrroloquinoline quinone (PQQ), and two genes (sup5 and sup6) which seem to be organized in a putative operon. This operon (named supX) consists of five genes, one of which encodes a non-ribosomal peptide synthase. A unique binding site for a GntR-type transcriptional factor is localized upstream of the supX putative operon. Synteny comparison of the genes in supX revealed that they are common in the genus Pseudomonas, but with a low degree of similarity. supX shows high similarity only to the mangotoxin operon of Ps. syringae pv. syringae UMAF0158. Quantitative real-time PCR analysis indicated that transcription of supX is strongly reduced in the gcd and PQQ-minus mutants of Ps. fluorescens strain X. On the contrary, transcription of supX in the wild type is enhanced by glucose and transcription levels that appear to be higher during the stationary phase. Gcd, which uses PQQ as a cofactor, catalyses the oxidation of glucose to gluconic acid, which controls the activity of the GntR family of transcriptional factors. The genes in the supX putative operon have not been implicated before in the biocontrol of plant pathogens by pseudomonads. They are involved in the biosynthesis of an antimicrobial compound by Ps. fluorescens strain X and their transcription is controlled by glucose, possibly through the activity of a GntR-type transcriptional factor binding upstream of this putative operon

Arrangement of the genes in the genomic locus of <i>sup</i>5 and <i>sup</i>6, compared to other <i>Pseudomonas</i> strains, and complementation analysis of the region.

<p>The lines beneath the genomic of <i>Ps. fluorescens</i> X represent regions of this locus that were PCR-amplified, cloned into pBBR1MCS5 and tested for complementation. Ability to complement is noted with plus (+) or minus (−). Putative ORFs are indicated by thick coloured arrows on a line. Genes that might be organised in a putative operon are enclosed by a grey frame. The direction of the plasposon Tn<i>5</i>-RL27 insertion in mutants δ40 and ρ93 is indicated with a black arrow beneath the sequence (▸). Predicted sites for the unique putative promoter and operator are also marked (;). Size, genomic location and locus tag of the different ORFs sequenced in <i>Ps. aeruginosa</i> PA7, <i>Ps. fluorescens</i> Pf0–1, <i>Ps. fluorescens</i> SBW25, <i>Ps. entomophila</i> L48, <i>Ps. syringae</i> pv. <i>syringae</i> B728a,<i>Ps. syringae</i> pv. <i>tomato</i> DC3000 and <i>Ps. syringae</i> pv. <i>syringae</i> UMAF0158 are indicated.</p

Complementation analysis of the PQQ biosynthesis region in <i>Ps. fluorescens</i> strain X.

<p>PCR fragments of this region (1–5), with different sets of genes from <i>Ps. fluorescens</i> X, and their ability to complement sup⁻ mutants. Ability to complement is noted with plus (+) or minus (−). The direction of the plasposon Tn<i>5</i>-RL27 insertion in the derivative mutants B91, B163, A150 and ρ26 is indicated with a flag beneath the sequence. The predicted site for the unique putative promoter is also marked.</p

Characteristics of sup⁻ mutants and complementation analysis.

<p>ND: Not Detected.</p>a<p>Acidification was observed on solid minimal medium M9 supplemented with 2% w/v glucose, as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061808#pone.0061808-Pujol1" target="_blank">[14]</a>.</p>b<p>Enzyme treatment was performed for filtrates from incubation in PDB, as described before <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0061808#pone.0061808-Arrebola2" target="_blank">[47]</a>.</p