Temperature-mediated biosynthesis of the phytotoxin phaseolotoxin by Pseudomonas syringae pv. phaseolicola depends on the autoregulated expression of the phtABC genes

Abstract

Pseudomonas syringae pv. phaseolicola produces phaseolotoxin in a temperature dependent manner, being optimally synthesized between 18°C and 20°C, while no detectable amounts are present above 28°C. The Pht cluster, involved in the biosynthesis of phaseolotoxin, contains 23 genes that are organized in five transcriptional units. The function of most of the genes from the Pht cluster is still unknown and little information about the regulatory circuitry leading to expression of these genes has been reported. The purpose of the present study was to investigate the participation of pht genes in the regulation of the operons coded into the Pht cluster. We conducted Northern blot, uidA fusions and reverse transcription-PCR assays of pht genes in several mutants unable to produce phaseolotoxin. This allowed us to determine that, in P. syringae pv. phaseolicola NPS3121, genes phtABC are essential to prevent their own expression at 28°C, a temperature at which no detectable amounts of the toxin are present. We obtained evidence that the phtABC genes also participate in the regulation of the phtD, phtM and phtL operons. According to our results, we propose that PhtABC and other Pht product activities could be involved in the synthesis of the sulfodiaminophosphinyl moiety of phaseolotoxin, which indirectly could be involved in the transcriptional regulation of the phtA operon.

Fig 1. Phaseolotoxin production by P. syringae pv. phaseolicola strains.

(A) Structure of phaseolotoxin (top), which is cleaved by plant peptidases (arrow) to release octicidin, with indication of the inorganic moiety (N’-sulfodiaminophosphinyl). Octicidin is a transition state analog, structurally similar to carbamoylphosphate and ornithine (bottom), substrates of OCTase during biosynthesis of citrulline. (B) Evaluation of the production of phaseolotoxin by the wild type strain NPS3121 and mutant 3121phtD using the E. coli growth inhibition assay.

Fig 2. Reverse transcription-PCR of the Pht cluster of P. syringae pv. phaseolicola NPS3121 and mutants.

(A) Graphic representation of the Pht cluster of P. syringae pv. phaseolicola NPS3121. Genes are represented by arrows that indicate the direction of transcription. The Pht cluster contains five transcriptional units, including two monocistronic (argK and phtL) and three polycistronic (phtA, phtD and phtM) [18]. Mutated genes are indicated by an X over the corresponding gene. (B) Analysis by RT-PCR of the transcriptional pattern of the Pht-cluster in the wild type strain NPS3121 and derivative mutants. Pictures show the RT-PCR product corresponding to each analyzed locus, as indicated by the black bars, separated by electrophoresis on agarose gels; mutated genes are indicated by an X over the corresponding picture of the RT-PCR gel. Numbers under the pictures represent the temperature at which expression was assayed: 1 = 18°C; 2 = 28°C.

Fig 3. Expression of transcriptional uidA reporter gene fusions in P. syringae pv. phaseolicola NPS3121 and mutant backgrounds.

3121phtA and 3121phtD correspond to phtA^- and phtD^- polar mutants, whereas NPS3121 indicates the wild type strain. As negative control, NPS3121 harboring pRG960sd was used. (A) GUS activity from pPphtA::GUS, which corresponds to the promoter of phtA operon cloned into vector pRG960sd. (B) GUS activity from pPphtD::GUS, corresponding to the promoter of phtD operon cloned into pRG960sd. The small numbers under the bars represent the temperatures at which expression was assayed: 1 indicates 18°C and 2 indicates 28°C. Bars represent mean values with standard deviations; bars in each panel topped with different letters indicate means that are significantly different according to a two-way ANOVA (P< 0.01) followed by the Duncan’s test.

Fig 4. Expression of genes phtABC in Tox+ and Tox- strains.

Northern blot hybridization of RNA isolated from P. syringae pv. phaseolicola strains NPS3121 (Tox+) and CYL233 (Tox-) containing the indicated plasmid constructs. Internal gene fragments used as hybridization probes as well as the biological source of total RNA are indicated above the gel pictures. Strain CYL233 containing the empty vector plasmid pUCP20 was used as negative control of gene expression, whereas the wild type strain NPS3121 was used as a positive control. Numbers on top of the Northern blots represent the temperatures at which expression was assayed: 1 indicates 18°C and 2 indicates 28°C.