Pyrrolnitrin and Hydrogen Cyanide Production by Pseudomonas chlororaphis Strain PA23 Exhibits Nematicidal and Repellent Activity against Caenorhabditis elegans

Abstract

Pseudomonas chlororaphis strain PA23 is a biocontrol agent able to suppress growth of the fungal pathogen Sclerotinia sclerotiorum. This bacterium produces an arsenal of exometabolites including pyrrolnitrin (PRN), phenazine (PHZ), hydrogen cyanide (HCN), and degradative enzymes. Production of these compounds is controlled at both the transcriptional and posttranscriptional levels by the Gac-Rsm system, RpoS, PsrA, and the Phz quorum-sensing system. Beyond pathogen-suppression, the success of a biocontrol agent is dependent upon its ability to establish itself in the environment where predation by bacterivorous organisms, including nematodes, may threaten persistence. The focus of this study was to investigate whether PA23 is able to resist grazing by Caenorhabditis elegans and to define the role played by exoproducts in the bacterial-nematode interaction. We discovered that both PRN and HCN contribute to fast- and slow-killing of C. elegans. HCN is well-established as having lethal effects on C. elegans; however, PRN has not been reported to be nematicidal. Exposure of L4 stage nematodes to purified PRN reduced nematode viability in a dose-dependent fashion and led to reduced hatching of eggs laid by gravid adults. Because bacterial metabolites can act as chemoattractants or repellents, we analyzed whether PA23 exhibited attractant or repulsive properties towards C. elegans. Both PRN and HCN were found to be potent repellents. Next we investigated whether the presence of C. elegans would elicit changes in PA23 gene activity. Co-culturing the two organisms increased expression of a number of genes associated with biocontrol, including phzA, hcnA, phzR, phzI, rpoS and gacS. Exoproduct analysis showed that PHZ and autoinducer signals were upregulated, consistent with the gene expression profiles. Collectively, these findings indicate that PA23 is able to sense the presence of C. elegans and it is able to both repel and kill the nematodes, which should facilitate environmental persistence and ultimately biocontrol.

Pseudomonas brassicacearum Strain DF41 Kills Caenorhabditis elegans through Biofilm-Dependent and Biofilm-Independent Mechanisms

“…Consequently, several different media were employed as part of our DF41-C. elegans interaction studies. On nutrient-rich BHI, which has been previously reported to support high exometabolite production and fast killing of C. elegans (6,7), none of the strains tested led to 100% killing at 24 h (Fig. 1A).…”

“…When DF41 and derivative strains, including DF41-1278, DF41hcn, autoinducer (AI)-deficient DF41, DF41rfiA, and DF41gacS, were grown on BHI, which supports fast killing by pseudomonads (7,8), differences in lethality were observed; however, none elicited 100% nematode killing at 24 h (Fig. 1A).…”

“…We discovered that DF41 is capable of killing C. elegans through two distinct modes; the first involves exposure to toxic metabolites, while the second entails biofilm formation on the nematode surface. It has been reported that bacteria are capable of responding to predator cues through altered expression of toxin-encoding genes (7,22,23). To see if the same holds true for DF41, we cocultured bacteria with C. elegans and monitored the expression of genes associated with exometabolite production.…”

“…For pseudomonads that exhibit pathogenicity toward C. elegans, killing ensues through two distinct mechanisms. On rich media, which support the production of high levels of toxic metabolites, death occurs through intoxication, a mechanism known as fast killing (5)(6)(7)(8). Conversely, low-nutrient media result in the production of sublethal levels of exometabolites, and slow killing occurs over the course of several days.…”

A decade of understanding secondary metabolism in Pseudomonas spp. for sustainable agriculture and pharmaceutical applications

“…A pathogenic strain of P. fluorescens, NZ17 successfully repelled grazing by bacterivore C. elegans naturally present in mushroom farms (Burlinson et al 2013). Furthermore, nematocidal activity of pyrrolnitrin producing P. chlororaphis biocontrol strain PA23 was also investigated against C. elegans (Nandi et al 2015). Rhizoxin was originally identified from Burkholderia rhizoxina and Rhizopus microsporus, however, later its production was also shown by P. protegens strain Pf-5, reported by Loper et al (2008).…”