Biological activity and regulation of cyclic lipopeptides and phenazines produced by biocontrol strain Pseudomonas CMR12a

Abstract

Biological control of plant diseases is a promising alternative to classical pesticides. Fluorescent pseudomonads have a lot of potential for biocontrol applications, partly because they produce various antimicrobial metabolites. Knowledge about the properties, roles and regulation of these metabolites is crucial for optimization and innovation of biocontrol strategies. Pseudomonas CMR12a was isolated from the rhizosphere of the tropical crop cocoyam (Xanthosoma sagittifolium) in Cameroon, and probably represents a new species. This strain secretes several interesting metabolites, such as phenazines, namely phenazine-1-carboxylic acid (PCA) and phenazine-1-carboxamide (PCN), and cyclic lipopeptide (CLP) type biosurfactants, which have not been characterized as yet. The structure of the biosurfactants produced by CMR12a was investigated in detail by a combination of chemical analysis and in silico analysis of the nonribosomal gene clusters responsible for CLP biosynthesis. To obtain these gene clusters, the whole genome of CMR12a was sequenced. CMR12a synthesizes two distinct types of CLPs, which were designated sessilin and motilin. Sessilin consists of an 18 amino acid peptide linked to a 3-hydroxyoctanoyl fatty acid, and is most related to tolaasin, a CLP produced by mushroom pathogen Pseudomonas tolaasii. Motilin is made up of a 10 amino acid peptide coupled to a 3-hydroxydodecanoyl or 3-hydroxytetradecanoyl fatty acid moiety, and is very similar to orfamide produced by biocontrol strain Pseudomonas fluorescens Pf-5. We investigated the importance of sessilin and phenazines for biocontrol by CMR12a of root rot on bean (Phaseolus vulgaris) caused by Rhizoctonia solani AG 2-2 and AG 4 HGI. The wild type CMR12a provided excellent control of the disease, while mutants in phenazine or sessilin biosynthesis lost part of their biocontrol ability, indicating that these compounds both play a role in antagonism and have an additive effect. Mutants deficient for both phenazines and sessilin were no longer capable of controlling the disease at all. Bacteria lost part of their effectiveness when the inoculum was washed before application, thus removing metabolites produced during incubation on a plate. In addition, microscopic observations showed that sessilin induced branching of R. solani mycelium, while phenazines had no obvious effect in vitro. The involvement of phenazines and sessilin in biocontrol of root rot disease on cocoyam caused by Pythium myriotylum was also tested. However, results were not straightforward in this case because the composition of the substrate appeared to influence the outcome of the experiments, which is possibly due to an effect on in situ production of phenazines and sessilin. To investigate physiological roles of phenazines and CLPs for Pseudomonas CMR12a, we compared swarming motility and biofilm formation by sessilin, motilin, and phenazine biosynthesis mutants. This revealed that sessilin and motilin play contrasting roles in the behavior of CMR12a. Sessilin is important for the development of a biofilm but it inhibits swarming, hence favoring a sessile lifestyle. On the other hand, motilin is indispensable for swarming, but has a negative effect on biofilm formation, thus promoting motility. In addition, phenazines also have a small but significant positive effect on biofilm formation, which may be correlated with a putative role in iron uptake. Analysis of quorum sensing (QS) regulation in CMR12a revealed the presence of two QS systems; PhzI/PhzR and CmrI/CmrR. The conserved PhzI/R system is responsible for production of four N-acyl-L-homoserine lactones (AHLs) with a (3-hydroxy)hexanoic or (3-hydroxy)octanoic fatty acid moiety, and controls phenazine production as in other fluorescent pseudomonads. However, the CmrI/CmrR QS system is novel. Its AHL signal is the uncommon 3-hydroxydodecanoyl-HSL, which has plant growth promoting activity. The regulon of this QS system remains to be investigated in detail, but it appears to regulate the ratio of sessilin and motilin production by CMR12a through the action of a QS regulated glutamine amidotransferase. To find novel regulators of CLP synthesis, a transposon library of CMR12a was constructed, which resulted in the discovery of GidA, a protein involved in tRNA modification. We studied the phenotype of GidA mutants in Pseudomonas CMR12a and related strain CMR5c, which showed that a GidA mutation affects CLP and pyoverdin synthesis, growth, and motility. However, biofilm formation by CMR12a-GidA was found to be greatly enhanced. Together, our results indicated that GidA is a global regulator in CMR12a and CMR5c, with different activity compared to that in other pseudomonads. In conclusion, this work has resulted in a complete characterization of the CLPs produced by Pseudomonas CMR12a, sessilin and motilin. In addition, we gained insight in possible biological roles of sessilin, motilin and phenazines, and in regulation mechanisms controlling production of these compounds. We also acquired knowledge about the involvement of phenazines and CLPs in the biocontrol capacity of CMR12a. These data contribute to insight into the physiology and ecology of Pseudomonas CMR12a and biocontrol agents in general. They underline the importance of interactions of these organisms with the environment for the efficacy of biocontrol. Finally, we obtained the complete genome sequence of Pseudomonas CMR12a, which can provide unlimited inspiration for further investigation of this remarkable strain