Two Homologues of the Global Regulator Csr/Rsm Redundantly Control Phaseolotoxin Biosynthesis and Virulence in the Plant Pathogen Pseudomonas amygdali pv. phaseolicola 1448A.

Diana Ramírez-Zapata,Selene Aguilera,Marta Martínez-Gil,Leire Bardaji,Jesús Murillo,Cayo Ramos

doi:10.3390/microorganisms8101536

Abstract

The widely conserved Csr/Rsm (carbon storage regulator/repressor of stationary-phase metabolites) post-transcriptional regulatory system controls diverse phenotypes involved in bacterial pathogenicity and virulence. Here we show that Pseudomonas amygdali pv. phaseolicola 1448A contains seven rsm genes, four of which are chromosomal. In RNAseq analyses, only rsmE was thermoregulated, with increased expression at 18 °C, whereas the antagonistic sRNAs rsmX1, rsmX4, rsmX5 and rsmZ showed increased levels at 28 °C. Only double rsmA-rsmE mutants showed significantly altered phenotypes in functional analyses, being impaired for symptom elicitation in bean, including in planta growth, and for induction of the hypersensitive response in tobacco. Double mutants were also non-motile and were compromised for the utilization of different carbon sources. These phenotypes were accompanied by reduced mRNA levels of the type III secretion system regulatory genes hrpL and hrpA, and the flagellin gene, fliC. Biosynthesis of the phytotoxin phaseolotoxin by mutants in rsmA and rsmE was delayed, occurring only in older cultures, indicating that these rsm homologues act as inductors of toxin synthesis. Therefore, genes rsmA and rsmE act redundantly, although with a degree of specialization, to positively regulate diverse phenotypes involved in niche colonization. Additionally, our results suggest the existence of a regulatory molecule different from the Rsm proteins and dependent on the GacS/GacA (global activator of antibiotic and cyanide production) system, which causes the repression of phaseolotoxin biosynthesis at high temperatures.

Highlights

The ability of free-living bacteria to respond and adapt to environmental changes relies on various interconnected regulatory networks, from which the Csr/Rsm is one of the most intensively studied post-transcriptional regulatory systems [1,2,3]
We show that double mutants in genes rsmA and rsmE, but not individual mutants, show alterations in diverse phenotypes relevant for the life cycle of Pph 1448A, including virulence in bean, motility, metabolism of carbon sources and biosynthesis of the phytotoxin phaseolotoxin
The different derivatives of strain 1448A, either mutants or strains overexpressing rsm genes, grew at slightly different rates in HSC; we evaluated the production of phaseolotoxin after 14-21 h, when the cultures were growing exponentially and had reached an optical density at 600 nm (OD600) of 0.2-0.3, and after 48 h of growth, in stationary phase (OD600 >1)

Summary

Introduction

The ability of free-living bacteria to respond and adapt to environmental changes relies on various interconnected regulatory networks, from which the Csr/Rsm (carbon storage regulator/ repressor of stationary-phase metabolites) is one of the most intensively studied post-transcriptional regulatory systems [1,2,3]. This system is widely present and conserved among nearly 3,000 species of bacteria and act as a global regulator of gene expression of hundreds of genes including, among others, those supporting robust growth and a diverse collection of virulence genes in animal and plant pathogens. The exact role of the different homologues is often unclear, some of them were shown to be redundant and/or have unique regulatory roles, which likely provide genetic flexibility and helps to fine-tune the regulatory responses [6,7,8,9,10]

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Results

Conclusion