Unusual extracellular appendages deployed by the model strain Pseudomonas fluorescens C7R12.

Pseudomonas fluorescens is considered to be a typical plant-associated saprophytic bacterium with no pathogenic potential. Indeed, some P. fluorescens strains are well-known rhizobacteria that promote plant growth by direct stimulation, by preventing the deleterious effects of pathogens, or both. Pseudomonas fluorescens C7R12 is a rhizosphere-competent strain that is effective as a biocontrol agent and promotes plant growth and arbuscular mycorrhization. This strain has been studied in detail, but no visual evidence has ever been obtained for extracellular structures potentially involved in its remarkable fitness and biocontrol performances. On transmission electron microscopy of negatively stained C7R12 cells, we observed the following appendages: multiple polar flagella, an inducible putative type three secretion system typical of phytopathogenic Pseudomonas syringae strains and densely bundled fimbria-like appendages forming a broad fractal-like dendritic network around single cells and microcolonies. The deployment of one or other of these elements on the bacterial surface depends on the composition and affinity for the water of the microenvironment. The existence, within this single strain, of machineries known to be involved in motility, chemotaxis, hypersensitive response, cellular adhesion and biofilm formation, may partly explain the strong interactions of strain C7R12 with plants and associated microflora in addition to the type three secretion system previously shown to be implied in mycorrhizae promotion.

Type III secretion system and virulence markers highlight similarities and differences between human- and plant-associated pseudomonads related to Pseudomonas fluorescens and P. putida.

Pseudomonas fluorescens is commonly considered a saprophytic rhizobacterium devoid of pathogenic potential. Nevertheless, the recurrent isolation of strains from clinical human cases could indicate the emergence of novel strains originating from the rhizosphere reservoir, which could be particularly resistant to the immune system and clinical treatment. The importance of type three secretion systems (T3SSs) in the related Pseudomonas aeruginosa nosocomial species and the occurrence of this secretion system in plant-associated P. fluorescens raise the question of whether clinical isolates may also harbor T3SSs. In this study, isolates associated with clinical infections and identified in hospitals as belonging to P. fluorescens were compared with fluorescent pseudomonads harboring T3SSs isolated from plants. Bacterial isolates were tested for (i) their genetic relationships based on their 16S rRNA phylogeny, (ii) the presence of T3SS genes by PCR, and (iii) their infectious potential on animals and plants under environmental or physiological temperature conditions. Two groups of bacteria were delineated among the clinical isolates. The first group encompassed thermotolerant (41°C) isolates from patients suffering from blood infections; these isolates were finally found to not belong to P. fluorescens but were closely related and harbored highly conserved T3SS genes belonging to the Ysc-T3SS family, like the T3SSs from P. aeruginosa. The second group encompassed isolates from patients suffering from cystic fibrosis; these isolates belonged to P. fluorescens and harbored T3SS genes belonging to the Hrp1-T3SS family found commonly in plant-associated P. fluorescens.

A type VI secretion system is involved in Pseudomonas fluorescens bacterial competition.

Protein secretion systems are crucial mediators of bacterial interactions with other organisms. Among them, the type VI secretion system (T6SS) is widespread in Gram-negative bacteria and appears to inject toxins into competitor bacteria and/or eukaryotic cells. Major human pathogens, such as Vibrio cholerae, Burkholderia and Pseudomonas aeruginosa, express T6SSs. Bacteria prevent self-intoxication by their own T6SS toxins by producing immunity proteins, which interact with the cognate toxins. We describe here an environmental P. fluorescens strain, MFE01, displaying an uncommon oversecretion of Hcp (hemolysin-coregulated protein) and VgrG (valine-glycine repeat protein G) into the culture medium. These proteins are characteristic components of a functional T6SS. The aim of this study was to attribute a role to this energy-consuming overexpression of the T6SS. The genome of MFE01 contains at least two hcp genes (hcp1 and hcp2), suggesting that there may be two putative T6SS clusters. Phenotypic studies have shown that MFE01 is avirulent against various eukaryotic cell models (amebas, plant or animal cell models), but has antibacterial activity against a wide range of competitor bacteria, including rhizobacteria and clinical bacteria. Depending on the prey cell, mutagenesis of the hcp2 gene in MFE01 abolishes or reduces this antibacterial killing activity. Moreover, the introduction of T6SS immunity proteins from S. marcescens, which is not killed by MFE01, protects E. coli against MFE01 killing. These findings suggest that the protein encoded by hcp2 is involved in the killing activity of MFE01 mediated by effectors of the T6SS targeting the peptidoglycan of Gram-negative bacteria. Our results indicate that MFE01 can protect potato tubers against Pectobacterium atrosepticum, which causes tuber soft rot. Pseudomonas fluorescens is often described as a major PGPR (plant growth-promoting rhizobacterium), and our results suggest that there may be a connection between the T6SS and the PGPR properties of this bacterium.

In Planta Biocontrol of Pectobacterium atrosepticum by Rhodococcus erythropolis Involves Silencing of Pathogen Communication by the Rhodococcal Gamma-Lactone Catabolic Pathway.

The virulence of numerous Gram-negative bacteria is under the control of a quorum sensing process based on synthesis and perception of N-acyl homoserine lactones. Rhodococcus erythropolis, a Gram-positive bacterium, has recently been proposed as a biocontrol agent for plant protection against soft-rot bacteria, including Pectobacterium. Here, we show that the γ-lactone catabolic pathway of R. erythropolis disrupts Pectobacterium communication and prevents plant soft-rot. We report the first characterization and demonstration of N-acyl homoserine lactone quenching in planta. In particular, we describe the transcription of the R. erythropolis lactonase gene, encoding the key enzyme of this pathway, and the subsequent lactone breakdown. The role of this catabolic pathway in biocontrol activity was confirmed by deletion of the lactonase gene from R. erythropolis and also its heterologous expression in Escherichia coli. The γ-lactone catabolic pathway is induced by pathogen communication rather than by pathogen invasion. This is thus a novel and unusual biocontrol pathway, differing from those previously described as protecting plants from phytopathogens. These findings also suggest the existence of an additional pathway contributing to plant protection.