Biocontrol of Biofilm Formation: Jamming of Sessile-Associated Rhizobial Communication by Rhodococcal Quorum-Quenching.

Biofilms are complex structures formed by a community of microbes adhering to a surface and/or to each other through the secretion of an adhesive and protective matrix. The establishment of these structures requires a coordination of action between microorganisms through powerful communication systems such as quorum-sensing. Therefore, auxiliary bacteria capable of interfering with these means of communication could be used to prevent biofilm formation and development. The phytopathogen Rhizobium rhizogenes, which causes hairy root disease and forms large biofilms in hydroponic crops, and the biocontrol agent Rhodococcus erythropolis R138 were used for this study. Changes in biofilm biovolume and structure, as well as interactions between rhizobia and rhodococci, were monitored by confocal laser scanning microscopy with appropriate fluorescent biosensors. We obtained direct visual evidence of an exchange of signals between rhizobia and the jamming of this communication by Rhodococcus within the biofilm. Signaling molecules were characterized as long chain (C14) N-acyl-homoserine lactones. The role of the Qsd quorum-quenching pathway in biofilm alteration was confirmed with an R. erythropolis mutant unable to produce the QsdA lactonase, and by expression of the qsdA gene in a heterologous host, Escherichia coli. Finally, Rhizobium biofilm formation was similarly inhibited by a purified extract of QsdA enzyme.

Recent Advances in Understanding the Roles of Pectin as an Active Participant in Plant Signaling Networks.

Pectin is an abundant cell wall polysaccharide with essential roles in various biological processes. The structural diversity of pectins, along with the numerous combinations of the enzymes responsible for pectin biosynthesis and modification, plays key roles in ensuring the specificity and plasticity of cell wall remodeling in different cell types and under different environmental conditions. This review focuses on recent progress in understanding various aspects of pectin, from its biosynthetic and modification processes to its biological roles in different cell types. In particular, we describe recent findings that cell wall modifications serve not only as final outputs of internally determined pathways, but also as key components of intercellular communication, with pectin as a major contributor to this process. The comprehensive view of the diverse roles of pectin presented here provides an important basis for understanding how cell wall-enclosed plant cells develop, differentiate, and interact.

Biosensors Used for Epifluorescence and Confocal Laser Scanning Microscopies to Study Dickeya and Pectobacterium Virulence and Biocontrol.

Promoter-probe vectors carrying fluorescent protein-reporter genes are powerful tools used to study microbial ecology, epidemiology, and etiology. In addition, they provide direct visual evidence of molecular interactions related to cell physiology and metabolism. Knowledge and advances carried out thanks to the construction of soft-rot Pectobacteriaceae biosensors, often inoculated in potato Solanum tuberosum, are discussed in this review. Under epifluorescence and confocal laser scanning microscopies, Dickeya and Pectobacterium-tagged strains managed to monitor in situ bacterial viability, microcolony and biofilm formation, and colonization of infected plant organs, as well as disease symptoms, such as cell-wall lysis and their suppression by biocontrol antagonists. The use of dual-colored reporters encoding the first fluorophore expressed from a constitutive promoter as a cell tag, while a second was used as a regulator-based reporter system, was also used to simultaneously visualize bacterial spread and activity. This revealed the chronology of events leading to tuber maceration and quorum-sensing communication, in addition to the disruption of the latter by biocontrol agents. The promising potential of these fluorescent biosensors should make it possible to apprehend other activities, such as subcellular localization of key proteins involved in bacterial virulence in planta, in the near future.

Crosstalk between the Type VI Secretion System and the Expression of Class IV Flagellar Genes in the Pseudomonas fluorescens MFE01 Strain.

Type VI secretion systems (T6SSs) are contractile bacterial multiprotein nanomachines that enable the injection of toxic effectors into prey cells. The Pseudomonas fluorescens MFE01 strain has T6SS antibacterial activity and can immobilise competitive bacteria through the T6SS. Hcp1 (hemolysin co-regulated protein 1), a constituent of the T6SS inner tube, is involved in such prey cell inhibition of motility. Paradoxically, disruption of the hcp1 or T6SS contractile tail tssC genes results in the loss of the mucoid and motile phenotypes in MFE01. Here, we focused on the relationship between T6SS and flagella-associated motility. Electron microscopy revealed the absence of flagellar filaments for MFE01Δhcp1 and MFE01ΔtssC mutants. Transcriptomic analysis showed a reduction in the transcription of class IV flagellar genes in these T6SS mutants. However, transcription of fliA, the gene encoding the class IV flagellar sigma factor, was unaffected. Over-expression of fliA restored the motile and mucoid phenotypes in both MFE01Δhcp1+fliA, and MFE01ΔtssC+fliA and a fliA mutant displayed the same phenotypes as MFE01Δhcp1 and MFE01ΔtssC. Moreover, the FliA anti-sigma factor FlgM was not secreted in the T6SS mutants, and flgM over-expression reduced both motility and mucoidy. This study provides arguments to unravel the crosstalk between T6SS and motility.

Close-up on a bacterial informational war in the geocaulosphere.

The geocaulosphere is home to microbes that establish communication between themselves and others that disrupt them. These cell-to-cell communication systems are based on the synthesis and perception of signaling molecules, of which the best known belong to the N-acyl-homoserine lactone (AHL) family. Among indigenous bacteria, certain Gram-positive actinobacteria can sense AHLs produced by soft-rot Gram-negative phytopathogens and can degrade the quorum-sensing AHL signals to impair the expression of virulence factors. We mimicked this interaction by introducing dual-color reporter strains suitable for monitoring both the location of the cells and their quorum-sensing and -quenching activities, in potato tubers. The exchange of AHL signals within the pathogen's cell quorum was clearly detected by the presence of bright green fluorescence instead of blue in a portion of Pectobacterium-tagged cells. This phenomenon in Rhodococcus cells was accompanied by a change from red fluorescence to orange, showing that the disappearance of signaling molecules is due to rhodococcal AHL degradation rather than the inhibition of AHL production. Rhodococci are victorious in this fight for the control of AHL-based communication, as their jamming activity is powerful enough to prevent the onset of disease symptoms.

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.

A Flavor Lactone Mimicking AHL Quorum-Sensing Signals Exploits the Broad Affinity of the QsdR Regulator to Stimulate Transcription of the Rhodococcal qsd Operon Involved in Quorum-Quenching and Biocontrol Activities.

In many Gram-negative bacteria, virulence, and social behavior are controlled by quorum-sensing (QS) systems based on the synthesis and perception of N-acyl homoserine lactones (AHLs). Quorum-quenching (QQ) is currently used to disrupt bacterial communication, as a biocontrol strategy for plant crop protection. In this context, the Gram-positive bacterium Rhodococcus erythropolis uses a catabolic pathway to control the virulence of soft-rot pathogens by degrading their AHL signals. This QS signal degradation pathway requires the expression of the qsd operon, encoding the key enzyme QsdA, an intracellular lactonase that can hydrolyze a wide range of substrates. QsdR, a TetR-like family regulator, represses the expression of the qsd operon. During AHL degradation, this repression is released by the binding of the γ-butyrolactone ring of the pathogen signaling molecules to QsdR. We show here that a lactone designed to mimic quorum signals, γ-caprolactone, can act as an effector ligand of QsdR, triggering the synthesis of qsd operon-encoded enzymes. Interaction between γ-caprolactone and QsdR was demonstrated indirectly, by quantitative RT-PCR, molecular docking and transcriptional fusion approaches, and directly, in an electrophoretic mobility shift assay. This broad-affinity regulatory system demonstrates that preventive or curative quenching therapies could be triggered artificially and/or managed in a sustainable way by the addition of γ-caprolactone, a compound better known as cheap food additive. The biostimulation of QQ activity could therefore be used to counteract the lack of consistency observed in some large-scale biocontrol assays.

Biocontrol of Soft Rot: Confocal Microscopy Highlights Virulent Pectobacterial Communication and Its Jamming by Rhodococcal Quorum-Quenching.

Confocal laser-scanning microscopy was chosen to observe the colonization and damage caused by the soft rot Pectobacterium atrosepticum and the protection mediated by the biocontrol agent Rhodococcus erythropolis. We developed dual-color reporter strains suited for monitoring quorum-sensing and quorum-quenching activities leading to maceration or biocontrol, respectively. A constitutively expressed cyan or red fluorescent protein served as a cell tag for plant colonization, while an inducible expression reporter system based on the green fluorescent protein gene enabled the simultaneous recording of signaling molecule production, detection, or degradation. The dual-colored pathogen and biocontrol strains were used to coinoculate potato tubers. At cellular quorum, images revealed a strong pectobacterial quorum-sensing activity, especially at the plant cell walls, as well as a concomitant rhodococcal quorum-quenching response, at both the single-cell and microcolony levels. The generated biosensors appear to be promising and complementary tools useful for molecular and cellular studies of bacterial communication and interference.

A Rhodococcal Transcriptional Regulatory Mechanism Detects the Common Lactone Ring of AHL Quorum-Sensing Signals and Triggers the Quorum-Quenching Response.

The biocontrol agent Rhodococcus erythropolis disrupts virulence of plant and human Gram-negative pathogens by catabolizing their N-acyl-homoserine lactones. This quorum-quenching activity requires the expression of the qsd (quorum-sensing signal degradation) operon, which encodes the lactonase QsdA and the fatty acyl-CoA ligase QsdC, involved in the catabolism of lactone ring and acyl chain moieties of signaling molecules, respectively. Here, we demonstrate the regulation of qsd operon expression by a TetR-like family repressor, QsdR. This repression was lifted by adding the pathogen quorum signal or by deleting the qsdR gene, resulting in enhanced lactone degrading activity. Using interactomic approaches and transcriptional fusion strategy, the qsd operon derepression was elucidated: it is operated by the binding of the common part of signaling molecules, the homoserine lactone ring, to the effector-receiving domain of QsdR, preventing a physical binding of QsdR to the qsd promoter region. To our knowledge, this is the first evidence revealing quorum signals as inducers of the suitable quorum-quenching pathway, confirming this TetR-like protein as a lactone sensor. This regulatory mechanism designates the qsd operon as encoding a global disrupting pathway for degrading a wide range of signal substrates, allowing a broad spectrum anti-virulence activity mediated by the rhodococcal biocontrol agent. Understanding the regulation mechanisms of qsd operon expression led also to the development of biosensors useful to monitor in situ the presence of exogenous signals and quorum-quenching activity.