Pseudomonas fluorescens MFE01 delivers a putative type VI secretion amidase that confers biocontrol against the soft-rot pathogen Pectobacterium atrosepticum.

The type VI secretion system (T6SS) is a contractile nanomachine widespread in Gram-negative bacteria. The T6SS injects effectors into target cells including eukaryotic hosts and competitor microbial cells and thus participates in pathogenesis and intermicrobial competition. Pseudomonas fluorescens MFE01 possesses a single T6SS gene cluster that confers biocontrol properties by protecting potato tubers against the phytopathogen Pectobacterium atrosepticum (Pca). Here, we demonstrate that a functional T6SS is essential to protect potato tuber by reducing the pectobacteria population. Fluorescence microscopy experiments showed that MFE01 displays an aggressive behaviour with an offensive T6SS characterized by continuous and intense T6SS firing activity. Interestingly, we observed that T6SS firing is correlated with rounding of Pectobacterium cells, suggesting delivery of a potent cell wall targeting effector. Mutagenesis coupled with functional assays then revealed that a putative T6SS secreted amidase, Tae3Pf , is mainly responsible for MFE01 toxicity towards Pca. Further studies finally demonstrated that Tae3Pf is toxic when produced in the periplasm, and that its toxicity is counteracted by the Tai3Pf inner membrane immunity protein.

Pseudomonas Flagella: Generalities and Specificities.

Flagella-driven motility is an important trait for bacterial colonization and virulence. Flagella rotate and propel bacteria in liquid or semi-liquid media to ensure such bacterial fitness. Bacterial flagella are composed of three parts: a membrane complex, a flexible-hook, and a flagellin filament. The most widely studied models in terms of the flagellar apparatus are E. coli and Salmonella. However, there are many differences between these enteric bacteria and the bacteria of the Pseudomonas genus. Enteric bacteria possess peritrichous flagella, in contrast to Pseudomonads, which possess polar flagella. In addition, flagellar gene expression in Pseudomonas is under a four-tiered regulatory circuit, whereas enteric bacteria express flagellar genes in a three-step manner. Here, we use knowledge of E. coli and Salmonella flagella to describe the general properties of flagella and then focus on the specificities of Pseudomonas flagella. After a description of flagellar structure, which is highly conserved among Gram-negative bacteria, we focus on the steps of flagellar assembly that differ between enteric and polar-flagellated bacteria. In addition, we summarize generalities concerning the fuel used for the production and rotation of the flagellar macromolecular complex. The last part summarizes known regulatory pathways and potential links with the type-six secretion system (T6SS).

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.

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.

Detoxification Response of Pseudomonas fluorescens MFAF76a to Gaseous Pollutants NO2 and NO.

Bacteria are often exposed to nitrosative stress from their environment, from atmospheric pollution or from the defense mechanisms of other organisms. Reactive nitrogen species (RNS), which mediate nitrosative stress, are notably involved in the mammalian immune response through the production of nitric oxide (NO) by the inducible NO synthase iNOS. RNS are highly reactive and can alter various biomolecules such as lipids, proteins and DNA, making them toxic for biological organisms. Resistance to RNS is therefore important for the survival of bacteria in various environments, and notably to successfully infect their host. The fuel combustion processes used in industries and transports are responsible for the emission of important quantities of two major RNS, NO and the more toxic nitrogen dioxide (NO2). Human exposure to NO2 is notably linked to increases in lung infections. While the response of bacteria to NO in liquid medium is well-studied, few data are available on their exposure to gaseous NO and NO2. This study showed that NO2 is much more toxic than NO at similar concentrations for the airborne bacterial strain Pseudomonas fluorescens MFAF76a. The response to NO2 involves a wide array of effectors, while the response to NO seemingly focuses on the Hmp flavohemoprotein. Results showed that NO2 induces the production of other RNS, unlike NO, which could explain the differences between the effects of these two molecules.

Bacterial Long-Range Warfare: Aerial Killing of Legionella pneumophila by Pseudomonas fluorescens.

Legionella pneumophila, the causative agent of Legionnaires' disease, is mostly found in man-made water systems and is one of the most closely monitored waterborne pathogens. With the aim of finding natural ways to control waterborne pathogens and thus further reduce the impact of disinfection by-products on human health, some studies have demonstrated the ability of bacteria to kill Legionella through the production of secondary metabolites or antimicrobial compounds. Here, we describe an unexpected growth inhibition of L. pneumophila when exposed to a physically separated strain of Pseudomonas fluorescens, designated as MFE01. Most of the members of the Legionellaceae family are sensitive to the volatile substances emitted by MFE01, unlike other bacteria tested. Using headspace solid-phase microextraction GC-MS strategy, a volatilome comparison revealed that emission of 1-undecene, 2-undecanone, and 2-tridecanone were mainly reduced in a Tn5-transposon mutant unable to inhibit at distance the growth of L. pneumophila strain Lens. We showed that 1-undecene was mainly responsible for the inhibition at distance in vitro, and led to cell lysis in small amounts, as determined by gas chromatography-mass spectrometry (GC-MS). Collectively, our results provide new insights into the mode of action of bacterial volatiles and highlight them as potent anti-Legionella agents to focus research on novel strategies to fight legionellosis. IMPORTANCE Microbial volatile compounds are molecules whose activities are increasingly attracting the attention of researchers. Indeed, they can act as key compounds in long-distance intrakingdom and interkingdom communication, but also as antimicrobials in competition and predation. In fact, most studies to date have focused on their antifungal activities and only a few have reported on their antibacterial properties. Here, we describe that 1-undecene, naturally produced by P. fluorescens, is a volatile with potent activity against bacteria of the genus Legionella. In small amounts, it is capable of inducing cell lysis even when the producing strain is physically separated from the target. This is the first time that such activity is described. This molecule could therefore constitute an efficient compound to counter bacterial pathogens whose treatment may fail, particularly in pulmonary diseases. Indeed, inhalation of these volatiles should be considered as a possible route of therapy in addition to antibiotic treatment.

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.

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.

The Type VI Secretion System: A Dynamic System for Bacterial Communication?

Numerous studies in Gram-negative bacteria have focused on the Type VI Secretion Systems (T6SSs), Quorum Sensing (QS), and social behavior, such as in biofilms. These interconnected mechanisms are important for bacterial survival; T6SSs allow bacteria to battle other cells, QS is devoted to the perception of bacterial cell density, and biofilm formation is essentially controlled by QS. Here, we review data concerning T6SS dynamics and T6SS-QS cross-talk that suggest the existence of inter-bacterial communication via T6SSs.