Two Different rpf Clusters Distributed among a Population of Stenotrophomonas maltophilia Clinical Strains Display Differential Diffusible Signal Factor Production and Virulence Regulation

The quorum-sensing (QS) system present in the emerging nosocomial pathogen Stenotrophomonas maltophilia is based on the signaling molecule diffusible signal factor (DSF). Production and detection of DSF are governed by the rpf cluster, which encodes the synthase RpfF and the sensor RpfC, among other components. Despite a well-studied system, little is known about its implication in virulence regulation in S. maltophilia. Here, we have analyzed the rpfF gene from 82 S. maltophilia clinical isolates. Although rpfF was found to be present in all of the strains, it showed substantial variation, with two populations (rpfF-1 and rpfF-2) clearly distinguishable by the N-terminal region of the protein. Analysis of rpfC in seven complete genome sequences revealed a corresponding variability in the N-terminal transmembrane domain of its product, suggesting that each RpfF variant has an associated RpfC variant. We show that only RpfC–RpfF-1 variant strains display detectable DSF production. Heterologous rpfF complementation of ΔrpfF mutants of a representative strain of each variant suggests that RpfF-2 is, however, functional and that the observed DSF-deficient phenotype of RpfC–RpfF-2 variant strains is due to permanent repression of RpfF-2 by RpfC-2. This is corroborated by the ΔrpfC mutant of the RpfC–RpfF-2 representative strain. In line with this observations, deletion of rpfF from the RpfC–RpfF-1 strain leads to an increase in biofilm formation, a decrease in swarming motility, and relative attenuation in the Caenorhabditis elegans and zebrafish infection models, whereas deletion of the same gene from the representative RpfC–RpfF-2 strain has no significant effect on these virulence-related phenotypes.     

Xanthomonas campestris RpfB is a fatty Acyl‐CoA ligase required to counteract the thioesterase activity of the RpfF diffusible signal factor (DSF) synthase

In Xanthomonas campestris pv. campestris (Xcc), the proteins encoded by the rpf (regulator of pathogenicity factor) gene cluster produce and sense a fatty acid signal molecule called diffusible signalling factor (DSF, 2(Z)‐11‐methyldodecenoic acid). RpfB was reported to be involved in DSF processing and was predicted to encode an acyl‐CoA ligase. We report that RpfB activates a wide range of fatty acids to their CoA esters in vitro. Moreover, RpfB can functionally replace the paradigm bacterial acyl‐CoA ligase, Escherichia coli FadD, in the E. coli ß‐oxidation pathway and deletion of RpfB from the Xcc genome results in a strain unable to utilize fatty acids as carbon sources. An essential RpfB function in the pathogenicity factor pathway was demonstrated by the properties of a strain deleted for both the rpfB and rpfC genes. The ΔrpfB ΔrpfC strain grew poorly and lysed upon entering stationary phase. Deletion of rpfF, the gene encoding the DSF synthetic enzyme, restored normal growth to this strain. RpfF is a dual function enzyme that synthesizes DSF by dehydration of a 3‐hydroxyacyl‐acyl carrier protein (ACP) fatty acid synthetic intermediate and also cleaves the thioester bond linking DSF to ACP. However, the RpfF thioesterase activity is of broad specificity and upon elimination of its RpfC inhibitor RpfF attains maximal activity and its thioesterase activity proceeds to block membrane lipid synthesis by cleavage of acyl‐ACP intermediates. This resulted in release of the nascent acyl chains to the medium as free fatty acids. This lack of acyl chains for phospholipid synthesis results in cell lysis unless RpfB is present to counteract the RpfF thioesterase activity by catalysing uptake and activation of the free fatty acids to give acyl‐CoAs that can be utilized to restore membrane lipid synthesis. Heterologous expression of a different fatty acid activating enzyme, the Vibrio harveyi acyl‐ACP synthetase, replaced RpfB in counteracting the effects of high level RpfF thioesterase activity indicating that the essential role of RpfB is uptake and activation of free fatty acids.     

Diffusible Signal Factor (DSF) Synthase RpfF of Xylella fastidiosa Is a Multifunction Protein Also Required for Response to DSF

Xylella fastidiosa, like related Xanthomonas species, employs an Rpf cell-cell communication system consisting of a diffusible signal factor (DSF) synthase, RpfF, and a DSF sensor, RpfC, to coordinate expression of virulence genes. While phenotypes of a ΔrpfF strain in Xanthomonas campestris could be complemented by its own DSF, the DSF produced by X. fastidiosa (XfDSF) did not restore expression of the XfDSF-dependent genes hxfA and hxfB to a ΔrpfF strain of X. fastidiosa, suggesting that RpfF is involved in XfDSF sensing or XfDSF-dependent signaling. To test this conjecture, rpfC and rpfF of X. campestris were replaced by those of X. fastidiosa, and the contribution of each gene to the induction of a X. campestris DSF-dependent gene was assessed. As in X. fastidiosa, XfDSF-dependent signaling required both X. fastidiosa proteins RpfF and RpfC. RpfF repressed RpfC signaling activity, which in turn was derepressed by XfDSF. A mutated X. fastidiosa RpfF protein with two substitutions of glutamate to alanine in its active site was incapable of XfDSF production yet enabled a response to XfDSF, indicating that XfDSF production and the response to XfDSF are two separate functions in which RpfF is involved. This mutant was also hypervirulent to grape, demonstrating the antivirulence effects of XfDSF itself in X. fastidiosa. The Rpf system of X. fastidiosa is thus a novel example of a quorum-sensing signal synthase that is also involved in the response to the signal molecule that it synthesizes.     

Ralfuranones contribute to mushroom‐type biofilm formation by Ralstonia solanacearum strain OE1‐1

After invasion into intercellular spaces of tomato plants, the soil‐borne, plant‐pathogenic Ralstonia solanacearum strain OE1‐1 forms mushroom‐shaped biofilms (mushroom‐type biofilms, mBFs) on tomato cells, leading to its virulence. The strain OE1‐1 produces aryl‐furanone secondary metabolites, ralfuranones (A, B, J, K and L), dependent on the quorum sensing (QS) system, with methyl 3‐hydroxymyristate (3‐OH MAME) synthesized by PhcB as a QS signal. Ralfuranones are associated with the feedback loop of the QS system. A ralfuranone productivity‐deficient mutant (ΔralA) exhibited significantly reduced growth in intercellular spaces compared with strain OE1‐1, losing its virulence. To analyse the function of ralfuranones in mBF formation by OE1‐1 cells, we observed cell aggregates of R. solanacearum strains statically incubated in tomato apoplast fluids on filters under a scanning electron microscope. The ΔralA strain formed significantly fewer microcolonies and mBFs than strain OE1‐1. Supplementation of ralfuranones A, B, J and K, but not L, significantly enhanced the development of mBF formation by ΔralA. Furthermore, a phcB‐ and ralA‐deleted mutant (ΔphcB/ralA) exhibited less formation of mBFs than OE1‐1, although a QS‐deficient, phcB‐deleted mutant formed mBFs similar to OE1‐1. Supplementation with 3‐OH MAME significantly reduced the formation of mBFs by ΔphcB/ralA. The application of each ralfuranone significantly increased the formation of mBFs by ΔphcB/ralA supplied with 3‐OH MAME. Together, our findings indicate that ralfuranones are implicated not only in the development of mBFs by strain OE1‐1, but also in the suppression of QS‐mediated negative regulation of mBF formation.     

A quorum sensing‐defective mutant of Pectobacterium carotovorum ssp. brasiliense 1692 is attenuated in virulence and unable to occlude xylem tissue of susceptible potato plant stems

Pectobacterium carotovorum ssp. brasiliense 1692 (Pcb1692) is an important emerging pathogen of potatoes causing blackleg in the field and soft rot during post‐harvest storage. Blackleg diseases involve the bacterial colonization of vascular tissue and the formation of aggregates, also known as biofilms. To understand the role of quorum sensing in vascular colonization by Pcb1692, we generated a Pcb1692ΔexpI mutant strain. Inactivation of expI led to the reduced production of plant cell wall‐degrading enzymes (PCWDEs), the inability to produce acyl homoserine lactone (AHL) and reduced virulence in potato tubers and stems. Complementation of the mutant strain with the wild‐type expI gene in trans successfully restored AHL and PCWDE production as well as virulence. Transmission electron microscopy and in vitro motility assays demonstrated hyperpiliation and loss of flagella and swimming motility in the mutant strain compared with the wild‐type Pcb1692. Furthermore, we noted that, in the early stages of infection, Pcb1692 wild‐type cells had intact flagella which were shed at the later stages of infection. Confocal laser microscopy of PcbΔexpI‐inoculated plants showed that the mutant strain tended to aggregate in intercellular spaces, but was unable to transit to xylem tissue. On the contrary, the wild‐type strain was often observed forming aggregates within xylem tissue of potato stems. Gene expression analyses confirmed that flagella are part of the quorum sensing regulon, whereas fimbriae and pili appear to be negatively regulated by quorum sensing. The relative expression levels of other important putative virulence genes, such as those encoding different groups of PCWDEs, were down‐regulated in the mutant compared with the wild‐type strain.     

Contribution of quorum‐sensing system to hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1

Aims: To investigate roles of quorum‐sensing (QS) system in Acinetobacter sp. strain DR1 and rifampicin‐resistant variant (hereinafter DR1R). Methods and Results: The DR1 strain generated three putative acyl homoserine lactones (AHLs), while the DR1R produced only one signal and QS signal production was abrogated in the aqsI (LuxI homolog) mutant. The hexadecane‐degradation and biofilm‐formation capabilities of DR1, DR1R, and aqsI mutants were compared, along with their proteomic data. Proteomics analysis revealed that the AHL lactonase responsible for degrading QS signal was highly upregulated in both DR1R and aqsI mutant, also showed that several proteins, including ppGpp synthase, histidine kinase sensors, might be under the control of QS signalling. Interestingly, biofilm‐formation and hexadecane‐biodegradation abilities were reduced more profoundly in the aqsI mutant. These altered phenotypes of the aqsI mutant were restored via the addition of free wild‐type cell supernatant and exogenous C₁₂‐AHL. Conclusions: The QS system in strain DR1 contributes to hexadecane degradation and biofilm formation. Significance and Impact of the Study: This is the first report to demonstrate that a specific QS signal appears to be a critical factor for hexadecane degradation and biofilm formation in Acinetobacter sp. strain DR1.     

Intercellular and intracellular signalling systems that globally control the expression of virulence genes in plant pathogenic bacteria

Plant pathogenic bacteria utilize complex signalling systems to control the expression of virulence genes at the cellular level and within populations. Quorum sensing (QS), an important intercellular communication mechanism, is mediated by different types of small molecules, including N‐acyl homoserine lactones (AHLs), fatty acids and small proteins. AHL‐mediated signalling systems dependent on the LuxI and LuxR family proteins play critical roles in the virulence of a wide range of Gram‐negative plant pathogenic bacteria belonging to the Alphaproteobacteria, Betaproteobacteria and Gammaproteobacteria. Xanthomonas spp. and Xylella fastidiosa, members of the Gammaproteobacteria, however, possess QS systems that are mediated by fatty acid‐type diffusible signal factors (DSFs). Recent studies have demonstrated that Ax21, a 194‐amino‐acid protein in Xanthomonas oryzae pv. oryzae, plays dual functions in activating a rice innate immune pathway through binding to the rice XA21 pattern recognition receptor and in regulating bacterial virulence and biofilm formation as a QS signal molecule. In xanthomonads, DSF‐mediated QS systems are connected with the signalling pathways mediated by cyclic diguanosine monophosphate (c‐di‐GMP), which functions as a second messenger for the control of virulence gene expression in these bacterial pathogens.     

The diffusible signal factor synthase,RpfF, in Xanthomonas oryzae pv. oryzae is required for the maintenance of membrane integrity and virulence

The Xanthomonas group of phytopathogens communicate with a fatty acid‐like cell–cell signalling molecule, cis‐11‐2‐methyl‐dodecenoic acid, also known as diffusible signal factor (DSF). In the pathogen of rice, Xanthomonas oryzae pv. oryzae, DSF is involved in the regulation of several virulence‐associated functions, including production and secretion of several cell wall hydrolysing type II secretion effectors. To understand the role of DSF in the secretion of type II effectors, we characterized DSF synthase‐deficient (rpfF) and DSF‐deficient, type II secretion (xpsE) double mutants. Mutant analysis by expression analysis, secretion assay, fatty acid analysis, and physiological studies indicated that rpfF mutants exhibit hypersecretion of several type II effectors due to a perturbed membrane and DSF is required for maintaining membrane integrity. The rpfF mutants exhibited significantly higher uptake of 1‐N‐phenylnapthylamine and ethidium bromide, and up‐regulation of r poE (σ^E). Increasing the osmolarity of the medium could rescue the hypersecretion phenotype of the rpfF mutant. The rpfF mutant exhibited highly reduced virulence. We report for the first time that in X. oryzae pv. oryzae RpfF is involved in the maintenance of membrane integrity by playing a regulatory role in the fatty acid synthesis pathway.     

The acyl-homoserine lactone (AHL)-type quorum sensing system affects growth rate,swimming motility and virulence in <Emphasis Type="Italic">Acidovorax avenae</Emphasis> subsp. <Emphasis Type="Italic">citrulli</Emphasis>

Acidovorax avenae subsp. citrulli is a Gram-negative bacterium and is the causal agent of bacterial fruit blotch (BFB) in cucurbits. In this study, the role played by the acyl-homoserine lactone (AHL)-type quorum sensing (QS) system in growth, swimming motility and virulence was characterized in A. avenae subsp. citrulli strain XJL12. The AHL synthase gene of the QS system from strain XJL12, defined as aacI, was cloned and characterized, and an aacI disruption mutant was generated. The aacI mutant XJL13 abolished the ability to produce AHL molecules, whereas the corresponding complemented strain CPXJL13 produced wild-type levels of AHL. The aacI mutant exhibited a significant decrease in growth rate relative to the wild type in minimal medium, and was partially impaired in swimming motility. In plants, the aacI mutant showed a significant reduction of virulence in watermelon fruits and melon seedlings when compared to the wild-type strain. However, the aacI mutation in strain XJL12 had no effects on biofilm formation, exopolysaccharide production, or induction of hypersentitive response in Nicotiana tabacum. Our data suggest that the AHL-type QS may play a key role in pathogen virulence and this may provide an opportunity to explore novel approaches for managing BFB in cucurbits by QS interference.     

Plant‐like bacterial expansins play contrasting roles in two tomato vascular pathogens

Expansin proteins, which loosen plant cell walls, play critical roles in normal plant growth and development. The horizontal acquisition of functional plant‐like expansin genes in numerous xylem‐colonizing phytopathogenic bacteria suggests that bacterial expansins may also contribute to virulence. To investigate the role of bacterial expansins in plant diseases, we mutated the non‐chimeric expansin genes (CmEXLX2 and RsEXLX) of two xylem‐inhabiting bacterial pathogens, the Actinobacterium Clavibacter michiganensis ssp. michiganensis (Cmm) and the β‐proteobacterium Ralstonia solanacearum (Rs), respectively. The Cmm Δ CmEXLX2 mutant caused increased symptom development on tomato, which was characterized by more rapid wilting, greater vascular necrosis and abundant atypical lesions on distant petioles. This increased disease severity correlated with larger in planta populations of the Δ CmEXLX2 mutant, even though the strains grew as well as the wild‐type in vitro. Similarly, when inoculated onto tomato fruit, Δ CmEXLX2 caused significantly larger lesions with larger necrotic centres. In contrast, the Rs Δ RsEXLX mutant showed reduced virulence on tomato following root inoculation, but not following direct petiole inoculation, suggesting that the RsEXLX expansin contributes to early virulence at the root infection stage. Consistent with this finding, Δ RsEXLX attached to tomato seedling roots better than the wild‐type Rs, which may prevent mutants from invading the plant's vasculature. These contrasting results demonstrate the diverse roles of non‐chimeric bacterial expansins and highlight their importance in plant–bacterial interactions.     

Rice bacterial blight pathogen <Emphasis Type="Italic">Xanthomonas oryzae</Emphasis> pv. <Emphasis Type="Italic">oryzae</Emphasis> produces multiple DSF-family signals in regulation of virulence factor production

Background  

Xanthomonas oryzae pv. oryzae (Xoo) is the causal agent of rice bacterial blight disease. Xoo produces a range of virulence factors, including EPS, extracellular enzyme, iron-chelating siderophores, and type III-secretion dependent effectors, which are collectively essential for virulence. Genetic and genomics evidence suggest that Xoo might use the diffusible signal factor (DSF) type quorum sensing (QS) system to regulate the virulence factor production. However, little is known about the chemical structure of the DSF-like signal(s) produced by Xoo and the factors influencing the signal production.