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.     

应用竞争力指数分析III型效应子SKWP对青枯菌在寄主植物体内增殖能力的影响

[目的]研究Ⅲ型效应子SKWP对青枯菌OE1-1在寄主植物体内增殖能力的影响。[方法]构建青枯菌RK7197（野生型突变体,带Gm抗性）和SKWP单基因缺失突变体（带PB抗性）,通过竞争力指数分析SKWP各效应子对青枯菌OE1-1在叶片组织内增殖能力的影响。[结果]竞争力指数适合在寄主植物茄子上分析各效应子功能,6个SKWP效应子对OE1-1细菌增殖能力影响不同,SKWP4影响最明显。[结论]竞争力指数可提供一个新视野来分析SKWP各效应子对青枯菌OE1-1在寄主茄子上增殖能力的影响。     

An Amino Acid Substitution at Position 740 in σ70 of Ralstonia solanacearum Strain OE1-1 Affects Its In Planta Growth

Growth of Ralstonia solanacearum strain OE1-1 in roots after invasion is required for virulence. An Arg740Cys substitution in σ⁷⁰ of OE1-1 resulted in loss of in planta growth and virulence. The negative dominance of mutant σ⁷⁰ over the wild-type protein suggested that the amino acid substitution may affect the in planta growth of OE1-1, leading to a lack of virulence.     

The vascular plant‐pathogenic bacterium Ralstonia solanacearum produces biofilms required for its virulence on the surfaces of tomato cells adjacent to intercellular spaces

The mechanism of colonization of intercellular spaces by the soil‐borne and vascular plant‐pathogenic bacterium Ralstonia solanacearum strain OE1‐1 after invasion into host plants remains unclear. To analyse the behaviour of OE1‐1 cells in intercellular spaces, tomato leaves with the lower epidermis layers excised after infiltration with OE1‐1 were observed under a scanning electron microscope. OE1‐1 cells formed microcolonies on the surfaces of tomato cells adjacent to intercellular spaces, and then aggregated surrounded by an extracellular matrix, forming mature biofilm structures. Furthermore, OE1‐1 cells produced mushroom‐type biofilms when incubated in fluids of apoplasts including intercellular spaces, but not xylem fluids from tomato plants. This is the first report of biofilm formation by R. solanacearum on host plant cells after invasion into intercellular spaces and mushroom‐type biofilms produced by R. solanacearum in vitro. Sugar application led to enhanced biofilm formation by OE1‐1. Mutation of lecM encoding a lectin, RS‐IIL, which reportedly exhibits affinity for these sugars, led to a significant decrease in biofilm formation. Colonization in intercellular spaces was significantly decreased in the lecM mutant, leading to a loss of virulence on tomato plants. Complementation of the lecM mutant with native lecM resulted in the recovery of mushroom‐type biofilms and virulence on tomato plants. Together, our findings indicate that OE1‐1 produces mature biofilms on the surfaces of tomato cells after invasion into intercellular spaces. RS‐IIL may contribute to biofilm formation by OE1‐1, which is required for OE1‐1 virulence.     

A phcA− marker-free mutant of Ralstonia solanacearum as potential biocontrol agent of tomato bacterial wilt

A phcA⁻ mutant of Ralstonia solanacearum strain ZJ3721 was created in a marker-free method. Expression of virulence-associated genes such as xpsR, egl, tek and epsE was significantly suppressed in the phcA⁻ mutant. The ability of the mutant to control tomato bacterial wilt was evaluated by potting experiment. Results showed that application of mutant with wild type (WT) at the same time only delayed the development of wilt for about one day and the population of WT in tomato rhizosphere soil was nearly 70-fold higher than that of the mutant, resulting in a 90% disease incidence at last, as high as that of control. If the phcA⁻ mutant was applied three days earlier than WT pathogen, tomato wilt disease incidence was only 6%, 80% lower than that of control and population of WT was about 0.5-fold as much as that of mutant. Under hydroponic conditions, phcA⁻ mutant significantly triggered the expressions of genes in salicylic acid pathway but inhibited the expressions of genes in jasmonic acid (JA) and ethylene (ET) pathways. The expressions of PR-1a and GluA genes (salicylic acid pathway) in phcA⁻ mutant were 66-fold and 7.5-fold higher than in WT pathogen after three days of inoculation.     

Ⅲ型效应子GALA对青枯菌在两种寄主植物上致病性的影响

[目的]研究Ⅲ型效应子GALAs对青枯菌OE1-1在不同寄主植物致病性上的影响。[方法]构建青枯菌OE1-1的多种GALA缺失突变体,通过根切和叶片注射等方法研究GALAs对青枯菌OE1-1致病力和细胞内增殖能力的影响。[结果]GALA多基因缺失突变体对寄主烟草的致病力减弱,在烟草体内细菌繁殖能力较野生型明显降低,但在寄主番茄上不影响其致病性。[结论]GALA效应子对青枯菌OE1-1在烟草植株致病性上展现协同作用。     

Studies on the biosynthesis of ralfuranones in Ralstonia solanacearum

Ralfuranones, aryl-furanone secondary metabolites, are involved in the virulence of Ralstonia solanacearum in solanaceous plants. Ralfuranone I (6) has been suggested as a biosynthetic precursor for other ralfuranones; however, this conversion has not been confirmed. We herein investigate the biosynthesis of ralfuranones using feeding experiments with ralfuranone I (6) and its putative metabolite, ralfuranone B (2). The results obtained demonstrated that the biosynthesis of ralfuranones proceeded in enzymatic and non-enzymatic manners.     

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.     

Interplay between two quorum sensing-regulated pathways,violacein biosynthesis and VacJ/Yrb,dictates outer membrane vesicle biogenesis in Chromobacterium violaceum

Outer membrane vesicles (OMVs) are lipid nanoparticles released by Gram-negative bacteria, which play multiple roles in bacterial physiology and adaptation to diverse environments. In this work, we demonstrate that OMVs released by the environmental pathogen Chromobacterium violaceum deliver the antimicrobial compound violacein to competitor bacteria, mediating its toxicity in vivo at a long distance. OMVs purified by ultracentrifugation from the wild-type strain, but not from a violacein-abrogated mutant ΔvioABCDE, contained violacein and inhibited several Gram-positive bacteria. Competition tests using co-culture and transwell assays indicated that the C. violaceum wild-type strain killed Staphylococcus aureus better than the ΔvioABCDE mutant strain. We found that C. violaceum achieves growth phase-dependent OMV release by the concerted expression of two quorum sensing (QS)-regulated pathways, namely violacein biosynthesis and VacJ/Yrb system. Although both pathways were activated at high cell density in a QS-dependent manner, the effect on vesiculation was the opposite. While the ΔvioABCDE mutant produced twofold fewer vesicles than the wild-type strain, indicating that violacein induces OMV biogenesis for its own delivery, the ΔvacJ and ΔyrbE mutants were hypervesiculating strains. Our findings uncovered QS-regulated pathways involved in OMV biogenesis used by C. violaceum to package violacein into OMVs for interbacterial competition.     

Bacterial quorum sensing in symbiotic and pathogenic relationships with hosts*

Gram-negative bacteria communicate with each other by producing and sensing diffusible signaling molecules. This mechanism is called quorum sensing (QS) and regulates many bacterial activities from gene expression to symbiotic/pathogenic interactions with hosts. Therefore, the elucidation and control of bacterial QS systems have been attracted increasing attention over the past two decades. The most common QS signals in Gram-negative bacteria are N-acyl homoserine lactones (AHLs). There are also bacteria that employ different QS systems, for example, the plant pathogen Ralstonia solanacearum utilizes 3-hydroxy fatty acid methyl esters as its QS signals. The QS system found in the endosymbiotic bacterium associated with the fungus Mortierella alpina, the development of an affinity pull-down method for AHL synthases, and the elucidation of a unique QS circuit in R. solanacearum are discussed herein.