Secondary-structure switch regulates the substrate binding of a YopJ family acetyltransferase.

The Yersinia outer protein J (YopJ) family effectors are widely deployed through the type III secretion system by both plant and animal pathogens. As non-canonical acetyltransferases, the enzymatic activities of YopJ family effectors are allosterically activated by the eukaryote-specific ligand inositol hexaphosphate (InsP6). However, the underpinning molecular mechanism remains undefined. Here we present the crystal structure of apo-PopP2, a YopJ family member secreted by the plant pathogen Ralstonia solanacearum. Structural comparison of apo-PopP2 with the InsP6-bound PopP2 reveals a substantial conformational readjustment centered in the substrate-binding site. Combining biochemical and computational analyses, we further identify a mechanism by which the association of InsP6 with PopP2 induces an α-helix-to-ß-strand transition in the catalytic core, resulting in stabilization of the substrate recognition helix in the target protein binding site. Together, our study uncovers the molecular basis governing InsP6-mediated allosteric regulation of YopJ family acetyltransferases and further expands the paradigm of fold-switching proteins.

Genome- and Mass Spectrometry-Guided Discovery of Ralstoamides A and B from Ralstonia solanacearum Species Complex.

Strains of Ralstonia solanacearum species complex (RSSC) are devastating plant pathogens distributed globally with a wide host range and genetic diversity. Many RSSC strains harbor the polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) hybrid gene rmyA/rmyB for ralstonin production. We report that ralstoamides A (1) and B (2), which are ralstonin-like but shorter lipopeptides, were discovered from the Japanese strains using accumulated RSSC genome data and LC/MS-based metabolite analysis. Their structures, including absolute configurations, were elucidated by spectroscopic analysis and chemical techniques. ramA, a PKS-NRPS gene for ralstoamide production, was identified from the producer strains by genome sequencing and gene-deletion experiments. Based on the analysis of biosynthetic genes of ralstoamides and ralstonins, we suggest the occurrence of NRPS-module reduction of rmyA/rmyB genes in some RSSC strains. This possible molecular evolution changed not only the structures, but also the biological activity of RSSC lipopeptides.

Antibacterial activity of medicinal plant extracts against Ralstonia solanacearum (Smith) that causes bacterial wilt in hot pepper (Capsicum annuum L. )

This study was conducted to evaluate the in vitro antibacterial activity of some medicinal plants against Ralstonia solanacearum. Bioactive chemicals were extracted from Burcea antidysenterica, Eucalyptus citriodora, Justicia schimperiana, Lantana camara, Melia azedarach and Ricinus communis leaves using maceration method. The bioassay was evaluated by disc diffusion method. The pathogen was isolated from infected Capsicum annuum plants using Casamino acid-Peptone-Glucose agar (CPG) medium. The isolate was identified using cultural, biochemical characteristics, pathogenicity test and found to be R. solanacearum. The methanol extracts had different composition, percentage extract yield, antibacterial activity and relative percentage inhibition. Unlike others, extracts of E. citriodora and R. communis consisted of all the tested secondary metabolites. All species showed antibacterial activity except M. azedarach. Significant differences were recorded in antibacterial activity among species and test concentrations. The highest antibacterial activity and the lowest bacteriostatic and bactericidal concentrations were found from E. citriodora and R. communis extracts. The higher potency of E. citriodora and R. communis extracts suggested the potential of the two species as a biocide to control bacterial wilt. However, further in vivo studies on these species extracts are compulsory.

Ralstonia solanacearum novel E3 ubiquitin ligase (NEL) effectors RipAW and RipAR suppress pattern-triggered immunity in plants.

Ralstonia solanacearum is the causal agent of bacterial wilt in solanaceous crops. This pathogen injects more than 70 effector proteins into host plant cells via the Hrp type III secretion system to cause a successful infection. However, the function of these effectors in plant cells, especially in the suppression of plant immunity, remains largely unknown. In this study, we characterized two Ralstonia solanacearum effectors, RipAW and RipAR, which share homology with the IpaH family of effectors from animal and plant pathogenic bacteria, that have a novel E3 ubiquitin ligase (NEL) domain. Recombinant RipAW and RipAR show E3 ubiquitin ligase activity in vitro. RipAW and RipAR localized to the cytoplasm of plant cells and significantly suppressed pattern-triggered immunity (PTI) responses such as the production of reactive oxygen species and the expression of defence-related genes when expressed in leaves of Nicotiana benthamiana. Mutation in the conserved cysteine residue in the NEL domain of RipAW completely abolished the E3 ubiquitin ligase activity in vitro and the ability to suppress PTI responses in plant leaves. These results indicate that RipAW suppresses plant PTI responses through the E3 ubiquitin ligase activity. Unlike other members of the IpaH family of effectors, RipAW and RipAR had no leucine-rich repeat motifs in their amino acid sequences. A conserved C-terminal region of RipAW is indispensable for PTI suppression. Transgenic Arabidopsis plants expressing RipAW and RipAR showed increased disease susceptibility, suggesting that RipAW and RipAR contribute to bacterial virulence in plants.

Host and non-host plant response to bacterial wilt in potato: role of the lipopolysaccharide isolated from Ralstonia solanacearum and molecular analysis of plant-pathogen interaction.

Ralstonia solanacearum is one of the most devastating phytopathogenic bacteria, in particular its race 3. This microorganism is the causal agent of destructive diseases of different crops including tomato and potato. An important aspect of the interaction between this pathogen, and the host and non-host plants was its biochemical and molecular basis. Thus, the lipopolysaccharides (LPS) were extracted from the R. solanacearum cell wall, purified, and the O-specific polysaccharide (OPS) was isolated and chemically characterized by compositional analyses and NMR spectroscopy. The OPS was constituted of two linear polymers of an approximate ratio of 3 : 1, both of which were built up from three rhamnose and one N-acetylglucosamine residues and differed only in the substitution of one rhamnose residue. The LPS inhibited the hypersensitivity reaction (HR) in non-host tobacco plants and induced localized resistance in host potato plants, both of which were pre-treated with the LPS before being inoculated with the pathogen. A cDNA-AFLP approach was used to study transcriptome variation during the resistant and susceptible interactions. This revealed the presence of metabolites specifically expressed in the S. commersonii-resistant genotypes, which could be involved in the plant-pathogen incompatible reaction. Furthermore, a specific EST collection of the Ralstonia-potato interaction has been built up.

Engineering of PA-IIL lectin from Pseudomonas aeruginosa - Unravelling the role of the specificity loop for sugar preference.

BACKGROUND: Lectins are proteins of non-immune origin capable of binding saccharide structures with high specificity and affinity. Considering the high encoding capacity of oligosaccharides, this makes lectins important for adhesion and recognition. The present study is devoted to the PA-IIL lectin from Pseudomonas aeruginosa, an opportunistic human pathogen capable of causing lethal complications in cystic fibrosis patients. The lectin may play an important role in the process of virulence, recognizing specific saccharide structures and subsequently allowing the bacteria to adhere to the host cells. It displays high values of affinity towards monosaccharides, especially fucose--a feature caused by unusual binding mode, where two calcium ions participate in the interaction with saccharide. Investigating and understanding the nature of lectin-saccharide interactions holds a great potential of use in the field of drug design, namely the targeting and delivery of active compounds to the proper site of action. RESULTS: In vitro site-directed mutagenesis of the PA-IIL lectin yielded three single point mutants that were investigated both structurally (by X-ray crystallography) and functionally (by isothermal titration calorimetry). The mutated amino acids (22-23-24 triad) belong to the so-called specificity binding loop responsible for the monosaccharide specificity of the lectin. The mutation of the amino acids resulted in changes to the thermodynamic behaviour of the mutants and subsequently in their relative preference towards monosaccharides. Correlation of the measured data with X-ray structures provided the molecular basis for rationalizing the affinity changes. The mutations either prevent certain interactions to be formed or allow formation of new interactions--both of afore mentioned have strong effects on the saccharide preferences. CONCLUSION: Mutagenesis of amino acids forming the specificity binding loop allowed identification of one amino acid that is crucial for definition of the lectin sugar preference. Altering specificity loop amino acids causes changes in saccharide-binding preferences of lectins derived from PA-IIL, via creation or blocking possible binding interactions. This finding opens a gate towards protein engineering and subsequent protein design to refine the desired binding properties and preferences, an approach that could have strong potential for drug design.