Suppression of MYC transcription activators by the immune cofactor NPR1 fine-tunes plant immune responses.

Plants tailor immune responses to defend against pathogens with different lifestyles. In this process, antagonism between the immune hormones salicylic acid (SA) and jasmonic acid (JA) optimizes transcriptional signatures specifically to the attacker encountered. Antagonism is controlled by the transcription cofactor NPR1. The indispensable role of NPR1 in activating SA-responsive genes is well understood, but how it functions as a repressor of JA-responsive genes remains unclear. Here, we demonstrate that SA-induced NPR1 is recruited to JA-responsive promoter regions that are co-occupied by a JA-induced transcription complex consisting of the MYC2 activator and MED25 Mediator subunit. In the presence of SA, NPR1 physically associates with JA-induced MYC2 and inhibits transcriptional activation by disrupting its interaction with MED25. Importantly, NPR1-mediated inhibition of MYC2 is a major immune mechanism for suppressing pathogen virulence. Thus, NPR1 orchestrates the immune transcriptome not only by activating SA-responsive genes but also by acting as a corepressor of JA-responsive MYC2.

Mass spectral analysis of acetylated peptides: Implications in proteomics.

Sequence determination of peptides using mass spectrometry plays a crucial role in the bottom-up approaches for the identification of proteins. It is crucially important to minimise false detection and validate sequence of the peptides in order to correctly identify a protein. Chemical modification of peptides followed by mass spectrometry is an option for improving the spectral quality. In silico-derived tryptic peptides with different N-terminal amino acids were designed from human proteins and synthesized. The effect of acetylation on the fragmentation of peptides was studied. N-terminal acetylation of the tryptic peptides was shown to form b1-ions, improve the abundance and occurrence of b-ions. In some cases, the intensity and occurrence of some y-ions also varied. Thus, it is demonstrated that acetylation plays an important role in improving the de novo sequencing efficiency of the peptides. The acetylation method was extended to tryptic peptides generated from the proteome of an Antarctic bacterium Pseudomonas syringae Lz4W using the proteomics work flow and mass spectra of the peptides were analysed. Comparison of the MS/MS spectra of the acetylated and unacetylated peptides revealed that acetylation helped in improving the spectral quality and validated the peptide sequences. Using this method, 673 proteins of the 1070 proteins identified were validated.

Microbial production of lipid-protein vesicles using enveloped bacteriophage phi6.

BACKGROUND: Cystoviruses have a phospholipid envelope around their nucleocapsid. Such a feature is unique among bacterial viruses (i.e., bacteriophages) and the mechanisms of virion envelopment within a bacterial host are largely unknown. The cystovirus Pseudomonas phage phi6 has an envelope that harbors five viral membrane proteins and phospholipids derived from the cytoplasmic membrane of its Gram-negative host. The phi6 major envelope protein P9 and the non-structural protein P12 are essential for the envelopment of its virions. Co-expression of P9 and P12 in a Pseudomonas host results in the formation of intracellular vesicles that are potential intermediates in the phi6 virion assembly pathway. This study evaluated the minimum requirements for the formation of phi6-specific vesicles and the possibility to localize P9-tagged heterologous proteins into such structures in Escherichia coli. RESULTS: Using transmission electron microscopy, we detected membranous structures in the cytoplasm of E. coli cells expressing P9. The density of the P9-specific membrane fraction was lower (approximately 1.13 g/cm3 in sucrose) than the densities of the bacterial cytoplasmic and outer membrane fractions. A P9-GFP fusion protein was used to study the targeting of heterologous proteins into P9 vesicles. Production of the GFP-tagged P9 vesicles required P12, which protected the fusion protein against proteolytic cleavage. Isolated vesicles contained predominantly P9-GFP, suggesting selective incorporation of P9-tagged fusion proteins into the vesicles. CONCLUSIONS: Our results demonstrate that the phi6 major envelope protein P9 can trigger formation of cytoplasmic membrane structures in E. coli in the absence of any other viral protein. Intracellular membrane structures are rare in bacteria, thus making them ideal chasses for cell-based vesicle production. The possibility to locate heterologous proteins into the P9-lipid vesicles facilitates the production of vesicular structures with novel properties. Such products have potential use in biotechnology and biomedicine.

The influence of the essential oil extracted from hops on monolayers and bilayers imitating plant pathogen bacteria membranes.

Many plant-derived compounds possess antimicrobial, antioxidant and even anticancer activities. Therefore, they are considered as substances that can be used instead of synthetic compounds in various applications. In this work, the essential oil from hop cones was extracted and analyzed, and then its effects on model bacteria membranes were studied to verify whether the hop essential oils could be used as ecological pesticides. The experiments involved surface pressure-area measurements, penetration studies and Brewster angle microscopy (BAM) imaging of lipid monolayers as well as hydrodynamic diameter, zeta potential, steady-state fluorescence anisotropy and Cryo-Transmission Electron Microscopy (cryo-TEM) measurements of liposomes. Finally the bactericidal tests on plant pathogen bacteria Pseudomonas syringae pv. lachrymans PCM 1410 were performed. The obtained results showed that the components of the essential oils from hop cones incorporate into lipid monolayers and bilayers and alter their fluidity. However, the observed effect is determined by the system composition, its condensation and the oil concentration. Interestingly, at a given dose, the effect of the essential oil on membranes was found to stabilize. Moreover, BAM images proved that hop oil prevents the formation of a large fraction of a condensed phase at the interface. Both the studies on model membranes as well as the in vitro tests allow one to conclude that the hop essential oil could likely be considered as the candidate to be used in agriculture as a natural pesticide.

The spent culture supernatant of Pseudomonas syringae contains azelaic acid.

BACKGROUND: Pseudomonas syringae pv. actinidiae (PSA) is an emerging kiwifruit bacterial pathogen which since 2008 has caused considerable losses. No quorum sensing (QS) signaling molecule has yet been reported from PSA and the aim of this study was to identify possible intercellular signals produced by PSA. RESULTS: A secreted metabolome analysis resulted in the identification of 83 putative compounds, one of them was the nine carbon saturated dicarboxylic acid called azelaic acid. Azelaic acid, which is a nine-carbon (C9) saturated dicarboxylic acid, has been reported in plants as a mobile signal that primes systemic defenses. In addition, its structure,(which is associated with fatty acid biosynthesis) is similar to other known bacterial QS signals like the Diffusible Signal Facor (DSF). For these reason it could be acting as s signal molecule. Analytical and structural studies by NMR spectroscopy confirmed that in PSA spent supernatants azelaic acid was present. Quantification studies further revealed that 20 µg/L of were present and was also found in the spent supernatants of several other P. syringae pathovars. The RNAseq transcriptome study however did not determine whether azelaic acid could behave as a QS molecule. CONCLUSIONS: This study reports of the possible natural biosynthesis of azelaic acid by bacteria. The production of azelaic acid by P. syringae pathovars can be associated with plant-bacteria signaling.

Split Green Fluorescent Protein System to Visualize Effectors Delivered from Bacteria During Infection.

Bacteria, one of the most important causative agents of various plant diseases, secrete a set of effector proteins into the host plant cell to subvert the plant immune system. During infection cytoplasmic effectors are delivered to the host cytosol via a type III secretion system (T3SS). After delivery into the plant cell, the effector(s) targets the specific compartment(s) to modulate host cell processes for survival and replication of the pathogen. Although there has been some research on the subcellular localization of effector proteins in the host cells to understand their function in pathogenicity by using fluorescent proteins, investigation of the dynamics of effectors directly injected from bacteria has been challenging due to the incompatibility between the T3SS and fluorescent proteins. Here, we describe our recent method of an optimized split superfolder green fluorescent protein system (sfGFPOPT) to visualize the localization of effectors delivered via the bacterial T3SS in the host cell. The sfGFP11 (11th ß-strand of sfGFP)-tagged effector secreted through the T3SS can be assembled with a specific organelle targeted sfGFP1-10OPT (1-10th ß-strand of sfGFP) leading to fluorescence emission at the site. This protocol provides a procedure to visualize the reconstituted sfGFP fluorescence signal with an effector protein from Pseudomonas syringae in a particular organelle in the Arabidopsis and Nicotiana benthamiana plants.

Heterologous expression and antitumor activity analysis of syringolin from Pseudomonas syringae pv. syringae B728a.

BACKGROUND: Syringolin, synthesized by a mixed non-ribosomal peptide synthetase/polyketide synthetase in Pseudomonas syringae pv. syringae (Pss) B728a, is a novel eukaryotic proteasome inhibitor. Meanwhile, directly modifying large fragments in the PKS/NRPS gene cluster through traditional DNA engineering techniques is very difficult. In this study, we directly cloned the syl gene cluster from Pss B301D-R via Red/ET recombineering to effectively express syringolin in heterologous hosts. RESULTS: A 22 kb genomic fragment containing the sylA-sylE gene cluster was cloned into the pASK vector, and the obtained recombinant plasmid was transferred into Streptomyces coelicolor and Streptomyces lividans for the heterologous expression of syringolin. Transcriptional levels of recombinant syl gene in S. coelicolor M145 and S. lividans TK24 were evaluated via RT-PCR and the production of syringolin compounds was detected via LC-MS analysis. The extracts of the engineered bacteria showed cytotoxic activity to B16, 4T1, Meth-A, and HeLa tumor cells. It is noteworthy that the syringolin displayed anticancer activity against C57BL/6 mice with B16 murine melanoma tumor cells. Together, our results herein demonstrate the potential of syrinolin as effective antitumor agent that can treat various cancers without apparent adverse effects. CONCLUSIONS: This present study is the first to report the heterologous expression of the entire syl gene cluster in Streptomyces strains and the successful expression of syringolin in both S. coelicolor M145 and S. lividans TK24. Syringolin derivatives demonstrated high cytotoxicity in vitro and in vivo. Hence, this paper provided an important foundation for the discovery and production of new antitumor compounds.

Structures and Mechanisms of the Non-Heme Fe(II)- and 2-Oxoglutarate-Dependent Ethylene-Forming Enzyme: Substrate Binding Creates a Twist.

The ethylene-forming enzyme (EFE) from Pseudomonas syringae pv. phaseolicola PK2 is a member of the mononuclear nonheme Fe(II)- and 2-oxoglutarate (2OG)-dependent oxygenase superfamily. EFE converts 2OG into ethylene plus three CO2 molecules while also catalyzing the C5 hydroxylation of l-arginine (l-Arg) driven by the oxidative decarboxylation of 2OG to form succinate and CO2. Here we report 11 X-ray crystal structures of EFE that provide insight into the mechanisms of these two reactions. Binding of 2OG in the absence of l-Arg resulted in predominantly monodentate metal coordination, distinct from the typical bidentate metal-binding species observed in other family members. Subsequent addition of l-Arg resulted in compression of the active site, a conformational change of the carboxylate side chain metal ligand to allow for hydrogen bonding with the substrate, and creation of a twisted peptide bond involving this carboxylate and the following tyrosine residue. A reconfiguration of 2OG achieves bidentate metal coordination. The dioxygen binding site is located on the metal face opposite to that facing l-Arg, thus requiring reorientation of the generated ferryl species to catalyze l-Arg hydroxylation. Notably, a phenylalanyl side chain pointing toward the metal may hinder such a ferryl flip and promote ethylene formation. Extensive site-directed mutagenesis studies supported the importance of this phenylalanine and confirmed the essential residues used for substrate binding and catalysis. The structural and functional characterization described here suggests that conversion of 2OG to ethylene, atypical among Fe(II)/2OG oxygenases, is facilitated by the binding of l-Arg which leads to an altered positioning of the carboxylate metal ligand, a resulting twisted peptide bond, and the off-line geometry for dioxygen coordination.

Bacterial Ice Nucleation in Monodisperse D2O and H2O-in-Oil Emulsions.

Ice nucleation is of fundamental significance in many areas, including atmospheric science, food technology, and cryobiology. In this study, we investigated the ice-nucleation characteristics of picoliter-sized drops consisting of different D2O and H2O mixtures with and without the ice-nucleating bacteria Pseudomonas syringae. We also studied the effects of commonly used cryoprotectants such as ethylene glycol, propylene glycol, and trehalose on the nucleation characteristics of D2O and H2O mixtures. The results show that the median freezing temperature of the suspension containing 1 mg/mL of a lyophilized preparation of P. syringae is as high as -4.6 °C for 100% D2O, compared to -8.9 °C for 100% H2O. As the D2O concentration increases every 25% (v/v), the profile of the ice-nucleation kinetics of D2O + H2O mixtures containing 1 mg/mL Snomax shifts by about 1 °C, suggesting an ideal mixing behavior of D2O and H2O. Furthermore, all of the cryoprotectants investigated in this study are found to depress the freezing phenomenon. Both the homogeneous and heterogeneous freezing temperatures of these aqueous solutions depend on the water activity and are independent of the nature of the solute. These findings enrich our fundamental knowledge of D2O-related ice nucleation and suggest that the combination of D2O and ice-nucleating agents could be a potential self-ice-nucleating formulation. The implications of self-nucleation include a higher, precisely controlled ice seeding temperature for slow freezing that would significantly improve the viability of many ice-assisted cryopreservation protocols.

The Hexapeptide Repeated Segment LIAGY is a Hot Spot of Aggregation of the Pseudomonas syringae Ice Nucleation Protein.

Ice nucleation proteins (INPs) form oligomeric structures by self-assembly and aggregation. We looked for the presence of potential aggregating sequences inside the INP from Pseudomonas syringae by a computational approach with the AGGRESCAN, FOMDAMYLOID and TANGO softwares. A total of 38 hot spots of aggregation were predicted in the INP sequence: 7 localized in the Nterminal domain, 2 in the C-terminal region, 28 in the highly repetitive central (HRC) region and 1 shared between the HRC and the Carboxyl-terminus regions of the protein. All the hot spots of aggregation identified in the HRC domain overlapped a 8-residue low fidelity repeat including a LIAGYrelated sequence. We confirmed the predictions by an experimental approach using synthetic peptides corresponding to different parts of the INP central sequence, absorbance spectroscopy and fluorescence spectroscopy in the presence of Congo red (CR) or Thioflavin T (ThT), respectively. Peptide 620-SFIIAGYG-627 predicted to aggregate by the three softwares induced an increase in fluorescence of ThT. Peptide 729-GFKSILTAGY-738 predicted to aggregate by AGGRESCAN and FOLDAMYLOID induced a shift in the maximum of absorbance of CR. Peptide 1124-SVLTAGA-1130 predicted to aggregate only by TANGO did not interfere with CR absorbance or ThT fluorescence. In conclusion, the use of three aggregation prediction algorithms and two biochemical assays showed that the hexapeptide repeated segment LIAGY, previously shown to form a hairpin loop may be involved in the aggregation of the P. syringae INP.

A lectin S-domain receptor kinase mediates lipopolysaccharide sensing in Arabidopsis thaliana.

The sensing of microbe-associated molecular patterns (MAMPs) triggers innate immunity in animals and plants. Lipopolysaccharide (LPS) from Gram-negative bacteria is a potent MAMP for mammals, with the lipid A moiety activating proinflammatory responses via Toll-like receptor 4 (TLR4). Here we found that the plant Arabidopsis thaliana specifically sensed LPS of Pseudomonas and Xanthomonas. We isolated LPS-insensitive mutants defective in the bulb-type lectin S-domain-1 receptor-like kinase LORE (SD1-29), which were hypersusceptible to infection with Pseudomonas syringae. Targeted chemical degradation of LPS from Pseudomonas species suggested that LORE detected mainly the lipid A moiety of LPS. LORE conferred sensitivity to LPS onto tobacco after transient expression, which demonstrated a key function in LPS sensing and indicated the possibility of engineering resistance to bacteria in crop species.

Determination of the self-association residues within a homomeric and a heteromeric AAA+ enhancer binding protein.

The σ(54)-dependent transcription in bacteria requires specific activator proteins, bacterial enhancer binding protein (bEBP), members of the AAA+ (ATPases Associated with various cellular Activities) protein family. The bEBPs usually form oligomers in order to hydrolyze ATP and make open promoter complexes. The bEBP formed by HrpR and HrpS activates transcription from the σ(54)-dependent hrpL promoter responsible for triggering the Type Three Secretion System in Pseudomonas syringae pathovars. Unlike other bEBPs that usually act as homohexamers, HrpR and HrpS operate as a highly co-dependent heterohexameric complex. To understand the organization of the HrpRS complex and the HrpR and HrpS strict co-dependence, we have analyzed the interface between subunits using the random and directed mutagenesis and available crystal structures of several closely related bEBPs. We identified key residues required for the self-association of HrpR (D32, E202 and K235) with HrpS (D32, E200 and K233), showed that the HrpR D32 and HrpS K233 residues form interacting pairs directly involved in an HrpR-HrpS association and that the change in side-chain length at position 233 in HrpS affects self-association and interaction with the HrpR and demonstrated that the HrpS D32, E200 and K233 are not involved in negative regulation imposed by HrpV. We established that the equivalent residues K30, E200 and E234 in a homo-oligomeric bEBP, PspF, are required for the subunit communication and formation of an oligomeric lock that cooperates with the ATP γ-phosphate sensing PspF residue R227, providing insights into their roles in the heteromeric HrpRS co-complex.

Suppression of plant defense responses by extracellular metabolites from Pseudomonas syringae pv. tabaci in Nicotiana benthamiana.

BACKGROUND: Pseudomonas syringae pv. tabaci (Pstab) is the causal agent of wildfire disease in tobacco plants. Several pathovars of Pseudomonas syringae produce a phytotoxic extracellular metabolite called coronatine (COR). COR has been shown to suppress plant defense responses. Interestingly, Pstab does not produce COR but still actively suppresses early plant defense responses. It is not clear if Pstab produces any extracellular metabolites that actively suppress early defense during bacterial pathogenesis. RESULTS: We found that the Pstab extracellular metabolite extracts (Pstab extracts) remarkably suppressed stomatal closure and nonhost hypersensitive response (HR) cell death induced by a nonhost pathogen, P. syringae pv. tomato T1 (Pst T1), in Nicotiana benthamiana. We also found that the accumulation of nonhost pathogens, Pst T1 and P. syringae pv. glycinea (Psgly), was increased in N. benthamiana plants upon treatment with Pstab extracts . The HR cell death induced by Pathogen-Associated Molecular Pattern (INF1), gene-for-gene interaction (Pto/AvrPto and Cf-9/AvrCf-9) and ethanol was not delayed or suppressed by Pstab extracts. We performed metabolite profiling to investigate the extracellular metabolites from Pstab using UPLC-qTOF-MS and identified 49 extracellular metabolites from the Pstab supernatant culture. The results from gene expression profiling of PR-1, PR-2, PR-5, PDF1.2, ABA1, COI1, and HSR203J suggest that Pstab extracellular metabolites may interfere with SA-mediated defense pathways. CONCLUSIONS: In this study, we found that Pstab extracts suppress plant defense responses such as stomatal closure and nonhost HR cell death induced by the nonhost bacterial pathogen Pst T1 in N. benthamiana.

Ethylene synthesis and regulated expression of recombinant protein in Synechocystis sp. PCC 6803.

The ethylene-forming enzyme (EFE) from Pseudomonas syringae catalyzes the synthesis of ethylene which can be easily detected in the headspace of closed cultures. A synthetic codon-optimized gene encoding N-terminal His-tagged EFE (EFEh) was expressed in Synechocystis sp. PCC 6803 (Synechocystis) and Escherichia coli (E. coli) under the control of diverse promoters in a self-replicating broad host-range plasmid. Ethylene synthesis was stably maintained in both organisms in contrast to earlier work in Synechococcus elongatus PCC 7942. The rate of ethylene accumulation was used as a reporter for protein expression in order to assess promoter strength and inducibility with the different expression systems. Several metal-inducible cyanobacterial promoters did not function in E. coli but were well-regulated in cyanobacteria, albeit at a low level of expression. The E. coli promoter P(trc) resulted in constitutive expression in cyanobacteria regardless of whether IPTG was added or not. In contrast, a Lac promoter variant, P(A1lacO-1), induced EFE-expression in Synechocystis at a level of expression as high as the Trc promoter and allowed a fine level of IPTG-dependent regulation of protein-expression. The regulation was tight at low cell density and became more relaxed in more dense cultures. A synthetic quorum-sensing promoter system was also constructed and shown to function well in E. coli, however, only a very low level of EFE-activity was observed in Synechocystis, independent of cell density.

Genetic analysis of genes involved in synthesis of modified 4-amino-4,6-dideoxyglucose in flagellin of Pseudomonas syringae pv. tabaci.

Glycosylation of flagellin contributes to swimming and swarming motilities, adhesion ability, and consequently virulence in Pseudomonas syringae pv. tabaci 6605. Glycans attached to six serine residues are located in the central region of the flagellin polypeptide. The glycan structure at position Ser 201 was recently revealed to consist of two L-rhamnoses and one modified 4-amino-4,6-dideoxyglucose (viosamine). To clarify the mechanisms for glycosylation of modified viosamine, genes encoding dTDP-viosamine aminotransferase (vioA), dTDP-viosamine acetyltransferase (vioB), and viosamine-derivative transferase (vioT) were isolated and defective mutants were generated. MALDI-TOF-MS analysis of a lysyl endopeptidase-digested peptide including all six glycosylation sites from each flagellin indicated that the molecular masses of the three flagellin mutants were reduced with highly heterogeneous patterns at regular intervals of 146 Da in the mass range from m/z 13,819 to 15,732. The data indicated that the glycopeptides obtained from mutants had glycans consisting only of deoxyhexose instead of the flagellin glycans including the viosamine derivatives determined previously. The motility and virulence on host tobacco leaves were strongly impaired in the Delta vioA mutant and were weakly reduced in the Delta vioB and Delta vioT mutant strains. These results suggest that the genes vioA, vioB, and vioT are essential for glycosylation of flagellin, and accordingly are required for bacterial virulence.

[Cloning and expressing of a harpin-encoding gene from Pseudomonas syringae pv. glycinea].

UNLABELLED: METHODS, OBJECTIVE: We amplified the 1026 bp hrp (hypersensitive response and pathogenicity) gene from Pseudomonas syringae pv. glycinea isolate Psg12 genomic DNA by PCR technique, and then constructed expression vector pGEX-hrpZ(Psg12) with regular molecular cloning operation. The recombinant plasmid was transformed into BL21(DE3). Recombinant protein was induced by Isopropylthio-beta-D-Galacgoside (IPTG). RESULTS: The molecular mass of the fusion protein is 61kDa analyzed by SDS-PAGE. The protein, similar to the other known harpins, was heat-stable, which contained abundant glycine(G), but had no cysteine. Furthermore, this protein was sensitive to protease K and able to trigger hypersensitive response (HR) in common tobacco. The HR elicitation by the protein in tobacco was inhibited by eukayotic metabolic inhibitors, NH4 VO3 and LaCl3. The hrpZ gene showed 79% identity to hrpZ(Psg) which cloned from P. syringae pv. glycinea (Psg r0) in Japan and 79 - 99% identity to other hrpZ in GenBank. However, it did not show any sequence identity with those of other genus of gram-negative plant pathogenic bacteria. CONCLUSION: In summary, hrpZ(Psg12) was a novel gene that was cloned by us from P. syringae pv. glycinea, and this is the first report to express hrpZ(Psg12) gene in BL21.

3-Methylarginine from Pseudomonas syringae pv. syringae 22d/93 suppresses the bacterial blight caused by its close relative Pseudomonas syringae pv. glycinea.

The epiphyte Pseudomonas syringae pv. syringae 22d/93 (Pss22d) produces a toxin that strongly inhibits the growth of its relative, the plant pathogen P. syringae pv. glycinea. The inhibition can be overcome by supplementing the growth medium with the essential amino acid, L-arginine; this suggests that the toxin acts as an inhibitor of the arginine biosynthesis. The highly polar toxin was purified by bioassay-guided fractionation using ion-exchange chromatography and subsequent RP-HPLC fractionation. The structure of the natural product was identified by HR-ESI-MS, HR-ESI-MS/MS, and NMR spectroscopy experiments as 3-methylarginine. This amino acid has previously only been known in nature as a constituent of the peptide lavendomycin from Streptomyces lavendulae. Results of experiments in which labeled methionine was fed to Pss22d indicated that the key step in the biosynthesis of 3-methylarginine is the introduction of the methyl group by a S-adenosylmethionine (SAM)-dependent methyltransferase. Transposon mutagenesis of Pss22d allowed the responsible SAM-dependent methyltransferase of the 3-methylarginine biosynthesis to be identified.

A plant pathogen virulence factor inhibits the eukaryotic proteasome by a novel mechanism.

Pathogenic bacteria often use effector molecules to increase virulence. In most cases, the mode of action of effectors remains unknown. Strains of Pseudomonas syringae pv. syringae (Pss) secrete syringolin A (SylA), a product of a mixed non-ribosomal peptide/polyketide synthetase, in planta. Here we identify SylA as a virulence factor because a SylA-negative mutant in Pss strain B728a obtained by gene disruption was markedly less virulent on its host, Phaseolus vulgaris (bean). We show that SylA irreversibly inhibits all three catalytic activities of eukaryotic proteasomes, thus adding proteasome inhibition to the repertoire of modes of action of virulence factors. The crystal structure of the yeast proteasome in complex with SylA revealed a novel mechanism of covalent binding to the catalytic subunits. Thus, SylA defines a new class of proteasome inhibitors that includes glidobactin A (GlbA), a structurally related compound from an unknown species of the order Burkholderiales, for which we demonstrate a similar proteasome inhibition mechanism. As proteasome inhibitors are a promising class of anti-tumour agents, the discovery of a novel family of inhibitory natural products, which we refer to as syrbactins, may also have implications for the development of anti-cancer drugs. Homologues of SylA and GlbA synthetase genes are found in some other pathogenic bacteria, including the human pathogen Burkholderia pseudomallei, the causative agent of melioidosis. It is thus possible that these bacteria are capable of producing proteasome inhibitors of the syrbactin class.

Syringolin A, a new plant elicitor from the phytopathogenic bacterium Pseudomonas syringae pv. syringae, inhibits the proliferation of neuroblastoma and ovarian cancer cells and induces apoptosis.

Syringolin A is a new plant elicitor produced by the plant pathogen Pseudomonas syringae pv. syringae. The goal of this study was to investigate whether syringolin A exhibits anti-proliferative properties in cancer cells. The treatment of human neuroblastoma (NB) cells (SK-N-SH and LAN-1) and human ovarian cancer cells (SKOV3) with syringolin A (0-100 microm) inhibited cell proliferation in a dose-dependent manner. The IC(50) (50% inhibition) for each cell line ranged between 20 microm and 25 microm. In SK-N-SH cells, the treatment with 20 microm syringolin A led to a rapid (24 h) increase of the apoptosis-associated tumour suppressor protein p53. In addition, we found that the treatment of SK-N-SH cells caused severe morphological changes after 48 h such as rounding of cells and loss of adherence, both conditions observed during apoptosis. The induction of apoptosis by syringolin A was confirmed by both poly (ADP-ribose) polymerase (PARP) cleavage and annexin V assay. Taken together, we show for the first time that the natural product syringolin A exhibits anti-proliferative activity and induces apoptosis. Syringolin A and structurally modified syringolin A derivatives may serve as new lead compounds for the development of novel anticancer drugs.

Identification of glycosylation genes and glycosylated amino acids of flagellin in Pseudomonas syringae pv. tabaci.

A glycosylation island is a genetic region required for glycosylation. The glycosylation island of flagellin in Pseudomonas syringae pv. tabaci 6605 consists of three orfs: orf1, orf2 and orf3. Orf1 and orf2 encode putative glycosyltransferases, and their deletion mutants, Deltaorf1 and Deltaorf2, exhibit deficient flagellin glycosylation or produce partially glycosylated flagellin respectively. Digestion of glycosylated flagellin from wild-type bacteria and non-glycosylated flagellin from Deltaorf1 mutant using aspartic N-peptidase and subsequent HPLC analysis revealed candidate glycosylated amino acids. By generation of site-directed Ser/Ala-substituted mutants, all glycosylated amino acid residues were identified at positions 143, 164, 176, 183, 193 and 201. Matrix-assisted laser desorption/ionization time of flight (MALDI-TOF) mass spectrometry (MS) analysis revealed that each glycan was about 540 Da. While all glycosylation-defective mutants retained swimming ability, swarming ability was reduced in the Deltaorf1, Deltaorf2 and Ser/Ala-substituted mutants. All glycosylation mutants were also found to be impaired in the ability to adhere to a polystyrene surface and in the ability to cause disease in tobacco. Based on the predicted tertiary structure of flagellin, S176 and S183 are expected to be located on most external surface of the flagellum. Thus the effect of Ala-substitution of these serines is stronger than that of other serines. These results suggest that glycosylation of flagellin in P. syringae pv. tabaci 6605 is required for bacterial virulence. It is also possible that glycosylation of flagellin may mask elicitor function of flagellin molecule.