Novel succinylated and large-sized osmoregulated periplasmic glucans of Pseudomonas syringae pv. syringae

Osmoregulated periplasmic glucans (OPGs) are intrinsic components of the Gram-negative bacterial envelope and are important for bacterial-host interactions. The OPGs of Pseudomonas syringae pv. syringae have been known to be highly branched linear glucans ranging from 6 to 13 glucose residues devoid of any substituents, while having backbone structure similar to those of Escherichia coli and Erwinia chrysanthemi. Here, we report for the first time succinylated and large-sized OPGs from P. syringae pv. syringae. The glucans were isolated with trichloroacetic acid treatment and various chromatographic techniques. These were further characterized by thin-layer chromatography, matrix-assisted laser desorption/ionization time of flight mass spectrometer, and 1D ¹H nuclear magnetic resonance spectroscopy. The results demonstrate that novel anionic glucans with one succinyl residue at the C-6 position of the glucose unit as well as neutral glucans including large-sized glucans with up to 28 degrees of polymerization are produced in P. syringae pv. syringae. Furthermore, the succinylated and large-sized OPGs of P. syringae pv. syringae are necessary for hypoosmotic adaptation.     

Crystal structure and metal binding properties of the periplasmic iron component EfeM from Pseudomonas syringae EfeUOB/M iron-transport system

BioMetals - EfeUOB/M has been characterised in Pseudomonas syringae pathovar. syringae as a novel type of ferrous-iron transporter, consisting of an inner-membrane protein (EfeUPsy) and three...     

Virulence factors are released in association with outer membrane vesicles of Pseudomonas syringae pv. tomato T1 during normal growth

Outer membrane vesicles (OMVs) are released from Pseudomonas syringae pv. tomato T1 (Pst T1) during their normal growth. These extracellular compartments are comprised of a complete set of biological macromolecules that includes proteins, lipids, lipopolysaccharides, etc. It is evident from proteomics analyses the OMVs of Pst T1 contain membrane- and virulence-associated proteins. In addition, OMVs of this organism are also associated with phytotoxin, coronatine. Therefore, OMVs of Pst T1 must play a significant role during pathogenicity to host plant. However, further studies are required whether these structures can serve as “vehicles” for the transport of virulence factors into the host membrane.     

Accumulation of phytuberin and phytuberol in tobacco callus inoculated with Pseudomonas solanacearum or Pseudomonas syringae pv. Tabaci

Tobacco callus tissues inoculated with Pseudomonas solanacearum or Pseudomonas syringae pv. tabaci accumulated phytuberin and phytuberol. Accumulation of the two sesquiterpenoids was dependent upon: the source (cultivar) of the explant; the number of transfers; the period which had elapsed after transfer; the bacterial species, and the time after inoculation.     

The role of a putative peroxisomal-targeted epoxide hydrolase of Nicotiana benthamiana in interactions with Colletotrichum destructivum, C. orbiculare or Pseudomonas syringae pv. tabaci

Motif analysis among 30 EH1 and EH2 epoxide hydrolases from Solanaceaeous plants showed differences primarily in the lid region around the catalytic site. Based on in silico models of 3D structures, EH1 proteins lack a catalytic triad because of the orientation of one of the conserved lid tyrosines, while the orientation of that tyrosine in EH2 proteins fomed a catalytic triad inside a hydrophobic tunnel. Two similar EH2 protein genes from Nicotiana benthamiana, NbEH2.1 and NbEH2.2, have a predicted peroxisomal targeting sequence, catalytic triad, and structural similarities to a potato cutin monomer-synthesizing epoxide hydrolase. NbEH2.1 expression increased with infections by the hemibiotrophs, Colletotrichum destructivum, Colletotrichum orbiculare or Pseudomonas syringae pv. tabaci only during their biotrophic phases, while there was only a slight increase during the hypersensitive response to P. syringae pv. tabaci (avrPto). In contrast, among the four pathogens, NbEH2.2 expression increased only in response to P. syringae pv. tabaci. Virus-induced gene silencing of NbEH2.1 significantly affected only the interaction with C. destructivum, resulting in a delay in the appearance of necrosis that may be related to its biotrophic phase being restricted to single epidermal cells, which is unique among these pathogens. These results differed from that of a previously reported EH1 gene of N. benthamiana for these interactions, demonstrating specialization among EH genes in basal resistance.     

Malonichrome,a new iron chelate from Fusarium roseum

The predominant iron chelates, or siderochromes, produced by the fungus, Fusarium roseum during culture periods up to seven days are the ester type fusarinine compounds. During longer periods of incubation, the fusarinine compounds completely disappear from the culture medium and are replaced by a new siderochrome. The new compound has been isolated, purified, and its structure determined. It is a cyclic hexapeptide containing one residue of l-alanine, two residues of glycine and three residues of δ-N-hydroxyornithine. The hydroxylamino groups of the ornithine residues are acylated with 3 mol of malonic acid to form a negatively charged ferrichrome type chelate. The circular dichroism spectrum indicates that the stereochemistry about the iron is Λ-cis. This compound, which we name malonichrome, is not an efficient iron donor to F. roseum nor does it show growth factor activity towards Arthrobacter flavescens.     

Quercetin-induced H2O2 mediates the pathogen resistance against Pseudomonas syringae pv. Tomato DC3000 in Arabidopsis thaliana

Quercetin is a potent antioxidant and has been extensively used as a therapy intervention to prevent age-associated diseases. However, emerging studies showed it can also act as a prooxidant and induce H₂O₂ under certain conditions. In the current study, our results showed that quercetin contributed to the pathogen resistance in Arabidopsis thaliana (Arabidopsis) in response to the infection of virulent strain Pseudomonas syringae pv. Tomato DC3000 (Pst). Various defense responses, such as H₂O₂ burst, callose deposition, cell death, PR1 (pathogenesis-related 1) and PAL1 (Phe ammonia-lyase 1) gene expression, have been investigated in quercetin-pretreated Pst-inoculated Arabidopsis Col-0 and there was a strong defensive response in quercetin-pretreated Arabidopsis against virulent Pst. However, with the presence of catalase, the protective effects of quercetin on pathogen resistance to virulent Pst disappeared in Arabidopsis, suggesting that H₂O₂ may play a key role in plant defense responses. In addition, we confirmed that quercetin did not show any beneficial effect on pathogen-free leaves in Arabidopsis, indicating that pathogen challenge is also required to induce the defense responses in quercetin-pretreated Arabidopsis. Furthermore, strong defense responses have been observed in quercetin-pretreated Arabidopsis mutant jar1, ein2, and abi1-2 under Pst challenge, whereas no protective effect has been observed in quercetin-pretreated Arabidopsis mutant NahG and npr1. These findings indicate that quercetin induces the resistance to Pst in Arabidopsis via H₂O₂ burst and involvement of SA and NPR1.     

Norcoronatine and N-coronafacoyl-L-valine,phytotoxic analogues of coronatine produced by a strain of Pseudomonas syringae pv. glycinea

Compounds in liquid cultures of the phytopathogenic bacterium Pseudomonas syringae pv. glycinea that cause chlorosis after application to young bean leaves have been investigated. Known compounds that were isolated and identified were coronatine, the major component, and N-coronafacoyl-L-valine, which are both biologically active, and coronafacic acid which is inactive. In addition a new minor component was isolated and purified. Mass spectrometry indicated that this was an amide of coronafacic acid, bearing one less methylene group than coronatine. Mass spectral and NMR data, together with a study of the products from acid hydrolysis of the new compound, established its structure to be norcoronatine (i.e. a methyl substituent in place of the 2-ethyl substituent on the cyclopropyl moiety of coronatine). The probable biosynthetic derivation of norcoronatine is discussed.     

Promoter activity of a putative pollen monosaccharide transporter in Petunia hybrida and characterisation of a transposon insertion mutant

Summary. For the growth of the male reproductive cells of plants, the pollen, the presence of sufficient sucrose or monosaccharides is of vital importance. From Petunia hybrida a pollen-specific putative monosaccharide transporter designated PMT1 (for petunia monosaccharide transporter) has been identified previously. The present work provides an in-depth analysis and characterisation of PMT1 in the context of pollen development with the GUS reporter gene and an insertion mutant. The promoter of the pollen-specific putative PMT1 gene has been isolated by inverse PCR and sequenced. Analysis of plants transformed with the promoter-GUS fusion confirmed the specificity of this gene, belonging to the late pollen-specific expressed genes. GUS activity was detected even after 24 h of in vitro pollen germination, at the pollen tube tip. To elucidate the importance of PMT1 for gametophyte development and fertilisation, we isolated a mutant plant containing a transposon insertion in the PMT1 gene by the dTph1 transposon-tagging PCR-based assay. The PMT1 mutant contained a dTph1 insertion in position 1474 bp of the transcribing part of the gene, before the last two transmembrane-spanning domains. Analysis of the progeny of the heterozygous mutant after selfing revealed no alterations in pollen viability and fertility. Mature pollen grains of a plant homozygous for the transposon insertion were able to germinate in vitro in a medium containing sucrose, glucose, or fructose, which indicates that PMT1 is not essential for pollen survival. Several explanations for these results are discussed in the present work. Correspondence and reprints (present address): Department of Plant Biology, University of Granada. Fuentenueva s/n, 18001 Granada, Spain. Present address: Swammerdam Institute for Life Sciences, Amsterdam, the Netherlands.     

Isolation of human proteasomes and putative proteasome-interacting proteins using a novel affinity chromatography method

The proteasome is the primary subcellular organelle responsible for protein degradation. It is a dynamic assemblage of 34 core subunits and many differentially expressed, transiently interacting, modulatory proteins. This paper describes a novel affinity chromatography method for the purification of functional human holoproteasome complexes using mild conditions. Human proteasomes purified by this simple procedure maintained the ability to proteolytically process synthetic peptide substrates and degrade ubiquitinated parkin. Furthermore, the entire purification fraction was analyzed by mass spectrometry in order to identify proteasomal proteins and putative proteasome-interacting proteins. The mild purification conditions maintained transient physical interactions between holoproteasomes and a number of known modulatory proteins. In addition, several classes of putative interacting proteins co-purified with the proteasomes, including proteins with a role in the ubiquitin proteasome system for protein degradation or DNA repair. These results demonstrate the efficacy of using this affinity purification strategy for isolating functional human proteasomes and identifying proteins that may physically interact with human proteasomes.     

Isolation and general characterization of a heat-stable proteinase from Pseudomonas fluorescens AFT 36

An extracellular proteinase from Pseudomonas fluorescens, strain AFT 36, was isolated to homogeneity by chromatography on DEAE-cellulose and Sephadex G-150; a 230-fold increase in specific activity with a recovery of 53% was obtained. The enzyme was optimally active at pH 6.5 and 45°C; activity declined rapidly at higher temperatures but significant activity persisted down to 4°C. Activity was strongly inhibited by 10^?3 M EDTA and was partially restored by addition of Zn²⁺, Ca²⁺ or Co²⁺. The K_m values on methylated casein and sodium caseinate were 18.2 and 7.1 mg/ml, respectively. The enzyme was very labile in phosphate buffer and in a milk salts buffer at 55°C but was very stable in the latter at more than 80°C.     

Schistosoma mansoni: Tyrosine,a putative in vivo substrate of phenol oxidase

l-Tyrosine, l-[3,4]dihydroxyphenylalanine (l-DOPA), and dopamine are known to be in vitro substrates for Schistosoma mansoni phenol oxidase. Since all three compounds are present in the female schistosome, it is not clear which one serves as the substrate for phenol oxidase in intact S. mansoni. However, the concentration of l-tyrosine in the female schistosome (252 ng/mg worm) is 4-fold higher than the K_m of phenol oxidase for this amino acid while the concentrations of l-DOPA and dopamine (0.954 and 0.790 ng/mg worm, respectively) are 100- and 500-fold lower than the K_m of these substrates. Tri-l-tyrosine methyl ester is oxidized at less than 3% of the rate of l-tyrosine methyl ester. A tyrosine:lysine peptide and chymotrypsinogen are not oxidized. Female S. mansoni do not incorporate l-tyrosine into proteins to a significantly greater extent than l-leucine. The results suggest that free l-tyrosine is the substrate for S. mansoni phenol oxidase in vivo.     

Synthesis, X-ray structure and redox properties of the macrobicyclic iron(II) N2- and S2-containing vic-dioximates

Nucleophilic substitution of the reactive chlorine atoms of the boron-capped macrobicyclic vic-di- and hexahalogen-containing iron(II) precursors with 1,2-ethanedithiol and 1,2-benzenedithiol in dichloromethane as a solvent in the presence of triethylamine as a strong organic base afforded the corresponding di- and hexasulfide mono- and triribbed-functionalized clathrochelates, respectively, in relatively high yields. In the case of the low-reactive tin-capped clathrochelate [Fe(Cl₂Gm)₃(SnCl₃)₂]²⁻ dianion this reaction was performed in DMF with the potassium salt of 1,2-benzenedithiol. The reaction of the dichlorine-containing FeBd₂(Cl₂Gm)(BF)₂ precursor with an excess of ethylenediamine in DMF led to the clathrochelate with N₂-containing vic-dioximate ribbed fragment. The complexes obtained were characterized using elemental analysis, MALDI-TOF mass spectrometry, IR, UV-vis, ¹H and ¹³C{¹H} NMR, ⁵⁷Fe Mössbauer spectroscopies, and X-ray crystallography.The nature and number of the ribbed substituents affect the geometry of a clathrochelate framework, first of all, the distortion of the trigonal prismatic-trigonal antiprismatic iron(II) coordination polyhedra, whereas the apical substituents at the capping boron atoms influe on the B-O distances in the apical RBO₃ fragments. The geometry of the tin-capped hexasulfide clathrochelate complex was deduced from EXAFS data using the scattering both on the encapsulated iron(II) and capping tin(IV) ions.The electrochemical properties of the iron(II) complexes obtained were studied by cyclic voltammetry. The electrochemically generated unstable reduced anionic forms are destabilized by the electron-donating ribbed substituents, whereas the oxidation led to the formation of the cationic macrobicycles, the stability of which depends on the nature of the apical capping groups and ribbed substituents as well. The pseudo-aromatic disulfide ribbed fragments stabilize the oxidized forms of the clathrochelate complexes.     

Isolation, cloning and expression analysis of EcPMA1, a putative plasma membrane H-ATPase transporter gene from the biotrophic pathogenic fungus Erysiphe cichoracearum

Little is known at the molecular level about the transporters involved in nutrient transfer in the plant/powdery mildew interaction. A PCR-based approach was used to identify and isolate a partial-length cDNA coding for an isoform of the plasma membrane H⁺-ATPase (EcPMA1) in the biotrophic pathogenic fungus Erysiphe cichoracearum. Southern analysis suggests that EcPMA1 exists as a single-copy gene. Sequence analysis indicated a high similarity of EcPMA1 to other fungal H⁺-ATPases. Expression of EcPMA1 increases in infected Arabidopsis leaves as the disease progresses, correlating with the growth of the pathogen.     

Identification, cloning and characterization of a derepressible Na+-coupled phosphate transporter in Saccharomyces cerevisiae

Based on the high sequence homology between the yeast ORF YBR296c (accession number P38361 in the SWISS-PROT database) and the PHO4 gene of Neurospora crassa, which codes for a Na⁺/P_i cotransporter with twelve putative transmembrane domains, the YBR296c ORF was considered to be a promising candidate gene for a plasma membrane-bound phosphate transporter in Saccharomyces cerevisiae. Therefore, this gene, here designated PHO89, was cloned and a set of deletion mutants was constructed. We then studied their P_i uptake activity under different conditions. We show here that a transport activity displayed by PHO89 strains under alkaline conditions and in the presence of Na⁺ is absent in pho89 null mutants. Moreover, when the pH was lowered to pH 4.5 or when Na⁺ was omitted, this activity decreased significantly, reaching values close to those exhibited by the Δpho89 mutant. Studies of the acid phosphatase activity of these strains, as well as promoter sequence analysis, suggest that expression of the PHO89 gene is under the control of the PHO regulatory system. Northern analysis shows that this gene is only transcribed under conditions of P_i limitation. This is, to our knowledge, the first demonstration that the PHO89 gene codes for the Na⁺/P_i cotransporter previously characterized by kinetic studies, and represents the only Na+-coupled secondary anion transport system so far identified in S. cerevisiae. Pho89p has been shown to have an apparent K_m of 0.5 μM and a pH optimum of 9.5, and is highly specific for Na⁺; activation of transport is maximal at a Na⁺ concentration of 25 mM. Received: 2 November 1997 / Accepted: 20 February 1998     

Isolation and characterization of a new gene, sre , which encodes a GATA-type regulatory protein that controls iron transport in Neurospora crassa

Multiple GATA factors – regulatory proteins with consensus zinc finger motifs that bind to DNA elements containing a GATA core sequence – exist in the filamentous fungus Neurospora crassa. One GATA factor, NIT2, controls nitrogen metabolism, whereas two others, WC-1 and WC-2, regulate genes responsive to blue light induction. A gene encoding a new GATA factor, named SRE, was isolated from Neurospora using a PCR-mediated method. Sequence analysis of the new GATA factor gene revealed an ORF specifying 587 amino acids, which is interrupted by two small introns. Unlike all previously known Neurospora GATA factors, which possess a single zinc-finger DNA-binding motif, SRE contains two GATA-type zinc fingers. The deduced amino acid sequence of SRE shows significant similarity to URBS1 of Ustilago and SREP of Penicillium. A loss-of-function mutation was created by the RIP procedure. Analysis of sre ⁺ and sre ⁻ strains revealed that SRE acts as a negative regulator of iron uptake in Neurospora by controlling the synthesis of siderophores. Siderophore biosynthesis is repressed by high iron concentrations in the wild-type strain but not in sre ⁻ mutant cells. The sre promoter contains a number of GATA sequences; however, expression of sre mRNA occurs in a constitutive fashion and is not regulated by the concentration of iron available to the cells. Received: 20 January 1998 / Accepted: 23 April 1998     

Quantification of the rate of CO2 formation in the periplasmic space of microalgae during photosynthesis. A comparison of whole-cell rate constants for CO2 and HCO3– uptake among three species of the green alga Chlorella

As previously described, the absolute rate of photosynthesis due to a limited concentration of dissolved inorganic carbon at alkaline pH, where the rate of CO₂ formation is strictly limited, plotted as a function of chlorophyll (Chl) concentration, will take the form of a rectangular hyperbola combined with a linear rate directly proportional to [Chl], which are, respectively, due to the contribution of CO₂ and HCO₃^– to photosynthesis. This model represents that the mathematical asymptote of absolute rate of photosynthesis versus cell density is described by the whole-cell rate constant for HCO₃^– uptake and the maximum rate of CO₂ formation in the extracellular space. This means that any trace modification of the CO₂ formation rate outside the cell will alter the photosynthetic rate and should be detectable experimentally. In air-grown Chlorella ellipsoidea and C. kessleri and in high CO₂-grown C. saccharophila, the graph of the absolute rate of photosynthesis against [Chl] clearly followed the mathematical model described above and the actual CO₂ formation rates outside the cells were not significantly different from the calculated rates. It also indicated that the whole-cell rate constants for CO₂ and HCO₃^– uptake in air-grown C. ellipsoidea and C. saccharophila were similar at ≈ 300 and 2·0 mm³μg^–1 Chl min^–1, respectively, whereas those in air-grown C. kessleri were ≈ 550 and 15 mm³μg^–1 Chl min^–1. These results indicate that no acidification of the periplasmic space occurs, and there is no trace activity of external carbonic anhydrase in these microalgae.     

Large-scale purification, dissociation and functional reassembly of the maltose ATP-binding cassette transporter (MalFGK2) of Salmonella typhimurium

The maltose ATP-binding cassette (ABC) transporter of Salmonella typhimurium is composed of a membrane-associated complex (MalFGK₂) and a periplasmic substrate binding protein. To further elucidate protein-protein interactions between the subunits, we have studied the dissociation and reassembly of the MalFGK₂ complex at the level of purified components in proteoliposomes. First, we optimized the yield in purified complex protein by taking advantage of a newly constructed expression plasmid that carries the malK, malF and malG genes in tandem orientation. Incorporated in proteoliposomes, the complex exhibited maltose binding protein/maltose-dependent ATPase activity with a V_max of 1.25 μmol P_i/min/mg and a K_m of 0.1 mM. ATPase activity was sensitive to vanadate and enzyme IIA^Glc, a component of the enterobacterial glucose transport system. The proteoliposomes displayed maltose transport activity with an initial rate of 61 nmol/min/mg. Treatment of proteoliposomes with 6.6 M urea resulted in the release of medium-exposed MalK subunits concomitant with the complete loss of ATPase activity. By adding increasing amounts of purified MalK to urea-treated proteoliposomes, about 50% of vanadate-sensitive ATPase activity relative to the control could be recovered. Furthermore, the phenotype of MalKQ140K that exhibits ATPase activity in solution but not when associated with MalFG was confirmed by reassembly with MalK-depleted proteoliposomes.