Characterization of the phytopathogen Pseudomonas syringae pathovar ribicola NCPPB 963

In 1939, a bacterial spot caused severe defoliation of Ribes aureum (Golden Currant) The causal agent is now recognized as Pseudomonas syringae pathovar ribicola. This communication extends the phenotype of the only identified strain of P. syringae pv. ribicola, which is reminiscent of those of other pathovars, and provides a molecular biological characterization. A minimum size of 5.55 Mb for the bacterial genome was obtained using pulsed-field electrophoresis. The SDS-PAGE outer-membrane profile contained seven major bands, and has obvious similarities to that of P. aeruginosa. SDS-PAGE of concentrated mid-log phase culture supernatants revealed large amounts of a single, cryptic 24.0 kD protein. The amino acid composition and 57 residues in the N-terminus of this protein. were determined. The protein sequence was nearly identical to the translation of a region of unknown function in the P. aeruginosa genome. Extensive similarity in N-terminal sequence, composition and subunit size to a secreted hydrophilic Vibrio cholerae protein of unknown function was also found. Neither protein has been directly associated with disease development.     

Erwinia amylovora secretes DspE, a pathogenicity factor and functional AvrE homolog, through the Hrp (type III secretion) pathway

Erwinia amylovora was shown to secrete DspE, a pathogenicity factor of 198 kDa and a functional homolog of AvrE of Pseudomonas syringae pv. tomato. DspE was identified among the supernatant proteins isolated from cultures grown in an hrp gene-inducing minimal medium by immunodetection with a DspE-specific antiserum. Secretion required an intact Hrp pathway.     

Two native plasmids of Pseudomonas syringae pathovar tomato strain PT23 share a large amount of repeated DNA, including replication sequences

Strain PT23 of Pseudomonas syringae pv. tomato contains four native plasmids, designated A, B, C, and D. By DNA hybridization of genomic and plasmid DNA digests from the wild type and a plasmid-cured strain, we determined that c. 61 kb (c. 74%) of pPT23B is repeated in pPT23 A and only c. 17 kb (c. 21%) is in single copy in strain PT23. pPT23B also contains DNA repeated in the chromosome that occurs in three DNA fragments of 0.6, 4.6, and 9.6 kb that might be transposable elements. Additionally, the 9.6 kb fragment also shares sequences with the three other plasmids of strain PT23. By DNA hybridization with the origin of replication from a native plasmid of P. syringae pv. syringae and in vivo replication tests, we identified the origins of replication of plasmids A, B, and D and showed that they cross-hybridize. The putative par region from pPT23 A has also been identified and is not conserved in the other three native plasmids from strain PT23. By using the defined minimal origin of replication from pPT23 A as a probe, we showed that it is highly conserved in 14 strains belonging to nine different pathovars of P. syringae and that as many as five different native plasmids with closely related origins of replication coexist in the same cell. The duplication and reorganization of plasmids might therefore occur at high frequency and could be responsible for the existence of large numbers of native plasmids in P. syringae strains.     

Identification and characterization of the Erwinia amylovora rpoS gene: RpoS is not involved in induction of fireblight disease symptoms

The Erwinia amylovora rpoS gene, encoding the alternative sigma factor RpoS, has been cloned and characterized. Though highly sensitive to a number of environmental stresses, an E. amylovora rpoS mutant was not compromised in its ability to grow or cause disease symptoms within apple seedlings or in an overwintering model.     

Cloning, nucleotide sequence, and expression in Escherichia coli of levansucrase genes from the plant pathogens Pseudomonas syringae pv. glycinea and P. syringae pv. phaseolicola

Plant-pathogenic bacteria produce various extracellular polysaccharides (EPSs) which may function as virulence factors in diseases caused by these bacteria. The EPS levan is synthesized by the extracellular enzyme levansucrase in Pseudomonas syringae, Erwinia amylovora, and other bacterial species. The lsc genes encoding levansucrase from P. syringae pv. glycinea PG4180 and P. syringae pv. phaseolicola NCPPB 1321 were cloned, and their nucleotide sequences were determined. Heterologous expression of the lsc gene in Escherichia coli was found in four and two genomic library clones of strains PG4180 and NCPPB 1321, respectively. A 3. 0-kb PstI fragment common to all six clones conferred levan synthesis on E. coli when further subcloned. Nucleotide sequence analysis revealed a 1,248-bp open reading frame (ORF) derived from PG4180 and a 1,296-bp ORF derived from NCPPB 1321, which were both designated lsc. Both ORFs showed high homology to the E. amylovora and Zymomonas mobilis lsc genes at the nucleic acid and deduced amino acid sequence levels. Levansucrase was not secreted into the supernatant but was located in the periplasmic fraction of E. coli harboring the lsc gene. Expression of lsc was found to be dependent on the vector-based Plac promoter, indicating that the native promoter of lsc was not functional in E. coli. Insertion of an antibiotic resistance cassette in the lsc gene abolished levan synthesis in E. coli. A PCR screening with primers derived from lsc of P. syringae pv. glycinea PG4180 allowed the detection of this gene in a number of related bacteria.     

Inactivation of the sapA to sapF locus of Erwinia chrysanthemi reveals common features in plant and animal bacterial pathogenesis

We investigated the role in pathogenesis of bacterial resistance to plant antimicrobial peptides. The sapA to sapF (for sensitive to antimicrobial peptides) operon from the pathogenic bacterium Erwinia chrysanthemi has been characterized. It has five open reading frames that are closely related (71% overall amino acid identity) and are in the same order as those of the sapA to sapF operon from Salmonella typhimurium. An E. chrysanthemi sap mutant strain was constructed by marker exchange. This mutant was more sensitive than was the wild type to wheat alpha-thionin and to snakin-1, which is the most abundant antimicrobial peptide from potato tubers. This mutant was also less virulent than was the wild-type strain in potato tubers: lesion area was 37% that of the control, and growth rate was two orders of magnitude lower. These results indicate that the interaction of antimicrobial peptides from the host with the sapA to sapF operon from the pathogen plays a similar role in animal and in plant bacterial pathogenesis.     

Characterization of dapB, a gene required by Pseudomonas syringae pv. tabaci BR2.024 for lysine and tabtoxinine-beta-lactam biosynthesis

The dapB gene, which encodes L-2,3-dihydrodipicolinate reductase, the second enzyme of the lysine branch of the aspartic amino acid family, was cloned and sequenced from a tabtoxin-producing bacterium, Pseudomonas syringae pv. tabaci BR2.024. The deduced amino acid sequence shared 60 to 90% identity to known dapB gene products from gram-negative bacteria and 19 to 21% identity to the dapB products from gram-positive bacteria. The consensus sequence for the NAD(P)H binding site [(V/I)(A/G)(V/I)XGXXGXXG)] and the proposed substrate binding site (HHRHK) were conserved in the polypeptide. A BR2.024 dapB mutant is a diaminopimelate auxotroph and tabtoxin negative. The addition of a mixture of L-,L-, D,D-, and meso-diaminopimelate to defined media restored growth but not tabtoxin production. Cloned DNA fragments containing the parental dapB gene restored the ability to grow in defined media and tabtoxin production to the dapB mutant. These results indicate that the dapB gene is required for both lysine and tabtoxin biosynthesis, thus providing the first genetic evidence that the biosynthesis of tabtoxin proceeds in part along the lysine biosynthetic pathway. These data also suggest that L-2,3,4,5-tetrahydrodipicolinate is a common intermediate for both lysine and tabtoxin biosynthesis.     

The cloning and characterization of phage promoters, directing high expression of luciferase in Pseudomonas syringae pv. phaseolicola, allowing single cell and microcolony detection

Regions of DNA containing promoter sequences from a Pseudomonas syringae pv. phaseolicola-specific phage (phi 11P) were identified by shotgun cloning into a broad-host-range promoter-probe vector (pQF70). When used in conjunction with the luciferase reporter genes, one of these DNA fragments, 19H, directed gene expression at a level which enabled the subsequent light output (bioluminescence) of single cells of P. syringae pv. phaseolicola to be detected and visualized using a charge-coupled device (CCD). The P. syringae pv. phaseolicola phi 11P, 19H and P. aeruginosa phi PLS27, HcM promoters gave a 50-fold increase in bioluminescence (maximum relative light output) compared to similar constructs containing other well-characterized promoters, for example, tetracycline. Similar bioluminescent characteristics of the transformed bacterium, were observed during growth with and without antibiotic-selection. When lux+ bacteria were inoculated onto French bean leaf (Phaseolus vulgaris L.), the resultant secondary halo blight lesions were bioluminescent and during phylloplane colonization by the lux+ bacterium, bioluminescence on leaf surfaces was detected and imaged by the CCD. Use of these newly identified promoters, combined with the greatly increased sensitivity of bioluminescence detection by the CCD, thus provided a new dimension for the study of natural ecological populations during the bacterial colonization of plants.     

Use of translational fusions to the maltose-binding protein to produce and purify proteins in Pseudomonas syringae and assess their activity in vivo

A simple approach is described for the production and purification of proteins in Pseudomonas syringae. The strategy involves the use of the tac promoter, the maltose-binding protein, and the broad-host-range vector, pRK415. This approach was used to partially purify two proteins involved in coronatine biosynthesis from P. syringae. The activity of the fusions was demonstrated in vivo in complementation experiments using the appropriate mutants.     

大豆细菌性斑点病菌hrpZPsg12基因增强病原菌对大豆的致病性并引起烟草过敏性反应

[目的]明确hrpZ<,Psg12>基因对大豆细菌性斑点病菌致病性的影响.[方法]采用PCR方法从大豆细菌性斑点病菌中克隆hrpZ<,Psg12>基因,利用具有自杀特性的敲除质粒pKNOCK-Cm和具有功能互补作用的粘粒pUFR034.构建了hrpZ<,Psg12>基因的突变载体pKNOCK477-7和互补载体pUFR1026-68,并筛选出hrpZ<,Psg12>基因的突变体477-1及其功能互补子1026-5.进一步将野生型Psg12、突变体477-1和互补子1026-5等3个菌株同时接种大豆叶片和烟草叶片,进行致病性测定和过敏性反应分析.[结果]所有被接种的大豆和烟草叶片都产生了反应斑.但是,反应斑的大小有差异,Psg12菌株接种的病斑较大,477-1的较小,互补子1026-5的接近野生型菌株Psg12.病斑中细菌繁殖量分析表明,野生型菌株Psg12繁殖量最高,突变体477-1的最低,互补子1026-5的接近野生型菌株Psg12的繁殖量.[结论]hrpZ<,Psg12>基因能够增强大豆细菌性斑点病菌对大豆的致病性,并且能够在烟草上产生过敏性反应.     

Homology modelling and molecular dynamics aided analysis of ligand complexes demonstrates functional properties of lipid-transfer proteins encoded by the barley low-temperature-inducible gene family, blt4

In the present study an investigation of the structure-activity relationships in 9-ethylpurine derivatives, aimed at preparing A1, A2A, A2B, and A3 selective adenosine receptor antagonists, was undertaken. Our synthetic approach was to introduce various substituents (amino, alkoxy and alkynyl groups) into the 2-, 6-, or 8-positions of the purine ring. The starting compounds for each series of derivatives were respectively: 2-iodo-9-ethyladenine (9), obtained from 2-amino-6-chloropurine (5); 9-ethyl-6-iodo-9H-purine (11), 8-bromo-9-ethyl-adenine (3) and 8-bromo-9-ethyl-6-iodo-9H-purine (13), obtained from 9-ethyl-adenine (2). The synthesized compounds were tested in in vitro radioligand binding assays at A1, A2A, and A3 human adenosine receptor subtypes. Due to the lack of a suitable radioligand the affinity of the 9-ethyladenine derivatives at A2B adenosine receptors was determined in adenylyl cyclase experiments. In general, the series of 9-ethylpurine derivatives exhibited a similar pharmacological profile at A1 and A2A receptors whereas some differences were found for the A3 and the A2B subtypes. 8-Bromo-9-ethyladenine (3) showed higher affinity for all receptors in comparison to the parent compound 2, and the highest affinity in the series for the A2A and A2B subtypes (Ki = 0.052 and 0.84 microM, respectively). Analyzing the different substituents, a phenethoxy group in 2-position (10a) gave the highest A2A versus A2B selectivity (near 400-fold), whereas a phenethylamino group in 2- and 6-position (10b and 12b, respectively) improved the affinity at A2B receptors, compared to the parent compound 2. The presence of a hexynyl substituent in 8-position led to a compound with good affinity at the A3 receptor (4d, Ki = 0.62 microM), whereas (ar)alkynyl groups are detrimental for the potency at the A2B subtype. These differences give raise to the hope that further modifications will result in the development of currently unavailable leads with good affinity and selectivity for A2B adenosine receptors.     

Cloning and sequencing of the gene for a lactococcal endopeptidase, an enzyme with sequence similarity to mammalian enkephalinase

The gene specifying an endopeptidase of Lactococcus lactis, named pepO, was cloned from a genomic library of L. lactis subsp. cremoris P8-2-47 in lambda EMBL3 and was subsequently sequenced. pepO is probably the last gene of an operon encoding the binding-protein-dependent oligopeptide transport system of L. lactis. The inferred amino acid sequence of PepO showed that the lactococcal endopeptidase has a marked similarity to the mammalian neutral endopeptidase EC 3.4.24.11 (enkephalinase), whereas no obvious sequence similarity with any bacterial enzyme was found. By means of gene disruption, a pepO-negative mutant was constructed. Growth and acid production of the mutant strain in milk were not affected, indicating that the endopeptidase is not essential for growth of L. lactis in milk.     

The length of the retention interval, forgetting, and subjective similarity.

A long retention interval tends to result in the poor retention known as forgetting. A high subjective similarity between stimuli frequently produces their poor retention. Thus, a long retention interval may increase the subjective similarity between stimuli (the RIISS hypothesis), and this increase may produce forgetting. To examine this hypothesis, college students made speeded same-different discriminations between two lines or tones of different lengths or frequencies that were 400 ms or 3,300 ms apart, and they rated the similarity of these stimuli. The long interval produced poorer overall performance as expected, but also produced poorer performance on different than same stimuli, implying that it increased the subjective similarity between the initial and subsequent stimuli, and it also increased rated similarity, in support of the RIISS hypothesis. The position that stored stimuli lose less common information than distinctive information explains RIISS evidence better than does perturbation theory. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Further studies on the blue and near ultraviolet reversible photoreaction with an intracellular particulate fraction of the fungus, Alternaria tomato

Further studies have been made on the blue and near ultraviolet-reversible photoreaction in a cell-free system of the fungus Alternaria tomato, prepared by two-layer sucrose density gradient centrifugation. A light-minus-dark difference spectrum of the supernatant fraction showed a dip in the near ultraviolet region, mainly at 280 nm, when irradiated with near ultraviolet light which could not be reversed by following the initial irradiation with blue light. The difference spectrum of the dialyzed particulate fraction, located in the 68% sucrose band, showed a peak absorbance in the blue region, mainly at 400 nm, when irradiated with near ultraviolet light, and also could not be reversed by blue light. When the particulate fraction plus the supernatant fraction was irradiated with near ultraviolet light, the increment rate of peak absorbance in the blue region increased compared to that of the particulate fraction lacking the supernatant, and both peak absorbance in the blue region and the dip in the near ultraviolet region were reversed by irradiation with blue light following the application of near ultraviolet light. Thus, it was concluded that two photoresponsive systems are involved in blue and near ultraviolet reversible photoreaction on an intracellular particulate fraction.     

Immunochemical characterization of O polysaccharides composing the alpha-D-rhamnose backbone of lipopolysaccharide of Pseudomonas syringae and classification of bacteria into serogroups O1 and O2 with monoclonal antibodies

Murine monoclonal antibodies (MAbs) reacting with Pseudomonas syringae lipopolysaccharide (LPS) O polysaccharides (OPS) composed of tetra- and tri-alpha-D-rhamnose repeats in the backbone [3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1-2)D-Rha(alpha1] and [3)D-Rha(alpha1-3)D-Rha(alpha1-2)D-Rha(alpha1] were generated and used for immunochemical analysis and for serological classification of the bacteria. A total of 195 of 358 P. syringae strains tested representing 21 pathovars were shown to share a common epitope, 1a, and were classified into serogroup O1. All strains with pathovars aptata, glycinea, japonica, phaseolicola, and pisi, most of the strains with pathovars atrofaciens and striafaciens, and half of the strains with pathovar syringae were classified into serotypes O1a', O1b, O1c, and O1d within serogroup O1. Serogroup-specific epitope 1a was inferred to be related to the (alpha1-2)D-Rha(alpha1-3) site of the OPS backbone. The serotype-specific epitopes 1b, 1c, 1d, and 1a' were inferred as relating to the immunodominant lateral (alpha1-3)D-Rha, (beta1-4)D-GlcNAc, and (alpha1-4)D-Fuc substituents and backbone-located site (alpha1-3)D-Rha(alpha1-2), respectively, of OPSs that share the common tetra-D-rhamnose repeats in the backbone. A total of 7.3% of the strains studied, all with pathovars morsprunorum and lapsa, were classified as serotypes O2a and O2d within serogroup 02. Serotype-specific epitope 2a was inferred as being related to the backbone-located site D-Rha(alpha1-3)D-Rha and epitope 2d to the immunodominant lateral (alpha1-4)D-Fuc residue of OPS consisting of tri-D-rhamnose repeats in the backbone. Epitope 2d alternated with 2a within the same LPS molecule and did not cross-react with epitope 1d. Serotypes O2a and O2d were observed in some strains correlating with the coexpression of the two chemotypes of OPS by the same strain. The serogroup O1-specific MAb Ps1a reacted weakly but definitely with all strains from serogroup 02. We propose serological formulas for serogroups O1 and 02 as well as for individual strains within these serogroups.