Lipopeptide phytotoxins produced by Pseudomonas syringae pv. syringae: comparison of the biosurfactant and ion channel-forming activities of syringopeptin and syringomycin

The maturational status of Adelta and C-fibers in the fetal rat spinal cord was examined using formalin-induced c-fos expression as a marker for neuronal activities. Awake 19-, 20-, and 21-day fetuses (FD) were injected ex utero with 5 microl of 10% formalin either into the ventral aspect of the forepaw or the hindpaw. FD 19 fetuses showed little response to the injection, but with increasing age, the fetuses exhibited more specific behaviors following injury of the paw. By FD 21, fetuses treated with formalin injection showed body curls and twitches, mouth opening, face wiping, and withdrawal of the injected paw. The anatomical data paralleled that of behavior; FD 19 animals expressed a small number of Fos labeled nuclei following the formalin injection that was not statistically different from control animals. The formalin-induced increase in Fos staining was first observed at FD 20 with a large increase in the number of Fos labeled cell occurring between FD 20 and 21. By FD 21, the pattern of Fos stained nuclei resembled that found in neonatal rats. There was constitutive bilateral staining in all untreated, saline and formalin injected fetuses that is unique to prenatal animals. Formalin treated fetuses showed constitutive level of staining in addition to the increase in the c-fos expression caused by formalin. We have thus demonstrated that, as indexed both by behavioral response and by Fos immunoreactivity, rat fetuses are capable of transmitting and responding to noxious input before birth.     

Ecological and genetic analysis of copper and streptomycin resistance in Pseudomonas syringae pv. syringae

Strains of Pseudomonas syringae pv. syringae resistant to copper, streptomycin, or both compounds were recovered from symptomless and diseased tissue of four woody hosts in three nurseries in Oklahoma. In strains resistant to copper and streptomycin (Cur Smr), resistance to both compounds was cotransferred with a single plasmid which was either 68, 190, or 220 kilobase pairs (kb). All Cus Smr strains contained a 68-kb conjugative plasmid. Cur Sms strains contained one plasmid which varied in size from 60 to 73 kb. All conjugative plasmids which transferred streptomycin resistance contained sequences homologous to the strA and strB Smr genes from the broad-host-range plasmid RSF1010. The Smr determinant was subsequently cloned from a 68-kb Cur Smr plasmid designated pPSR1. A restriction map detailing the organization of the homologous Smr genes from pPSR1 and RSF1010 and cloned Smr genes from P. syringae pv. papulans and Xanthomonas campestris pv. vesicatoria revealed the conservation of all sites studied. The Cur genes cloned from P. syringae pv. tomato PT23 and X. campestris pv. vesicatoria XV10 did not hybridize to the Cur plasmids identified in the present study, indicating that copper resistance in these P. syringae pv. syringae strains may be conferred by a distinct genetic determinant.     

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.     

Characterization of type IV pilus genes in Pseudomonas syringae pv. tomato DC3000

Many strains of Pseudomonas syringae produce retractile pili that act as receptors for lytic bacteriophage phi 6. As these are also characteristics of type IV pili, it was postulated that P. syringae may possess genes for type IV pilus biogenesis. A cosmid clone bank of P. syringae pv. tomato DC3000 genomic DNA was used to complement a mutant of Pseudomonas aeruginosa defective in the PilD (XcpA) prepilin peptidase gene by selection for restoration of extracellular protein secretion, a function also known to require PilD. A cosmid able to complement this mutant was also able to complement mutations in the pilB and pilC genes, suggesting that, if the organization of these genes is similar to that of P. aeruginosa, the cosmid may contain the P. syringae pilA. This was confirmed by sequencing a region from this plasmid that was shown to hybridize at low stringency to the P. aeruginosa pilA gene. The deduced P. syringae PilA polypeptide possesses the characteristic properties of the type IV pilins. Heterologous expression of the P. syringae pilA in P. aeruginosa was also shown, conferring not only phi 6 phage sensitivity to P. aeruginosa pilA mutants but also sensitivity to PO4, a lytic bacteriophage specific for the pilus of P. aeruginosa. This suggests that additional components might be present in the mature pilus of P. aeruginosa that are the true receptors for this phage. Chromosomal mutations in P. syringae pv. tomato DC3000 pilA and pilD genes were shown to abolish its sensitivity to bacteriophage phi 6. To determine the importance of P. syringae pilus in plant leaf interactions, these mutations were tested under laboratory and field conditions. Although little effect was seen on pathogenicity, culturable leaf-associated population sizes of the pilA mutant were significantly different from those of the wild-type parent. In addition, the expression of the DC3000 pilA gene appears to contribute to the UV tolerance of P. syringae and may play a role in survival on the plant leaf surface.     

A race-specific insertion of transposable element IS801 in Pseudomonas syringae pv. phaseolicola

To detect free zinc ions in the rat testes four rats were transcardially perfused with Na2S, and the seminiferous tubules from two other rats were incubated in Na2S. Sections from the two sources were autometallographically (AMG) developed, whereby zinc sulphide crystal lattices created in the tissue by the sulphide treatment were silver enhanced. Light microscopical analysis showed zinc ions in primary spermatogonia until the zygotene primary spermatocytes (stage I), in late pachytene spermatocytes (stages XII and XIII), and in late spermatids from step 15 to step 19 (stages I-VIII). The highest intensity of AMG grains was detected in the residual bodies and tails of step 19 spermatids. Grains were occasionally found in the cytoplasm of Leydig cells. Sections from animals treated with the chelator diethyldithiocarbamate prior to sulphide treatment showed a complete lack of AMG staining. At ultrastructural levels the AMG grains were found in smooth-surfaced endoplasmic reticulum of all spermatogonial stages, and in the acrosome, midpiece, and tail of late spermatids. The presence of zinc ions in preleptotene spermatocytes and cytoplasmic lobes of late spermatids suggests a specific role of free zinc at the onset of meiosis and at spermiation.     

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.     

In vitro inhibition of the growth of Gardnerella vaginalis by bacteriocins produced by strains of Pseudomonas aeruginosa

BACKGROUND: Pseudomonas aeruginosa with inhibitory capacity in vitro was studied on Gardnerella vaginalis strains. METHODS: Antimicrobial activity was demonstrated by inhibitory halos of bacterial growth on solid media by two methods: crossed streak and agar well diffusion. The inhibitory activity of this substance produced by P. aeruginosa was characterized as bacteriocin by: activity spectrum sensitivity proteolytic enzyme, chloroform, heat, pH, ultraviolet, irradiation effect and molecular weight. RESULTS: Four strains of P. aeruginosa producers of bacteriocins were chosen for this study and contacted with 40 strains of G. vaginalis. The producing strain D inhibited 70% of these G. vaginalis strains. The strains B and C inhibited 55% and 52.5%, respectively. The 3 strains presented a wide rank of action but the strain A had effect on a few strains of G. vaginalis. CONCLUSIONS: This work showed the inhibitory in vitro effect of bacteriocins of P. aeruginosa on strains of G. vaginalis. The results obtained suggest the probable topic application of bacteriocins as an alternative of conventional therapeutic on this infection biological control.     

Cloning of genes by mRNA differential display induced during the hypersensitive reaction of soybean after inoculation with Pseudomonas syringae pv. glycinea

Soybean (Glycine max [L.] Merr.) cell suspension cultures (cv. Williams 82) inoculated with the pathogenic bacteria Pseudomonas syringae pv. glycinea respond with a hypersensitive reaction (HR) when the bacteria express the avirulence gene avrA. A mRNA differential display was established for this system to allow the identification of genes induced during the HR. Six PCR-fragments (DD1-DD6) from the differential display analysis were identified, which are induced during the HR. Database searches revealed that the fragment DD1 encodes chalcone isomerase and DD2 was identified as ubiquitin. The fragment DD3 shares significant homology to the signalling molecule 14-3-3. The partial DD4 product is homologous to the enhancer of rudimentary from Drosophila and an uncharacterized homologue of it from Arabidopsis. The fragment DD5 is similar to glucose-6-phosphate dehydrogenase which provides NADPH to the cell. The PCR-product DD6 seems to be a new leucine-rich-repeat disease resistance gene from soybean, which is significantly induced during the HR. All of the identified genes are clearly induced during a HR in infected plants of the same cultivar, indicating that results from the cell culture model system can be transferred to intact plants. These studies show that complex mRNA differential display is a powerful tool to identify new induced gene in plant-pathogen interactions.     

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.     

In vitro efficacy and fungicidal activity of voriconazole against Aspergillus and Fusarium species

Twenty-nine Aspergillus isolates and 25 Fusarium isolates underwent in vitro antifungal susceptibility testing by a broth macrodilution procedure adapted from the National Committee for Clinical Laboratory Standards guidelines. The MIC50s of both voriconazole and amphotericin B were 0.5 microg/ml and 1 microg/ml against species of Aspergillus and Fusarium, respectively, while the MIC90s of both agents were 1 and 2 microg/ml. Voriconazole was more active in vitro than amphotericin B: the geometric mean MICs of voriconazole and amphotericin B against Aspergillus spp. were 0.36 microg/ml and 0.64 microg/ml, respectively. Voriconazole also demonstrated fungicidal activity against Aspergillus spp., with 86% (24/29) of isolates exhibiting minimum lethal concentrations of < or = 4 microg/ml.     

Biological activities of pseudomycin A, a lipodepsinonapeptide from Pseudomonas syringae MSU 16H

Similarly to other Pseudomonas lipodepsinonapeptides, pseudomycin A inhibits proton extrusion from maize roots, promotes closure of stomata in Vicia faba, necrosis of tobacco leaves, haemolysis of human erythrocytes, affects H(+)-ATPase activity and proton translocation in plasma membrane vesicles, and stimulates succinate respiration in pea mitochondria. In general, the biological activities of pseudomycin A are lower than those of syringomycin-E, the prototype member of this family of bacterial metabolities. This difference might depend on the diverse number and distribution of charged residues in the peptide moiety of these compounds.     

陕西关中地区猕猴桃溃疡病调查及其生物防治研究

[目的]掌握陕西关中地区猕猴桃溃疡病发病特点,筛选出对猕猴桃溃疡病有防治作用的植物内生放线菌.[方法]对陕西关中地区(杨陵、周至、眉县)13个村65个猕猴桃园中猕猴桃溃疡病发病特点进行调查分析,同时在242株植物内生放践菌中筛选猕猴桃溃疡病菌拮抗菌株并进行田间病害防治试验.[结果]陕西关中地区猕猴桃溃疡病的平均发病率达7.95%;猕猴桃品种与发病率密切相关.对猕猴桃溃疡菌有抑制活性的菌株有76株,其中35株的发酵液对猕猴桃溃疡菌的抑菌圈直径≥20 mm,TTASA5菌株对4种靶标细菌均具有抑制活性,而gCLA4菌株不仅对4种靶标细菌具有抑制活性,还对10种植物病原真菌有较强的抑制活性.田间试验结果表明,gcLA4菌株发酵液对猕猴桃溃疡病具有明显防效,21 d的相对防效可达66.7%.[结论]gclA4菌发酵液的抑菌谱广,gclA4菌具有开发成为一种广谱农用杀菌剂的潜力.     

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.     

In vivo effect of growth-inhibitor produced by Pseudomonas aeruginosa isolates and anti-pseudomonal drugs on model infection due to Pseudomonas aeruginosa

Pseudomonas aeruginosa Nos. 1 and 5, each co-existing growth-inhibitor-producing and -nonproducing cells, were used in this study. An equal number of both cells (each 10(8) CFU/mouse) was challenged intraperitoneally to mice, and these cells in the heart blood and kidneys of mice were determined. Furthermore, the effect of piperacillin, ceftazidime and sisomicin on the cell distribution in mice was studied in the model infection due to P. aeruginosa Nos. 1 and 5. As a control experiment both cells of P. aeruginosa No. 1 were each challenged intraperitoneally at a dose of 10(8) CFU/mouse to mice of two groups, but there were no marked differences between the two types in cell counts of the heart blood or kidneys 9 hours after challenge. When a concomitant challenge of both cells (each 10(8) CFU/mouse) was performed in mice, the number of growth-inhibitor-producing cells of the heart blood and kidneys was about 100 times greater than that of the non-producing cells. These in vivo results were well comparable to the previous in vitro results and indicated that the inhibitor affected the invasion of the non-producing bacteria in the body in the model infection due to P. aeruginosa isolates consisting of the two types of cells. Similar results were obtained in mice with the model infection due to P. aeruginosa No. 5. Anti-pseudomonal drugs such as piperacillin (50 mg/mouse) and ceftazidime (50 mg/mouse) and sisomicin (1 mg/mouse) were given intramuscularly to mice infected concomitantly with both cells of P. aeruginosa No. 1.(ABSTRACT TRUNCATED AT 250 WORDS)     

The Pseudomonas syringae pv. tomato HrpW protein has domains similar to harpins and pectate lyases and can elicit the plant hypersensitive response and bind to pectate

The host-specific plant pathogen Pseudomonas syringae elicits the hypersensitive response (HR) in nonhost plants and secretes the HrpZ harpin in culture via the Hrp (type III) secretion system. Previous genetic evidence suggested the existence of another harpin gene in the P. syringae genome. hrpW was found in a region adjacent to the hrp cluster in P. syringae pv. tomato DC3000. hrpW encodes a 42. 9-kDa protein with domains resembling harpins and pectate lyases (Pels), respectively. HrpW has key properties of harpins. It is heat stable and glycine rich, lacks cysteine, is secreted by the Hrp system, and is able to elicit the HR when infiltrated into tobacco leaf tissue. The harpin domain (amino acids 1 to 186) has six glycine-rich repeats of a repeated sequence found in HrpZ, and a purified HrpW harpin domain fragment possessed HR elicitor activity. In contrast, the HrpW Pel domain (amino acids 187 to 425) is similar to Pels from Nectria haematococca, Erwinia carotovora, Erwinia chrysanthemi, and Bacillus subtilis, and a purified Pel domain fragment did not elicit the HR. Neither this fragment nor the full-length HrpW showed Pel activity in A230 assays under a variety of reaction conditions, but the Pel fragment bound to calcium pectate, a major constituent of the plant cell wall. The DNA sequence of the P. syringae pv. syringae B728a hrpW was also determined. The Pel domains of the two predicted HrpW proteins were 85% identical, whereas the harpin domains were only 53% identical. Sequences hybridizing at high stringency with the P. syringae pv. tomato hrpW were found in other P. syringae pathovars, Pseudomonas viridiflava, Ralstonia (Pseudomonas) solanacearum, and Xanthomonas campestris. DeltahrpZ::nptII or hrpW::OmegaSpr P. syringae pv. tomato mutants were little reduced in HR elicitation activity in tobacco, whereas this activity was significantly reduced in a hrpZ hrpW double mutant. These features of hrpW and its product suggest that P. syringae produces multiple harpins and that the target of these proteins is in the plant cell wall.     

In vitro and in vivo hematopoietic activities of Betafectin PGG-glucan

Betafectin PGG-glucan is a novel beta-(1,3)glucan that has broad-spectrum anti-infective activities without cytokine induction. Here we report that PGG-glucan also has both in vitro and in vivo hematopoietic activities. In vitro studies with bone marrow target cells from the C3H/HeN mouse revealed that although PGG-glucan alone had no direct effect on hematopoietic colony-forming cell (CFC) growth, when combined with granulocyte colony-stimulating factor (CSF) or granulocyte-macrophage CSF, it increased CFC numbers 1.5- to 2.0-fold over those obtained with CSFs alone. Bone marrow cells cultured for high-proliferative-potential CFCs in the presence of interleukin (IL)-1, IL-3, macrophage CSF, and stem cell factor (SCF), or cultured for erythroid burst-forming units in the presence of IL-3, SCF, and erythropoietin, also exhibited enhanced growth in the presence of PGG-glucan. The synergistic effect of PGG-glucan was specific and could be abrogated by anti-PGG-glucan antibody. The ability of PGG-glucan to modulate hematopoiesis in vivo was evaluated in myelosuppressed rodents and primates. C3H/HeN female mice were intravenously administered saline solution or PGG-glucan (0.5 mg/kg) 24 hours before the intraperitoneal administration of cyclophosphamide (200 mg/kg), and the recovery of bone marrow cellularity and granulocyte-macrophage progenitor cells was evaluated on days 4 and 8 after cyclophosphamide treatment. At both time points, enhanced hematopoietic recovery was observed in PGG-glucan-treated mice compared with saline-treated control mice. In a final series of in vivo experiments, we evaluated the ability of therapeutically administered PGG-glucan to enhance hematopoietic recovery in cyclophosphamide-treated cynomolgus monkeys. Monkeys received intravenous infusions of cyclophosphamide (55 mg/kg) on days 1 and 2, followed on days 3 and 10 by intravenous infusion of PGG-glucan (0.5, 1.0, or 2.0 mg/kg). Compared with those in saline-treated monkeys, accelerated white blood cell recovery and a reduction in the median duration of neutropenia were observed in PGG-glucan-treated monkeys. These studies illustrate that PGG-glucan has both in vitro and in vivo hematopoietic activities and that this agent may be useful in the prevention and/or treatment of chemotherapy-associated myelosuppression.     

In vitro cytotoxic effects of vacuolating cytotoxin produced by clinical isolates of Helicobacter pylori

The vacuolating cytotoxin produced by Helicobacter pylori is considered to be one virulence factor causing peptic ulceration. In this study, we examined the activity of vacuolating cytotoxin in induction of intracellular vacuolation of rabbit gastric epithelial cells (RGECs). We used culture supernatants of H. pylori as a source of vacuolating cytotoxin and quantitated cytotoxic activity by the MTT method. Intracellular vacuolation of RGECs was observed in the presence of 36 of 57 (63%) clinically isolated H. pylori strains. However, there were no differences in the incidence of H. pylori strains with positive vacuolating cytotoxin (Tox+) among patients with gastritis, gastric ulcers or duodenal ulcers. The MTT assay showed that the cytotoxic activity of H. pylori supernatants obtained from patients with gastric ulcers was significantly higher than in patients with gastritis (p < 0.01), but was not different to duodenal ulcer patient supernatants. Similar results were also observed in Tox+ isolates, however, there were no significant differences between patients with regard to the incidence of vacuolating cytotoxin-negative isolates. Although our data may not indicate a clear correlation between prevalence of vacuolating cytotoxin and clinical manifestations, they suggest that H. pylori harboring vacuolating cytotoxin may particularly induce damage to the gastric epithelium in patients with gastric ulcers.