Ice Nucleation Induced by Pseudomonas syringae

Broth cultures of suspensions of Pseudomonas syringae isolated from decaying alder leaves (Alnus tenuifolia) were found to freeze at very warm (-1.8 to -3.8 C) temperatures. The initiation of freezing appears associated with the intact cell and not with extracellular material. Chemical treatments and physical destruction of the cell destroy activity. Bacteria must be in concentrations of approximately 10(6)/ml before freezing at warm temperatures occurs.     

Apoplexy of Peach Trees Caused by Pseudomonas viridiflava

A sudden apoplexy of young nectarine trees was observed during early autumn 1995 in Piedmont (northern Italy). At collar level, extensive wet necrosis of the tissues beneath the bark was observed. When the infection girdled the trunk, the tree died. LOPAT tests, fluorescence on single-carbon sources and comparison of whole-cell protein profiles with type-strains indicated that the bacterial isolates belong to Pseudomonas viridiflava and P. syringae pv. syringae. In the pathogenicity tests, the first bacterium incited symptoms similar to those observed in the field. This is the first record of P. viridiflava as a pathogen of peach.     

Effect of Plant Species and Environmental Conditions on Ice Nucleation Activity of Pseudomonas syringae on Leaves

Selected plant species and environmental conditions were investigated for their influences on expression of ice nucleation activity by 15 Pseudomonas syringae strains grown on plants in constant-temperature growth chamber studies. Ice nucleation frequencies (INFs), the fraction of cells that expressed ice nucleation at −5 or −9°C, of individual strains varied greatly, both on plants and in culture. This suggests that the probability of frost injury, which is proportional to the number of ice nuclei on leaf surfaces, is strongly determined by the particular bacterial strains that are present on a leaf surface. The INFs of strains were generally higher when they were grown on plants than when they were grown in culture. In addition, INFs in culture did not correlate closely with INFs on plants, suggesting that frost injury prediction should be based on INF measurements of cells grown on plants rather than in culture. The relative INFs of individual strains varied with plant host and environment. However, none of seven plant species tested optimized the INFs of all 15 strains. Similarly, incubation for 48 h at near 100% relative humidity with short photoperiods did not always decrease the INF when compared with a 72 h, 40% relative humidity, long-photoperiod incubation. Pathogenic strains on susceptible hosts were not associated with higher or lower INFs relative to their INFs on nonsusceptible plant species. The ice nucleation activity of individual bacterial strains on plants therefore appears to be controlled by complex and interacting factors such as strain genotype, environment, and host plant species.     

Diel Variation in Population Size and Ice Nucleation Activity of Pseudomonas syringae on Snap Bean Leaflets

The extent to which diel changes in the physical environment affect changes in population size and ice nucleation activity of Pseudomonas syringae on snap bean leaflets was determined under field conditions. To estimate bacterial population size and ice nucleation activity, bean leaflets were harvested at 2-h intervals during each of three 26-h periods. A tube nucleation test was used to assay individual leaflets for ice nuclei. Population sizes of P. syringae were determined by dilution plating of leaflet homogenates. The overall diel changes in P. syringae population sizes differed during each of the 26-h periods. In one 26-h period, there was a continuous increase in the logarithm of P. syringae population size despite intense solar radiation, absence of free moisture on leaf surfaces, and low relative humidity during the day. A mean doubling time of approximately 4.9 h was estimated for the 28-fold increase in P. syringae population size that occurred from 0900 to 0900 h during the 26-h period. However, doubling times of 3.3 and 1.9 h occurred briefly during this period from 1700 to 2300 h and from 0100 to 0700 h, respectively. Thus, growth rates of P. syringae in association with leaves in the field were of the same order of magnitude as optimal rates measured in the laboratory. The frequency with which leaflets bore ice nuclei active at −2.0, −2.2, and −2.5°C varied greatly within each 26-h period. These large diel changes were inversely correlated primarily with the diel changes in air temperature and reflected changes in nucleation frequency rather than changes in population size of P. syringae. Thus, the response of bacterial ice nucleation activity to the physical environment was distinct from the changes in population size of ice nucleation-active P. syringae.     

Pseudomonas viridiflava and P. syringae--natural pathogens of Arabidopsis thaliana

We report the isolation and identification of two natural pathogens of Arabidopsis thaliana, Pseudomonas viridiflava and Pseudomonas syringae, in the midwestern United States. P. viridiflava was found in six of seven surveyed Arabidopsis thaliana populations. We confirmed the presence in the isolates of the critical pathogenicity genes hrpS and hrpL. The pathogenicity of these isolates was verified by estimating in planta bacterial growth rates and by testing for disease symptoms and hypersensitive responses to A. thaliana. Infection of 21 A. thaliana ecotypes with six locally collected P. viridiflava isolates and with one P. syringae isolate showed both compatible (disease) and incompatible (resistance) responses. Significant variation in response to infection was evident among Arabidopsis ecotypes, both in terms of symptom development and in planta bacterial growth. The ability to grow and cause disease symptoms on particular ecotypes also varied for some P. viridiflava isolates. We believe that these pathogens will provide a powerful system for exploring coevolution in natural plant-pathogen interactions.     

冰核活性基因及其作报告基因的应用研究进展

国内外已经克隆了8个冰核活性基因,可用作报告基因,主要应用在植物－微生物互作研究、营养元素利用研究、病原微生物高敏检测和各种用途的细菌细胞表面展示等领域。     

Epiphytic survival of Pseudomonas viridiflava on tomato and selected weed species

A rifampicin-nalidixic acid mutant of Pseudomonas viridiflava (PV) was studied in the field and greenhouse with respect to its epiphytic survival on the roots and foliage of a susceptible (FM 6203) and resistant (Ontario 7710) tomato cultivar and 16 weed species. In the field, populations varied between years, which was attributed to differences in environmental conditions. Hot, dry conditions caused rapid decline or elimination of populations. Some hosts were more conducive than others in promoting epiphytic growth, and generally, roots were better survival sites than foliage. Some hosts such as johnsongrass, lambsquarters, pigweed, prickly sida, and red sorrel had no detectable populations of PV on foliage 2 weeks after inoculation. (Plants had been misted with a 10⁸ cfu/ml suspension until run off occurred.) PV was recovered at week 4 on the foliage of the two tomato cultivars, beggarweed, jimsonweed, morning glory, smooth vetch, and wild mustard, and was recovered until week 16 on roots of buckhorn plantain in the field and for the same period on the ground cherry in the field and greenhouse. In scanning electron microscopy studies, PV was observed to survive as microcolonies in depressions between epidermal cells, around trichomes, along veins, and sometimes around stomates of tomato and beggarweed. Bacterial cells sometimes were held together and to the leaf surface by fibril-like strands. These studies show that PV does have an epiphytic stage on both tomato and certain weed species. However, the epidemiological significance of the epiphytic stage is probably dependent on environmental conditions.     

Large-scale Production and Purification of an Erwinia ananas Ice Nucleation Protein and Evaluation of Its Ice Nucleation Activity

The ice nucleation-active protein of Erwinia ananas IN-10 (inaA protein) was over-expressed as inclusion bodies in Escherichia coli in a yield of 15.3 mg of inaA protein from 60 mg of bacterial cells on a dry-matter basis. The inaA protein was purified from the inclusion bodies by solubilization with detergents to obtain a protein preparation free from sugar and lipid. This preparation had a distinct ice nucleation activity, indicating that the inaA protein per se is able to act as a nucleus.     

Ice-nucleation Gene of Pseudomonas viridiflava KUIN-2 Coded on a Plasmid

Mepanipyrim, N-(4-methyl-6-prop-1-ynylpyrimidin-2-yl)aniline, diminished the cell surface expression of envelope glycoproteins of Newcastle disease and vesicular stomatitis viruses at concentrations where their synthesis was not profoundly affected. Intoxication by diphtheria toxin and ricin and recycling of transferrin were not affected even when cells were treated with mepanipyrim for 2 h before the addition of these probes, indicating that mepanipyrim does not act on the endocytic and recycling pathways of these proteins. Metabolic conversion of C₆-NBD-ceramide to sphingomyelin and its back-exchange to the medium was also not affected, but synthesis and back-exchange of C₆-NBD-glucosylceramide were greatly influenced, and an accumulation of LDL-derived, unesterified cholesterol was induced by the drug. These results are discussed relating to the site(s) of action of mepanipyrim.     

The Ice Nucleation Gene from Pseudomonas syringae as a Sensitive Gene Reporter for Promoter Analysis in Zymomonas mobilis

The expression of the ice nucleation gene inaZ from Pseudomonas syringae in Zymomonas mobilis strains under the control of three different promoters was investigated to establish the utility of the gene as a reporter and examine the possible use of the organism as a source of ice nuclei for biotechnological applications. A promoterless version of the inaZ gene was placed under the control of three different promoters: P(infpdc) (pyruvate decarboxylase), a homologous strong promoter from Z. mobilis; P(infbla) ((beta)-lactamase) of plasmid pBR325; and P(infhrpR), the promoter of hrpR, a regulatory gene from P. syringae pv. phaseolicola. The apparent strengths of all three promoters, measured by quantifying the ice nucleation activity at -9 deg C, were lower in Z. mobilis than in Escherichia coli. The levels of ice nucleation activity expressed under the P(infpdc) promoter were significantly higher than those obtained with the two heterologous promoters in Z. mobilis. Plasmid pCG4521 (RK2 replicon) gave much lower levels of ice nucleation activity when propagated in strain uvs-51, a plasmid instability mutant of Z. mobilis, compared with the wild-type strain. The ice nucleation activity in Z. mobilis cultures showed unusual partitioning in that the culture supernatants obtained after low-speed centrifugation contained the majority of ice nuclei. Analysis of the ice nucleation spectra revealed that the cell pellets contained both "warm" and "cold" nuclei, while the culture supernatant contained primarily cold nuclei, suggesting that the cold nucleus activity may be extracellular. However, all nucleation activity was retained by 0.22-(mu)m-pore-size filters.     

Inactivation of Kanamycin,Neomycin, and Streptomycin by Enzymes Obtained in Cells of Pseudomonas aeruginosa

Ten strains of Pseudomonas aeruginosa were disrupted and centrifuged. The supernatant fluids from centrifugation at 105,000 × g contained enzymes inactivating kanamycin, neomycin, and streptomycin in the presence of adenosine triphosphate. Kanamycin-inactivating enzyme was precipitated with ammonium sulfate at 66% of saturated concentration, and the inactivated kanamycin was shown to be kanamycin-3′-phosphate in which the C-3 hydroxyl group of 6-amino-6-deoxy-d-glucose moiety was phosphorylated. This is identical with kanamycin inactivated by Escherichia coli carrying R factor. Streptomycin-inactivating enzyme was precipitated with ammonium sulfate at 33% of saturated concentration.     

Survival of the Halophilic Archaeon Halovarius luteus after Desiccation,Simulated Martian UV Radiation and Vacuum in Comparison to Bacillus atrophaeus

Origins of Life and Evolution of Biospheres - Extraterrestrial environments influence the biochemistry of organisms through a variety of factors, including high levels of radiation and vacuum,...     

Genetic diversity, recombination and cryptic clades in Pseudomonas viridiflava infecting natural populations of Arabidopsis thaliana

Species-level genetic diversity and recombination in bacterial pathogens of wild plant populations have been nearly unexplored. Pseudomonas viridiflava is a common natural bacterial pathogen of Arabidopsis thaliana, for which pathogen defense genes and mechanisms are becoming increasing well known. The genetic variation contained within a worldwide sample of P. viridiflava collected from wild populations of A. thaliana was investigated using five genomic sequence fragments totaling 2.3 kb. Two distinct and deeply diverged clades were found within the P. viridiflava sample and in close proximity in multiple populations, each genetically diverse with synonymous variation as high as 9.3% in one of these clades. Within clades, there is evidence of frequent recombination within and between each sequenced locus and little geographic differentiation. Isolates from both clades were also found in a small sample of other herbaceous species in Midwest populations, indicating a possibly broad host range for P. viridiflava. The high levels of genetic variation and recombination together with a lack of geographic differentiation in this pathogen distinguish it from other bacterial plant pathogens for which intraspecific variation has been examined.     

The ice Nucleation Activity of Pseudomonas fluorescens Migula and its Inhibition by Various Chemicals

The ice nucleation activity (INA) of three strains of Pseudomonas fluorecens, nos 553, 554 and 606, isolated by the Institute for Pathogen Diagnostics in Ascherleben, Germany, was determined. Under equal growth conditions and at given test temperatures the ice nucleation frequency spectra of the isolates differed slightly. The fraction of cells which acted as ice nuclei increased with falling temperatures. Below ?5°C the nucleation frequency rose from 10^-8 to 10^-3. Between, 0 and ?10°C only a fraction of approximately 2 to 5 × 10^-3 cells performed ice nucleation activity. Fifteen newly synthesized chemicals showed no or only a very slight intrinsic INA at ?5°C and ?7°C. The compounds were used as antinucleators against INA-exhibiting bacteria. In INA-exhibiting suspensions of isolate 553 bacterial ice nuclei were reduced after treatment with the 15 compounds. Dependent on the compounds, a nucleation frequency of ?8.32 to ?5.10 was detected at ?5°C. At ?7°C, the frequency amounted to ?7.89 to ?5.05. As the temperature was lowered to ?10°C in bacterial suspensions which were treated with 9 (of the 15) compounds, a remainder of 1.79 to 5.91 × 10^-6 cells retained ice nucleation activity. The most pronounced inhibitory effect was noted for the compounds 1989/6255, 1989/6436 and 1990/6158. In a 10-fold dilution of isolate 553 the compound 1989/6153 inhibited ice nucleation between 0 and 10°C so strongly that it was about 100 times below the control. The ‘tube-freezing’ method showed that on excised corn leaves treated with 1989/6259 and 1990/6155, the bacterial INA decreased while the super-cooling was more pronounced. ‘Frostgard’, 1986/6205, 1986/6199 and 1989/6259 inhibited most INA-exhibiting bacteria on corn seedlings. Compared to inoculated plants, a significantly higher percentage of treated plants survived at ?2 and ?3°C.     

The role of pectate lyase and the jasmonic acid defense response in Pseudomonas viridiflava virulence

Pseudomonas viridiflava is a common pathogen of Arabidopsis thaliana in wild populations, yet very little is known about mechanisms of resistance and virulence in this interaction. We examined the induced defense response of A. thaliana to several strains of P. viridiflava collected from this host by quantifying the expression of PR-1 and LOX2/PDF1.2, which serve as markers for induction of the salicylic and jasmonic acid (JA) pathways, respectively. Growth of these strains then was assessed on Col-0, the fad3/7/8 and coil-1 mutants deficient in JA- and ethylene (ET)-induced defense responses, and the sid2-1 mutant deficient in salicylic acid-induced defense responses. All strains of P. viridiflava induced high expression of LOX2 and PDF1.2 on Col-0. In contrast, PR-1 expression was delayed and reduced relative to PDF1.2 expression. Additionally, three of four P. viridiflava strains were more virulent on fad3/7/8 relative to Col-0, whereas all strains were more virulent on coil-1 relative to Col-0, indicating that P. viridiflava generally may be suppressed by JA/ET-mediated defense responses. In contrast, no increase in the growth of P. viridiflava strains was observed in the sid2-1 mutant relative to Col-0. Parallel experiments were performed with the closely related P. syringae pv. tomato for comparative purposes. In addition, we assessed the role of pectate lyase and the alternative sigma factor HrpL in P. viridiflava virulence on A. thaliana and found that pectate lyase activity is correlated with virulence, whereas the removal of pectate lyase or HrpL significantly reduced virulence.     

Effects of UV-B Radiation and Periodic Desiccation on the Morphogenesis of the Edible Terrestrial Cyanobacterium Nostoc flagelliforme

The terrestrial cyanobacterium Nostoc flagelliforme Berk. et M. A. Curtis has been a popular food and herbal ingredient for hundreds of years. To meet great market demand and protect the local ecosystem, for decades researchers have tried to cultivate N. flagelliforme but have failed to get macroscopic filamentous thalli. In this study, single trichomes with 50 to 200 vegetative cells were induced from free-living cells by low light and used to investigate the morphogenesis of N. flagelliforme under low UV-B radiation and periodic desiccation. Low-fluence-rate UV-B (0.1 W m(-2)) did not inhibit trichome growth; however, it significantly increased the synthesis of extracellular polysaccharides and mycosporine-like amino acids and promoted sheath formation outside the trichomes. Under low UV-B radiation, single trichomes developed into filamentous thalli more than 1 cm long after 28 days of cultivation, most of which grew separately in liquid BG11 medium. With periodic desiccation treatment, the single trichomes formed flat or banded thalli that grew up to 2 cm long after 3 months on solid BG11 medium. When trichomes were cultivated on solid BG11 medium with alternate treatments of low UV-B and periodic desiccation, dark and scraggly filamentous thalli that grew up to about 3 cm in length after 40 days were obtained. In addition, the cultivation of trichomes on nitrogen-deficient solid BG11 medium (BG11(0)) suggested that nitrogen availability could affect the color and lubricity of newly developed thalli. This study provides promising techniques for artificial cultivation of N. flagelliforme in the future.