Effects of aqueous ozone on Pseudomonas syringae viability and ice nucleating activity

This study reports the impact of different ozone treatments on a Pseudomonas syringae strain known for its ice nucleation activity (INA). Ozone is a very powerful germicidal agent used for water treatment. The effect of ozone on viability and on cultivability of P. syringae was determined by flow cytometry analysis and by plate counting respectively. The impact of ozone on the outer membrane using the INA as marker was investigated by the drop freezing technique.The destruction curve followed a shoulder pattern with a slight reduction in population with CT values between 0 and 8 min. For an initial population of 9.3 log CFU mL^?1, the cultivability was lost starting at 14 min and a loss of viability was observed after 16 min of ozone treatment at 0.45 mg L^?1. Microscopic observations at this point revealed whole but aggregated bacilli. INA decreased after 8 min of ozone treatment but did not disappear. This decrease could be due to the progressive disruption of ice nucleating sites in the outer membrane. It was however partially restored after long storage at 4 °C of dead cells treated for 16 min.     

Ice nucleating activity of Pseudomonas syringae and Erwinia herbicola.

Chemical and biological properties of the ice nucleating sites of Pseudomonas syringae, strain C-9, and Erwinia herbicola have been characterized. The ice nucleating activity (INA) for both bacteria was unchanged in buffers ranging from pH 5.0 to 9.2, suggesting that there were no essential groups for which a change in charge in this range was critical. The INA of both bacteria was also unaffected by the addition of metal chelating compounds. Borate compounds and certain lectins markedly inhibited the INA of both types of bacterial cells. Butyl borate was not an inhibitor, but borate, phenyl borate, and m-nitrophenyl borate were, in order, increasingly potent inhibitors. These compounds have a similar order of affinity for cis hydroxyls, particularly for those found on sugars. Lentil lectin and fava bean lectin, which have binding sites for mannose or glucose, inhibited the INA of both bacteria. All other lectins examined had no effect. The inhibition of INA by these two types of reagents indicate that sugar-like groups are at or near the ice nucleating site. Sulfhydryl reagents were potent inhibitors of the INA of both bacteria. When treated with N-ethylmaleimide, p-hydroxymercuribenzoate, or iodoacetamide, the INA was irreversibly inhibited by 99%. The kinetics of inactivation with N-ethylmaleimide suggested that E. herbicola cells have at least two separate ice nucleating sites, whereas P. syringae cells have possibly four or more separate sites. The effect of infection with a virulent phage (Erh 1) on the INA of E. herbicola was examined. After multiple infection of a bacterial culture the INA was unchanged until 40 to 45 min, which was midway through the 95-min latent period. At that time, the INA activity began falling and 99% of the INA was lost by 55 min after infection, well before any cells had lysed. This decrease in INA before lysis is attributed to phage-induced changes in the cell wall.     

A method for quantitative determination of ice nucleating agents in insect hemolymph

The relationship between the concentration of insect hemolymph ice nucleators in samples of 0.9% NaCl solution and the supercooling points of the samples was determined by using a dilution technique. The supercooling points were only moderately reduced following dilution by a factor of up to 10³, whereas dilution beyond this point caused a marked drop in the supercooling points. The dilution factor corresponding to a 50% reduction in the nucleating activity of native hemolymph is taken as a measure of the concentration of ice nucleators in native hemolymph.This method was used to determine the concentration of ice nucleators in the hemolymph of Eurosta solidaginis larvae from Minnesota and Texas, acclimated to different temperatures. Significant levels of nucleators were found only in larvae from Minnesota, and +5 °C was found to be the optimal temperature for nucleator formation. This comparatively high temperature optimum is interpreted as a physiological adaptation, ensuring sufficient nucleator levels in the hemolymph by the time of the first exposure to freezing temperatures in the winter.     

Phospholipid requirement for expression of ice nuclei in Pseudomonas syringae and in vitro

Delipidation of partially purified outer membranes of Pseudomonas syringae by various delipidating agents resulted in a significant loss of ice nucleation activity associated with the cell envelopes of this and other ice nucleation active bacteria. Lipopolysaccharide depletion of such membranes caused no reduction in ice nucleation activity. Both phospholipid content and ice nucleation activity of membranes were decreased by a similar fractional amount with time after treatment with phospholipase A2. A proportional quantitative relationship between loss of ice nucleation activity and lipid removal with increasing concentrations of sodium cholate and sodium dodecyl sulfate (SDS) was also observed. Significant linear relationships between the amount of lipid removed by phospholipase A2, sodium cholate, and SDS and the loss of ice nucleation activity in P. syringae outer membranes were observed. However, the slopes of these linear relationships for membranes treated with phospholipase A2 (m = 0.80), SDS (m = 0.94), and sodium cholate (m = 0.53) differed. The lower slope value for cholate-treated membranes indicated a partial substitution of sodium cholate for the phospholipids removed. The ice nucleation activity of delipidated outer membranes was restored by reconstitution with various phospholipids in a cholate dialysis procedure. Lipid classes differed in their ability to restore ice nucleation activity to sodium cholate-treated outer membranes. These results suggest that a hydrophobic environment provided either by lipids or certain detergent micelles is required for proper assembly and structural organization of an oligomeric ice protein complex enabling its expression as an ice nucleus.     

The consensus sequence of ice nucleation proteins from Erwinia herbicola, Pseudomonas fluorescens and Pseudomonas syringae

The consensus sequence of three bacterial ice nucleation proteins was determined by extrapolation from the nucleotide (nt) sequences of three ice nucleation-encoding genes, iceE (presented here), inaW and inaZ. The three proteins possess considerable similarity, so that a preferred amino acid is shown in most positions of the consensus. The corresponding genes show considerable divergence in the third nt positions of synonymous codons, suggesting that the proteins' conserved features have been maintained by selection. Therefore, the consensus sequence is likely to represent the components of primary structure most important to the ice nucleation function.     

Immunological characterization of ice nucleation proteins from Pseudomonas syringae, Pseudomonas fluorescens, and Erwinia herbicola.

Antibodies were raised against the InaW protein, the product of the ice nucleation gene of Pseudomonas fluorescens MS1650, after protein isolation from an Escherichia coli clone. On Western blots (immunoblots), these antibodies recognized InaW protein and InaZ protein (the ice nucleation gene product of Pseudomonas syringae S203), produced by both E. coli clones and the source organisms. The InaZ protein appeared in P. syringae S203 during stationary phase; its appearance was correlated with the appearance of the ice nucleation-active phenotype. In contrast, the InaW protein occurred at relatively constant levels throughout the growth phases of P. fluorescens MS1650; the ice nucleation activity was also constant. Western analyses of membrane preparations of P. syringae PS31 and Erwinia herbicola MS3000 with this antibody revealed proteins which were synthesized with development of the nucleating phenotype. In these species the presence or absence of the nucleating phenotype was controlled by manipulation of culture conditions. In all nucleation-positive cultures examined, cross-reacting low-molecular-weight bands were observed; these bands appeared to be products of proteolytic degradation of ice nucleation proteins. The proteolysis pattern of InaZ protein seen on Western blots showed a periodic pattern of fragment sizes, suggesting a highly repetitive site for protease action. A periodic primary structure is predicted by the DNA sequence of the inaZ gene.     

Localization of ice nucleation activity and the iceC gene product in Pseudomonas syringae and Escherichia coli

Ice nucleation activity and the iceC gene product were quantified in different subcellular fractions of the Pseudomonas syringae source strain and in Escherichia coli containing the cloned iceC gene to determine the activity of this protein in different subcellular locations. Ice nuclei were nearly completely retained during isolation of cell envelopes but exhibited a decrease in the temperature at which they were expressed. Ice nucleation activity was found in Triton X-100 insoluble membrane fragments as well as in slowly sedimenting and high-density membrane fragments. Nearly all ice nucleation activity was associated with the outer membrane because the partitioning of 3-ketodeoxyoctonate (a lipopolysaccharide component) and ice nuclei in cell fractions were similar to and opposite that of NADH oxidase (a cytoplasmic membrane component). The iceC gene product had an apparent mass of 150,000 Da based on migration in SDS-polyacrylamide gels. This protein was not found in soluble cell components. Nearly all of the iceC gene product, which occurred in low abundance, was associated with the outer membrane of both P. syringae and E. coli. Therefore, the iceC gene product is located at and is maximally active in or on the outer membrane of cells of the source strain and heterologous strains.     

Comparison of strains of Pseudomonas syringae pv. savastanoi from olive leaves and knots

A collection of strains of Pseudomonas syringae pv. savastanoi was subjected to numeric phenetic analysis of 60 characters using unweighted average linkage on the simple matching coefficient. Most strains recovered by washing random leaves in April and October shared lower similarity values between themselves than with the majority of those isolated from 6-month-old knots in October and April, respectively.     

Differences between Pseudomonas syringae pv. syringae B728a and Pantoea agglomerans BRT98 in epiphytic and endophytic colonization of leaves

The leaf colonization strategies of two bacterial strains were investigated. The foliar pathogen Pseudomonas syringae pv. syringae strain B728a and the nonpathogen Pantoea agglomerans strain BRT98 were marked with a green fluorescent protein, and surface (epiphytic) and subsurface (endophytic) sites of bean and maize leaves in the laboratory and the field were monitored to see if populations of these strains developed. The populations were monitored using both fluorescence microscopy and counts of culturable cells recovered from nonsterilized and surface-sterilized leaves. The P. agglomerans strain exclusively colonized epiphytic sites on the two plant species. Under favorable conditions, the P. agglomerans strain formed aggregates that often extended over multiple epidermal cells. The P. syringae pv. syringae strain established epiphytic and endophytic populations on asymptomatic leaves of the two plant species in the field, with most of the P. syringae pv. syringae B728a cells remaining in epiphytic sites of the maize leaves and an increasing number occupying endophytic sites of the bean leaves in the 15-day monitoring period. The epiphytic P. syringae pv. syringae B728a populations appeared to originate primarily from multiplication in surface sites rather than from the movement of cells from subsurface to surface sites. The endophytic P. syringae pv. syringae B728a populations appeared to originate primarily from inward movement through the stomata, with higher levels of multiplication occurring in bean than in maize. A rainstorm involving a high raindrop momentum was associated with rapid growth of the P. agglomerans strain on both plant species and with rapid growth of both the epiphytic and endophytic populations of the P. syringae pv. syringae strain on bean but not with growth of the P. syringae pv. syringae strain on maize. These results demonstrate that the two bacterial strains employed distinct colonization strategies and that the epiphytic and endophytic population dynamics of the pathogenic P. syringae pv. syringae strain were dependent on the plant species, whereas those of the nonpathogenic P. agglomerans strain were not.     

A simple and efficient PCR method for the specific detection of Pseudomonas syringae pv phaseolicola in bean seeds

Using Southern blot hybridizations, it was found that the gene encoding the phaseolotoxin-insensitive ornithyl carbamoyl transferase (argK) was specific for Pseudomonas syringae pv. phaseolicola, the causal agent of the halo-blight disease. Based on these findings, a PCR protocol was developed for the specific detection of P.syringae. pv. phaseolicola in water-extracts of soaked bean seed. For this PCR protocol, two oligonucleotide primers were designed, based on the sequence of argK, which allowed the detection of a specific 1kb fragment. The protocol is simple since PCR was directly applied to bacterial suspensions, thus avoiding DNA extraction. The sensitivity of detection was increased by allowing the bacteria present in seed extracts to multiply in semi-selective media for 18h prior to PCR amplification. The detection threshold by visual detection using ethidium bromide staining was one naturally infected seed in lots of 400 to 600 seeds.     

Up-regulation and localization of asparagine synthetase in tomato leaves infected by the bacterial pathogen Pseudomonas syringae

Nitrogen metabolism is one aspect of basic metabolism, which is still quite unknown in the field of plant-pathogen interactions. Evidence derived from previous studies conducted in our laboratory strongly suggests that during microbial pathogenesis an important nitrogen mobilization process takes place in diseased tissues. Here we describe the expression pattern of asparagine synthetase (AS; EC 6.3.5.4) in tomato leaves infected by the bacterial pathogen Pseudomonas syringae pv. tomato. Using an homologous AS cDNA probe isolated by RT-PCR from infected leaves, we have observed a high level induction of AS expression during the course of infection. Concomitantly, a single AS polypeptide also accumulated in response to bacterial infection. Furthermore, immunohistochemical analysis of AS in infected leaves revealed a strong immunostaining in phloem cells of the main vascular bundles and in secondary veins of the leaf blade. These data correlate with those previously reported for expression of a cytosolic isoform of glutamine synthetase (GS1) also induced during development of the infectious process. Taken together, our results suggest the existence of a GS1/AS pathway representing a metabolic route for transferring ammonium released from protein catabolism into asparagine, an amino acid that may have a major role in nitrogen mobilization from diseased tissues.     

A note on Pseudomonas syringae pv. berberidis infections of Berberis: comparative infection studies in attached and detached leaves

Results of infectivity titrations were compared for attached and detached leaves of Berberis gagnepainii (evergreen) and B. vulgaris (deciduous) inoculated with Pseudomonas syringae pv. berberidis . In B. vulgaris results for attached and detached leaves were not comparable because of difficulties in maintaining detached leaves in a viable state. In B. gagnepainii infection of detached leaves occurred following inoculation with as few as 1.1 cfu. The slopes ( b ) of probit response/log dose curves were < 2 suggesting that cells of Ps. syringae pv. berberidis act individually and independently of each other following inoculation, which is further supported by values obtained for response time (RT₅₀) and their distributions. No significant differences (P = 0.05) were found between attached and detached leaves of B. gagnepainii , for values of RT₅₀, ED₅₀ or b, suggesting that detached leaves of B. gagnepainii might be useful for the study of this leaf spot disease.     

Regulation of syringomycin synthesis in Pseudomonas syringae pv. syringae and defined conditions for its production

Production of the phytotoxin, syringomycin (SR), by Pseudomonas syringae pv. syringae strain B301D was regulated by both iron and inorganic phosphate similar to that of many bacterial secondary metabolites. Iron concentrations of 2 µmol/1 or more in deferrated potato-dextrose broth (PDB) resulted in the production of 1024 SR units/ml, a yield comparable to that produced in non-deferrated PDB. Moreover, production of one SR unit required approximately 0†4 ng of available FeCl₃. No SR was produced by strain B301D in deferrated PDB despite growth nearly identical with that of B301D in deferrated PDB supplemented with 10 µmol/1 FeCl₃. Furthermore, a phosphate concentration of 1 mmol/1 or more was suppressive to SR production. Of the amino acids tested, L-histidine at a concentration of ca 20 mmol/1 was the most effective nitrogen source for SR synthesis under defined conditions. Based on these observations, a synthetic medium, SR minimal, was formulated for SR or syringotoxin production by representative strains of Ps. syringae pv. syringae. The regulation of phytotoxin production is discussed in relation to pathogen survival and virulence.     

Differential expression of genes of Pseudomonas syringae on leaves and in culture evaluated with random genomic lux fusions

Differential expression of genes of Pseudomonas syringae strain B728a on plants and in culture was assessed by measuring light production by a large collection of mutant strains containing random genomic insertions of a promoterless lux operon. Reporter gene fusions were made using Tn4431 containing lux CDABE from Vibrio fisheri. Light production reproducibly increased seven-fold when n-decanal was added to cells harvested from plant surfaces, to over 800-fold when added to cells cultured on a solidified culture medium, thus increasing the sensitivity of this reporter gene system. One of the 173 mutants tested exhibited significantly higher light production on plants than on solidified culture media compared to other mutants, while one lux fusion-containing strain produced significantly more light on culture media than on plants relative to the other mutants. The plant-inducible genes identified were not required for pathogenicity of this strain. Approximately 2% of the genes of P. syringae are apparently transcribed more actively in cells growing epiphytically on plants than in common culture media indicating that bacterial cells on plants may have substantially different behaviours than that of cultured cells.     

High-level expression of ice nuclei in a Pseudomonas syringae strain is induced by nutrient limitation and low temperature.

Attempts were made to maximize the expression of ice nuclei in Pseudomonas syringae T1 isolated from a tomato leaf. Nutritional starvation for nitrogen, phosphorous, sulfur, or iron but not carbon at 32 degrees C, coupled to a shift to 14 to 18 degrees C, led to the rapid induction of type 1 ice nuclei (i.e., ice nuclei active at temperatures warmer than -5 degrees C). Induction was most pronounced in stationary-phase cells that were grown with sorbitol as the carbon source and cooled rapidly, and under optimal conditions, the expression of type 1 ice nuclei increased from < 1 per 10(7) cells (i.e., not detectable) to 1 in every cell in 2 to 3 h. The induction was blocked by protein and RNA synthesis inhibitors, indicative of new gene expression. Pulse-labeling of nongrowing cultures with [35S]methionine after a shift to a low temperature demonstrated that the synthesis of a new set of "low-temperature" proteins was induced. Induced ice nuclei were stable at a low temperature, with no loss in activity at 4 degrees C after 8 days, but after a shift back to 32 degrees C, type 1 ice nuclei completely disappeared, with a half-life of approximately 1 h. Repeated cycles of low-temperature induction and high-temperature turnover of these ice nuclei could be demonstrated with the same nongrowing cells. Not all P. syringae strains from tomato or other plants were fully induced under the same culture conditions as strain T1, but all showed increased expression of type 1 ice nuclei after the shift to the low temperature. In support of this view, analysis of the published DNA sequence preceding the translational start site of the inaZ gene (R. L. Green and G. Warren, Nature [London] 317:645-648, 1985) suggests the presence of a gearbox-type promoter (M. Vincente, S. R. Kushner, T. Garrido, and M. Aldea, Mol. Microbiol. 5:2085-2091, 1991).     

A simple and nondestructive method for estimation of worker population size in Myrmica ant nests

A method to estimate the number of workers in Myrmica ant nests on abandoned meadows was developed based on removal of workers. Ant workers have a tendency to climb up on wooden sticks put into their nests, therefore, assuming that the number of workers removed on sticks is related to the total number of workers within the nests, regression models for Myrmica rubra, M. ruginodis and M. scabrinodis may be built. We used a general regression model to perform a backward stepwise elimination of explanatory variables. These were the number of workers removed on sticks, temperature at the nest and site (a categorical variable). In case of each species the final model contained only the number of workers removed as a significant variable. The method is apparently non-destructive as we did not observe decreased survival of nests surveyed as compared to control nests. The method can be a very useful tool in population studies of ants as well as in biodiversity projects, where ants are used as bioinidcators. Received 10 February 2005; revised 4 August 2005; accepted 24 August 2005.     

A newly identified regulator is required for virulence and toxin production in Pseudomonas syringae

The genes lemA (which we here redesignate gacS ) and gacA encode members of a widely conserved two-component regulatory system. In Pseudomonas syringae strain B728a, gacS and gacA are required for lesion formation on bean, as well as for the production of protease and the toxin syringomycin. A gene, designated salA , was discovered that restored syringomycin production to a gacS mutant when present on a multiple-copy plasmid. Disruption of chromosomal salA resulted in loss of syringomycin production and lesion formation in laboratory assays. Sequence analysis of salA suggests that it encodes a protein with a DNA-binding motif but without other significant similarity to proteins in current databases. Chromosomal reporter fusions revealed that gacS and gacA positively regulate salA , that salA upregulates its own expression and that salA positively regulates the expression of a syringomycin biosynthetic gene, syrB . Loss of syringomycin production does not account for the salA mutant's attenuated pathogenicity, as a syrB mutant was found to retain full virulence. The salA gene did not similarly suppress the protease deficient phenotype of gacS mutants, nor were salA mutants affected for protease production. A gacS/gacA -dependent homoserine lactone activity as detected by bioassay was also unaffected by the disruption of salA . Thus, salA appears to encode a novel regulator that activates the expression of at least two separate genetic subsets of the gacS/gacA regulon, one pathway leading to syringomycin production and the other resulting in plant disease.