Pseudomonas Marginalis Research Articles

Production, Purification, and Properties of a Pectin Lyase fromPseudomonas marginalisN6301

Pseudomonas marginalis N6301 produced pectin lyase (EC 4.2.2.10) in the medium with cell lysis when the culture was treated with mitomycin C. We purified the enzyme by carboxymethylcellulose, hydroxylapatite, and gel-filtration column chromatographies. The enzyme had a molecular weight of 34,000 by SDS polyacrylamide gel electrophoresis and was mostly stable around pH 6.5. The optimum pH and temperature for the enzyme activity were 8.0 and 30°C, respectively. The activity was inhibited severely by 2 mM N-ethylmaleimide, maleic anhydride, and p-chloromercuriphenylsulfonic acid. Further, the pectin lyase produced in a medium containing glycerol was purified. The molecular weight of the enzyme was identical to that of the enzyme produced in the presence of mitomycin C.

Characterisation and pathogenicity of bacteria from shoot tips of the globe artichoke (Cynara scolymus L.)

SummaryBacteria have been isolated from shoot tips of symptomless globe artichoke plants. These were identified as Pseudomonas fluorescens, Pseudomonas marginalis, Pseudomonas spp., Serratia liquefaciens, Enterobacter agglomerans/Erwinia, Agrobacterium radiobacter, an unidentified member of Rhizobiaceae and another classified in the “corynebacteria” group. The most frequently isolated species was P. fluorescens, biovars II and III. The endogenous character of these bacteria was studied in plants growing in vitro and in the open field. P. fluorescens, P. marginalis, S. liquefaciens and E. agglomerans/Erwinia caused symptoms in plants growing in vitro, but only P. fluorescens biovar II and P. marginalis produced symptoms in plants growing in open fields. Differences in pathogenicity were observed on inoculated plants growing in vitro or in the open field. This suggests that several endophytic bacterial species may be responsible for the high levels of contaminants found during the micropropagation of globe artichoke.

Leaf spot and blight of Dieffenbachia amoena caused by Pseudomonas marginalis pv. marginalis

Leaf spot and blight of Dieffenbachia amoena caused by Pseudomonas marginalis pv. marginalis are described for the first time. Symptoms were noticed during winter on plants in the greenhouse, the first signs were small, elongated, light brown lesions on the surface of the leaves. As the infection spreads, leaves become completely brown and collapse. The identity of the causal agent was determined by biochemical, nutritional and pathogenecity tests as well as by comparison of whole-cell protein profiles of the isolates under study with those of type strains.

Enantioselective degradation of the herbicide mecoprop [2-(2-methyl-4-chlorophenoxy) propionic acid] by mixed and pure bacterial cultures

A consortium of three bacteria was isolated from top soil through their capacity to utilise the chlorinated, aromatic herbicide mecoprop as a single growth substrate. The consortium constituted a tight association of Alcaligenes denitrificans, Pseudomonas glycinea and Pseudomonas marginalis. The culture exclusively degraded the (R)-(+)-isomer of the herbicide while the (S)-(−)-enantiomer remained unaffected. The mecoprop-degrading community could also degrade 2,4-dichlorophenoxyacetic acid, 2-methyl-4-chlorophenoxyacetic acid and racemic 2-phenoxypropionic acid. Initially, no single member of the consortium was able to degrade mecoprop as a pure culture but after prolonged incubation, A. denitrificans was able to grow on the herbicide as the sole source of carbon and energy.

In situ transfer of an exogenously isolated plasmid between Pseudomonas spp. in sugar beet rhizosphere

Summary: A method for studying plasmid transfer in the rhizosphere is described. This work demonstrates plasmid transfer in an unenclosed rhizosphere under field conditions. The donor (Pseudomonas marginalis 376N) and recipient (Pseudomonas aureofaciens 381R) bacteria and the conjugative mercury resistance plasmid (pQBR11) studied were all isolated from the bacterial community indigenous to sugar beet rhizosphere. Spontaneous nalidixic acid and rifampicin resistant mutants of these bacteria were used as donors and recipients of pQBR11 for in situ matings. Fresh field soil was mixed with donors and recipients to give a soil mating mix (SMM) which was placed underground on the surface of a sugar beet root storage organ. Plasmid transfer in the SMM was determined after 24 h at frequencies between 5.1 × 10-5 and 1.3 × 10-8 transconjugants per recipient. Higher transfer frequencies (1.3 × 10-2 to 1.7 × 10-6) were recorded on the peel adjacent to the SMM. No transfer of mercury resistance was detected in SMM controls incubated at 20°C in vitro or placed in soil at distances of more than 5 cm from plants.

Expression of a pectin lyase gene in Escherichia coli from Pseudomonas marginalis N6301.

We constructed deletion mutant clones of a pectin lyase gene, and measured their pectin lyase activities in Escherichia coli. Pectin lyase activities were detected only in a recA+ strain but not in a recA- strain of E. coli. We also cloned and sequenced recA from Pseudomonas marginalis N6301. The recA from P. marginalis N6301 can complement recA- to form recA+ in the phenotype of E. coli. Highly conserved sequences of recA are observed among E. coli, P. fluorescens, and P. marginalis. From these results, we presume that recA is required for the expression of the pectin lyase gene in P. marginalis N6301.

Fungi and Gram‐negative bacteria as soilborne minor pathogens of goat's rue (Galega orientalis Lam.)

SummaryFungi and Gram‐negative bacteria were isolated from inside the roots of field‐grown goat's rue (Galega orientalis). Fungi were isolated from three plants out of a total of 45 tested. Two multinuclear Rhizoctonia solani isolates were identified to the anastomosis group 5 (R. solani AG‐5‐Gal) using pairings with known AG test cultures. One fungal isolate was identified to Phoma chrysanthemicola. Gram‐negative bacteria were isolated from three plants out of 25 tested. They were identified using classical methods, the BIOLOG identification system based on the utilisation of 95 different carbon sources, and the MIDI system for the analysis of whole cell fatty acids. The two latter systems were computer‐associated and utilised an extensive reference library of isolates. One bacterial isolate was identified as Enterobacter agglomerans and two isolates as Pseudomonas marginalis. R. solani AG‐5‐Gal reduced the emergence of Lupinus luteus, L. polyphyllus and french bean (Phaseolus vulgaris) and the growth of broad bean (Viciafaba), L. luteus and french bean, but did not cause obvious damage in goat's rue and pea (Pisum sativum). However, R. solani AG‐5‐Gal was re‐isolated from the roots of all the test plant species following inoculation. P. chrysanthemicola reduced the emergence of L. polyphyllus and the growth of goat's rue, french bean and broad bean, and it was re‐isolated from all of the test plant species (except for french bean) following inoculation.All the bacteria reduced the emergence of french bean, but not that of goat's rue and pea, when applied to the soil. When the roots were dipped into bacterial suspension, all the bacteria damaged french bean and L. polyphyllus. Additionally, P. marginalis JV3 damaged goat's rue and red clover. The pathogenicity of the fungi and bacteria were not changed when they were double‐inoculated in pairs, except for R. solani AG‐5‐Gal and P. marginalis JV2 which reduced the emergence of goat's rue when inoculated together but not when inoculated separately.

Molecular cloning and nucleotide sequence of the pectate lyase gene from Pseudomonas marginalis N6301.

Pectate lyase was purified approximately 29-fold to electrophoretic homogeneity from Pseudomonas marginalis N6301. A pectate lyase (PL; EC4.2.2.2) gene of the strain was cloned and expressed in Escherichia coli. The nucleotides of the PL gene (pel) were sequenced. An open reading frame that encodes a polypeptide (molecular weight: 40,812) composed of 380 amino acids including a 29 amino acid signal peptide was assigned. The structural gene of pel consisted of 1140 base pairs. The nucleotide sequence of the 5'-flanking region of pel showed a consensus sequence of the promoter region of the pectin lyase gene (pnl) in P. marginalis N6301, a Pribnow box, and a ribosome binding site as found in E. coli.

Classification of Fluorescent Soft Rot Pseudomonas Bacteria, Including P. marginalis Strains, Using Whole Cell Fatty Acid Analysis

In this study the taxonomy of the opportunistic plant pathogenic soft rot Pseudomonas bacteria has been investigated, also in order to clarify the status of the species name Pseudomonas marginalis which is used either for soft rot strains resembling P. fluorescens biovar 2 (= P. marginalis sensu stricto) or for any fluorescent, oxidase positive soft rot pseudomonad (= P. marginalis sensu lato). It was also tried to verify the possible soft rot activity of P. aeruginosa, isolated from plant, animal and man. In total 164 saprophytic and pathogenic strains, including 14 type or pathotype strains were studied. Conventional physiological and biochemical tests, pathogenicity tests on potato disks and chicory heads and whole cell fatty acid analysis (FAA) were applied. Of the non-reference strains 25 were identified phenotypically as P. aeruginosa, 81 with some difficulty, due to many variations, as biovars, intermediates of these biovars or new forms of P. fluorescens, two as P. aureofaciens, two as P. cichorii, 11 as P. putida, 21 as unknown fluorescent pseudomonads, two as P. stutzeri and seven as P. viridiflava. Oxidase positive soft rot strains could not be distinguished by biochemical tests from P. fluorescens, P. putida or P. aureofaciens. There was only a low correlation between pectolytic activity and ability to cause soft rot. In multivariate analysis of fatty acid patterns four clusters could be distinguished, viz.: one heterogeneous (super) cluster containing strains of P. aureofaciens, P. chlororaphis, P. fluorescens, P. putida and P. tolaasii and strains received as P. marginalis; one cluster comprising the oxidase negative P. syringae and P. viridiflava and the related oxidase positive P. cichorii; one containing P. stutzeri and P. mendocina; one tight cluster with all but one P. aeruginosa strains. All oxidase positive soft rot strains were found in the P. fluorescens supercluster and all the oxidase negative ones (phenotypically P. viridiflava) in the cluster containing P. syringae, P. viridiflava and P. cichorii. Within the P. fluorescens supercluster there were no clusters discriminating soft rot strains (P. marginalis sensu stricto or sensu lato), they were found scattered among saprophytic strains. The absence of clustering of soft rot strains as a separate taxon in phenotypic or fatty acid analysis renders the species name P. marginalis artificial and its use should be abandoned. The taxonomic implications of finding a continuum of strains within the P. fluorescens complex (including P. aureofaciens, P. chlororaphis, P. putida and P. tolaasii) using FAA or phenotypic tests by us and other authors is discussed, also in relation to future naming of soft rot pseudomonads. The possible soft rot activity of P. aeruginosa could not be confirmed, only a few strains occasionally caused some necrotizing action on plant tissue. Finally pathogenic strains of P. fluorescens not able to produce soft rot, causing bacterial leaf stripe of Iris spec. are described.

A field and storage rot of onion caused byPseudomonas marginalis

Abstract Pseudomonas marginalis (Brown 1918) Stevens 1925 isolated from infected leaves of onion (Allium cepa L.) in the field and from diseased bulbs in store, was confirmed as a soft rot pathogen of onion by Koch's postulates. The first official record of P. marginalis on onion in New Zealand is attributed to Hale in Pennycook 1989. This paper documents the evidence for that record.

Effects of Nitrogen Fertilization on Bacterial Soft Rot in Two Broccoli Cultivars, One Resistant and One Susceptible to the Disease

Sidedress applications of ammonium nitrate increased the incidence and severity of bacterial soft rot (caused by Pseudomonas marginalis) in a susceptible broccoli cultivar, Premium Crop, but had no effect on a resistant one, Shogun. Plots received N in amounts ranging from 19 to 152 kg/ha and were sprinkler-irrigated two to five times a day during head maturation to create conditions conducive to the disease. The length of time necessary for heads to reach harvest maturity decreased with increased N (.)

Resistance in Broccoli to Bacterial Soft Rot Caused byPseudomonas marginalisand FluorescentPseudomonasSpecies

Significant differences in susceptibility of brocoli (Brassica oleracea var. italica) genotypes to bacterial soft rot (heat rot), caused by Pseudomonas marginalis (P. fluorescens biovar II) and other fluorescent Pseudomonas spp., were observed in four field studies from 1986 to 1989. Cultivars Green Defender and Shogun consistently appeared highly resistant to bacterial soft rot. High disease resistance was observed in 1989 in experimental line NVH 521 (...)

Erwinia pel gene homology survey in selected bacteria

A 2.1 kb Bam H1 DNA fragment encoding a pectate lyase (PL) enzyme was isolated from an Erwinia carotovora subsp. atroseptica (Eca) cosmid library. The fragment was labeled with 32P-CTP and hybridized to total DNA digests from selected bacteria which included plant-invasive as well as plant associative organisms. The pel gene probe hybridized to E. carotovora subsp. carotovora (Ecc) DNA under all conditions tested. Hybridization to DNAs from Agrobacterium tumefaciens and Pseudomonas marginalis was observed at low stringency conditions (45°C). No hybridization was observed between the pel gene probe and six other DNA samples.

The structure of the acidic exopolysaccharide of Pseudomonas marginalis strains PF-05-2 and PM-LB-1

The structure of an acidic exopolysaccharide of two strains of Pseudomonas marginalis, a bacterium which causes soft rots of various vegetables, has been determined to consist of a repeating unit of: →4) β- d-Man p-(1→3) a- d-Glc p-(1→4) a- l-Rha p-(1-. The glucose is pyruvated at O-4 and O-6 and the mannose is acetylated at either O-2 or O-3.

Involvement of pseudomonads in deterioration or ‘ready‐to‐use’ salads

SummaryMicroorganisms involved in the deterioration of ‘ready‐to‐use’ salads were investigated and bacteria able to cause soft rot of endive (Chicorum endiva) leaves were frequently found. These soft rot bacteria are fluorescent pectinolytic pseudomonads, specifically Pseudomonas marginalis, which is the most frequently isolated Pseudomonas sp. and present in the greatest numbers in stored ‘ready‐to‐use’ salads. A clear relationship exists between the incidence of ‘ready‐to‐use’ salads deterioration and the count of P. marginalis, after 10 days of storage at 10°C. Apart from the fluorescent pseudomonads, Erwinia herbicola is the next most commonly isolated organism.

Production and properties of pectin lyase in Pseudomonas marginalis induced by mitomycin C.

Production and properties of pectin lyase in Pseudomonas marginalis induced by mitomycin C.

The etiology of head rot disease of broccoli

Transmission and scanning electron microscope studies of broccoli florets affected by head rot, at various stages of disease development, strongly indicated a bacterial etiology for the disease. Nevertheless, the different species of bacteria isolated from diseased heads, using standard techniques, failed to reproduce symptoms in pathogenicity tests conducted in the glasshouse and in the field. However, a modified isolation technique, using broccoli heads showing incipient watersoaking symptoms, yielded a fluorescent pseudomonad which reproduced disease symptoms readily in glasshouse and field tests. On the basis of physiological and biochemical characters, the pathogenic bacterium was identified as a highly pectolytic pathovar of Pseudomonas marginalis. The bacterium also caused the rotting of potato, tomato and swede turnip slices, and also of intact and detached tomato fruit. However, it was not pathogenic on lettuce, parsnip or lucerne, and also failed to rot carrot slices.

Bacterial Growth Rates and Competition Affect Nodulation and Root Colonization by Rhizobium meliloti

The addition of streptomycin to nonsterile soil suppressed the numbers of bacterial cells in the rhizosphere of alfalfa (Medicago sativa L.) for several days, resulted in the enhanced growth of a streptomycin-resistant strain of Rhizobium meliloti, and increased the numbers of nodules on the alfalfa roots. A bacterial mixture inoculated into sterile soil inhibited the colonization of alfalfa roots by R. meliloti, caused a diminution in the number of nodules, and reduced plant growth. Enterobacter aerogenes, Pseudomonas marginalis, Acinetobacter sp., and Klebsiella pneumoniae suppressed the colonization by R. meliloti of roots grown on agar and reduced nodulation by R. meliloti, the suppression of nodulation being statistically significant for the first three species. Bradyrhizobium sp. and "Sarcina lutea" did not suppress root colonization nor nodulation by R. meliloti. The doubling times in the rhizosphere for E. aerogenes, P. marginalis, Acinetobacter sp., and K. pneumoniae were less and the doubling times for Bradyrhizobium sp. and "S. lutea" were greater than the doubling time of R. meliloti. Under the same conditions, Arthrobacter citreus injured alfalfa roots. We suggest that competition by soil bacteria reduces nodulation by rhizobia in soil and that the extent of inhibition is related to the growth rates of the rhizosphere bacteria.

Effect of heating on the sensitivity and specificity of an enzyme immunoassay (EIA) forErwinia carotovorasubsp.atrosepticaserogroup I

A double sandwich enzyme immunoassay (EIA) standardized for detection of Erwinia carotovora subsp. atroseptica serogroup I was serogroup specific only at the lower limit of sensitivity (105 to 106 cells/mL). At concentrations > 107 cells/mL strains representing subsp. atroseptica serogroups XVIII, XX, and XXII and subsp. carotovora serogroups II, III, IV, and V also gave positive but less strong reactions. Strains from 16 other E. carotovora serogroups and strains of Corynebacterium sepedonicum and Pseudomonas marginalis were all similar to the buffer controls. Heating suspensions of serogroup I strains prior to assay for at least 3 min at temperatures > 60°C enhanced A405 values. However the lower limit for detection was not reliably reduced below 105 cells/mL for all serogroup I strains. A405 values for strains known to share a common flagellar antigen (serogroups III and V) were reduced but not eliminated by heating. Strains having a related cell wall antigen (serogroups II and XVIII) were either unaffected by heating or had increased A405 values similar to the homologous strains. Heat-increased A405 values for serogroup XX and XXII strains suggest that they also have a heat-stable cell wall antigen in common with serogroup 1 strains.

Detection of β-carbanion formation during kynurenine hydrolysis catalyzed by Pseudomonas marginalis kynureninase

2-Amino-4-hydroxy-4-phenylbutyric acid has been shown to be formed during the Pseudomonas marginalis kynureninase-catalyzed hydrolysis of kynurenine in the presence of benzaldehyde and pyridoxal phosphate. The formation of 2-amino-4-hydroxy-4-phenylbutyric acid is the first demonstration, to our knowledge, of the controlled trapping of an amino acid β-carbanion generated either chemically or enzymatically, and is perhaps the best empirical evidence to date that enzyme mechanisms can proceed through a β-carbanionic intermediate. The lifetime of the β-carbanionic alanyl intermediate generated by kynureninase is of sufficient duration to allow reaction with benzaldehyde. Other aromatic, but no aliphatic, aldehydes will undergo electrophilic addition with kynureninase-generated β-carbanionic alanyl intermediates to form the corresponding amino acid.