Pseudomonas Phaseolicola Research Articles

Pseudomonas frederiksbergensis sp. nov., isolated from soil at a coal gasification site.

Phenotypic and genotypic characterization indicated that a group of 29 closely related phenanthrene-degrading bacteria from a coal gasification site in Frederiksberg, Copenhagen, Denmark, belonged to the genus Pseudomonas. The strains were isolated at two sampling occasions 2 years apart. The isolates were phenotypically different from any known species of the genus Pseudomonas and were therefore subject to further identification. Colonies were smooth and pale yellowish and did not produce pigments fluorescent in UV light when grown on King's B agar. Cells were rod-shaped, approximately 0.5-0.8 x 1.5-3.0 microm, and grew at 4 and 30 degrees C, but not 37 degrees C. The bacteria were oxidase- and catalase-positive, accumulated poly-beta-hydroxybutyrate and denitrified, but did not utilize D-xylose. The mean G+C content was 59.6 mol%. Phenotypic data and 16S rDNA sequence data information for Pseudomonas amygdali and Pseudomonas corrugata, and 16S rDNA sequence data for Pseudomonas chlororaphis and Pseudomonas syringae showed close relationships to these strains. However, DNA-DNA hybridization data showed that the isolates belong to a new species, for which the name Pseudomonas frederiksbergensis sp. nov. is proposed. The type strain is JAJ28T (DSM 13022T).

DNA relatedness among the pathovars of Pseudomonas syringae and description of Pseudomonas tremae sp. nov. and Pseudomonas cannabina sp. nov. (ex Sutic and Dowson 1959).

A total of 48 pathovars of Pseudomonas syringae and eight related species were studied by DNA-DNA hybridization (S1 nuclease method) and ribotyping. The existence of nine discrete genomospecies was indicated. Genomospecies 1 corresponded to P. syringae sensu stricto and included P. syringae pathovars syringae, aptata, lapsa, papulans, pisi, atrofaciens, aceris, panici, dysoxyli and japonica. Genomospecies 2 included P. syringae pathovars phaseolicola, ulmi, mori, lachrymans, sesami, tabaci, morsprunorum, glycinea, ciccaronei, eriobotryae, mellea, aesculi, hibisci, myricae, photiniae and dendropanacis and nomenspecies Pseudomonas savastanoi, Pseudomonas ficuserectae, Pseudomonas meliae and Pseudomonas amygdali, which are thus synonymous. P. amygdali is the earliest valid name for this genomospecies. Genomospecies 3 included P. syringae pathovars tomato, persicae, antirrhini, maculicola, viburni, berberidis, apii, delphinii, passiflorae, philadelphi, ribicola and primulae. We recommend strain CFBP 2212 of P. syringae pv. tomato to serve as the type strain. Genomospecies 4 included 'Pseudomonas coronafaciens' and P. syringae pathovars porri, garcae, striafaciens, atropurpurea, oryzae and zizaniae and corresponds to 'P. coronafaciens'. Genomospecies 5 included P. syringae pv. tremae and corresponds to Pseudomonas tremae sp. nov. Genomospecies 6 included Pseudomonas viridiflava and the presently misidentified pathotype strains of P. syringae pv. ribicola and P. syringae pv. primulae and thus corresponds to P. viridiflava. Genomospecies 7 included P. syringae pv. tagetis and P. syringae pv. helianthi. We recommend strain CFBP 1694 of P. syringae pv. tagetis to serve as a reference strain. Genomospecies 8 included P. syringae pv. these and Pseudomonas avellanae and thus corresponds to P. avellanae. Genomospecies 9 included P. syringae pv. cannabina and corresponds to Pseudomonas cannabina sp. nov. Ribotyping (SmaI and HincII endonucleases) could separate seven of the nine genomospecies. The unnamed genomospecies 3 and 7 will be named when phenotypic data are available for identification. Two species are described, P. tremae sp. nov. and P. cannabina sp. nov. Other species will be named when phenotypic data are available for identification.

Sex and the evolution of intrahost competition in RNA virus phi6.

Sex allows beneficial mutations that occur in separate lineages to be fixed in the same genome. For this reason, the Fisher-Muller model predicts that adaptation to the environment is more rapid in a large sexual population than in an equally large asexual population. Sexual reproduction occurs in populations of the RNA virus phi6 when multiple bacteriophages coinfect the same host cell. Here, we tested the model's predictions by determining whether sex favors more rapid adaptation of phi6 to a bacterial host, Pseudomonas phaseolicola. Replicate populations of phi6 were allowed to evolve in either the presence or absence of sex for 250 generations. All experimental populations showed a significant increase in fitness relative to the ancestor, but sex did not increase the rate of adaptation. Rather, we found that the sexual and asexual treatments also differ because intense intrahost competition between viruses occurs during coinfection. Results showed that the derived sexual viruses were selectively favored only when coinfection is common, indicating that within-host competition detracts from the ability of viruses to exploit the host. Thus, sex was not advantageous because the cost created by intrahost competition was too strong. Our findings indicate that high levels of coinfection exceed an optimum where sex may be beneficial to populations of phi6, and suggest that genetic conflicts can evolve in RNA viruses.

Hyperplastic canker – a perennial disease of almond caused by Pseudomonas amygdali

The history of hyperplastic canker of almond, caused by Pseudomonas amygdali, its evolution and the present situation in Greece are reviewed. Its main aspects, causal agent, symptomatology, epidemiology and control measures are described. The main characteristics differentiating the pathogen from some other pseudomonads pathogenic to fruit trees are also reported.

Lipopolysaccharides from three phytopathogenic pseudomonads

Analysis of the polysaccharide and lipid moieties of the lipopolysaccharides (LPSs) of the phytopathogenic bacteria, Pseudomonas amygdali and P. syringae pv. ciccaronei has demonstrated that for both bacteria the O-chain consists of a tetrasaccharide repeating unit of three α- l-Rha p and one terminal nonreducing α- d-Fuc p3NAc. Two of the rhamnosyl residues are 3-linked, the third one 2,3-linked. This structure had been previously found for the O-chains of three phytopathogenic strains of P. syringae subsp. savastanoi, but this is the first report on its occurrence in P. amygdali and P. syringae pv. ciccaronei. The results of the LPS lipid residue analysis made it possible to make some chemotaxonomic considerations and therefore classify P. amygdali as a chemotype, which is different from that of the other two bacteria examined.

Local and Systemic Biosynthesis of Salicylic Acid in Infected Cucumber Plants.

Radiolabeling studies showed that salicylic acid (SA), an essential component in the signal transduction pathway leading to systemic acquired resistance, is synthesized from phenylalanine (Phe) and benzoic acid in cucumber (Cucumis sativus L.) plants inoculated with pathogens. Leaf discs from plants inoculated with either tobacco necrosis virus or Pseudomonas lachrymans incorporated more [14C]Phe into [14C]SA than mock-inoculated controls. The identity of SA was confirmed by gas chromatography-mass spectrometry. No reduction in specific activity of [14C]SA was observed for either free or bound SA between control and infected plants after feeding [14C]Phe. A specific inhibitor of Phe ammonia-lyase, 2-aminoindan-2-phosphonic acid, completely inhibited the incorporation of [14C]Phe into [14C]SA, although plants treated with 2-aminoindan-2-phosphonic acid could still produce [14C]SA from [14C]benzoic acid. Biosynthesis of SA in tissue inoculated with tobacco necrosis virus followed a transient pattern with the highest induction occurring 72 h postinoculation. Uninfected tissues from an infected plant synthesized de novo more SA than did controls. This suggests the involvement of a systemic signal triggering SA synthesis in tissue distant from the site of infection that display systemic acquired resistance.

A New Small Low-Abundant Nonstructural Protein Encoded by the L Segment of the dsRNA Bacteriophage Φ6

A new small low-abundant protein encoded by the large genome segment of bacteriophage Φ6 has been detected in extracts of Escherichia coli bearing Φ6 cDNA clones and in extracts of phage-infected Pseudomonas phaseolicola. This 62 amino acid protein, designated P14, has a net charge of +2 at pH 7.0. Gene 14 has been located by deletion analysis and N-terminal protein sequence. Expression of P14 is down-regulated in E. coli when the complete L segment clone is expressed. Polyclonal antibodies to P14 detected the new protein in small amounts in phage-infected cells but not in Φ6 virions or nucleocapsids, or in procapsids assembled in E. coli. Procapsids assembled in E. coli after expression of Φ6 L segment clones with or without gene 14 had essentially similar protein composition and activity in an in vitro packaging and replication system. Thus P14 does not appear to be essential for the structure or assembly of functional procapsids. P14 might, however, facilitate packaging in vivo. Alternatively this protein could play a role in repair of host membranes following vital penetration.

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.

Ortho-Anisic Acid as Internal Standard for the Simultaneous Quantitation of Salicylic Acid and Its Putative Biosynthetic Precursors in Cucumber Leaves

Salicylic acid (SA) acts as an endogenous signal for the induction of systemic acquired resistance (SAR) in plants infected by pathogens. In order to study SA biosynthesis in plants, a quantitative method using an internal standard was developed for the simultaneous measurement of SA and three of its putative bioprecursors, namely trans-cinnamic acid (CA), trans-ortho-hydroxycinnamic acid (oHCA), and benzoic acid (BA). ortho-Anisic acid (oANI) was found to be a suitable internal standard for all four compounds. It was not present as endogenous compound in cucumber leaves, no substance interfered with its detection, and it followed identical losses as SA, CA, oHCA, and BA during the extraction procedure. Baseline separation of these four compounds and of eight other plant phenolics was achieved in 35 min using deactivated reversed-phase HPLC. On-line detection was performed fluorimetrically for SA, oANI, and oHCA (with the excitation and emission wavelengths optimized for each compound) and with uv detection for CA and BA. Correlation equations for SA, CA, oHCA, and BA using oANI as internal standard were linear for a broad range of concentrations. The limits of detection (in ng/g fresh weight, FW) obtained during routine analyses were as follows: SA, 4; oHCA, 100; CA, 150; and BA, 500. The method was used to quantify levels of free and acid-hydrolyzed bound SA in cucumber plants either 5 days postinoculation with Pseudomonas lachrymans or after mock-inoculation with water on their first leaf. Free SA increased 33-fold locally (to 382.0 ng/g FW) in the infected leaf and 4.2-fold systemically (to 21.3 ng/g FW) in the second leaf compared to controls. Bound SA reached 4399 and 287.3 ng/g FW in the first and second leaves of infected plants, respectively (not detectable in controls).

Broad host range, regulated expression system utilizing bacteriophage T7 RNA polymerase and promoter

An IPTF-regulated broad host range expression system was constructed using compatible broad host range plasmids, the T7 RNA polymerase, and T7 promoter sequences. The system is implemented by the coexistence of two plasmids. The first contains the T7 RNA polymerase gene under the control of lacl or lacl(q) genes and lacUV5 promoter. The second encodes the T7 promoter upstream of a multicloning site. IncP1 or IncP4 T7 promoter plasmids, and IncP1, IncP4 or IncW T7 RNA polymerase plasmids were constructed. The expression from the IncP1 promoter plasmids in the presence of the IncP4 polymerase plasmids was tested by in vivo lacZ fusions and vivo labeling of proteins. In this combination, the use of lac(q) improves the regulation levels in Escherichia coli, whereas, in Pseudomonas phaseolicola, a 28.5-fold regulation was obtained with lacl, Although the level of lacZ expression from the T7 promoter in P. phaseolicola is low compared with E. coli, it is similar to levels obtained with the pm promoter in Pseudomonas putida when the differences in the copy number of the expression vectors are taken into consideration.

Response of 25 Bean Cultivars to Pseudomonas Phaseolicola (Burk) Dows.

Response of 25 Bean Cultivars to <i>Pseudomonas Phaseolicola</i> (Burk) Dows.

Oxalate and phosphates induce systemic resistance against diseases caused by fungi, bacteria and viruses in cucumber

Spraying aqueous solutions (20 or 50 m m) of oxalate, potassium phosphate dibasic (K 2HPO 4) or tribasic (K 3PO 4) on the upper surface of leaf 1 of cucumber plants, or inoculating leaf 1 with a spore suspension of Colletotrichum lagenarium induced systemic resistance to C. lagenarium, Cladosporium cucumerinum, Dydimella bryoniae, Sphaerotheca fuliginea, Pseudomonas lachrymans, Erwinia tracheiphila, tobacco necrosis virus (TNV) and cucumber mosaic virus (CMV) when plants were challenged on leaf 2 with the test pathogens 7 days after treatment of leaf 1. Plants were rated for disease 5–15 days after challenge-inoculation, depending on the pathogen. The level of protection induced by the test solutions varied and generally was lower than that induced by C. lagenarium. Each chemical solution tested caused chlorotic stippling on the induced leaf within 48 h after application. The stippling later developed into restricted necrotic lesions. The nature of damage in the inducer leaf was related to the level of protection. Plants with distinct but restricted necrotic lesions on inducer leaves were better protected than those with extensive damage or few lesions. These experiments provide further evidence that induced resistance is due to a low level of persistent stress which can be induced chemically and not the result of a specific component of the inducing pathogen.

RNA-Protein complexes responsible for replication and transcription of the double-stranded RNA bacteriophage φ6

RNA-protein complexes active for transcription and replication of the double-stranded RNA bacteriophage φ6 have been partially purified from lysates of infected Pseudomonas phaseolicola. Transcribing particles (filled procapsids) contain the three viral dsRNAs and all four procapsid proteins P1, P2, P4, and P7. Particles with replicase activity contain the same four proteins as well as single plus RNA strands duplexed with various extents of minus strands initiated in vivo. The in vitro replication reaction is insensitive to RNaseA. Sarkosyl destroys transcription complexes but does not reduce the activity of replication complexes, although the latter lose 80% of their P4 and the single-strand RNA template becomes sensitive to RNase. The detection of complexes that replicate small only, or both small and medium, RNA suggests that the RNAs are packaged sequentially in the order small, medium, large.

Synthesis of the naturally occurring cytokinin 2′-deoxyzeatin riboside

2′-Deoxyzeatin riboside, previously isolated together with other three cytokinins from culture filtrates of the pathogenic bacterium Pseudomonas amygdali, has been synthesized by reaction of 6-chloro-9-(2-deoxy-β- d-ribofuranosyl)purine with ( E)-4-amino-2-methyl-but-2-en-1-ol. The identity of the synthetic and the naturally occurring cytokinin has been established.

A calcium flux at the termination of replication triggers cell devision in Escherichia coli: Hypothesis

Cell division in Escherichia coli is coupled to chromosome replication. Even in the absence of known inducible division inhibitors, perturbations of chromosome replication affect cell division. Early studies suggested that a signal at the termination of replication might trigger subsequent division. Although later studies have suggested that fork encounter during termination is an active process involving specific termination sites and the tus protein, the coupling mechanism between termination and cell division remains to be elucidated. Recently it has been shown that the chromosome of a bacterium, Pseudomonas tabaci, contains a high proportion of calcium. E. coli maintains an intracellular concentration of free calcium identical to that of higher organisms and in dividing cells of E. coli a twenty-fold increase in the level of total calcium in the cytoplasm, a flux, occurs. In this article I propose that during the replication of the chromosome calcium entry balances calcium binding to DNA. At the termination of replication, there is a brief interval between the end of calcium binding to the chromosome and the end of calclum entry or release into the cytoplasm. During this interval the level of free calcium therefore rises. This rise may result in the observed flux by triggering the entry of calcium directly via voltage-gated calcium channels or indirectly via changes in phospholipid configurations. Mechanisms whereby these changes in calcium levels might be coupled to cell division and to a phospholipid control of the cell cycle are discussed.

Serological Comparison of Antigens Related to Local Virus and Bacterial Infections and Aspecific Stresses in Tobacco Leaves

AbstractTwo “new” precipitin bands (antigens) detected by the immunodiffusion test were demon strated in leaf extracts of tobacco inoculated with tobacco mosaic virus (TMV), Pseudomonas tabaci or treated with mercuric chloride, sodium azide or sodium hypochlorite. One of the precipitin bands was stronger, than the other, These antigens were also detected in the upper, non‐infected leaves of tobacco plants when the lower leaves were locally stressed (necrotized) either by TMV or by chemical injury. The “new” antigens formed in the upper leaves were detected even if the TMV‐inoculated lower leaves were removed one day after inoculation. The “new” antigens were identical both in the lower and upper leaves and their induction was independent from the stress whether pathogenic or chemical.A coincidence exists between the appearance of “new” antigens and acquired resistance, but this does not mean necessarily a cause‐and‐effect relationship between the two phenomena. Our experiments indicate that the induction of the synthesis of “new” stress proteins in tobacco is aspecific and the proteins formed are related to the aspecific stress itself rather than to pathogenesis.

Induction of chitinase and β-1,3-glucanase in tobacco plants infected with Pseudomonas tabaci and Phytophthora parasitica var. Nicotianae

We have measured by rocket immunoassay the glucan endo-1,3-β-glucosidase (β-1,3-glucanase, EC 3.2.1.39) and endochitinase (chitinase, EC 3.2.1.14) contents of Nicotiana tabacum cv. “Havana 425” plants following infection with the bacterial pathogen Pseudomonas tabaci and the fungal pathogen Phytophtorara parasitica var. nicotianae. Both enzymes were induced, relative to non-infected controls, in leaves infected with P. tabaci and in stems and leaves infected with P. parasitica. Increases in enzyme content were confined to infected parts of the plant indicating that this response is local rather than systemic. The β-1,3-glucanase- and chitinase-mRNA content of tissues was measured by RNA blot hybridization using the inserts of cDNA clones for the tobacco mRNAs as probes. P. tabaci and P. parasitica infection increased the mRNA content relative to controls indicating that both pathogens act at least in part at the level of mRNA accumulation. As judged by immunological tests and RNA hybridization, the β-1,3-glucanase and chitinase induced by P. tabaci and P. parasitica are the same basic isoforms known to be induced by tobacco mosaic virus. Therefore the induction appears to be a non-specific response of the host to pathogens.

In vitro replication and transcription of the segmented double-stranded RNA bacteriophage φ6

In vitro conditions that support viral-specific replication and transcription have been developed from Pseudomonas phaseolicola cells infected with the segmented double-stranded RNA bacteriophage φ6. Transcription activity, previously shown to occur by semiconservative strand displacement, labeled (+) strands of all three genome segments and produced all three corresponding genome length messenger RNAs. Replication activity for each of the three double-stranded RNA segments is observed. Our criteria for replication were formation of genomic length double-stranded RNA products and at least (-) strand synthesis activity. Mn 2+ and Sarkosyl together selectively inhibited transcription. Analysis of replication alone suggested that replication templates are the viral (+) messenger RNAs.

Studies on the Extracellular Polysaccharides (EPS) Produced in vitro by Pseudomonas phaseolicola III. Kinetics of Levan and Alginate Formation in Batch Culture and Demonstration of Levansucrase Activity in Crude EPS

AbstractIn batch culture Pseudomonas phaseolicola converted ca. 8–11% of the substrate (sucrose) into extracellular polysaccharides (EPS). The percentage of conversion was independent of the concentration of sucrose in the medium. However, the increase of total EPS observed with enhanced substrate concentration was largely due to a drastic stimulation of the levan produced. Production of alginate, in contrast, was stimulated to a much lower extent. EPS production was highest during the middle phaseof exponential growth and gradually terminated as the cultures grew older. With sucrose as substrate, the formation of alginate followed the levan synthesis. With glucose or gluconate, however, there was no production of levan, and the alginate synthesis, started immediately after the lag‐phase of the culture.Levansucrase activity was detected in the protein fraction of crude EPS produced by a culture grown in sucrose containing medium. Enzyme activity was demonstrated both by the release of glucose and the formation of levan from sucrose. Upon ion exchange chromatography on DEAE‐fractogel levansucrase behaved as a rather acidic protein. Native polyacrylamide gel electrophoresis of the enzyme fraction revealed a major band with an apparent molecular weight of ca. 68,000 d. Several grams of levan were synthesized with a partly purified enzyme preparation in the absence of bacteria.

Cloning and characterization of Saccharomyces cerevisiae genes that confer L-methionine sulfoximine and tabtoxin resistance

Pseudomonas tabaci produces a toxin, tabtoxin, that causes wildfire disease in tobacco. The primary target of tabtoxin is presumed to be glutamine synthetase. Some effects of tabtoxin in tobacco can be mimicked by methionine sulfoximine (MSO), a compound that is known to inactivate glutamine synthetase. To understand how organisms can be made resistant to tabtoxin and MSO, we used Saccharomyces cerevisiae. We demonstrate that yeast strains carrying the glutamine synthetase gene, GLN1, on a multicopy plasmid overproduced glutamine synthetase and showed increased drug resistance. These and other data indicate that glutamine synthetase is the primary target of tabtoxin and MSO in S. cerevisiae. We also isolated three S. cerevisiae DNA inserts of 2.1, 2.3, and 2.8 kilobases that conferred tabtoxin and MSO resistance when the inserts were present on a multicopy plasmid. These plasmids conferred resistance to MSO by blocking intracellular transport of the drug. Transport appeared to occur by one or more methionine permeases. Resistance to tabtoxin could also occur by blockage of intracellular transport, but the drug was transported by some permease other than a methionine permease. These drug resistance plasmids did not block transport of citrulline, indicating that they did not affect the general amino acid permease.