Production and comparison of peptide siderophores from strains of distantly related pathovars of Pseudomonas syringae and Pseudomonas viridiflava LMG 2352

The production of peptide siderophores and the variation in siderophore production among strains of Pseudomonas syringae and Pseudomonas viridiflava were investigated. An antibiose test was used to select a free amino acid-containing agar medium favorable for production of fluorescent siderophores by two P. syringae strains. A culture technique in which both liquid and solid asparagine-containing culture media were used proved to be reproducible and highly effective for inducing production of siderophores in a liquid medium by the fluorescent Pseudomonas strains investigated. Using asparagine as a carbon source appeared to favor siderophore production, and relatively high levels of siderophores were produced when certain amino acids were used as the sole carbon and energy sources. Purified chelated siderophores of strains of P. syringae pv. syringae, P. syringae pv. aptata, P. syringae pv. morsprunorum, P. syringae pv. tomato, and P. viridiflava had the same amino acid composition and spectral characteristics and were indiscriminately used by these strains. In addition, nonfluorescent strains of P. syringae pv. aptata and P. syringae pv. morsprunorum were able to use the siderophores in biological tests. Our results confirmed the proximity of P. syringae and P. viridiflava; siderotyping between pathovars of P. syringae was not possible. We found that the spectral characteristics of the chelated peptide siderophores were different from the spectral characteristics of typical pyoverdins. Our results are discussed in relation to the ecology of the organisms and the conditions encountered on plant surfaces.     

Evaluation of Pseudosel Agar as an Aid in the Identification of Pseudomonas aeruginosa

Growth of Pseudomonas aeruginosa and thirty-five other species of gramnegative bacilli was observed on 0.03% cetrimide in heart infusion agar medium and Pseudosel agar (BBL). The 0.03% cetrimide agar was more selective for growth of P. aeruginosa than was Pseudosel agar; however, certain bacteria other than P. aeruginosa also grew on the former medium. Although Pseudosel agar was not a highly selective medium for P. aeruginosa, it was preferable to technicolor agar for detection of the pyocyanin and pyorubin pigments produced by P. aeruginosa.     

Isolation and characteristics of new nonlysogenic bacteriophages of Pseudomonas aeruginosa

A large group of nonlysogenic bacteriophages specific for Pseudomonas aeruginosa was studied. According to their absorption characteristics and serological properties, the phages were subdivided into four groups: luminal diameter k, luminal diameter m, luminal diameter mnP78 and luminal diameter mnF82. Within each of the groups, the phages were similar in the morphology of their particles and certain physiological characteristics. The luminal diameter m phages were similar to the P. aeruginosa bacteriophage E79 in their adsorption properties and antigenic specificity. The phages of the other groups differed in the above characteristics from the known P. aeruginosa bacteriophages. The effect of some plasmids on the growth of bacteriophages luminal diameter k, luminal diameter m, luminal diameter mnP78 and luminal diameter mnF82 was studied. The growth of new bacteriophages on certain plasmid-containing strains was inhibited in some cases.     

Pseudomonas diversity in crude-oil-contaminated intertidal sand samples obtained after the Prestige oil spill

The Galicia seashore, in northwestern Spain, was one of the shorelines affected by the Prestige oil spill in November 2002. The diversity of autochthonous Pseudomonas populations present at two beaches (Carnota municipality) was analyzed using culture-independent and culture-dependent methods. The first analysis involved the screening of an rpoD gene library. The second involved the isolation of 94 Pseudomonas strains that were able to grow on selective media by direct plating or after serial enrichments on several carbon sources: biphenyl, gentisate, hexadecane, methylnaphthalene, naphthalene, phenanthrene, salicylate, xylene, and succinate. Eight denitrifying Pseudomonas strains were also isolated by their ability to grow anaerobically with nitrate. The calculated coverage index for Pseudomonas species was 89% when clones and isolates were considered together, and there were 29 phylospecies detected. The most abundant were members of the species P. stutzeri, P. putida, P. anguilliseptica, and P. oleovorans. Thirty-one isolates could not be identified at the species level and were considered representatives of 16 putative novel Pseudomonas species. One isolate was considered representative of a novel P. stutzeri genomovar. Concordant results were obtained when the diversities of the cloned DNA library and the cultured strains were compared. The clone library obtained by the rpoD PCR method was a useful tool for evaluating Pseudomonas communities and also for microdiversity studies of Pseudomonas populations.     

松材线虫对其携带的细菌繁殖的影响

1.引言松材线虫病（Bursaphelenchus xylophilus）是松树的一种毁灭性病害,在日本、中国、韩国和北美、尼日利亚和葡萄牙等国家蔓延,造成了巨大经济损失,其中以日本和中国受害最重.一直认为松材线虫是引起该病的唯一病原,但近十几年来的研究发现,细菌在致病过程中可能起着重要作用,相继从病木和松材线虫体上分离到能对黑松苗有致萎活性的细菌.赵博光等首次根据实验提出松材线虫病是线虫和细菌共同侵染引起的复合侵染病害的假说,并在以后的试验中得到了验证.关于松材线虫对其细菌繁殖的影响研究鲜有报道.本试验采用从感病松树上分离并鉴定了的细菌菌株中选取假单胞属7株、其它属的细菌菌株3株,     

Isolation and Characterization of the algD gene of Pseudomonas syringae pv. phaseolicola and its distribution among other Pseudomonads and related Organisms

The gene coding for GDP-mannose dehydrogenase ( algD ) was isolated from a Pseudomonas syringae pv. phaseolicola genomic library using a polymerase chain reaction-generated heterologous DNA-probe from Pseudomonas aeruginosa . A total of 2123 base pairs were sequenced (accession number AF001555) and analysed for homologies to the alginate gene cluster of P. aeruginosa . Downstream from algD an alg8 homologue was found suggesting a similar arrangement of the alginate gene cluster in P. syringae pv. phaseolicola to that in P. aeruginosa . Also, the deduced amino acid sequence of algD shows high similarity to that of P. aeruginosa (0.9) and Azotobacter vinelandii (0.88). Southern hybridization experiments revealed that algD is widely distributed among members of the Pseudomonas rRNA homology group I. Among others, sequences homologous to algD were detected in the P. syringae pathovars lachrymans , mori , morsprunorum, pisi , savastanoi, tabaci and tomato as well as in Pseudomonas amygdali . For most of the algD positive organisms synthesis of alginate has been reported by other studies. However, algD homologues were also detected for the species Pseudomonas corrugata , Pseudomonas marginalis and Pseudomonas avenae ( Acidovorax avenae ), for which alginate biosynthesis has not yet been reported.     

An explanation for the apparent host specificity of Pseudomonas plasmid R91 expression.

Pseudomonas aeruginosa strain 9169 has been reported to contain a plasmid that expresses resistance to carbenicillin (Cb), kanamycin (Km), and tetracycline (Tc) in Escherichia coli but resistance only to Cb in certain Pseudomonas recipients. The triply resistant plasmid in E. coli belonged to incompatibility (Inc) group P or P-1, whereas the singly resistant plasmid in P. aeruginosa was compatible with IncP-1 plasmids and other plasmids of established Inc specificity but incompatible with plasmid pSR1 that is here used to define a new Pseudomonas Inc group P-10. Additional physical and genetic studies showed that strain 9169 contained not one but two plasmids: IncP-1 plasmid R91a, determining the Cb Km Tc phenotype, and IncP-10 plasmid R91, determining Cb that differed in molecular weight and in EcoRI and BamHI restriction endonuclease recognition sites. Plasmid multiplicity rather than host effects on plasmid gene expression can account for differences in the phenotype of strain 9169 transconjugants to E. coli and P. aeruginosa.     

Identification of an emergent and atypical Pseudomonas viridiflava lineage causing bacteriosis in plants of agronomic importance in a Spanish region

Pseudomonas strains with an atypical LOPAT profile (where LOPAT is a series of determinative tests: L, levan production; O, oxidase production; P, pectinolitic activity; A, arginine dihydrolase production; and T, tobacco hypersensibility) can be regarded as emergent pathogens in the Principality of Asturias (Spain), where they have been causing, since 1999, severe damage in at least three taxonomically unrelated orchard plants of agronomic importance: common bean (Phaseolus vulgaris), kiwifruit (Actinidia deliciosa), and lettuce (Lactuca sativa). These strains are mainly differentiated by production of yellowish mucoid material in hypersucrose medium, used for the levan test, and by a variable pectinolytic activity on different potato varieties. The atypical organisms were identified as Pseudomonas viridiflava based on their 16S rRNA sequences. Among them a certain intraspecies genetic heterogeneity was detected by randomly amplified polymorphic DNA (RAPD) typing. To differentiate between isolates of P. viridiflava and Pseudomonas syringae pathovars, a 16S ribosomal DNA restriction fragment length polymorphism method employing the restriction endonucleases SacI and HinfI was developed. This could be used as a means of reliable species determination after the usual phenotypical characterization, which includes the LOPAT tests.     

Specific ion concentration as a factor in barotolerant protein synthesis in bacteria.

The degree of barotolerance exhibited by Pseudomonas fluorescens and Pseudomonas bathycetes in vitro polyphenylalanine-synthesizing systems can be modified by altering the concentrations of specific ions in the reaction mixture. Hybrid-protein-synthesizing systems, utilizing all the possible S-100 supernatant fluid and ribosome combinations from Escherichia coli, P. fluorescens, and P. bathycetes, were tested for barotolerance under conditions of low (16 mM Mg2+ plus 0 mM Na+) and high (150 mM Na+ plus 60 mM Mg2+) ion concentrations. The results reveal that barotolerant synthesis is a characteristic determined by the origin of the ribosome. Systems utilizing E. coli ribosomes are barosensitive at both low and high ion concentrations, P. fluorescens ribosomes barotolerant under both conditions, and P. bathycetes ribosomes barosensitive at low and barotolerant at high ion concentrations. Therefore, certain concentrations of specific ions will increase barotolerance, but only if the ribosomes are capable of functioning at high pressures.     

Identification and characterization of the emhABC efflux system for polycyclic aromatic hydrocarbons in Pseudomonas fluorescens cLP6a

The hydrocarbon-degrading environmental isolate Pseudomonas fluorescens LP6a possesses an active efflux mechanism for the polycyclic aromatic hydrocarbons phenanthrene, anthracene, and fluoranthene but not for naphthalene or toluene. PCR was used to detect efflux pump genes belonging to the resistance-nodulation-cell division (RND) superfamily in a plasmid-cured derivative, P. fluorescens cLP6a, which is unable to metabolize hydrocarbons. One RND pump, whose gene was identified in P. fluorescens cLP6a and was designated emhB, showed homology to the multidrug and solvent efflux pumps in Pseudomonas aeruginosa and Pseudomonas putida. The emhB gene is located in a gene cluster with the emhA and emhC genes, which encode the membrane fusion protein and outer membrane protein components of the efflux system, respectively. Disruption of emhB by insertion of an antibiotic resistance cassette demonstrated that the corresponding gene product was responsible for the efflux of polycyclic aromatic hydrocarbons. The emhB gene disruption did not affect the resistance of P. fluorescens cLP6a to tetracycline, erythromycin, trimethoprim, or streptomycin, but it did decrease resistance to chloramphenicol and nalidixic acid, indicating that the EmhABC system also functions in the efflux of these compounds and has an unusual selectivity. Phenanthrene efflux was observed in P. aeruginosa, P. putida, and Burkholderia cepacia but not in Azotobacter vinelandii. Polycyclic aromatic hydrocarbons represent a new class of nontoxic, highly hydrophobic compounds that are substrates of RND efflux systems, and the EmhABC system in P. fluorescens cLP6a has a narrow substrate range for these hydrocarbons and certain antibiotics.     

Phenotypic Characterization and Deoxyribonucleic Acid Homologies of Pseudomonas solanacearum

Twenty-six strains and colony variants of Pseudomonas solanacearum belonging to four described biotypes were characterized, by using 169 phenotypic characters previously found useful in distinguishing among strains of other Pseudomonas species. Deoxyribonucleic acid (DNA) hybridization (intra- and interspecific DNA-DNA hybridizations) was performed by using the in vitro "DNA competition" technique. P. solanacearum appears to be a moderately homogeneous species, which is, at most, only remotely related to all other species of the genus studied to date. The four biotypes are not clearly distinct from one another with respect to nutritional characters or DNA homologies. Discrepancies between acid production and growth with some carbohydrates were noted. Difficulties were encountered in certain DNA competition experiments and some problems of the methodology are discussed.     

Primary alkylsulphatase activities of the detergent-degrading bacterium Pseudomonas C12B. Purification and properties of the P1 enzyme.

The P1 primary alkylsulphatase of Pseudomonas C12B was purified 1500-fold to homogeneity by a combination of streptomycin sulphate precipitation of nucleic acids, (NH4)2SO4 fractionation and chromatography on columns of DEAE-cellulose, Sephacryl S-300 and butyl-agarose. The protein was tetrameric with an Mr of 181000-193000, and exhibited maximum activity at pH 6.1. Primary alkyl sulphates of carbon-chain length C1-C5 or above C14 were not substrates, but the intermediate homologues were shown to be substrates, either by direct assay (C6-C9 and C12) or by gel zymography (C10, C11, C13 and C14). Increasing the chain length from C6 to C12 led to diminishing Km. Values of delta G0' for binding substrates to enzyme were dependent linearly on chain length, indicating high dependence on hydrophobic interactions. Vmax./Km values increased with increasing chain length. Inhibition by alk-2-yl sulphates and alkane-sulphonates was competitive and showed a similar dependence on hydrophobic binding. The P1 enzyme was active towards several aryl sulphates, including o-, m- and p-chlorophenyl sulphates, 2,4-dichlorophenyl sulphate, o-, m- and p-methoxyphenyl sulphates, m- and p-hydroxyphenyl sulphates and p-nitrophenyl sulphate, but excluding bis-(p-nitrophenyl) sulphate and the O-sulphate esters of tyrosine, nitrocatechol and phenol. The arylsulphatase activity was weak compared with alkylsulphatase activity, and it was distinguishable from the de-repressible arylsulphatase activity of Pseudomonas C12B reported previously. Comparison of the P1 enzyme with the inducible P2 alkylsulphatase of this organism, and with the Crag herbicide sulphatase of Pseudomonas putida, showed that, although there are certain similarities between any two of the three enzymes, very few properties are common to all three.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent.

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

Slime Production by Pseudomonas aeruginosa

Adequate experimental conditions for slime production by Pseudomonas aeruginosa were investigated using a cellophane plate method. Definite slime production was observed on heart infusion agar, brain heart infusion agar, yeast extract agar and synthetic agar, but not on nutrient agar. The addition of phosphate to the nutrient agar above 0.05% caused visible slime formation. Incubation at 37 C resulted in a higher yield of slime than at 25 C. Longer incubation seemed more favorable for slime production, while the pH reaction of the test media did not effect the slime yield. All the test cultures of P. aeruginosa produced large amounts of slime by this procedure. Cultures of Pseudomonas fluorescens, other Pseudomonas spp. and certain vibrios also produced slime under these experimental conditions.     

A note on Pseudomonas syringae pv. berberidis infections of Berberis: aetiology of a leaf spot and leaf fall disease in England

R oberts , S.J. & P reece , T.F. 1984. A note on Pseudomonas syringae pv. berberidis infections of Berberis : aetiology of a leaf spot and leaf fall disease in England. Journal of Applied Bacteriology 56 , 507–513.
A leaf spot and leaf fall disease is currently causing problems among certain Berberis spp. in England. Evergreens affected include B. julianae, B. gagnepainii, B. candidula, B. hookeri , and deciduous spp. include B. x ottawensis and B. thunbergii . The pathogen, Pseudomonas syringae pv. berberidis , which was confirmed as a distinct pathover by biochemical and host tests, was isolated from less than half of leaves showing typical symptoms. Isolates of Ps. syringae pv. berberidis from England, New Zealand and USA differed in cultural characteristics and in patho-genicity to certain Berberis spp.     

Comparison of enzymatic and immunochemical properties of 2,3-dihydroxybiphenyl-1,2-dioxygenases from four Pseudomonas strains

Abstract A 2,3-dihydroxybiphenyl-1,2-dioxygenase gene has been cloned from chromosomal DNA of Pseudomonas sp. DJ-12 which can grow on biphenyl or 4-chlorobiphenyl as the sole carbon and energy source. Enzymatic and immunochemical properties of the cloned 2,3-dihydroxybiphenyl-1,2-dioxygenase were characterized, and compared with those of P. pseudoalcaligenes KF707, Pseudomonas sp. KKS102, and P. putida OU83. The dioxygenase of Pseudomonas sp. DJ-12 was similar to those of P. pseudoalcaligenes KF707, and Pseudomonas sp. KKS102, but significantly different from that of P. putida OU83 in electrophoretic mobilities on native PAGE and SDS-PAGE. The dioxygenases of Pseudomonas sp. DJ-12 and P. putida OU83 exhibited the highest ring-fission activity to 3-methylcatechol, and those of P. pseudoalcaligenes KF707 and Pseudomonas sp. KKS102 to 2,3-dihydroxybiphenyl among 2,3-dihydroxybiphenyl, catechol, 3-methylcatechol, 4-methylcatechol, and 4-chlorocatechol as substrates. 2,3-dihydroxybiphenyl-1,2-dioxygenase of P. pseudoalcaligenes KF707 was immunochemically related to that of Pseudomonas sp. KKS102, but was different from those of Pseudomonas sp. DJ-12 and P. putida OU83.     

Selective medium for Pseudomonas aeruginosa that uses 1,10-phenanthroline as the selective agent

The MIC of 1,10-phenanthroline for 35 Pseudomonas aeruginosa strains was 128 micrograms/ml, whereas 32 micrograms or less per ml inhibited all other microorganisms tested. On the basis of these results, a selective agar for P. aeruginosa which contained 15 g of Trypticase soy broth (BBL Microbiology Systems), 15 g of agar, and 0.1 g of phenanthroline per liter was formulated. Forty-four P. aeruginosa strains yielded a mean efficiency of plating on this medium of 79% of the counts obtained on Trypticase soy agar, which was significantly higher than that obtained with pseudomonas isolation agar or Pseudosel agar. Pseudomonas cepacia, Pseudomonas fluorescens, Pseudomonas putida, Pseudomonas stutzeri, representatives of 13 other genera (including gram-negative rods, gram-positive rods, and cocci), and a yeast were not recovered within 48 h at 35 degrees C when approximately 10(7) CFU were plated on this medium. Only small colonies from one strain each of P. fluorescens and P. putida could be seen at 3 and 7 days, respectively, and they had an efficiency of plating of only less than 0.001%. When 10(7) CFU of either of these strains was plated with 10(2) CFU of P. aeruginosa, it did not interfere with the quantitative recovery of P. aeruginosa.     

The esterase activity of a pigmented bacterial group belonging to the Pseudomonas genus

Bacteria belonging to the Pseudomonas genus and isolated from zonal soils in different geographical zones of the USSR as well as from the rhizosphere of cultivated and wild plants were tested for their esterase activity. The studied collection of cultures included 205 strains of different pigmented Pseudomonas species which, according to the conventional taxonomy, were assigned to the so-called "Pseudomonas fluorescens complex". As was shown in this study, many Pseudomonas species are potential producers of nonspecific esterases. P. maltophilia and P. geniculata synthesizing pyomelanin have the highest activity of esterase. The activity of esterase correlates with the formation of a melanin-like pigment in Pseudomonas cultures. It also correlates with the species to which a culture belongs, which makes it possible to use this property as an additional criterion for the identification of Pseudomonas species.     

Effects of external stimuli on environmental bacterial strains harboring analgD-lux bioluminescent reporter plasmid for the study of corrosive biofilms

An alginic acid biosynthesis bioluminescent reporter plasmid, pUTK50, was transconjugated into environmental strains ofPseudomonas putida, Pseudomonas fluorescens, andStenotrophomonas maltophilia. Bioluminescent transconjugates were selected from each strain for investigation of environmental stress factors that promote alginic acid exopolymer biosynthesis in developing biofilms. Environmental stimuli associated with increased levels of alginate synthesis, in a previously developed organism,P. aeruginosa FRD1, were applied to the environmental strains. Increased salt concentrations and higher ratios of nitrate vs ammonium ions as the limiting nitrogen source induced bioluminescence in FRD1 and the environmental strains. However, for environmental strains ofP. putida, P. fluorescens andS. maltophilia, polysaccharides were detected with low uronic acids content and different structural components. When tested within a biofilm,S. maltophilia O46 demonstrated exceptional adhesive and corrosive properties while alginic acid synthesis was not high. In most of the environmental strains, periods of increased bioluminescence were induced by external stimuli, but exopolysaccharides other than alginic acid were expressed. It is hypothesized that the environmental strains have homologous but nonidentical promoter sequences which are responsive to certain environmental stimuli and may control genes necessary for the production of alternative exopolysaccharides.