Tryptophan 7-Halogenase from Pseudomonas aureofaciens ACN Strain: Gene Cloning and Sequencing and the Enzyme Expression

The gene of tryptophan 7-halogenase was isolated from the Pseudomonas aureofaciens ACN strain producing pyrrolnitrin, a chlorocontaining antibiotic, and sequenced. A high homology degree (over 95%) was established for the genes and the corresponding halogenases from P. aureofaciens ACN and P. fluorescens BL915. The tryptophan 7-halogenase gene was amplified by PCR, and the corresponding enzyme was expressed in Escherichia coli cells using the pBSII SK+ vector.     

The Effect of Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense on Induction of Defense Enzymes and Phenolics in Banana

The effect of Pseudomonas fluorescens treatment and Fusarium oxysporum f. sp. cubense inoculation on induction of phenylalanine ammonia-lyase (PAL), peroxidase (POX), chitinase, -1,3-glucanase and accumulation of phenolics in banana (Musa sp.) was studied. When banana roots were treated with P. fluorescens strain Pf10, a two-fold increase in phenolic content in leaf tissues was recorded 3 – 6 d after treatment. Challenge inoculation with F. oxysporum, the wilt pathogen, steeply increased the phenolic content in P. fluorescens-treated banana plants. Significant increase in POX activity was detected 6 – 9 d after P. fluorescens treatment. PAL, chitinase and -1,3-glucanase activities increased significantly from 3 d after P. fluorescens treatment and reached the maximum 6 d after treatment. Challenge inoculation with F. oxysporum further increased the enzyme activities. These results suggest that the enhanced activities of defense enzymes and elevated content of phenolics may contribute to bioprotection of banana plants against F. oxysporum.     

The structure of a pyoverdine produced by a Pseudomonas tolaasii-like isolate

Cultures of Agaricus bisporus, the most extensively cultivated mushroom, can be infected severely by Pseudomonas tolaasii. This pathogen is characterized by the so-called white line reaction, a precipitate formed on agar plates between its colonies and those of P. reactans, both belonging to the collective species P. fluorescens. A recent study has shown that a group of P. tolaasii isolates can be subdivided into two groups or 'siderovars', based on the pyoverdines they produce (Munsch et al. 2000). One group of strains is characterized by the pyoverdine described by Demange et al. (1990). A representative of the second group (strain Ps3a) was found to produce the same pyoverdine as a strain which had been classified before as P. aureofaciens. However, based mainly on 16S rRNA gene sequence comparisons and REP-PCR generated fingerprints, the two strains are not identical. They are also distinguishable from the P. tolaasii type strain.     

Aerobic degradation of phthalic acid by Comamonas acidovoran Fy-1 and dimethyl phthalate ester by two reconstituted consortia from sewage sludge at high concentrations

Microbial degradation of phthalic acid (PA) and dimethyl phthalate ester (DMPE) under aerobic conditions was investigated using a pure species of bacteria and two consortia from sewage sludge. Five morphologically distinct microorganisms were obtained in pure culture and identified, and tested for the capability of degrading phthalate and DMPE. Comamonas acidovorans strain Fy-1 showed the highest ability to degrade high concentrations of phthalate (2600 mg/l) within 48 h. Two reconstituted consortia of microorganisms, one comprising Pseudomonas fluorescens, P. aureofaciens and Sphingomonas paucimobilis, and the other of Xanthomonas maltophilia and S. paucimobilis, were effective in completely degrading DMPE (400 mg/l) in 48–96 h. The three-species consortium appeared to be more effective in the degradation of DMPE, and both consortia proceeded via formation of mono-methyl phthalate (MMP) and then phthalatic acid before mineralization. This study suggests that high concentrations of the endocrine-disrupting chemicals phthalate and DMPE can be mineralized in wastewater treatment systems by indigenous microorganisms.     

Structural characterization of the O-chain polysaccharide from an environmentally beneficial bacterium Pseudomonas chlororaphis subsp. aureofaciens strain M71

Pseudomonas chlororaphis subsp. aureofaciens strain M71 was isolated from the root of a tomato plant and it was able to control in vivo Fusarium oxysporum f. sp. radicis-lycopersici responsible for the tomato crown and root rot. Recently, strain M71 was evaluated even for its efficacy in controlling Seiridium cardinale, the causal agent of bark canker of common cypress (Cupressus sempervirens L.). Strain M71 ability to persist on the tomato rhizosphere and on the aerial part of cypress plants could be related to the nature of the lipopolysaccharides (LPS) present on the outer membrane and in particular to the O-specific polysaccharide.A neutral O-specific polysaccharide was obtained by mild acid hydrolysis of the lipopolysaccharide from P. chlororaphis subsp. aureofaciens strain M71. By means of compositional analyses and NMR spectroscopy, the chemical repeating unit of the polymer was identified as the following linear trisaccharide.     

Biological control ofFusarium oxysporum f.sp.cubense in banana by inoculation withPseudomonas fluorescens

Native strains ofPseudomonas fluorescens exhibitedin vitro antibiosis towards isolates of races 1 and 4 ofFusarium oxysporum f.sp.cubense, the Panama wilt pathogen of banana. The seedlings ofMusa balbisiana seedlings treated withP. fluorescens showed less severe wilting and internal discolouration due toF. oxysporum f.sp.cubense infection in greenhouse experiments. In addition to suppressing Panama wilt, bacterized seedlings ofM. balbisiana also showed better root growth and enhanced plant height.     

Induction of pathogenesis-related proteins during biocontrol of <Emphasis Type="Italic">Rhizoctonia solani</Emphasis> with <Emphasis Type="Italic">Pseudomonas aureofaciens</Emphasis> in soybean (<Emphasis Type="Italic">Glycine max</Emphasis> L. Merr.) plants

To investigate the biocontrol effectiveness of the antibiotic producing bacterium, Pseudomonas aureofaciens 63–28 against the phytopathogen Rhizoctonia solani AG-4 on Petri plates and in soybean roots, growth response and induction of PR-proteins were estimated after inoculation with P. aureofaciens 63–28 (P), with R. solani AG-4 (R), or with P. aureofaciens 63–28 + R. solani AG-4 (P + R). P. aureofaciens 63–28 showed strong antifungal activity against R. solani AG-4 pathogens in Petri plates. Treatment with P. aureofaciens 63–28 alone increased the emergence rate, shoot fresh weight, shoot dry weight and root fresh weight at 7 days after inoculation, when compared to R. solani AG-4; P + R treatment showed similar effects. Peroxidase (POD) and β-1,3-glucanase activity of P. aureofaciens 63–28 treated roots increased by 41.1 and 49.9%, respectively, compared to control roots. POD was 26% greater in P + R treated roots than R. solani treated roots. Two POD isozymes (59 and 27 kDa) were strongly induced in P + R treated roots. The apparent molecular weight of chitinase from treated roots, as determined through SDS-PAGE separation and comparison with standards, was about 29 kDa. Five β-1,3-glucanase isozymes (80, 70, 50, 46 and 19 kDa) were observed in all treatments. These results suggest that inoculation of soybean plants with P. aureofaciens 63–28 elevates plant growth inhibition by R. solani AG-4 and activates PR-proteins, potentially through induction of systemic resistance mechanisms.     

Zwei neue Arten der GattungSaponaria (Caryophyllaceae) aus Afghanistan und Pakistan

Saponaria stenopetala sp.n. in Eastern Afghanistan is close toS. pachyphylla Rech. f. andS. subrosularis Rech. f.—The nearest allies ofS. makranica sp.n. from Western Pakistan and Southeastern Iran areS. kermanensis Bornm. andS. floribunda (Kar. & Kir.)Boiss.

Flora Iranicae praecursores 36–37. — Praecursores praecurrentes in Pl. Syst. Evol.139, 313–317 (1982).     

Enhanced nodulation of soybean by Bradyrhizobium in the presence ofPseudomonas fluorescens

Experiments were undertaken to determine the effect ofPseudomonas fluorescens on nodulation of soybean by two strains ofBradyrhizobium japonicum, USDA I-110 and 61A76.Pseudomonas fluorescens can enhance the nodulation ability ofB. japonicum. Preincubation ofB. japonicum withP. fluorescens before inoculation further increased the level of nodulation.     

Interaction of Fusarium Oxysporum f.sp. Cubense with Pseudomonas Fluorescens Precolonized to Banana Roots

Effect of precolonization of banana cv Neeypovan roots with Pseudomonas fluorescens on infection with Fusarium oxysporum f.sp. cubense was studied. Under in vitro conditions Pseudomonas fluorescens clearly inhibited Fusarium oxysporum f.sp. cubense. Fluorescein isothiocyanate-tagged antibodies raised in a rabbit system for Pseudomonas fluorescens and Fusarium oxysporum f.sp. cubense separately were used to study the spread of both organisms in banana root. It was observed that precolonization with Pseudomonas fluorescens could reduce Fusarium oxysporum f.sp. cubense colonization by 72%, and also correlated with a number of structural changes in the cortical cells, mainly with densely stained amorphous material and polymorphic wall thickenings as revealed by light and electron microscopic studies. Massive depositions of unusual structures at sites of fungal entry was also noticed, which clearly indicated that bacterized root cells were signalled to mobilize a number of defence structures for preventing the spread of pathogen in the tissue. This revised version was published online in July 2006 with corrections to the Cover Date.     

Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955

Summary The genetic organization of functions responsible for mannityl opine catabolism of the Ti plasmid of Agrobacterium tumefaciens strain 15955 was investigated. A partial HindIII digest of pTi15955 was cloned into a broad host range cosmid and the clones obtained were tested for ability to confer mannityl opine degradation upon Agrobacterium. Inserts containing genes for catabolism of mannopinic acid, mannopine, agropine, and agropinic acid were obtained, spanning a segment of 43 kb on the Ti plasmid. Two clones conferring upon Agrobacterium the ability to catabolize the mannityl opines were mobilized to several Rhizobium sp., to Pseudomonas putida and P. fluorescens and to Escherichia coli. The catabolic functions were phenotypically expressed in all Rhizobium sp. tested, and in P. fluorescens, but not in P. putida or in E. coli.     

Three native Pseudomonas fluorescens strains tested under growth chamber and field conditions as biocontrol agents against damping-off in alfalfa

Alfalfa (Medicago sativa) is one of the most important crops used in Uruguay for livestock feeding. Seedling diseases, particularly damping-off, are a critical factor which limits its establishment. Three native Pseudomonas fluorescens strains, UP61.2, UP143.8 and UP148.2, previously isolated from Lotus corniculatus, were evaluated to determine their efficacy as biological control agents for alfalfa seedling diseases in the field. Their compatibility with the alfalfa-Sinorhizobium meliloti symbiosis was also assessed. In growth chamber conditions seed inoculation with Pseudomonas strains did not affect different parameters of alfalfa-rhizobium symbiosis as shown by nodulation rate and shoot dry weight of plants. The presence of the commercial inoculant strains of S. meliloti did not impair colonization by the P. fluorescens and vice versa. In field trials the dynamics of rhizobial rhizospheric populations were not affected by the presence of P. fluorescens. Each P. fluorescens strain successfully colonized alfalfa roots at adequate densities for biocontrol activity. Results showed that P. fluorescens strains provided a 10–13% increase in the number of established plants relative to the control, an intermediate result compared to the fungicide treatment (24%). The alfalfa above-ground biomass was increased by 13% and 15–18% in the presence of the fungicide and P. fluorescens strains, respectively. Therefore, results from this study demonstrated that the three P. fluorescens strains provided effective control against soil-borne pathogens and suggest a potential use in the development of a commercial inoculant to be applied for the control of legume seedling diseases.     

The Production of Phenazine Antibiotics by the Pseudomonas aureofaciens Strain with Plasmid-Controlled Resistance to Cobalt and Nickel

Plasmid pBS501, responsible for the resistance of the wild-type Pseudomonas sp. BS501(pBS501) to cobalt and nickel ions, was conjugatively transferred to the rhizosphere Pseudomonas aureofaciens strain BS1393, which is able to synthesize phenazine antibiotics and to suppress a wide range of phytopathogenic microorganisms. The transconjugant P. aureofaciens BS1393(pBS501) turned out to be resistant to cobalt and nickel with an MIC of 8 mM. When grown in a synthetic medium with 0.25 mM cobalt, the transconjugant accumulated 6 times more cobalt than the wild-type strain BS501(pBS501) (1.2 versus 0.2 g Co/mg protein). Electron microscopic studies showed that cobalt accumulates on the surface of transconjugant cells in the form of electron-opaque granules. In a culture medium with 2 mM cobalt or nickel, strain BS1393 produced phenazine-1-carboxylic acid in trace amounts. The transconjugant P. aureofaciens BS1393(pBS501) produced this antibiotic in still smaller amounts. Unlike the parent strain BS1393, the transconjugant P. aureofaciens BS1393(pBS501) was able to suppress in vitro the growth of the phytopathogenic fungus Gaeumannomyces graminis var. tritici1818 in a medium containing 0.5 mM cobalt or nickel.     

Diel activity patterns in Metapenaeus and Penaeus juveniles

Small (5–10.9 mm carapace length), medium (11–15.9 mm), and large (16–20.9 mm) juveniles of Metapenaeus anchistus, Metapenaeus sp., Penaeus monodon and P. merguiensis were stocked individually in glass tanks provided with sand substrate, sea water, artificial bamboo shelter, aeration and food. The seven activity types (recorded for each shrimp hourly for 24 h) were classified as below (burrowing) or above substrate (swimming, walking, stationary, in shelter, feeding and cleaning). Shrimp juveniles exhibited a strong diel periodicity — emergence and activity at night and burrowing in the day. The chi-square test showed that type of activity (above/below substrate) was associated with period (light/dark). Diurnal burrowing was greater among Metapenaeus than Penaeus; inversely, above substrate activities were more frequent for Penaeus species compared to Metapenaeus. Feeding was the major above substrate and nocturnal activity for M. anchistus, Metapenaeus sp. and P. monodon. Only P. Monodon used the shelter consistently. Frequency of the 7 activity types was dependent on juvenile size for Penaeus, e.g., the preference for shelters shifted to burrowing with increase in size in P. monodon. Results are discussed in relation to the importance of mangrove habitats in providing shelter to penaeids, in particular the mangrove-associated P. monodon and P. merguiensis.     

Alga-lytic activity of Pseudomonas fluorescens against the red tide causing marine alga Heterosigma akashiwo (Raphidophyceae)

A bacterial strain, HAK-13, exhibited strongest activity against Heterosigma akashiwo and was capable of controlling this bloom forming phytoplankton. Based on 16S rDNA sequences and biochemical and morphological characteristics, the strain HAK-13 was determined to be Pseudomonas fluorescens on the basis of 99.9% similarity with reference strains in the DNA databases. The growth of H. akashiwo was strongly suppressed by HAK-13 in all growth phases, with the strongest alga-lytic activity noted against harmful bloom-forming species in the exponential stage (6–22 days). Host range tests showed that HAK-13 also significantly inhibited the growth of Alexandrium tamarense and Cochlodinium polykrikoides but could not destroy Gymnodinium catenatum. P. fluorescens HAK-13 indirectly attacked H. akashiwo by alga-lytic substances that might be located at the compartment of cytoplasmic membrane of the bacterium at a level of 45.86 units/mg of specific activity. The results indicated that P. fluorescens HAK-13 caused cell lysis and death of H. akashiwo, A. tamarense, and C. polykrikoides dramatically and Prorocentrum dentatum slightly. Therefore, P. fluorescens HAK-13 has potential for use as a selective biocontrol of harmful algal blooms.     

Chlorobenzoate catabolism and interactions between Alcaligenes and Pseudomonas species from Bloody Run Creek

A mixed community of bacteria from surface runoff waters of the Hyde Park industrial landfill was enriched on 3-chlorobenzoate. Alcaligenes and Pseudomonas species were dominant in the community. Alcaligenes sp. BR60 carried an unstable plasmid specifying 3-chlorobenzoate catabolism. Metabolites detected in culture supernatants included chlorocatechol and chloro-cis,cis-muconic acid. Oxygen uptake in the presence of 3- and 4-substituted methyl-catechols revealed a catechol-1,2-oxygenase activity specific for substituted catechols with very limited activity for catechol. The isolate grew very slowly on benzoate. Alcaligenes sp. BR60 was isolated in co-culture with Pseudomonas fluorescens NR52. The latter contained no detectable plasmids and did not grow on benzoate or any of the chlorobenzoates in pure culture. Growth of the co-culture in Bloody Run Creek water supplemented with 3-chlorobenzoate indicated that phosphate concentrations in the water severely limited biodegradation. Under phosphate limited conditions in continuous culture, Pseudomonas fluorescens NR52 effectively scavenged available phosphate when it was present at a ratio of 1 cell to 20 of Alcaligenes sp. BR60. Under these conditions the growth of Alcaligenes sp. BR60 on 3-chlorobenzoate was reduced 5 fold, the frequency of plasmid deletion mutants increased, and 96% of the contaminant remained in the outflow in the form of the starting material or metabolites. No evidence was found for conjugation of the plasmid determining chlorobenzoate catabolism in Alcaligenes sp. BR60 to P. fluorescens NR52.Abbreviations 3 and 4 Cba chlorobenzoic acid isomers and growth phenotypes - Ba benzoic acid     

Two newVeronica species (Scrophulariaceae) of Turkey and Iraq

Veronica davisii M. A.Fischer, sp. n., limited to the mountains of Kurdistan (S. E. Turkey and N. Iraq), is related to the Turkish-CaucasianV. gentianoides Vahl.—V. montbretii M. A.Fischer, sp. n., a local endemic of Erzincan prov. (E. Anatolia), shows affinities to the CaucasianV. liwanensis C. Koch and to the Turkish endemicV. oltensis Woron. & Schelk. from Erzurum province.     

Antimicrobial potentials of endophytic fungi residing in <Emphasis Type="Italic">Quercus variabilis</Emphasis> and brefeldin A obtained from <Emphasis Type="Italic">Cladosporium</Emphasis> sp.

Among 67 endophytic fungi isolated from Quercus variabilis, 53.7% of endophytic fungal fermentation broths displayed growth inhibition on at least one test microorganism, such as pathogenic fungi (Trichophyton rubrum, Candida albicans, Aspergillus niger, Epidermophyton floccosum, Microsporum canis) and bacteria (Escherichia coli, Bacillus subtilis, Pseudomonas fluorescens). Moreover, 19.4% of strains showed a broader antimicrobial spectrum, such as Aspergillus sp., Penicillium sp., Alternaria sp., 20.9% of strains showed strong inhibition (+++) to pathogenic bacteria, while only 7.5% displayed that to test fungi. The most active antifungal strain I(R)9-2, Cladosporium sp. was selected and fermented. From the broth, a secondary metabolite, brefeldin A was obtained. This is the first report on the antimicrobial potentials of endophytic fungi residing in Q. variabilis and isolation of brefeldin A produced by Cladosporium sp.     

Kinorhyncha from the stomach of the shrimp Pleoticus muelleri (Bate, 1888) from Comodoro Rivadavia, Argentina

Six kinorhynchs were found in the stomachs of the Argentine red shrimp, Pleoticus mulleri (Bate, 1888) from the Argentine coast of Patagonia. Three new species are described: Condyloderes storchi n. sp., Pycnophyes argentinensis n. sp. and P. neuhausi n. sp. A fourth species, Kinorhynchus anomalus Lang, 1953 was previously known only from the coast of Chile. This is the third known record of kinorhynchs documented as a food source. Condyloderes storchi, n. sp. is the fourth new species in this genus. It is distinguished by its paradorsal cuspidate spines on segments 7 and 9, lateral accessory and ventrolateral spines on segments 3, 6, 7, 10 and 11. P. argentinensis, n. sp. has nearly equal sternal width for segments 3–11 (about 7% of the trunk length), episternal plates with three distinct areas along the anterior margin, mid-sternal plate with even margin, mid-dorsal spinose protrusions along the terminal borders of segments 11 and 12, and lateral terminal spines 176 μm long, about 21% of trunk length. P. neuhausi, n. sp.has a prominent posterior elongation of the tergal plate of segment 3, uneven lateral margins of the mid-sternal plate, a maximum sternal width at segment 3, no mid-dorsal spinose processes and mid-ventral thickenings on segments 10–12.     

Comparative effect of methioninyl adenylate on the growth of Salmonella typhimurium and Pseudomonas aeruginosa

The bacteriostatic effect of methioninyl adenylate (MAMP)—a specific inhibitor of the enzyme methionyl-tRNA synthetase—was investigated on Salmonella typhimurium and Pseudomonas aeruginosa.0.1 mM of this molecule added to the culture, inhibits the growth of S. typhimurium. The inhibition is specifically reversible by 0.1 mM L-methionine. In the same conditions even 1–2 mM MAMP has a very slight effect on the growth rate of P. aeruginosa and only during the first two generations. The same observation was made with the two other members of the fluorescens group P. fluorescens and P. putida.The growth rate of P. testosteroni with 1 mM MAMP in the medium is similar to the growth rate of P. aeruginosa but the other member of the acidovorans group P. acidovorans is much more affected by the same concentration of the inhibitor. — P. multivorans is inhibited by MAMP like P. acidovorans but with a somewhat higher yield at the end of the culture.—MAMP has no effect on P. alcaligenes.The possible reasons for the weak bacteriostatic effect of MAMP on P. aeruginosa were investigated. It was established that the inhibitor enters the cells and is not used as a carbon and energy source. The intracellular methionine concentration in S. typhimurium and in P. aeruginosa is about the same and does not increase when bacteria are cultivated with MAMP. The MTS of the two microorganisms is inhibited by MAMP in vitro to about the same extent. Furthermore the tRNA^met from P. aeruginosa are fully acylated after 3 to 4 generations with this compound. Nevertheless MAMP elicits higher MTS activity in P. aeruginosa and in P. acidovorans after 1 h of incubation. The most striking difference between S. typhimurium and P. aeruginosa is that the intra and extracellular level of 5phosphodiesterase which degrades MAMP is 10–20 fold higher in the second than in the first species.Non Standard Abbreviations MAMP Methioninyl adenylate - MTS methionyl-tRNA synthetase (EC 6.1.1.10) - VTS valyl-tRNA synthetase (EC 6.1.1.0) - Tris trihydroxymethylaminomethane Dedicated to Prof. R. Y. Stanier on the occasion of his 60th birthday