Regulation of the transposase of Tn4652 by the transposon-encoded protein TnpC.
(33/1206)
Transposition is a DNA reorganization reaction potentially deleterious for the host. The frequency of transposition is limited by the amount of transposase. Therefore, strict regulation of a transposase is required to keep control over the destructive multiplication of the mobile element. We have shown previously that the expression of the transposase (tnpA) of the Pseudomonas putida PaW85 transposon Tn4652 is positively affected by integration host factor. Here, we present evidence that the amount of the transposase of Tn4652 in P. putida cells is controlled by the transposon-encoded protein (TnpC). Sequence analysis of the 120-amino-acid-long TnpC, coded just downstream of the tnpA gene, showed that it has remarkable similarity to the putative polypeptide encoded by the mercury resistance transposon Tn5041. As determined by quantitative Western blot analysis, the abundance of TnpA was reduced up to 10-fold in the intact tnpC background. In vivo experiments using transcriptional and translational fusions of the tnpA gene and the reporter gene gusA indicated that TnpC operates in the regulation of the transposase of Tn4652 at the post-transcriptional level. (+info)
Mutations of glutamate-84 at the putative potassium-binding site affect camphor binding and oxidation by cytochrome p450cam.
(34/1206)
Cytochrome P450cam (CYP101) from Pseudomonas putida is unusual among P450 enzymes in that it exhibits co-operative binding between the substrate camphor and a potassium ion. This behaviour has been investigated by mutagenesis of Glu84, a surface residue which forms part of the cation-binding site. Substitutions that neutralize or reverse the charge of this side chain are shown to disrupt the co-operativity of potassium and camphor binding by P450cam, and also to influence the catalytic activity. In particular, replacement of Glu84 by positively charged residues such as lysine results in increased high-spin haem fractions and camphor turnover activities in the absence of potassium, along with decreased camphor dissociation constants. However, in the presence of potassium the camphor dissociation constants of these mutants are significantly increased compared with the wild-type, although the camphor turnover activities remain marginally higher. In contrast, substitution by aspartate results in tighter binding of both potassium and camphor, but has little effect on the enzymatic activity. In all cases the reaction remains essentially 100% coupled and gives 5-exo-hydroxycamphor as the only product. These results suggest that an anionic side chain at the 84 position is crucial for the co-operativity of camphor and cation binding, and that the physiological role for potassium binding by cytochrome P450cam is to promote camphor binding even at the expense of turnover rate, thus allowing the organism to utilize low environmental concentrations of this substrate for growth. (+info)
Crystal structure of delta(5)-3-ketosteroid isomerase from Pseudomonas testosteroni in complex with equilenin settles the correct hydrogen bonding scheme for transition state stabilization.
(35/1206)
Delta(5)-3-Ketosteroid isomerase from Pseudomonas testosteroni has been intensively studied as a prototype to understand an enzyme-catalyzed allylic isomerization. Asp(38) (pK(a) approximately 4.7) was identified as the general base abstracting the steroid C4beta proton (pK(a) approximately 12.7) to form a dienolate intermediate. A key and common enigmatic issue involved in the proton abstraction is the question of how the energy required for the unfavorable proton transfer can be provided at the active site of the enzyme and/or how the thermodynamic barrier can be drastically reduced. Answering this question has been hindered by the existence of two differently proposed enzyme reaction mechanisms. The 2.26 A crystal structure of the enzyme in complex with a reaction intermediate analogue equilenin reveals clearly that both the Tyr(14) OH and Asp(99) COOH provide direct hydrogen bonds to the oxyanion of equilenin. The result negates the catalytic dyad mechanism in which Asp(99) donates the hydrogen bond to Tyr(14), which in turn is hydrogen bonded to the steroid. A theoretical calculation also favors the doubly hydrogen-bonded system over the dyad system. Proton nuclear magnetic resonance analyses of several mutant enzymes indicate that the Tyr(14) OH forms a low barrier hydrogen bond with the dienolic oxyanion of the intermediate. (+info)
Recruitment of RNA polymerase is a rate-limiting step for the activation of the sigma(54) promoter Pu of Pseudomonas putida.
(36/1206)
The activity of the sigma(54)-promoter Pu of Pseudomonas putida was examined in vitro with a DNA template lacking upstream activating sequences, such that RNA polymerase can be activated by the enhancer-binding protein XylR only from solution. Although the transcription activation pathway in this system lacked the step of integration host factor (IHF)-mediated looping of the XylR.DNA complex toward the prebound RNA polymerase, IHF still stimulated promoter activity. The positive effect of IHF became evident not only with XylR from solution, but also with other sigma(54)-dependent activators such as NtrC and NifA. Furthermore, an equivalent outcome was shown for the nonspecific DNA-binding protein HU. This stimulation of transcription in the absence of the enhancer was traced to the recruitment of RNA polymerase (i.e. increased efficiency of formation of closed complexes) brought about by IHF or HU binding. Thus, under limiting concentrations of the polymerase, the factor-mediated binding of the enzyme to Pu seems to enter a kinetic checkpoint in the system that prevents the XylR-mediated formation of an open complex. (+info)
A siderophore from Pseudomonas putida type A1: structural and biological characterization.
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A siderophore from a root-colonizing, plant-beneficial fluorescent Pseudomonas (P. putida type A1) isolated from chickpea rhizosphere was studied. Culture conditions required for optimal production of the chromophore by the organism were standardized. The compound was purified by gel filtration, ion exchange and RP-HPLC chromatographic procedures. The purified compound exhibited siderophore activity for P. putida and antifungal activity on phytopathogens, Fusarium oxysporum f. sp. ciceri and Helminthosporium oryzae. Growth inhibition of the pathogens was observed under iron-deficient conditions. Complete acid hydrolysis of the compound revealed that it is a peptide containing Asx, Thr, Glx, Val, His, Lys, Ser and Gly. Spectral analysis revealed that it contains hydroxyquinoline-based chromophore in addition to an aromatic residue and the molecular weight of the compound was 1.5 kDa. EPR analysis of the peptide-chromophore-iron complex showed that the compound binds to iron and the bound iron was in the Fe(3+) oxidation state having a high spin d(5) system. The peptide-chromophore-iron complex takes a turn structure in solution as shown by circular dichroism spectroscopy, a feature which was hitherto not known for other siderophores. The siderophore studied here is unique in this respect but otherwise strikingly similar to other pseudobactin-type siderophores of plant growth-promoting and plant-deleterious pseudomonads. The possible functional significance of the compound is discussed in relation to the secondary structure described earlier for siderophores. (+info)
Removal of mercury from chloralkali electrolysis wastewater by a mercury-resistant Pseudomonas putida strain.
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A mercury-resistant bacterial strain which is able to reduce ionic mercury to metallic mercury was used to remediate in laboratory columns mercury-containing wastewater produced during electrolytic production of chlorine. Factory effluents from several chloralkali plants in Europe were analyzed, and these effluents contained total mercury concentrations between 1.6 and 7.6 mg/liter and high chloride concentrations (up to 25 g/liter) and had pH values which were either acidic (pH 2.4) or alkaline (pH 13.0). A mercury-resistant bacterial strain, Pseudomonas putida Spi3, was isolated from polluted river sediments. Biofilms of P. putida Spi3 were grown on porous carrier material in laboratory column bioreactors. The bioreactors were continuously fed with sterile synthetic model wastewater or nonsterile, neutralized, aerated chloralkali wastewater. We found that sodium chloride concentrations up to 24 g/liter did not inhibit microbial mercury retention and that mercury concentrations up to 7 mg/liter could be treated with the bacterial biofilm with no loss of activity. When wastewater samples from three different chloralkali plants in Europe were used, levels of mercury retention efficiency between 90 and 98% were obtained. Thus, microbial mercury removal is a potential biological treatment for chloralkali electrolysis wastewater. (+info)
Utilization of heterologous siderophores enhances levels of iron available to Pseudomonas putida in the rhizosphere.
(39/1206)
Pseudomonas spp. have the capacity to utilize siderophores produced by diverse species of bacteria and fungi, and the present study was initiated to determine if siderophores produced by rhizosphere microorganisms enhance the levels of iron available to a strain of Pseudomonas putida in this natural habitat. We used a previously described transcriptional fusion (pvd-inaZ) between an iron-regulated promoter (pvd) and the ice nucleation reporter gene (inaZ) to detect alterations in iron availability to P. putida. Ice nucleation activity (INA) expressed from the pvd-inaZ fusion by P. putida N1R or N1R Pvd(-), a derivative deficient in the production of a pyoverdine siderophore, was inversely related to the concentration of ferric citrate in a culture medium. In culture, INA expressed by N1R Pvd(-) (pvd-inaZ) was reduced in the presence of the ferric complex of pseudobactin-358, a pyoverdine siderophore produced by P. putida WCS358 that can be utilized as a source of iron by N1R Pvd(-). In the rhizosphere of cucumbers grown in sterilized soil, N1R Pvd(-) (pvd-inaZ) expressed INA, indicating that iron availability was sufficiently low in that habitat to allow transcription of the iron-regulated pvd promoter. Coinoculation with WCS358 or N1R significantly decreased INA expressed by N1R Pvd(-) (pvd-inaZ) in the rhizosphere, whereas coinoculation with a pyoverdine-deficient mutant of WCS358 did not reduce INA expressed by N1R Pvd(-) (pvd-inaZ). These results indicate that iron availability to N1R Pvd(-) (pvd-inaZ) in the rhizosphere was enhanced by the presence of another strain of P. putida that produces a pyoverdine that N1R Pvd(-) (pvd-inaZ) was able to utilize as a source of iron. In culture, strain N1R Pvd(-) also utilized ferric complexes of the siderophores enterobactin and aerobactin as sources of iron. In the rhizosphere of cucumbers grown in sterilized soil, INA expressed by N1R Pvd(-) (pvd-inaZ) was reduced in the presence of strains of Enterobacter cloacae that produced enterobactin, aerobactin, or both siderophores, but INA expressed by N1R Pvd(-) (pvd-inaZ) was not altered in the presence of a mutant of E. cloacae deficient in both enterobactin and aerobactin production. Therefore, the iron status of P. putida was altered by siderophores produced by an unrelated bacterium coinhabiting the rhizosphere. Finally, we demonstrated that INA expressed by N1R containing pvd-inaZ in the rhizosphere differed between plants grown in sterilized versus nonsterilized field soil. The results of this study demonstrate that (i) P. putida expresses genes for pyoverdine production and uptake in the rhizosphere, but the level of gene expression is influenced by other bacteria that coexist with P. putida in this habitat, and (ii) diverse groups of microorganisms can alter the availability of chemical resources in microbial habitats on root surfaces. (+info)
Engineering of a stable whole-cell biocatalyst capable of (S)-styrene oxide formation for continuous two-liquid-phase applications.
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Recombinant strains of Pseudomonas putida KT2440 carrying genetic expression cassettes with xylene oxygenase- and styrene monooxygenase-encoding genes on their chromosomes could be induced in shaking-flask experiments to specific activities that rivaled those of multicopy-plasmid-based Escherichia coli recombinants. Such strains maintained the introduced styrene oxidation activity in continuous two-liquid-phase cultures for at least 100 generations, although at a lower level than in the shaking-flask experiments. The data suggest that placement of target genes on the chromosome might be a suitable route for the construction of segregationally stable and highly active whole-cell biocatalysts. (+info)