Inactivation of the Pseudomonas putida KT2440 dsbA gene promotes extracellular matrix production and biofilm formation

To identify genes essential to biofilm formation in Pseudomonas putida KT2440, 12 mutants defective in oxidative stress-related or metabolic pathway-related genes were evaluated. Of them, only the dsbA mutant lacking the disulfide bond isomerase exhibited significantly increased attachment to the polystyrene surface. Visual evaluation by extracellular matrix staining and scanning electron microscopy indicated that the KT2440-Δ dsbA strain displays enhanced extracellular matrix production, rugose colony morphology on agar plates and floating pellicles in static culture. Accordingly, we propose that deletion of the dsbA gene may stimulate production of the extracellular matrix, resulting in those phenotypes. In addition, the lack of detectable fluorescence in the KT2440-Δ dsbA under UV light as well as in both the wild type and the KT2440-Δ dsbA when grown on Luria–Bertani plates containing ferrous iron suggests that the fluorescent molecule may be a fluorescent siderophore with its synthesis/secretion controlled by DsbA in KT2440. These phenotypic defects observed in the dsbA mutant were complemented by the full-length KT2440 and Escherichia coli dsbA genes. In contrast to the role of DsbA in other bacteria, our results provide the first evidence that disruption of P. putida KT2440 dsbA gene overproduces the extracellular matrix and thus promotes biofilm formation.     

A two-partner secretion system is involved in seed and root colonization and iron uptake by Pseudomonas putida KT2440

We describe the first two-partner secretion system known to play a role in mutualistic plant-bacterial interactions, identified in the soil and rhizosphere-colonizing bacterium Pseudomonas putida KT2440. The genes coding for the two components of the system are organized in an operon, which we have named hlpBA. HlpA is a secreted protein that has similarities with iron-regulated haemolysins, while HlpB would be responsible for the activation and transport of HlpA across the outer membrane. Mutations in this novel two-partner secretion system result in reduced capacity to colonize corn seeds. When introduced in the rhizosphere, hlpA and hlpB mutants show no competitive disadvantage, but the number of cells attached to the root surface is reduced with respect to the wild type, suggesting this protein plays a role directly in the bacterial cell-root surface interaction. Under iron-limiting conditions, the presence of a truncated HlpA causes reduced viability and high levels of siderophore release. These data further strengthen our previous observations indicating the importance of iron acquisition for attachment of P. putida KT2440 to plant surfaces.     

Siderophore-mediated iron acquisition in the entomopathogenic bacterium Pseudomonas entomophila L48 and its close relative Pseudomonas putida KT2440

Pseudomonas entomophila L48 is a recently identified entomopathogenic bacterium which, upon ingestion, kills Drosophila melanogaster, and is closely related to P. putida. The complete genome of this species has been sequenced and therefore a genomic, genetic and structural analysis of the siderophore-mediated iron acquisition was undertaken. P. entomophila produces two siderophores, a structurally new and unique pyoverdine and the secondary siderophore pseudomonine, already described in P. fluorescens species. Structural analysis of the pyoverdine produced by the closely related P. putida KT2440 showed that this strain produces an already characterised pyoverdine, but different from P. entomophila, and no evidence was found for the production of a second siderophore. Growth stimulation assays with heterologous pyoverdines demonstrated that P. entomophila is able to utilize a large variety of structurally distinct pyoverdines produced by other Pseudomonas species. In contrast, P. putida KT2440 is able to utilize only its own pyoverdine and the pyoverdine produced by P. syringae LMG 1247. Our data suggest that although closely related, P. entomophila is a more efficient competitor for iron than P. putida.     

Enhanced tolerance to naphthalene and enhanced rhizoremediation performance for Pseudomonas putida KT2440 via the NAH7 catabolic plasmid

In this work, we explore the potential use of the Pseudomonas putida KT2440 strain for bioremediation of naphthalene-polluted soils. Pseudomonas putida strain KT2440 thrives in naphthalene-saturated medium, establishing a complex response that activates genes coding for extrusion pumps and cellular damage repair enzymes, as well as genes involved in the oxidative stress response. The transfer of the NAH7 plasmid enables naphthalene degradation by P. putida KT2440 while alleviating the cellular stress brought about by this toxic compound, without affecting key functions necessary for survival and colonization of the rhizosphere. Pseudomonas putida KT2440(NAH7) efficiently expresses the Nah catabolic pathway in vitro and in situ, leading to the complete mineralization of [(14)C]naphthalene, measured as the evolution of (14)CO(2), while the rate of mineralization was at least 2-fold higher in the rhizosphere than in bulk soil.     

Molecular characterization of the gallate dioxygenase from Pseudomonas putida KT2440. The prototype of a new subgroup of extradiol dioxygenases

In this work we have characterized the galA gene product from Pseudomonas putida KT2440, a ring-cleavage dioxygenase that acts specifically on gallate to produce 4-oxalomesaconate. The protein is a trimer composed by three identical subunits of 47.6 kDa (419 amino acids) that uses Fe2+ as the main cofactor. The gallate dioxygenase showed maximum activity at pH 7.0, and the Km and Vmax values for gallate were 144 microM and 53.2 micromol/min/mg of protein, respectively. A phylogenetic study suggests that the gallate dioxygenase from P. putida KT2440 is the prototype of a new subgroup of type II extradiol dioxygenases that share a common ancestor with protocatechuate 4,5-dioxygenases and whose two-domain architecture might have evolved from the fusion of the large and small subunits of the latter. A three-dimensional model for the N-terminal domain (residues 1-281) and C-terminal domain (residues 294-420) of the gallate dioxygenase from P. putida KT2440 was generated by comparison with the crystal structures of the large (LigB) and small (LigA) subunits of the protocatechuate 4,5-dioxygenase from Sphingomonas paucimobilis SYK-6. The expression of the galA gene was specifically induced when P. putida KT2440 cells grew in the presence of gallate. A P. putida KT2440 galA mutant strain was unable to use gallate as the sole carbon source and it did not show gallate dioxygenase activity, suggesting that the GalA protein is the only dioxygenase involved in gallate cleavage in this bacterium. This work points to the existence of a new pathway that is devoted to the catabolism of gallic acid and that remained unknown in the paradigmatic P. putida KT2440 strain.     

Influence of putative exopolysaccharide genes on Pseudomonas putida KT2440 biofilm stability

We report a study of the role of putative exopolysaccharide gene clusters in the formation and stability of Pseudomonas putida KT2440 biofilm. Two novel putative exopolysaccharide gene clusters, pea and peb, were identified, and evidence is provided that they encode products that stabilize P. putida KT2440 biofilm. The gene clusters alg and bcs, which code for proteins mediating alginate and cellulose biosynthesis, were found to play minor roles in P. putida KT2440 biofilm formation and stability under the conditions tested. A P. putida KT2440 derivative devoid of any identifiable exopolysaccharide genes was found to form biofilm with a structure similar to wild-type biofilm, but with a stability lower than that of wild-type biofilm. Based on our data, we suggest that the formation of structured P. putida KT2440 biofilm can occur in the absence of exopolysaccharides; however, exopolysaccharides play a role as structural stabilizers.     

Fitness in soil and rhizosphere of Pseudomonas fluorescens C7R12 compared with a C7R12 mutant affected in pyoverdine synthesis and uptake

Fluorescent pseudomonads have evolved an efficient strategy of iron uptake based on the synthesis of the siderophore pyoverdine and its relevant outer membrane receptor. The possible implication of pyoverdine synthesis and uptake on the ecological competence of a model strain (Pseudomonas fluorescens C7R12) in soil habitats was evaluated using a pyoverdine minus mutant (PL1) obtained by random insertion of the transposon Tn5. The Tn5 flanking DNA was amplified by inverse PCR and sequenced. The nucleotide sequence was found to show a high level of identity with pvsB, a pyoverdine synthetase. As expected, the mutant PL1 was significantly more susceptible to iron starvation than the wild-type strain despite its ability to produce another unknown siderophore. As with the wild-type strain, the mutant PL1 was able to incorporate the wild-type pyoverdine and five pyoverdines of foreign origin, but at a significantly lower rate despite the similarity of the outer membrane protein patterns of the two strains. The survival kinetics of the wild-type and of the pyoverdine minus mutant, in bulk and rhizosphere soil, were compared under gnotobiotic and non-gnotobiotic conditions. In gnotobiotic model systems, both strains, when inoculated separately, showed a similar survival in soil and rhizosphere, suggesting that iron was not a limiting factor. In contrast, when inoculated together, the bacterial competition was favorable to the pyoverdine producer C7R12. The efficient fitness of PL1 in the presence of the indigenous microflora, even when coinoculated with C7R12, is assumed to be related to its ability to uptake heterologous pyoverdines. Altogether, these results suggest that pyoverdine-mediated iron uptake is involved in the ecological competence of the strain P. fluorescens C7R12.     

Genetic analysis of functions involved in adhesion of Pseudomonas putida to seeds

Many agricultural uses of bacteria require the establishment of efficient bacterial populations in the rhizosphere, for which colonization of plant seeds often constitutes a critical first step. Pseudomonas putida KT2440 is a strain that colonizes the rhizosphere of a number of agronomically important plants at high population densities. To identify the functions involved in initial seed colonization by P. putida KT2440, we subjected this strain to transposon mutagenesis and screened for mutants defective in attachment to corn seeds. Eight different mutants were isolated and characterized. While all of them showed reduced attachment to seeds, only two had strong defects in their adhesion to abiotic surfaces (glass and different plastics). Sequences of the loci affected in all eight mutants were obtained. None of the isolated genes had previously been described in P. putida, although four of them showed clear similarities with genes of known functions in other organisms. They corresponded to putative surface and membrane proteins, including a calcium-binding protein, a hemolysin, a peptide transporter, and a potential multidrug efflux pump. One other showed limited similarities with surface proteins, while the remaining three presented no obvious similarities with known genes, indicating that this study has disclosed novel functions.     

Construction of a stable genetically engineered rhamnolipid-producing microorganism for remediation of pyrene-contaminated soil

One rhamnolipid-producing bacterial strain named Pseudomonas aeruginosa BSFD5 was isolated and characterized. Its rhlABRI cassette including necessary genes for rhamnolipid synthesis was cloned and transformed into the chromosome of P. putida KT2440 by a new random transposon vector without introducing antibiotic-resistance marker, generating a genetically engineered microorganism named P. putida KT2440-rhlABRI, which could stably express the rhlABRI cassette and produce rhamnolipid at a yield of 1.68?g?l(-1). In experiments using natural soil, it was shown that P. putida KT2440-rhlABRI could increase the dissolution of pyrene and thus promote its degradation by indigenous microorganisms. P. putida KT2440-rhlABRI thus demonstrated potential for enhancing the remediation of soils contaminated with polycyclic aromatic hydrocarbons.     

Cell surface display of organophosphorus hydrolase in Pseudomonas putida using an ice-nucleation protein anchor

A surface anchor system derived from the ice-nucleation protein (INP) from Pseudomonas syringe was used to localize organophosphorus hydrolase (OPH) onto the surface of Pseudomonas putida KT2440. Cells harboring the shuttle vector pPNCO33 coding for the INP-OPH fusion were capable of targeting OPH onto the cell surface as demonstrated by whole cell ELISA. The whole cell activity of P. putida KT2440 was shown to be 10 times higher than those of previous efforts expressing the same fusion protein in Escherichia coli. The capability of expressing enzymes on the surface of a robust and environmentally benign P. putida KT2440 should open up new avenues for a wide range of applications such as in situ bioremediation.     

The davDT operon of Pseudomonas putida, involved in lysine catabolism, is induced in response to the pathway intermediate delta-aminovaleric acid

Pseudomonas putida KT2440 is a soil microorganism that attaches to seeds and efficiently colonizes the plant's rhizosphere. Lysine is one of the major compounds in root exudates, and P. putida KT2440 uses this amino acid as a source of carbon, nitrogen, and energy. Lysine is channeled to delta-aminovaleric acid and then further degraded to glutaric acid via the action of the davDT gene products. We show that the davDT genes form an operon transcribed from a single sigma70-dependent promoter. The relatively high level of basal expression from the davD promoter increased about fourfold in response to the addition of exogenous lysine to the culture medium. However, the true inducer of this operon seems to be delta-aminovaleric acid because in a mutant unable to metabolize lysine to delta-aminovaleric acid, this compound, but not lysine, acted as an effector. Effective induction of the P. putida P(davD) promoter by exogenously added lysine requires efficient uptake of this amino acid, which seems to proceed by at least two uptake systems for basic amino acids that belong to the superfamily of ABC transporters. Mutants in these ABC uptake systems retained basal expression from the davD promoter but exhibited lower induction levels in response to exogenous lysine than the wild-type strain.     

Disruption of N-acyl homoserine lactone-mediated cell signaling and iron acquisition in epiphytic bacteria by leaf surface compounds

Since N-acyl homoserine lactones (AHLs) are key mediators of cell density-dependent regulation of traits involved in virulence and epiphytic fitness in gram-negative bacteria such as Pseudomonas syringae, a variety of plant species were examined to determine their production of leaf surface compounds that could interact with these signaling systems. Leaf washings of 17 of 52 plant species tested stimulated or inhibited AHL-dependent traits in at least one of the bacterial reporter strains used. The active compounds from most plants could be distinguished from known AHLs due to different patterns of mobility during C8 and C18 reverse-phase thin-layer chromatography (TLC) and normal-phase TLC compared to the patterns for authentic bacterial AHLs. All plant extracts were also tested to determine their abilities to sequester iron and trigger bacterial siderophore synthesis on a medium containing abundant iron. Leaf washings from 16 of the 52 plant species, as well as tannic acid solutions, stimulated pyoverdine synthesis in P. syringae in a high-iron medium. These preparations also inhibited the growth of a P. syringae mutant unable to produce pyoverdine siderophores but not the growth of the wild-type bacterium. The stimulation of siderophore production and the growth inhibition by plant extracts and purified tannins were both reversed by addition of ferric chloride to culture media, indicating that iron was made unavailable by the compounds released onto the leaf surface.     

Role of iron and the TonB system in colonization of corn seeds and roots by Pseudomonas putida KT2440

Iron, which is abundant in corn (Zea mays L.) seeds, plays an important role in the initial establishment of Pseudomonas putida KT2440 populations on seeds. Sequestration of seed-borne iron by chelators decreases the capacity of KT2440 to initiate attachment to corn seeds. The importance of iron for this plant-bacteria interaction is further supported by the fact that mutations in the TonB system, which is key for iron uptake, result in reduced seed colonization. TonB is also a primary determinant of the fitness of P. putida in the rhizosphere, as a deletion mutant shows a clear competitive disadvantage during colonization of corn roots.     

Quinolobactin, a new siderophore of Pseudomonas fluorescens ATCC 17400, the production of which is repressed by the cognate pyoverdine

Transposon mutant strain 3G6 of Pseudomonas fluorescens ATCC 17400 which was deficient in pyoverdine production, was found to produce another iron-chelating molecule; this molecule was identified as 8-hydroxy-4-methoxy-quinaldic acid (designated quinolobactin). The pyoverdine-deficient mutant produced a supplementary 75-kDa iron-repressed outer membrane protein (IROMP) in addition to the 85-kDa IROMP present in the wild type. The mutant was also characterized by substantially increased uptake of (59)Fe-quinolobactin. The 75-kDa IROMP was produced by the wild type after induction by quinolobactin-containing culture supernatants obtained from the pyoverdine-negative mutant or by purified quinolobactin. Conversely, adding purified wild-type pyoverdine to the growth medium resulted in suppression of the 75-kDa IROMP in the pyoverdine-deficient mutant; however, suppression was not observed when Pseudomonas aeruginosa PAO1 pyoverdine, a siderophore utilized by strain 3G6, was added to the culture. Therefore, we assume that the quinolobactin receptor is the 75-kDa IROMP and that the quinolobactin-mediated iron uptake system is repressed by the cognate pyoverdine.     

Involvement of cyclopropane fatty acids in the response of Pseudomonas putida KT2440 to freeze-drying

Pseudomonas putida KT2440, a saprophytic soil bacterium that colonizes the plant root, is a suitable microorganism for the removal of pollutants and a stable host for foreign genes used in biotransformation processes. Because of its potential use in agriculture and industry, we investigated the conditions for the optimal preservation of the strain and its derivatives for long-term storage. The highest survival rates were achieved with cells that had reached the stationary phase and which had been subjected to freeze-drying in the presence of disaccharides (trehalose, maltose, and lactose) as lyoprotectants. Using fluorescence polarization techniques, we show that cell membranes of KT2440 were more rigid in the stationary phase than in the exponential phase of growth. This is consistent with the fact that cells grown in the stationary phase exhibited a higher proportion of C17:cyclopropane as a fatty acid than cells in the exponential phase. Mutants for the cfaB gene, which encodes the main C17:cyclopropane synthase, and for the cfaA gene, which encodes a minor C17:cyclopropane synthase, were constructed. These mutants were more sensitive to freeze-drying than wild-type cells, particularly the mutant with a knockout in the cfaB gene that produced less than 2% of the amount of C17:cyclopropane produced by the parental strain.