Engineering siderophore production in Pseudomonas to improve asbestos weathering.

Iron plays a key role in microbial metabolism and bacteria have developed multiple siderophore-driven mechanisms due to its poor bioavailability for organisms in the environment. Iron-bearing minerals generally serve as a nutrient source to sustain bacterial growth after bioweathering. Siderophores are high-affinity ferric iron chelators, of which the biosynthesis is tightly regulated by the presence of iron. Pyoverdine-producing Pseudomonas have shown their ability to extract iron and magnesium from asbestos waste as nutrients. However, such bioweathering is rapidly limited due to repression of the pyoverdine pathway and the low bacterial requirement for iron. We developed a metabolically engineered strain of Pseudomonas aeruginosa for which pyoverdine production was no longer repressed by iron as a proof of concept. We compared siderophore-promoted dissolution of flocking asbestos waste by this optimized strain to that by the wild-type strain. Interestingly, pyoverdine production by the optimized strain was seven times higher in the presence of asbestos waste and the dissolution of magnesium and iron from the chrysotile fibres contained in flocking asbestos waste was significantly enhanced. This innovative mineral weathering process contributes to remove toxic iron from the asbestos fibres and may contribute to the development of an eco-friendly method to manage asbestos waste.

Biodeterioration of asbestos cement by siderophore-producing Pseudomonas.

Since the ban on the use of asbestos due to its carcinogenic properties, the removal of asbestos cement, representing the major asbestos-containing waste, has proven to be a challenge in most industrial countries. Asbestos-containing products are mainly disposed of in landfills and have remained untreated. Bioremediation involving bacteria previously reported the ability of Pseudomonas aeruginosa to release iron from flocking asbestos waste through a siderophore-driven mechanism. We examined the involvement of siderophore-producing Pseudomonas in the biodeterioration of asbestos cement. Iron and magnesium solubilization were evaluated by specific siderophore-producing mutants. The absence of one of the two siderophores affected iron extraction, whereas equivalent dissolution as that of the control was observed in the absence of siderophore. Both pyoverdine and pyochelin biosynthesis was repressed in the presence of asbestos cement, suggesting iron bioavailability from the waste. We compared the efficiency of various pyoverdines to scavenge iron from asbestos cement waste that revealed the efficiency of all pyoverdines. Pyoverdines were efficient in iron removal extracted continuously, with no evident extraction limit, in long-term weathering experiments with these pyoverdines. The optimization of pyoverdine-asbestos weathering may allow the development of a bioremediation process to avoid the disposal of such waste in landfills.

The pathogen Pseudomonas aeruginosa optimizes the production of the siderophore pyochelin upon environmental challenges.

Siderophores are iron chelators produced by bacteria to access iron, an essential nutrient. The pathogen Pseudomonas aeruginosa produces two siderophores, pyoverdine and pyochelin, the former with a high affinity for iron and the latter with a lower affinity. Furthermore, the production of both siderophores involves a positive auto-regulatory loop: the presence of the ferri-siderophore complex is essential for their large production. Since pyochelin has a lower affinity for iron it was hard to consider the role of pyochelin in drastic competitive environments where the host or the environmental microbiota produce strong iron chelators and may inhibit iron chelation by pyochelin. We showed here that the pyochelin pathway overcomes this difficulty through a more complex regulating mechanism for pyochelin production than previously described. Indeed, in the absence of pyoverdine, and thus higher difficulty to access iron, the bacteria are able to produce pyochelin independently of the presence of ferri-pyochelin. The regulation of the pyochelin pathway appeared to be more complex than expected with a more intricate tuning between repression and activation. Consequently, when the bacteria cannot produce pyoverdine they are able to produce pyochelin even in the presence of strong iron chelators. Such results support a more complex and varied role for this siderophore than previously described, and complexify the battle for iron during P. aeruginosa infection.

A Review of Asbestos Bioweathering by Siderophore-Producing Pseudomonas: A Potential Strategy of Bioremediation.

Asbestos, silicate minerals present in soil and used for building constructions for many years, are highly toxic due primarily to the presence of high concentrations of the transition metal iron. Microbial weathering of asbestos occurs through various alteration mechanisms. Siderophores, complex agents specialized in metal chelation, are common mechanisms described in mineral alteration. Solubilized metals from the fiber can serve as micronutrients for telluric microorganisms. The review focuses on the bioweathering of asbestos fibers, found in soil or manufactured by humans with gypsum (asbestos flocking) or cement, by siderophore-producing Pseudomonas. A better understanding of the interactions between asbestos and bacteria will give a perspective of a detoxification process inhibiting asbestos toxicity.

Efficiency of pyoverdines in iron removal from flocking asbestos waste: An innovative bacterial bioremediation strategy.

The use of asbestos-containing products has been banned in many countries since the beginning of the 80's due to its carcinogenic properties. However, asbestos is widely present in private and public buildings, resulting in the need to process a vast amount of asbestos-containing waste. Among the current technologies for the destruction of asbestos fibers, biodegradation by fungi, lichens, and, more recently, bacteria has been described. We previously reported the involvement of the bacterial siderophore pyoverdine in the release of iron from the two asbestos groups, serpentines and amphiboles. Among the large diversity encountered in the pyoverdine family, we examined whether these siderophores can alter flocking asbestos waste as well. All the tested pyoverdines were efficient in chrysotile-gypsum and amosite-gypsum weathering, although some exhibited higher iron dissolution. Iron was solubilized by pyoverdines from Pseudomonas aeruginosa and mandelii in a time-dependent manner from chrysotile-gypsum within 24 h. Renewal of pyoverdine-containing supernatant every 24 or 96 h allowed iron removal from chrysotile-gypsum at each cycle, until a limit was reached after 42 days of total incubation. Moreover, the dissolution was concentration-dependent, as demonstrated for the pyoverdine of P. mandelii. Pyoverdine-asbestos weathering could therefore become an innovative method to reduce anthropogenic waste.

Flocking asbestos waste, an iron and magnesium source for Pseudomonas.

Iron and magnesium are essential nutrients for most microorganisms. In the environment, the availability of iron is low relative to that of magnesium. Microorganisms have developed various iron acquisition systems, which have been well studied, whereas few studies have examined magnesium acquisition. The production of siderophores is one of the efficient strategies widely used to sustain iron nutritional requirements. Many studies have shown that minerals, such as clays, iron oxides, and silicates, can serve as nutrient sources for bacteria. Asbestos, a natural fibrous silicate present in soil contains iron and/or magnesium, depending on the species of asbestos. Our aim was to study the acquisition of iron and magnesium from flocking asbestos waste by Pseudomonas aeruginosa and the involvement of the siderophores, pyoverdine and pyochelin. Flocking asbestos waste promoted growth under iron- and magnesium-limited conditions, together with a decrease in pyoverdine production, correlating with the dissolution of iron from the waste. In long-term experiments, flocking asbestos waste provided these two essential elements for bacterial growth and resulted in a decrease of iron in asbestos fibers. Among the enzymes required for pyochelin and pyoverdine synthesis, PchA and PvdJ were tagged with the fluorescent protein mCherry to analyze the expression patterns of proteins involved in siderophore production. Both enzymes were produced in the presence of flocking asbestos waste, suggesting a role of the pyoverdine and pyochelin pathway in asbestos dissolution. We investigated the involvement of each siderophore in iron and magnesium removal using mutants in one or both siderophore pathways. We observed a significant increase in iron extraction in the presence of siderophores and the absence of one of the two siderophores could be compensated by the other. Flocking asbestos waste represents an iron and magnesium source for P. aeruginosa, with iron removal linked to a siderophore-driven mechanism.

Iron removal from raw asbestos by siderophores-producing Pseudomonas.

Asbestos, mineral present in soil, are highly toxic due to the presence of iron. Microbes-mineral interactions occur naturally through various processes leading to their alteration. We examined the effect of siderophore-producing Pseudomonas with a particular focus on the role of pyoverdine and pyochelin on raw asbestos fibers such as amosite, crocidolite and chrysotile. We compared the efficiency of pyoverdine to the iron chelating agent EDTA in the release of iron from raw asbestos fibers. Pyoverdine was able to extract iron from all the tested raw asbestos with the higher efficiency observed for chrysotile and crocidolite. When asbestos were grinded, the iron removal was more important for all types. We monitored the effect of bacterial growth and siderophores containing bacterial supernatant on raw asbestos dissolution by solution chemistry analysis and transmission electron microscopy. The siderophore-containing supernatant allowed a higher iron solubilisation than the one obtained after bacterial growth. Moreover, the iron dissolution was faster with pyoverdine-containing supernatant than pyochelin-containing supernatant, with approximately the same iron level for the maximum extraction with a delay of 48 h. Our study clearly showed the involvement of bacterial siderophores, pyoverdine and pyochelin on chrysotile, crocidolite and amosite fibers weathering.

Iron Uptake Analysis in a Set of Clinical Isolates of Pseudomonas putida.

Pseudomonas putida strains are frequent inhabitants of soil and aquatic niches and they are occasionally isolated from hospital environments. As the available iron sources in human tissues, edaphic, and aquatic niches are different, we have analyzed iron-uptake related genes in different P. putida strains that were isolated from all these environments. We found that these isolates can be grouped into different clades according to the genetics of siderophore biosynthesis and recycling. The pyoverdine locus of the six P. putida clinical isolates that have so far been completely sequenced, are not closely related; three strains (P. putida HB13667, HB3267, and NBRC14164T) are grouped in Clade I and the other three in Clade II, suggesting possible different origins and evolution. In one clinical strain, P. putida HB4184, the production of siderophores is induced under high osmolarity conditions. The pyoverdine locus in this strain is closely related to that of strain P. putida HB001 which was isolated from sandy shore soil of the Yellow Sea in Korean marine sand, suggesting their possible origin, and evolution. The acquisition of two unique TonB-dependent transporters for xenosiderophore acquisition, similar to those existing in the opportunistic pathogen P. aeruginosa PAO, is an interesting adaptation trait of the clinical strain P. putida H8234 that may confer adaptive advantages under low iron availability conditions.

Metallome of Pseudomonas aeruginosa: a role for siderophores.

In this paper, we describe the total metal composition (metallome) of Pseudomonas aeruginosa. Inductively coupled plasma atomic emission spectroscopy analyses showed that P. aeruginosa cells concentrate each metal of the metallome from the extracellular media with different efficiencies. Growth in nutrient-restricted media did not substantially affect the overall profile of the metallome; however, the uptake of some metals was strongly stimulated, showing the high potential of some metal acquisition pathways to adapt to changing growth conditions. We also investigated the role of the two major siderophores produced by P. aeruginosa, pyoverdine and pyochelin, in iron uptake and more generally in metallome homeostasis. In addition to their role in iron acquisition, siderophore production also significantly prevented the accumulation of toxic metals in P. aeruginosa cells, thus preserving the equilibrium of the metallome in a polluted environment.

Siderophore-promoted dissolution of smectite by fluorescent Pseudomonas.

Siderophores are organic chelators produced by microorganisms to fulfil their iron requirements. Siderophore-promoted dissolution of iron-bearing minerals has been clearly documented for some siderophores, but few studies have addressed metabolizing siderophore-producing bacteria. We investigated iron acquisition from clays by fluorescent Pseudomonads, bacteria that are ubiquitous in the environment. We focused on the interactions between smectite and Pseudomonas aeruginosa, a bacterium producing two structurally different siderophores: pyoverdine and pyochelin. The presence of smectite in iron-limited growth media promoted planktonic growth of P. aeruginosa and biofilm surrounding the smectite aggregates. Chemical analysis of the culture media indicated increases in the dissolved silicon, iron and aluminium concentrations following smectite supplementation. The use of P. aeruginosa mutants unable to produce either one or both of the two siderophores indicated that pyoverdine, the siderophore with the higher affinity for iron, was involved in iron and aluminium solubilization by the wild-type strain. However, in the absence of pyoverdine, pyochelin was also able to solubilize iron but with a twofold lower efficiency. In conclusion, pyoverdine and pyochelin, two structurally different siderophores, can solubilize structural iron from smectite and thereby make it available for bacterial growth.

Influence of bacteria on lanthanide and actinide transfer from specific soil components (humus, soil minerals and vitrified municipal solid waste incinerator bottom ash) to corn plants: Sr-Nd isotope evidence.

Experiments have been performed to test the stability of vitrified municipal solid waste (MSW) incinerator bottom ash under the presence of bacteria (Pseudomonas aeruginosa) and plants (corn). The substratum used for the plant growth was a humus-rich soil mixed with vitrified waste. For the first time, information on the stability of waste glasses in the presence of bacteria and plants is given. Results show that inoculated plant samples contained always about two times higher lanthanide and actinide element concentrations. Bacteria support the element transfer since plants growing in inoculated environment developed a smaller root system but have higher trace element concentrations. Compared with the substratum, plants are light rare earth element (LREE) enriched. The vitrified bottom ash has to some extent been corroded by bacteria and plant activities as indicated by the presence of Nd (REE) and Sr from the vitrified waste in the plants. (87)Sr/(86)Sr and (143)Nd/(144)Nd isotope ratios of plants and soil components allow the identification of the corroded soil components and confirm that bacteria accelerate the assimilation of elements from the vitrified bottom ash. These findings are of importance for landfill disposal scenarios, and similar experiments should be performed in order to better constrain the processes of microbially mediated alteration of the MSW glasses in the biosphere.

Yersiniabactin production requires the thioesterase domain of HMWP2 and YbtD, a putative phosphopantetheinylate transferase.

One requirement for the pathogenesis of Yersinia pestis, the causative agent of bubonic plague, is the yersiniabactin (Ybt) siderophore-dependent iron transport system that is encoded within a high-pathogenicity island (HPI) within the pgm locus of the Y. pestis chromosome. Nine gene products within the HPI have demonstrated functions in the nonribosomal peptide synthesis (NRPS)/polyketide (PK) synthesis or transport of Ybt. NRPS/PK synthetase or synthase enzymes are generally activated by phosphopantetheinylation. However, no products with similarities to known phosphopantetheinyl (P-pant) transferases were found within the pgm locus. We have identified a gene, ybtD, encoded outside the HPI and pgm locus, that is necessary for function of the Ybt system and has similarities to other P-pant transferases such as EntD of Escherichia coli. A deletion within ybtD yielded a strain (KIM6-2085+) defective in siderophore production. This strain was unable to grow on iron-deficient media at 37 degrees C but could be cross-fed by culture supernatants from Ybt-producing strains of Y. pestis. The promoter region of ybtD was fused to lacZ; beta-galactosidase expression from this reporter was not regulated by the iron status of the bacterial cells or by YbtA, a positive regulator of other genes of the ybt system. The ybtD mutant failed to express indicator Ybt proteins (high-molecular-weight protein 1 [HMWP1], HMWP2, and Psn), a pattern similar to those seen with several other ybt biosynthetic mutants. In contrast, cells containing a single amino acid substitution (S2908A) in the terminal thioesterase domain of HMWP2 failed to exhibit any ybt regulatory defects but did not elaborate extracellular Ybt under iron-deficient conditions.

Siderophore typing, a powerful tool for the identification of fluorescent and nonfluorescent pseudomonads.

A total of 301 strains of fluorescent pseudomonads previously characterized by conventional phenotypic and/or genomic taxonomic methods were analyzed through siderotyping, i.e., by the isoelectrophoretic characterization of their main siderophores and pyoverdines and determination of the pyoverdine-mediated iron uptake specificity of the strains. As a general rule, strains within a well-circumscribed taxonomic group, namely the species Pseudomonas brassicacearum, Pseudomonas fuscovaginae, Pseudomonas jessenii, Pseudomonas mandelii, Pseudomonas monteilii, "Pseudomonas mosselii," "Pseudomonas palleronii," Pseudomonas rhodesiae, "Pseudomonas salomonii," Pseudomonas syringae, Pseudomonas thivervalensis, Pseudomonas tolaasii, and Pseudomonas veronii and the genomospecies FP1, FP2, and FP3 produced an identical pyoverdine which, in addition, was characteristic of the group, since it was structurally different from the pyoverdines produced by the other groups. In contrast, 28 strains belonging to the notoriously heterogeneous Pseudomonas fluorescens species were characterized by great heterogeneity at the pyoverdine level. The study of 23 partially characterized phenotypic clusters demonstrated that siderotyping is very useful in suggesting correlations between clusters and well-defined species and in detecting misclassified individual strains, as verified by DNA-DNA hybridization. The usefulness of siderotyping as a determinative tool was extended to the nonfluorescent species Pseudomonas corrugata, Pseudomonas frederiksbergensis, Pseudomonas graminis, and Pseudomonas plecoglossicida, which were seen to have an identical species-specific siderophore system and thus were easily differentiated from one another. Thus, the fast, accurate, and easy-to-perform siderotyping method compares favorably with the usual phenotypic and genomic methods presently necessary for accurate identification of pseudomonads at the species level.

Siderophore-mediated iron uptake in fluorescent Pseudomonas: characterization of the pyoverdine-receptor binding site of three cross-reacting pyoverdines.

Two Pseudomonas fluorescens and one Pseudomonas aeruginosa strains, although producing structurally different pyoverdines, demonstrated highly efficient cross-reactions when tested for pyoverdine-mediated iron uptake. A ferripyoverdine receptor-deficient mutant of the P. aeruginosa strain was unable to use any of the three pyoverdines. Moreover, the three strains presented each a specific outer membrane siderophore-receptor pattern. Thus, the capacity of using heterologous pyoverdines was related not to the presence of supplementary specific ferripyoverdine receptors but to the existence within the respective pyoverdine-peptide chains of a common dipeptide motif which should act as the receptor-binding site for the three pyoverdines. Other pyoverdines sharing the same motif but at another position within the peptide chain were not efficient in iron transport, demonstrating the importance of the spatial position of the binding site.