Abiotic process for Fe(II) oxidation and green rust mineralization driven by a heterotrophic nitrate reducing bacteria (Klebsiella mobilis).

Green rusts (GRs) are mixed Fe(II)-Fe(III) hydroxides with a high reactivity toward organic and inorganic pollutants. GRs can be produced from ferric reducing or ferrous oxidizing bacterial activities. In this study, we investigated the capability of Klebsiella mobilis to produce iron minerals in the presence of nitrate and ferrous iron. This bacterium is well-known to reduce nitrate using an organic carbon source as electron donor but is unable to enzymatically oxidize Fe(II) species. During incubation, GR formation occurred as a secondary iron mineral precipitating on cell surfaces, resulting from Fe(II) oxidation by nitrite produced via bacterial respiration of nitrate. For the first time, we demonstrate GR formation by indirect microbial oxidation of Fe(II) (i.e., a combination of biotic/abiotic processes). These results therefore suggest that nitrate-reducing bacteria can potentially contribute to the formation of GR in natural environments. In addition, the chemical reduction of nitrite to ammonium by GR is observed, which gradually turns the GR into the end-product goethite. The nitrogen mass-balance clearly demonstrates that the total amount of ammonium produced corresponds to the quantity of bioreduced nitrate. These findings demonstrate how the activity of nitrate-reducing bacteria in ferrous environments may provide a direct link between the biogeochemical cycles of nitrogen and iron.

Unravelling the Role of Melanin in Cd and Zn Tolerance and Accumulation of Three Dark Septate Endophytic Species.

Dark septate endophytes (DSEs) are often trace element (TE)-tolerant fungi and are abundant in TE-polluted environments. The production of melanin, a black polymer found in cell walls, was hypothesized by several authors to play a role in the TE tolerance of DSEs. To test this hypothesis, we established a series of experiments using albino strains and melanin inhibitors and examined the responses to Cd and Zn. Six DSEs belonging to genera Cadophora sp., Leptodontidium sp. and Phialophora mustea, were evaluated. The strains mainly produced 1,8-dihydroxynaphthalene (DHN) melanin whereas 3,4-dihydroxyphenylalanin melanin was also synthetized. Cd and Zn decreased melanin synthesis in most of the strains. A reduction in melanin concentration in hyphae through the use of tricyclazole, an inhibitor of DHN-melanin synthesis, did not reduce the tolerance of the strains to Cd and Zn. Similarly, albino mutants of Leptodontidium sp. were not more sensitive to Cd and Zn than the WT strain. Moreover, tricyclazole-treated colonies accumulated less Cd but more Zn compared to untreated colonies. The Cd and Zn contents of Leptodontidium albino strains were variable and similar to that of the WT. The results suggest that melanin production is not an important functional trait that contributes to Cd and Zn tolerance, but might contribute to Cd accumulation.

Soil Particles and Phenanthrene Interact in Defining the Metabolic Profile of Pseudomonas putida G7: A Vibrational Spectroscopy Approach.

In soil, organic matter and mineral particles (soil particles; SPs) strongly influence the bio-available fraction of organic pollutants, such as polycyclic aromatic hydrocarbons (PAHs), and the metabolic activity of bacteria. However, the effect of SPs as well as comparative approaches to discriminate the metabolic responses to PAHs from those to simple carbon sources are seldom considered in mineralization experiments, limiting our knowledge concerning the dynamics of contaminants in soil. In this study, the metabolic profile of a model PAH-degrading bacterium, Pseudomonas putida G7, grown in the absence and presence of different SPs (i.e., sand, clays and humic acids), using either phenanthrene or glucose as the sole carbon and energy source, was characterized using vibrational spectroscopy (i.e., FT-Raman and FT-IR spectroscopy) and multivariate classification analysis (i.e., PLS-DA). The different type of SPs specifically altered the metabolic profile of P. putida, especially in combination with phenanthrene. In comparison to the cells grown in the absence of SPs, sand induced no remarkable change in the metabolic profile of the cells, whereas clays and humic acids affected it the most, as revealed by the higher discriminative accuracy (R 2, RMSEP and sensitivity) of the PLS-DA for those conditions. With respect to the carbon-source (phenanthrene vs. glucose), no effect on the metabolic profile was evident in the absence of SPs or in the presence of sand. On the other hand, with clays and humic acids, more pronounced spectral clusters between cells grown on glucose or on phenanthrene were evident, suggesting that these SPs modify the way cells access and metabolize PAHs. The macromolecular changes regarded mainly protein secondary structures (a shift from α-helices to ß-sheets), amino acid levels, nucleic acid conformation and cell wall carbohydrates. Our results provide new interesting evidences that SPs specifically interact with PAHs in defining bacteria metabolic profiles and further emphasize the importance of studying the interaction of bacteria with their surrounding matrix to deeply understand PAHs degradation in soils.

Functional Role of Lanthanides in Enzymatic Activity and Transcriptional Regulation of Pyrroloquinoline Quinone-Dependent Alcohol Dehydrogenases in Pseudomonas putida KT2440.

The oxidation of alcohols and aldehydes is crucial for detoxification and efficient catabolism of various volatile organic compounds (VOCs). Thus, many Gram-negative bacteria have evolved periplasmic oxidation systems based on pyrroloquinoline quinone-dependent alcohol dehydrogenases (PQQ-ADHs) that are often functionally redundant. Here we report the first description and characterization of a lanthanide-dependent PQQ-ADH (PedH) in a nonmethylotrophic bacterium based on the use of purified enzymes from the soil-dwelling model organism Pseudomonas putida KT2440. PedH (PP_2679) exhibits enzyme activity on a range of substrates similar to that of its Ca2+-dependent counterpart PedE (PP_2674), including linear and aromatic primary and secondary alcohols, as well as aldehydes, but only in the presence of lanthanide ions, including La3+, Ce3+, Pr3+, Sm3+, or Nd3+ Reporter assays revealed that PedH not only has a catalytic function but is also involved in the transcriptional regulation of pedE and pedH, most likely acting as a sensory module. Notably, the underlying regulatory network is responsive to as little as 1 to 10 nM lanthanum, a concentration assumed to be of ecological relevance. The present study further demonstrates that the PQQ-dependent oxidation system is crucial for efficient growth with a variety of volatile alcohols. From these results, we conclude that functional redundancy and inverse regulation of PedE and PedH represent an adaptive strategy of P. putida KT2440 to optimize growth with volatile alcohols in response to the availability of different lanthanides.IMPORTANCE Because of their low bioavailability, lanthanides have long been considered biologically inert. In recent years, however, the identification of lanthanides as a cofactor in methylotrophic bacteria has attracted tremendous interest among various biological fields. The present study reveals that one of the two PQQ-ADHs produced by the model organism P. putida KT2440 also utilizes lanthanides as a cofactor, thus expanding the scope of lanthanide-employing bacteria beyond the methylotrophs. Similar to the system described in methylotrophic bacteria, a complex regulatory network is involved in lanthanide-responsive switching between the two PQQ-ADHs encoded by P. putida KT2440. We further show that the functional production of at least one of the enzymes is crucial for efficient growth with several volatile alcohols. Overall, our study provides a novel understanding of the redundancy of PQQ-ADHs observed in many organisms and further highlights the importance of lanthanides for bacterial metabolism, particularly in soil environments.

Siderophore-Mediated Iron Dissolution from Nontronites Is Controlled by Mineral Cristallochemistry.

Bacteria living in oxic environments experience iron deficiency due to limited solubility and slow dissolution kinetics of iron-bearing minerals. To cope with iron deprivation, aerobic bacteria have evolved various strategies, including release of siderophores or other organic acids that scavenge external Fe(III) and deliver it to the cells. This research investigated the role of siderophores produced by Pseudomonas aeruginosa in the acquisition of Fe(III) from two iron-bearing colloidal nontronites (NAu-1 and NAu-2), comparing differences in bioavailability related with site occupancy and distribution of Fe(III) in the two lattices. To avoid both the direct contact of the mineral colloids with the bacterial cells and the uncontrolled particle aggregation, nontronite suspensions were homogenously dispersed in a porous silica gel before the dissolution experiments. A multiparametric approach coupling UV-vis spectroscopy and spectral decomposition algorithm was implemented to monitor simultaneously the solubilisation of Fe and the production of pyoverdine in microplate-based batch experiments. Both nontronites released Fe in a particle concentration-dependent manner when incubated with the wild-type P. aeruginosa strain, however iron released from NAu-2 was substantially greater than from NAu-1. The profile of organic acids produced in both cases was similar and may not account for the difference in the iron dissolution efficiency. In contrast, a pyoverdine-deficient mutant was unable to mobilize Fe(III) from either nontronite, whereas iron dissolution occurred in abiotic experiments conducted with purified pyoverdine. Overall, our data provide evidence that P. aeruginosa indirectly mobilize Fe from nontronites primarily through the production of pyoverdine. The structural Fe present on the edges of NAu-2 rather than NAu-1 particles appears to be more bio-accessible, indicating that the distribution of Fe, in the tetrahedron and/or in the octahedron sites, governs the solubilisation process. Furthermore, we also revealed that P. aeruginosa could acquire iron when in direct contact with mineral particles in a siderophore-independent manner.

Nitrate reduction by mixed iron(II-III) hydroxycarbonate green rust in the presence of phosphate anions: the key parameters influencing the ammonium selectivity.

The reduction of nitrate anions by a mixed Fe(II)-Fe(III) carbonated green rust (GR) in aqueous medium is studied as a function of the initial pH and the initial concentrations of iron, phosphate and nitrate. The influence of these parameters on the fraction of nitrate removed and the production of ammonium is investigated by the help of statistical experimental designs. The goal is to determine experimental conditions that maximize the fraction of NO3(-) removed and concomitantly minimize the production of NH4(+). Increasing the phosphate concentration relatively to the initial Fe(II) concentration inhibits the reduction of nitrate probably due to a surface saturation of the lateral sites of the GR crystals. The kinetics of the reaction is greatly enhanced by increasing the initial pH at 10.5, however it leads to a global increase of the NH4(+) production. A partial saturation of the surface sites by phosphate leads to a global decrease of selectivity of the reaction towards ammonium. The evolution of the ratio of the NH4(+) concentration to the Fe(II) concentration confirms that the NO3(-) species are only partially transformed into ammonium. Interestingly at an initial pH of 7.5, the selectivity of the reaction towards NH4(+) is often lower than ∼30%. The reduction of nitrate by carbonated GR differs from the behavior of other GRs incorporating Cl(-), F(-) and SO4(2-) anions that fully transform nitrate into ammonium. Finally, if GR is intended to be used during a passive water denitrification process, complementary dephosphatation and ammonium treatments should be considered.