Jean-Philippe Bellenger

Multiple roles of siderophores in free-living nitrogen-fixing bacteria

Free-living nitrogen-fixing bacteria in soils need to tightly regulate their uptake of metals in order to acquire essential metals (such as the nitrogenase metal cofactors Fe, Mo and V) while excluding toxic ones (such as W). They need to do this in a soil environment where metal speciation, and thus metal bioavailability, is dependent on a variety of factors such as organic matter content, mineralogical composition, and pH. Azotobacter vinelandii, a ubiquitous gram-negative soil diazotroph, excretes in its external medium catechol compounds, previously identified as siderophores, that bind a variety of metals in addition to iron. At low concentrations, complexes of essential metals (Fe, Mo, V) with siderophores are taken up by the bacteria through specialized transport systems. The specificity and regulation of these transport systems are such that siderophore binding of excess Mo, V or W effectively detoxifies these metals at high concentrations. In the topsoil (leaf litter layer), where metals are primarily bound to plant-derived organic matter, siderophores extract essential metals from natural ligands and deliver them to the bacteria. This process appears to be a key component of a mutualistic relationship between trees and soil diazotrophs, where tree-produced leaf litter provides a living environment rich in organic matter and micronutrients for nitrogen-fixing bacteria, which in turn supply new nitrogen to the ecosystem.

Essential metals for nitrogen fixation in a free-living N2-fixing bacterium: chelation, homeostasis and high use efficiency

Biological nitrogen fixation, the main source of new nitrogen to the Earths ecosystems, is catalysed by the enzyme nitrogenase. There are three nitrogenase isoenzymes: the Mo-nitrogenase, the V-nitrogenase and the Fe-only nitrogenase. All three types require iron, and two of them also require Mo or V. Metal bioavailability has been shown to limit nitrogen fixation in natural and managed ecosystems. Here, we report the results of a study on the metal (Mo, V, Fe) requirements of Azotobacter vinelandii, a common model soil diazotroph. In the growth medium of A. vinelandii, metals are bound to strong complexing agents (metallophores) excreted by the bacterium. The uptake rates of the metallophore complexes are regulated to meet the bacterial metal requirement for diazotrophy. Under metal-replete conditions Mo, but not V or Fe, is stored intracellularly. Under conditions of metal limitation, intracellular metals are used with remarkable efficiency, with essentially all the cellular Mo and V allocated to the nitrogenase enzymes. While the Mo-nitrogenase, which is the most efficient, is used preferentially, all three nitrogenases contribute to N₂ fixation in the same culture under metal limitation. We conclude that A. vinelandii is well adapted to fix nitrogen in metal-limited soil environments.

Photocatalytic degradation of carbamazepine and three derivatives using TiO2 and ZnO: Effect of pH, ionic strength, and natural organic matter

Removal of pharmaceuticals (PhCs) by photocatalysis is a promising avenue in water treatment. The efficiency of these treatments on PhC derivatives compared to their parent molecules remains poorly documented. The present study investigates the efficiency of photodegradation catalyzed by TiO₂ and ZnO nanoparticles on the removal of carbamazepine (CBZ) and three of its derivatives; carbamazepine epoxide (CBZ-E), acridine (AI), and acridone (AO). The effects of environmental parameters such as pH, ionic strength, and natural organic matter content on photodegradation efficiency (transformation after 6h and kinetics) were tested. We report that the efficiency of the catalysts (TiO2 and ZnO) can be very different when comparing CBZ and its derivatives (CBZ-E, AI and AO). TiO₂ was more efficient than ZnO at degrading CBZ and CBZ-E. For AI and AO, no significant differences were observed between the two catalysts. We also report that environmental parameters have contrasting effects on the efficiency of the photodegradation of CBZ compared to its derivatives. Changing pH and organic matter content had the most contrasted effects; the photodegradation of CBZ and CBZ-E was significantly affected by pH (especially in presence of TiO₂ NPs) and by the presence of natural organic matter. In contrast, the photodegradation of AI and AO was not affected by pH and organic matter. Only the photodegradation of CBZ was clearly affected by IS and solely at very high IS (1M). Overall, our results highlight that TiO₂ and ZnO catalysts present contrasted efficiency on the removal of CBZ when compared to its derivatives (CBZ-E, AI and AO). Our results also show that the effect of environmental parameters on the efficiency of the photodegradation of CBZ derivatives cannot be predicted based on the behavior of the parent molecule (CBZ).

Lichen-symbiotic cyanobacteria associated with Peltigera have an alternative vanadium-dependent nitrogen fixation system

In past decades, environmental nitrogen fixation has been attributed almost exclusively to the action of enzymes in the well-studied molybdenum-dependent nitrogen fixation system. However, recent evidence has shown that nitrogen fixation by alternative pathways may be more frequent than previously suspected. In this study, the nitrogen fixation systems employed by lichen-symbiotic cyanobacteria were examined to determine whether their diazotrophy can be attributed, in part, to an alternative pathway. The mining of metagenomic data (generated through pyrosequencing) and PCR assays were used to determine which nitrogen-fixation systems are present in cyanobacteria from the genus Nostoc associated with four samples from different geographical regions, representing different lichen-forming fungal species in the genus Peltigera. A metatranscriptomic sequence library from an additional specimen was examined to determine which genes associated with N2 fixation are transcriptionally expressed. Results indicated that both the standard molybdenum-dependent system and an alternative vanadium-dependent system are present and actively transcribed in the lichen symbiosis. This study shows for the first time that an alternative system is utilized by cyanobacteria associated with fungi. The ability of lichen-associated cyanobacteria to switch between pathways could allow them to colonize a wider array of environments, including habitats characterized by low temperature and trace metal (e.g. molybdenum) availability. We discuss the implications of these findings for environmental studies that incorporate acetylene-reduction assay data.

Vanadium requirements and uptake kinetics in the dinitrogen-fixing bacterium Azotobacter vinelandii.

ABSTRACT Vanadium is a cofactor in the alternative V-nitrogenase that is expressed by some N2-fixing bacteria when Mo is not available. We investigated the V requirements, the kinetics of V uptake, and the production of catechol compounds across a range of concentrations of vanadium in diazotrophic cultures of the soil bacterium Azotobacter vinelandii. In strain CA11.70, a mutant that expresses only the V-nitrogenase, V concentrations in the medium between 10−8 and 10−6 M sustain maximum growth rates; they are limiting below this range and toxic above. A. vinelandii excretes in its growth medium micromolar concentrations of the catechol siderophores azotochelin and protochelin, which bind the vanadate oxoanion. The production of catechols increases when V concentrations become toxic. Short-term uptake experiments with the radioactive isotope 49V show that bacteria take up the V-catechol complexes through a regulated transport system(s), which shuts down at high V concentrations. The modulation of the excretion of catechols and of the uptake of the V-catechol complexes allows A. vinelandii to precisely manage its V homeostasis over a range of V concentrations, from limiting to toxic.

Evaluation of the efficiency of Trametes hirsuta for the removal of multiple pharmaceutical compounds under low concentrations relevant to the environment.

An evaluation of the efficiency of the White-rot fungi (WRF) Trametes hirsuta to remove multi-classes pharmaceutical active compounds (17 PhACs) at low and environmentally realistic concentrations (20-500 ng L(-1)) was performed. The importance of biosorption over enzymatic activity on PhACs removal was also evaluated. Results highlight the importance to consider environmentally relevant PhACs concentrations while evaluating the removal capacities of WRF in wastewaters treatment processes, as PhACs concentration strongly influence both the enzymatic activity profile and the removal efficiency. Results also show that under tested experimental conditions, laccase was the only active extracellular lignin modifying enzyme and that biosorption and possibly intracellular enzymes also contribute to the removal of some PhACs.

Is vanadium a biometal for boreal cyanolichens

Molybdenum (Mo) nitrogenase has long been considered the predominant isoenzyme responsible for dinitrogen fixation worldwide. Recent findings have challenged the paradigm of Mo hegemony, and highlighted the role of alternative nitrogenases, such as the vanadium-nitrogenase. Here, we first characterized homeostasis of vanadium (V) along with other metals in situ in the dinitrogen fixing cyanolichen Peltigera aphthosa. These lichens were sampled in natural sites exposed to various levels of atmospheric metal deposition. These results were compared with laboratory experiments where Anabaena variabilis, which is also hosting the V-nitrogenase, and a relatively close relative of the lichen cyanobiont Nostoc, was subjected to various levels of V. We report here that V is preferentially allocated to cephalodia, specialized structures where dinitrogen fixation occurs in tri-membered lichens. This specific allocation is biologically controlled and tightly regulated. Vanadium homeostasis in lichen cephalodia exposed to various V concentrations is comparable to the one observed in Anabaena variabilis and other dinitrogen fixing organisms using V-nitrogenase. Overall, our findings support current hypotheses that V could be a more important factor in mediating nitrogen input in high latitude ecosystems than previously recognized. They invite the reassessment of current theoretical models linking metal dynamics and dinitrogen fixation in boreal and subarctic ecosystems.

Direct quantification of bacterial molybdenum and iron metallophores with ultra-high-performance liquid chromatography coupled to time-of-flight mass spectrometry

Metallophores are a unique class of organic ligands released, for example, by nitrogen fixing bacteria in their environment in order to recruit the micronutrients molybdenum (Mo) and iron (Fe). Mo and Fe are essential cofactors of nitrogenase that reduces atmospheric nitrogen into bioavailable ammonium. Upon release, these bacterial metallophores bind to both metal cations and oxo-anions in the extracellular medium increasing the bioavailability of the metals to the nitrogen fixers, which can subsequently recruit the complexes. The efficient quantification of those metal complexes is crucial for the understanding of the homeostasis of the metal cofactors of nitrogenase (e.g., Fe and Mo), the dynamics of nitrogen fixation and the nitrogen cycle. A novel direct ultra-high-performance liquid chromatography coupled to a time-of-flight mass spectrometer (UHPLC-ToF-MS) was developed to quantify and monitor the production of Fe and Mo complexes of the catecholate metallophores protochelin (Prot) and azotochelin (Azo) in the growth medium of the nitrogen fixer and model organism Azotobacter vinelandii. Chromatographic separations were carried on a reversed C18-phase with a mobile phase ramped from water to acetonitrile spiked with 1 mmol/L ammonium acetate (pH 6.6) to achieve stability of the metal complexes. Linearity for Mo-protochelin and Fe-protochelin was found at the concentration range between 5.0×10(-8) and 9.0×10(-7) mol/L with a limit of detection of 2.0×10(-8) and 3.0×10(-8) mol/L, respectively. The coefficient of variation of the procedure is in the range from 1.5 to 3.4%. The validation has hence demonstrated that the UHPLC-ToF-MS methodology is a fast, precise, specific, robust, and sensitive approach allowing the direct measurement of metallophores in growth medium without any sample preparation. The UHPLC-ToF-MS methodology was applied to the analysis of metallophores in our laboratory. Under lower Mo concentration, the Mo-protochelin concentration peaks in the middle lag phase, while the Fe-protochelin concentration rises to two maxima at the beginning of the exponential phase and during the stationary phase. The results indicate that the production of metallophores is highly dynamic throughout the growth and has to be monitored with high sensitivity and temporal resolution.

Determination of elemental baseline using peltigeralean lichens from Northeastern Canada (Québec): Initial data collection for long term monitoring of the impact of global climate change on boreal and subarctic area in Canada.

Northeastern Canada is mostly free of anthropogenic activities. The extent to which this territory has been impacted by anthropogenic atmospheric depositions remains to be studied. The main goal of our study was to establish background levels for metals in boreal muscicolous/terricolous macrolichens over non-urbanized areas of northeastern Canada (Québec). Concentrations of 18 elements (Na, Mg, Al, P, K, Ca, Ti, V, Cr, Mn, Fe, Co, Ni, Cu, Zn, Mo, Cd, and Pb) were determined for three species of the genus Peltigera (Peltigera aphthosa (L.) Willd. s.l., Peltigera neopolydactyla (Gyeln.) Gyeln. s.l., Peltigera scabrosa Th. Fr. s.l.), and Nephroma arcticum (L.) Torss., along a 1080 km south-north transect and along a of 730 km west-east transect. We report that elemental contents in the sampled lichen thalli are very low and similar to background levels found in other studies performed in pristine places (high elevation or remote ecosystems) throughout the world. Overall, our results demonstrate that most of the boreal and subarctic zone of Québec (northeastern Canada) is still pristine. The elemental baseline established in these lichen populations will contribute to monitor metal pollution in boreal and sub-polar ecosystems due to global climate change and future industrial expansion.

Biological nitrogen fixation by alternative nitrogenases in boreal cyanolichens: importance of molybdenum availability and implications for current biological nitrogen fixation estimates

Cryptogamic species and their associated cyanobacteria have attracted the attention of biogeochemists because of their critical roles in the nitrogen cycle through symbiotic and asymbiotic biological fixation of nitrogen (BNF). BNF is mediated by the nitrogenase enzyme, which, in its most common form, requires molybdenum at its active site. Molybdenum has been reported as a limiting nutrient for BNF in many ecosystems, including tropical and temperate forests. Recent studies have suggested that alternative nitrogenases, which use vanadium or iron in place of molybdenum at their active site, might play a more prominent role in natural ecosystems than previously recognized. Here, we studied the occurrence of vanadium, the role of molybdenum availability on vanadium acquisition and the contribution of alternative nitrogenases to BNF in the ubiquitous cyanolichen Peltigera aphthosa s.l. We confirmed the use of the alternative vanadium-based nitrogenase in the Nostoc cyanobiont of these lichens and its substantial contribution to BNF in this organism. We also showed that the acquisition of vanadium is strongly regulated by the abundance of molybdenum. These findings show that alternative nitrogenase can no longer be neglected in natural ecosystems, particularly in molybdenum-limited habitats.