The non-ribosomal peptide synthetase-independent siderophore (NIS) rhizobactin produced by Caballeronia mineralivorans PML1(12) confers the ability to weather minerals.

To mobilize nutrients entrapped into minerals and rocks, heterotrophic bacteria living in nutrient-poor environments have developed different mechanisms based mainly on acidolysis and chelation. However, the genetic bases of these mechanisms remain unidentified. To fill this gap, we considered the model strain Caballeronia mineralivorans PML1(12) known to be effective at weathering. Based on its transcriptomics and proteomics responses in Fe-depleted conditions, we pointed a cluster of genes differentially expressed and putatively involved in the production of siderophores. In this study, we report the characterization of this gene region coding for the production of a non-ribosomal peptide synthetase-independent siderophore (NIS). Targeted mutagenesis associated with functional assays and liquid chromatography coupled to high-resolution tandem mass spectrometry demonstrated the production of a single siderophore, identified as rhizobactin. This siderophore represents the first NIS containing malic acid in its structure. The evidence for the implication of rhizobactin in mineral weathering was demonstrated during a hematite dissolution assay. This study provides the first demonstration of the synthesis of a NIS in the genus Caballeronia and its involvement in mineral weathering. Our conclusions reinforce the idea that strain PML1(12) is particularly well adapted to nutrient-poor environments. IMPORTANCE This work deciphers the molecular and genetic bases used by strain PML1(12) of Caballeronia mineralivorans to mobilize iron and weather minerals. Through the combination of bioinformatics, chemical, and phylogenetic analyses, we characterized the siderophore produced by strain PML1(12) and the related genes. This siderophore was identified as rhizobactin and classified as a non-ribosomal peptide synthetase-independent siderophore (NIS). Contrary to the previously identified NIS synthetases that form siderophores containing citric acid, α-ketoglutarate, or succinic acid, our analyses revealed that rhizobactin contains malic acid in its structure, representing, therefore, the first identified NIS with such an acid and probably a new NIS category. Last, this work demonstrates for the first time the effectiveness at weathering minerals of a siderophore of the NIS family. Our findings offer relevant information for different fields of research, such as environmental genomics, microbiology, chemistry, and soil sciences.

The cytosolic Arabidopsis thaliana cysteine desulfurase ABA3 delivers sulfur to the sulfurtransferase STR18.

The biosynthesis of many sulfur-containing molecules depends on cysteine as a sulfur source. Both the cysteine desulfurase (CD) and rhodanese (Rhd) domain-containing protein families participate in the trafficking of sulfur for various metabolic pathways in bacteria and human, but their connection is not yet described in plants. The existence of natural chimeric proteins containing both CD and Rhd domains in specific bacterial genera, however, suggests a general interaction between these proteins. We report here the biochemical relationships between two cytosolic proteins from Arabidopsis thaliana, a Rhd domain-containing protein, the sulfurtransferase 18 (STR18), and a CD isoform referred to as ABA3, and compare these biochemical features to those of a natural CD-Rhd fusion protein from the bacterium Pseudorhodoferax sp. We observed that the bacterial enzyme is bifunctional exhibiting both CD and STR activities using l-cysteine and thiosulfate as sulfur donors but preferentially using l-cysteine to catalyze transpersulfidation reactions. In vitro activity assays and mass spectrometry analyses revealed that STR18 stimulates the CD activity of ABA3 by reducing the intermediate persulfide on its catalytic cysteine, thereby accelerating the overall transfer reaction. We also show that both proteins interact in planta and form an efficient sulfur relay system, whereby STR18 catalyzes transpersulfidation reactions from ABA3 to the model acceptor protein roGFP2. In conclusion, the ABA3-STR18 couple likely represents an uncharacterized pathway of sulfur trafficking in the cytosol of plant cells, independent of ABA3 function in molybdenum cofactor maturation.

The mineral weathering ability of Collimonas pratensis PMB3(1) involves a Malleobactin-mediated iron acquisition system.

Mineral weathering by microorganisms is considered to occur through a succession of mechanisms based on acidification and chelation. While the role of acidification is established, the role of siderophores is difficult to disentangle from the effect of the acidification. We took advantage of the ability of strain Collimonas pratensis PMB3(1) to weather minerals but not to acidify depending on the carbon source to address the role of siderophores in mineral weathering. We identified a single non-ribosomal peptide synthetase (NRPS) responsible for siderophore biosynthesis in the PMB3(1) genome. By combining iron-chelating assays, targeted mutagenesis and chemical analyses (HPLC and LC-ESI-HRMS), we identified the siderophore produced as malleobactin X and how its production depends on the concentration of available iron. Comparison with the genome sequences of other collimonads evidenced that malleobactin production seems to be a relatively conserved functional trait, though some collimonads harboured other siderophore synthesis systems. We also revealed by comparing the wild-type strain and its mutant impaired in the production of malleobactin that the ability to produce this siderophore is essential to allow the dissolution of hematite under non-acidifying conditions. This study represents the first characterization of the siderophore produced by collimonads and its role in mineral weathering.

Atypical Iron-Sulfur Cluster Binding, Redox Activity and Structural Properties of Chlamydomonas reinhardtii Glutaredoxin 2.

Glutaredoxins (GRXs) are thioredoxin superfamily members exhibiting thiol-disulfide oxidoreductase activity and/or iron-sulfur (Fe-S) cluster binding capacities. These properties are determined by specific structural factors. In this study, we examined the capacity of the class I Chlamydomonas reinhardtii GRX2 recombinant protein to catalyze both protein glutathionylation and deglutathionylation reactions using a redox sensitive fluorescent protein as a model protein substrate. We observed that the catalytic cysteine of the CPYC active site motif of GRX2 was sufficient for catalyzing both reactions in the presence of glutathione. Unexpectedly, spectroscopic characterization of the protein purified under anaerobiosis showed the presence of a [2Fe-2S] cluster despite having a presumably inadequate active site signature, based on past mutational analyses. The spectroscopic characterization of cysteine mutated variants together with modeling of the Fe-S cluster-bound GRX homodimer from the structure of an apo-GRX2 indicate the existence of an atypical Fe-S cluster environment and ligation mode. Overall, the results further delineate the biochemical and structural properties of conventional GRXs, pointing to the existence of multiple factors more complex than anticipated, sustaining the capacity of these proteins to bind Fe-S clusters.

Structural and Biochemical Analysis of OrfG: The VirB8-like Component of the Conjugative Type IV Secretion System of ICESt3 From Streptococcus thermophilus.

Conjugative transfer is a major threat to global health since it contributes to the spread of antibiotic resistance genes and virulence factors among commensal and pathogenic bacteria. To allow their transfer, mobile genetic elements including Integrative and Conjugative Elements (ICEs) use a specialized conjugative apparatus related to Type IV secretion systems (Conj-T4SS). Therefore, Conj-T4SSs are excellent targets for strategies that aim to limit the spread of antibiotic resistance. In this study, we combined structural, biochemical and biophysical approaches to study OrfG, a protein that belongs to Conj-T4SS of ICESt3 from Streptococcus thermophilus. Structural analysis of OrfG by X-ray crystallography revealed that OrfG central domain is similar to VirB8-like proteins but displays a different quaternary structure in the crystal. To understand, at a structural level, the common and the diverse features between VirB8-like proteins from both Gram-negative and -positive bacteria, we used an in silico structural alignment method that allowed us to identify different structural classes of VirB8-like proteins. Biochemical and biophysical characterizations of purified OrfG soluble domain and its central and C-terminal subdomains indicated that they are mainly monomeric in solution but able to form an unprecedented 6-mer oligomers. Our study provides new insights into the structural analysis of VirB8-like proteins and discusses the interplay between tertiary and quaternary structures of these proteins as an essential component of the conjugative transfer.

Arabidopsis thaliana 3-mercaptopyruvate sulfurtransferases interact with and are protected by reducing systems.

The formation of a persulfide group (-SSH) on cysteine residues has gained attention as a reversible posttranslational modification contributing to protein regulation or protection. The widely distributed 3-mercaptopyruvate sulfurtransferases (MSTs) are implicated in the generation of persulfidated molecules and H2S biogenesis through transfer of a sulfane sulfur atom from a suitable donor to an acceptor. Arabidopsis has two MSTs, named STR1 and STR2, but they are poorly characterized. To learn more about these enzymes, we conducted a series of biochemical experiments including a variety of possible reducing systems. Our kinetic studies, which used a combination of sulfur donors and acceptors revealed that both MSTs use 3-mercaptopyruvate efficiently as a sulfur donor while thioredoxins, glutathione, and glutaredoxins all served as high-affinity sulfane sulfur acceptors. Using the redox-sensitive GFP (roGFP2) as a model acceptor protein, we showed that the persulfide-forming MSTs catalyze roGFP2 oxidation and more generally trans-persulfidation reactions. However, a preferential interaction with the thioredoxin system and glutathione was observed in case of competition between these sulfur acceptors. Moreover, we observed that MSTs are sensitive to overoxidation but are protected from an irreversible inactivation by their persulfide intermediate and subsequent reactivation by thioredoxins or glutathione. This work provides significant insights into Arabidopsis STR1 and STR2 catalytic properties and more specifically emphasizes the interaction with cellular reducing systems for the generation of H2S and glutathione persulfide and reactivation of an oxidatively modified form.

A Redox-Sensitive Cysteine Is Required for PIN1At Function.

Parvulins are ubiquitous peptidyl-prolyl isomerases (PPIases) required for protein folding and regulation. Among parvulin members, Arabidopsis PIN1At, human PIN1, and yeast ESS1 share a conserved cysteine residue but differ by the presence of an N-terminal WW domain, absent in PIN1At. In this study, we have explored whether the cysteine residue of Arabidopsis PIN1At is involved in catalysis and subject to oxidative modifications. From the functional complementation of yeast ess1 mutant, we concluded that the cysteine at position 69 is mandatory for PIN1At function in vivo, unless being replaced by an Asp which is found in a few parvulin members. This result correlates with a decrease of the in vitro PPIase activity of non-functional PIN1At cysteinic variants. A decrease of PIN1At activity was observed upon H2O2 treatment. The in vitro oxidation of cysteine 69, which has an acidic pKa value of 4.9, leads to the formation of covalent dimers that are reduced by thioredoxins, or to sulfinic or sulfonic acid forms at higher H2O2 excess. These investigations highlight the importance of the sole cysteine residue of PIN1At for activity. The reversible formation of an intermolecular disulfide bond might constitute a protective or regulatory mechanism under oxidizing conditions.

Sinorhizobium meliloti YrbA binds divalent metal cations using two conserved histidines.

Sinorhizobium meliloti is a nitrogen-fixing bacterium forming symbiotic nodules with the legume Medicago truncatula. S. meliloti possesses two BolA-like proteins (BolA and YrbA), the function of which is unknown. In organisms where BolA proteins and monothiol glutaredoxins (Grxs) are present, they contribute to the regulation of iron homeostasis by bridging a [2Fe-2S] cluster into heterodimers. A role in the maturation of iron-sulfur (Fe-S) proteins is also attributed to both proteins. In the present study, we have performed a structure-function analysis of SmYrbA showing that it coordinates diverse divalent metal ions (Fe2+, Co2+, Ni2+, Cu2+ and Zn2+) using His32 and His67 residues, that are also used for Fe-S cluster binding in BolA-Grx heterodimers. It also possesses the capacity to form heterodimers with the sole monothiol glutaredoxin (SmGrx2) present in this species. Using cellular approaches analyzing the metal tolerance of S. meliloti mutant strains inactivated in the yrbA and/or bolA genes, we provide evidence for a connection of YrbA with the regulation of iron homeostasis. The mild defects in M. truncatula nodulation reported for the yrbA bolA mutant as compared with the stronger defects in nodule development previously observed for a grx2 mutant suggest functions independent of SmGrx2. These results help in clarifying the physiological role of BolA-type proteins in bacteria.

The plastidial Arabidopsis thaliana NFU1 protein binds and delivers [4Fe-4S] clusters to specific client proteins.

Proteins incorporating iron-sulfur (Fe-S) co-factors are required for a plethora of metabolic processes. Their maturation depends on three Fe-S cluster assembly machineries in plants, located in the cytosol, mitochondria, and chloroplasts. After de novo formation on scaffold proteins, transfer proteins load Fe-S clusters onto client proteins. Among the plastidial representatives of these transfer proteins, NFU2 and NFU3 are required for the maturation of the [4Fe-4S] clusters present in photosystem I subunits, acting upstream of the high-chlorophyll fluorescence 101 (HCF101) protein. NFU2 is also required for the maturation of the [2Fe-2S]-containing dihydroxyacid dehydratase, important for branched-chain amino acid synthesis. Here, we report that recombinant Arabidopsis thaliana NFU1 assembles one [4Fe-4S] cluster per homodimer. Performing co-immunoprecipitation experiments and assessing physical interactions of NFU1 with many [4Fe-4S]-containing plastidial proteins in binary yeast two-hybrid assays, we also gained insights into the specificity of NFU1 for the maturation of chloroplastic Fe-S proteins. Using bimolecular fluorescence complementation and in vitro Fe-S cluster transfer experiments, we confirmed interactions with two proteins involved in isoprenoid and thiamine biosynthesis, 1-hydroxy-2-methyl-2-(E)-butenyl-4-diphosphate synthase and 4-amino-5-hydroxymethyl-2-methylpyrimidine phosphate synthase, respectively. An additional interaction detected with the scaffold protein SUFD enabled us to build a model in which NFU1 receives its Fe-S cluster from the SUFBC2D scaffold complex and serves in the maturation of specific [4Fe-4S] client proteins. The identification of the NFU1 partner proteins reported here more clearly defines the role of NFU1 in Fe-S client protein maturation in Arabidopsis chloroplasts among other SUF components.

Iron-sulfur protein NFU2 is required for branched-chain amino acid synthesis in Arabidopsis roots.

Numerous proteins require a metallic co-factor for their function. In plastids, the maturation of iron-sulfur (Fe-S) proteins necessitates a complex assembly machinery. In this study, we focused on Arabidopsis thaliana NFU1, NFU2, and NFU3, which participate in the final steps of the maturation process. According to the strong photosynthetic defects observed in high chlorophyll fluorescence 101 (hcf101), nfu2, and nfu3 plants, we determined that NFU2 and NFU3, but not NFU1, act immediately upstream of HCF101 for the maturation of [Fe4S4]-containing photosystem I subunits. An additional function of NFU2 in the maturation of the [Fe2S2] cluster of a dihydroxyacid dehydratase was obvious from the accumulation of precursors of the branched-chain amino acid synthesis pathway in roots of nfu2 plants and from the rescue of the primary root growth defect by supplying branched-chain amino acids. The absence of NFU3 in roots precluded any compensation. Overall, unlike their eukaryotic and prokaryotic counterparts, which are specific to [Fe4S4] proteins, NFU2 and NFU3 contribute to the maturation of both [Fe2S2] and [Fe4S4] proteins, either as a relay in conjunction with other proteins such as HCF101 or by directly delivering Fe-S clusters to client proteins. Considering the low number of Fe-S cluster transfer proteins relative to final acceptors, additional targets probably await identification.

The thioredoxin-mediated recycling of Arabidopsis thaliana GRXS16 relies on a conserved C-terminal cysteine.

BACKGROUND: Glutaredoxins (GRXs) are oxidoreductases involved in diverse cellular processes through their capacity to reduce glutathionylated proteins and/or to coordinate iron­sulfur (Fe-S) clusters. Among class II GRXs, the plant-specific GRXS16 is a bimodular protein formed by an N-terminal endonuclease domain fused to a GRX domain containing a 158CGFS signature. METHODS: The biochemical properties (redox activity, sensitivity to oxidation, pKa of cysteine residues, midpoint redox potential) of Arabidopsis thaliana GRXS16 were investigated by coupling oxidative treatments to alkylation shift assays, activity measurements and mass spectrometry analyses. RESULTS: Activity measurements using redox-sensitive GFP2 (roGFP2) as target protein did not reveal any significant glutathione-dependent reductase activity of A. thaliana GRXS16 whereas it was able to catalyze the oxidation of roGFP2 in the presence of glutathione disulfide. Accordingly, Arabidopsis GRXS16 reacted efficiently with oxidized forms of glutathione, leading to the formation of an intramolecular disulfide between Cys158 and the semi-conserved Cys215, which has a midpoint redox potential of - 298 mV at pH 7.0 and is reduced by plastidial thioredoxins (TRXs) but not GSH. By promoting the formation of this disulfide, Cys215 modulates GRXS16 oxidoreductase activity. CONCLUSION: The reduction of AtGRXS16, which is mandatory for its oxidoreductase activity and the binding of Fe-S clusters, depends on light through the plastidial FTR/TRX system. Hence, disulfide formation may constitute a redox switch mechanism controlling GRXS16 function in response to day/night transition or oxidizing conditions. GENERAL SIGNIFICANCE: From the in vitro data obtained with roGFP2, one can postulate that GRXS16 would mediate protein glutathionylation/oxidation in plastids but not their deglutathionylation.

Mitochondrial Arabidopsis thaliana TRXo Isoforms Bind an Iron⁻Sulfur Cluster and Reduce NFU Proteins In Vitro.

In plants, the mitochondrial thioredoxin (TRX) system generally comprises only one or two isoforms belonging to the TRX h or o classes, being less well developed compared to the numerous isoforms found in chloroplasts. Unlike most other plant species, Arabidopsis thaliana possesses two TRXo isoforms whose physiological functions remain unclear. Here, we performed a structure⁻function analysis to unravel the respective properties of the duplicated TRXo1 and TRXo2 isoforms. Surprisingly, when expressed in Escherichia coli, both recombinant proteins existed in an apo-monomeric form and in a homodimeric iron⁻sulfur (Fe-S) cluster-bridged form. In TRXo2, the [4Fe-4S] cluster is likely ligated in by the usual catalytic cysteines present in the conserved Trp-Cys-Gly-Pro-Cys signature. Solving the three-dimensional structure of both TRXo apo-forms pointed to marked differences in the surface charge distribution, notably in some area usually participating to protein⁻protein interactions with partners. However, we could not detect a difference in their capacity to reduce nitrogen-fixation-subunit-U (NFU)-like proteins, NFU4 or NFU5, two proteins participating in the maturation of certain mitochondrial Fe-S proteins and previously isolated as putative TRXo1 partners. Altogether, these results suggest that a novel regulation mechanism may prevail for mitochondrial TRXs o, possibly existing as a redox-inactive Fe-S cluster-bound form that could be rapidly converted in a redox-active form upon cluster degradation in specific physiological conditions.

Chloroplast FBPase and SBPase are thioredoxin-linked enzymes with similar architecture but different evolutionary histories.

The Calvin-Benson cycle of carbon dioxide fixation in chloroplasts is controlled by light-dependent redox reactions that target specific enzymes. Of the regulatory members of the cycle, our knowledge of sedoheptulose-1,7-bisphosphatase (SBPase) is particularly scanty, despite growing evidence for its importance and link to plant productivity. To help fill this gap, we have purified, crystallized, and characterized the recombinant form of the enzyme together with the better studied fructose-1,6-bisphosphatase (FBPase), in both cases from the moss Physcomitrella patens (Pp). Overall, the moss enzymes resembled their counterparts from seed plants, including oligomeric organization-PpSBPase is a dimer, and PpFBPase is a tetramer. The two phosphatases showed striking structural homology to each other, differing primarily in their solvent-exposed surface areas in a manner accounting for their specificity for seven-carbon (sedoheptulose) and six-carbon (fructose) sugar bisphosphate substrates. The two enzymes had a similar redox potential for their regulatory redox-active disulfides (-310 mV for PpSBPase vs. -290 mV for PpFBPase), requirement for Mg(2+) and thioredoxin (TRX) specificity (TRX f > TRX m). Previously known to differ in the position and sequence of their regulatory cysteines, the enzymes unexpectedly showed unique evolutionary histories. The FBPase gene originated in bacteria in conjunction with the endosymbiotic event giving rise to mitochondria, whereas SBPase arose from an archaeal gene resident in the eukaryotic host. These findings raise the question of how enzymes with such different evolutionary origins achieved structural similarity and adapted to control by the same light-dependent photosynthetic mechanism-namely ferredoxin, ferredoxin-thioredoxin reductase, and thioredoxin.

Transcriptomic responses of Phanerochaete chrysosporium to oak acetonic extracts: focus on a new glutathione transferase.

The first steps of wood degradation by fungi lead to the release of toxic compounds known as extractives. To better understand how lignolytic fungi cope with the toxicity of these molecules, a transcriptomic analysis of Phanerochaete chrysosporium genes was performed in the presence of oak acetonic extracts. It reveals that in complement to the extracellular machinery of degradation, intracellular antioxidant and detoxification systems contribute to the lignolytic capabilities of fungi, presumably by preventing cellular damages and maintaining fungal health. Focusing on these systems, a glutathione transferase (P. chrysosporium GTT2.1 [PcGTT2.1]) has been selected for functional characterization. This enzyme, not characterized so far in basidiomycetes, has been classified first as a GTT2 compared to the Saccharomyces cerevisiae isoform. However, a deeper analysis shows that the GTT2.1 isoform has evolved functionally to reduce lipid peroxidation by recognizing high-molecular-weight peroxides as substrates. Moreover, the GTT2.1 gene has been lost in some non-wood-decay fungi. This example suggests that the intracellular detoxification system evolved concomitantly with the extracellular ligninolytic machinery in relation to the capacity of fungi to degrade wood.

Putative roles of glutaredoxin-BolA holo-heterodimers in plants.

Several genomic analyses, high-throughput or targeted interaction studies including the purification of protein complexes indicated a physical and functional link between BolAs and monothiol glutaredoxins (Grxs) that is conserved both in prokaryotes and eukaryotes. In a recent work, we confirmed that several Arabidopsis protein couples, used as plant representatives, also physically interact. More interestingly, we determined that two BolA proteins, BolA2 and SufE1, contain a conserved cysteine that is sensitive to oxidizing treatments, unraveling a possible redox-control of BolA2 and SufE1 by monothiol glutaredoxins. By coexpressing physiological partners in E. coli, Grx-BolA heterodimers binding a labile, oxygen sensitive iron-sulfur cluster were isolated. Altogether, these results illustrate the existence of different modes of interaction between monothiol glutaredoxins and BolA proteins in plants and probably in other organisms. Incidentally, the function of each partner could be differentially modulated depending on the type of interaction.

Monothiol glutaredoxin-BolA interactions: redox control of Arabidopsis thaliana BolA2 and SufE1.

A functional relationship between monothiol glutaredoxins and BolAs has been unraveled by genomic analyses and in several high-throughput studies. Phylogenetic analyses coupled to transient expression of green fluorescent protein (GFP) fusions indicated that, in addition to the sulfurtransferase SufE1, which contains a C-terminal BolA domain, three BolA isoforms exist in Arabidopsis thaliana, BolA1 being plastidial, BolA2 nucleo-cytoplasmic, and BolA4 dual-targeted to mitochondria and plastids. Binary yeast two-hybrid experiments demonstrated that all BolAs and SufE1, via its BolA domain, can interact with all monothiol glutaredoxins. Most interactions between protein couples of the same subcellular compartment have been confirmed by bimolecular fluorescence complementation. In vitro experiments indicated that monothiol glutaredoxins could regulate the redox state of BolA2 and SufE1, both proteins possessing a single conserved reactive cysteine. Indeed, a glutathionylated form of SufE1 lost its capacity to activate the cysteine desulfurase, Nfs2, but it is reactivated by plastidial glutaredoxins. Besides, a monomeric glutathionylated form and a dimeric disulfide-bridged form of BolA2 can be preferentially reduced by the nucleo-cytoplasmic GrxS17. These results indicate that the glutaredoxin-BolA interaction occurs in several subcellular compartments and suggest that a redox regulation mechanism, disconnected from their capacity to form iron-sulfur cluster-bridged heterodimers, may be physiologically relevant for BolA2 and SufE1.