Biology subjects

Rouhier, N.

Publications and source records attributed to Rouhier, N..

5 recordsLinked to original sources

bioRxiv2023

Correction of non-random mutational biases along a linear bacterial chromosome by the mismatch repair endonuclease NucS

The linear chromosome of Streptomyces exhibits a highly compartmentalized structure with a conserved central region flanked by variable arms. As double strand break (DSB) repair mechanisms play a crucial role in shaping the genome plasticity of Streptomyces, we investigated the role of EndoMS/NucS, a recently characterized endonuclease involved in a non-canonical Mismatch Repair (MMR) mechanism in archaea and actinobacteria, that singularly corrects mismatches by creating a DSB. We showed that Streptomyces mutants lacking NucS display a marked colonial phenotype and a drastic increase in spontaneous mutation rate. In vitro biochemical assays revealed that NucS cooperates with the replication clamp to efficiently cleave G/T, G/G and T/T mismatched DNA by producing DSBs. These findings are consistent with the transition-shifted mutational spectrum observed in the mutant strains and reveal that NucS-dependent MMR specific task is to eliminate G/T mismatches generated by the DNA polymerase during replication. Interestingly, our data unveil a crescent-shaped distribution of the transition frequency from the replication origin towards the chromosomal ends, shedding light on a possible link between NucS-mediated DSBs and Streptomyces genome evolution. GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=105 SRC="FIGDIR/small/572499v1_ufig1.gif" ALT="Figure 1"> View larger version (16K): org.highwire.dtl.DTLVardef@1aa491dorg.highwire.dtl.DTLVardef@a62657org.highwire.dtl.DTLVardef@1f1d2aorg.highwire.dtl.DTLVardef@b3b389_HPS_FORMAT_FIGEXP M_FIG C_FIG

microbiology↗

bioRxiv2022

The mitochondrial ferredoxin-like is essential for the formation of complex I-containing respiratory supercomplexes in Arabidopsis thaliana

In eukaryotes, mitochondrial ATP is mainly produced by the oxidative phosphorylation (OXPHOS) system, which is composed of five multiprotein complexes (complexes I to V). Analyses of the OXPHOS system by native gel electrophoresis revealed an organization of OXPHOS complexes in supercomplexes, but their roles and assembly pathways remain unclear. In this study, we characterized an atypical mitochondrial ferredoxin (mFDX-like). This protein was previously found associated with complex I, being part of the bridge domain linking the matrix and membrane arms of the complex. A phylogenetic analysis suggests that the Arabidopsis thaliana mFDX-like evolved from classical mitochondrial ferredoxin, but it lost one of the cysteines required for the coordination of the iron-sulfur (Fe-S) cluster essential for the electron transfer function of ferredoxins. Accordingly, our biochemical study shows that AtmFDX-like does not bind an Fe-S cluster, and is therefore unlikely to be involved in electron transfer reactions. To study the function of mFDX-like, we created deletion lines in Arabidopsis using a CRISPR/Cas9 approach. These lines do not show any growth phenotype under standard growth condition. However, the characterization of the OXPHOS system demonstrates that mFDX-like is important for the assembly of complex I, and essential for the formation of complex I-containing supercomplexes. We propose that mFDX-like and the bridge domain are required for the correct conformation of the membrane arm of complex I that is essential for the association of complex I with complex III to form supercomplexes.

plant biology↗

bioRxiv2021

The Fe-S cluster assembly factors NFU4 and NFU5 are primarily required for protein lipoylation in mitochondria

Plants have evolutionarily conserved NFU-domain proteins that are targeted to plastids or mitochondria. The plastid-type NFU1, NFU2 and NFU3 in Arabidopsis thaliana play a role in iron-sulfur (Fe-S) cluster assembly in this organelle, whereas the type-II NFU4 and NFU5 proteins have not been subjected to mutant studies in any plant species to determine their biological role. Here we confirm that NFU4 and NFU5 are targeted to the mitochondria. The proteins are constitutively produced in all parts of the plant, suggesting a housekeeping function. Double nfu4 nfu5 knockout mutants were embryonic lethal, and depletion of the proteins led to growth arrest of young seedlings. Biochemical analyses revealed that NFU4 and NFU5 are required for lipoylation of the H proteins of the glycine decarboxylase complex and the E2 subunits of other mitochondrial dehydrogenases, with little impact on Fe-S cluster-containing respiratory complexes and aconitase. Consequently, the Gly-to-Ser ratio was increased in mutant seedlings and early growth was improved by elevated CO2. In addition, pyruvate, 2-oxoglutarate and branched-chain amino acids accumulated in the nfu4 nfu5 mutants, further supporting defects in the other three mitochondrial lipoate-dependent enzyme complexes. NFU4 and NFU5 interacted with mitochondrial lipoyl synthase (LIP1) in yeast 2-hybrid and bimolecular fluorescence complementation assays. These data indicate that NFU4 and NFU5 have a more specific function than previously thought, in providing Fe-S clusters to lipoyl synthase. One sentence summaryA pair of evolutionarily conserved proteins involved in iron-sulfur cofactor assembly have a specific role in lipoate biosynthesis for mitochondrial dehydrogenases.

plant biology↗

bioRxiv2020

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. 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, however, containing both CD and Rhd domains in specific bacterial genera suggests a general interaction between both 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 uses L-cysteine to catalyze trans-persulfidation 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. Both proteins interact in planta and form an efficient sulfur relay system whereby STR18 catalyzes trans-persulfidation 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.

plant biology↗

bioRxiv2019

Structural genomic applied on the rust fungus Melampsora larici-populina reveals two candidate effector proteins adopting cystine-knot and nuclear transport factor 2-like protein folds

Rust fungi are plant pathogens that secrete an arsenal of effector proteins interfering with plant functions and promoting parasitic infection. Effectors are often species-specific, evolve rapidly, and display low sequence similarities with known proteins or domains. How rust fungal effectors function in host cells remains elusive, and biochemical and structural approaches have been scarcely used to tackle this question. In this study, we used a strategy based on recombinant protein production in Escherichia coli to study eleven candidate effectors of the leaf rust fungus Melampsora larici-populina. We successfully purified and solved the three-dimensional structure of two proteins, MLP124266 and MLP124017, using NMR spectroscopy. Although both proteins show no sequence similarity with known proteins, they exhibit structural similarities to knottin and nuclear transport factor 2-like proteins, respectively. Altogether, our findings show that sequence-unrelated effectors can adopt folds similar to known proteins, and encourage the use of biochemical and structural approaches to functionally characterize rust effector candidates.

microbiology↗