Structural microbiology
and enzymology
Our team is interested in elucidating action mechanisms of singles proteins/enzymes or within multi-molecular complexes.
Our work allows supplying fundamental basis describing molecular function at atomic level. Our team possesses a strong expertise in structural analysis by crystallography of (i) bacterial, plant, phage and human enzymatic mechanisms, (ii) bacterial transport systems for opines, metals, peptides (iii) bacterial and human proteins interacting with DNA such as DNA repair/modification enzymes, transcriptional factors. For short, we are specialized in the study of protein-protein interactions, protein-DNA and protein-ligand interactions, which are at the center of all the biological processes. Our main theme concern proteins involved in host-pathogen relationship, nutrient transport, virulence. Our team is open to collaborations with microbiologists and biologists interested in molecular level of their study model.
Experimental approach
We use protein crystallography to determine the 3D structure of proteins and analyze their function alone or within multimolecular assemblies. The structure-function analysis is completed by biochemical and biophysical studies in solution. Techniques usually used are:
- Production of proteins (cloning, mutagenesis, bacterial culture…)
- Purification of recombinant proteins (Chromatography of affinity, Gel filtration…)
- Enzymatic tests and inhibition measurement ( Spectrophotometry)
- Crystallization, X-rays crystallography and structural analysis
- Interactions determination (Microcalorimétrie, Fluorescence, BIAcore)
- Conformational analyses (Circular Dichroism, Ultracentrifugation, light scattering)
- Post-traductionnal modifications analysis (mass spectrometry)
- Bioinformatic and molecular modelisation
Two examples of our recent works
A- The transcriptional repressor Atu1419 from the bacterial pathogen A.tumefaciens (Vigouroux et al, (2021), NAR 49, 529-546 doi: 10.1093/nar/gkaa1181)
A species-specific region, denoted SpG8-1b allowing hydroxycinnamic acids (HCAs) degradation is important for the transition between the two lifestyles (rhizospheric versus pathogenic) of the plant pathogen Agrobacterium fabrum. Indeed, HCAs can be either used as trophic resources and/or as induced-virulence molecules. The SpG8-1b region is regulated by two transcriptional regulators, namely, HcaR (Atu1422) and Atu1419. Our structural study revealed that the tetrameric Atu1419 transcriptional regulator belongs to the VanR group of Pfam PF07729 subfamily of the large GntR superfamily. Until now, GntR regulators were described as dimers. We showed that Atu1419 represses three genes of the HCAs catabolic pathway. We characterized both the effector and DNA binding sites and identified key nucleotides in the target palindrome.
Figure legend: Interactions between Atu1419 repressor and Patu1418-1419 region of 370 bp containing two palindromes separated by 190 bp visualized by electron microscopy on the left, and corresponding models on the right. (A) Image of Patu1418-1419 region, (B) Image of Atu1419 tetramer, (C) Image of one tetramer bound to one palindrome, (D) Image of two tetramers bound each to one palindrome, (E) Image of one tetramer inducing a DNA loop. Scales bars: 20 nm.
B- Import pathways of the mannityl-opines into the bacterial pathogen A.tumefaciens (Vigouroux et al. 2020, Biochem J. 477, 615-628).
Agrobacterium tumefaciens pathogens use specific compounds namely opines as nutrients in their plant tumor niche. These opines are produced by the host plant cells genetically modified by agrobacteria. They are imported into bacteria via solute-binding proteins (SBPs) in association with ABC transporters. The mannityl-opine family encompasses mannopine, mannopinic acid, agropine and agropinic acid. Combining in vitro and in vivo approaches, our work finalizes the characterization of the mannityl-opines assimilation pathways, highlighting the important role of two dual imports of agropinic and mannopinic acids. Our data shed new light on how the mannityl-opines contribute to the establishment of the ecological niche of agrobacteria from the early to the late stages of tumor development.
Figure legend: Metabolism of mannityl-opines in transformed plant cells and in A. tumefaciens R10/B6 strains. In modified plant cell, the mas1 and mas2 genes responsible for the biosynthesis of mannopinic acid and mannopine are located on the T-DNA as well as ags gene product, which catalyzes the lactonization of mannopine to agropine. Agropinic acid results from a spontaneous lactamization of the three mannityl-opines: agropine, mannopine and mannopinic acid. In A. tumefaciens B6/R10 strain, two transport pathways for agropinic acid and mannopinic acid co-exist. The (moaADCB) genes (cyan) and the (motABDC) genes (pink) located on the Ti plasmid code for the selective and non-selective transport of mannopinic acid, respectively. The (agaACDB) genes (magenta) and (agtABC) genes (blue) code for the selective and non-selective transport of agropinic acid, respectively. The structure of the four SBPs MoaA-, AgaA-, AgtB- and MotA-mediated transport bound to their preferred mannityl-opine, which are mannopinic acid, agropinic acid, agropine and mannopine, respectively are shown. Affinity values are indicated. The (agaE), (agaFG), (agcA), (mocC) and (mocDE) genes products are involved in mannityl-opines degradation. The (moaR) and (mocR) genes code for a corresponding transcriptional regulator of each genes clusters.
team
David KOPECNY
Invited Researcher
Armelle VIGOUROUX
Engineer
Latest publications
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Partners
I2BC Facilities
Synchrotron SOLEIL