Biology subjects

Bourdin, T.

Publications and source records attributed to Bourdin, T..

3 recordsLinked to original sources

bioRxiv2026

The stereochemical mechanism of the B12-dependent radical SAM glutamine methyltransferase (QCMT): Novel insights and unprecedented post-translational modifications

Methyl-coenzyme M reductase (MCR) is a crucial enzyme for methanogenesis and harbors several unusual post-translational modifications. Recent studies have identified glutamine C-methyltransferase (QCMT), as a B12-dependent radical SAM enzyme responsible for methylating a glutamine residue within the MCR active site. B12-dependent radical SAM enzymes have the remarkable ability to alkylate unactivated Csp2- and Csp3-atoms in a stereoselective manner. However, the factors influencing the stereo-selectivity and catalytic properties of this emerging superfamily of enzymes remain poorly understood. In this study, we report the mechanistic, structural, and biochemical investigation of several QCMTs. Our findings reveal significant differences among them, notably in their ability to bind cobalamin. In addition, our data support that C H-atom abstraction and methyl transfer are not concerted but rather independent processes that require motion within the enzymes active site. We also demonstrate that QCMT can catalyze novel reactions, including the formation of unnatural C-methylated residues, peptide epimerization, reversible H-atom abstraction, and the direct conversion of glycine into O_SCPLOWDC_SCPLOW-alanine. Overall, our data are consistent with QCMT being a unique and versatile biocatalyst allowing for the installation of unnatural post-translational modifications and provide a structural and biochemical rationale for the control of the stereochemistry by B12-dependent radical SAM enzymes.

biochemistry↗

bioRxiv2023

High-Throughput Short Sequence Typing Schemes for Pseudomonas aeruginosa and Stenotrophomonas maltophilia pure culture and environmental DNA

Molecular typing techniques are employed to determine the genetic similarities between bacterial isolates. These methods primarily utilize specific genetic markers or analyze the complete genome sequence of pure bacterial cultures. However, the use of environmental DNA profiling to assess epidemiologic links between patients and their environment has not been explored in depth. This work reports on the development and validation of two High-Throughput Short Sequence Typing (HiSST) schemes targeting the opportunistic pathogens Pseudomonas aeruginosa and Stenotrophomonas maltophilia, along with a modified SM2I medium for specific isolation of S. maltophilia. Our HiSST schemes are based on four discriminative loci for each species and demonstrate high discrimination power, comparable to pairwise whole genomes comparison. Moreover, each scheme includes species-specific PCR primers, enabling precise differentiation from closely related taxa without the need for upstream culture-dependent methods. For example, the primers designed to target the bvgS locus allow to distinguish P. aeruginosa from the very closely related Pseudomonas paraeruginosa sp. nov. The selected loci included in the schemes for P. aeruginosa (pheT, btuB, sdaA, bvgS) and for S. maltophilia (yvoA, glnG, ribA, tycC), are within the range of 271 to 330 base pairs adapted to massive parallel amplicon sequencing technology. A R-based script implemented in the DADA2 pipeline was assembled to facilitate HiSST analysis for efficient and accurate genotyping of P. aeruginosa and S. maltophilia. The performance of both schemes was demonstrated through in-silico validations, assessments against reference culture collections, and a case study involving environmental samples.

molecular biology↗

bioRxiv2021

Development and Validation of a High-throughput Short Sequence Typing Scheme for Serratia marcescens Pure Culture and Environmental DNA

Molecular typing methods are used to characterize the relatedness between bacterial isolates involved in infections. These approaches rely mostly on discrete loci or whole genome sequences (WGS) analyses of pure cultures. On the other hand, their application to environmental DNA profiling to evaluate epidemiological relatedness amongst patients and environments has received less attention. We developed a specific, high-throughput short sequence typing (HiSST) method for the opportunistic human pathogen Serratia marcescens. Genes displaying the highest polymorphism were retrieved from the core genome of 60 S. marcescens strains. Bioinformatics analyses showed that use of only three loci (within bssA, gabR and dhaM) distinguished strains with the same level of efficiency than average nucleotide identity scores of whole genomes. This HiSST scheme was applied to an epidemiological survey of S. marcescens in a neonatal intensive care unit (NICU). In a first case study, a strain responsible for an outbreak in the NICU was found in a sink drain of this unit, by using HiSST scheme and confirmed by WGS. The HiSST scheme was also applied to environmental DNA extracted from sink-environment samples. Diversity of S. marcescens was modest, with 11, 6 and 4 different sequence types (ST) of gabR, bssA and dhaM loci amongst 19 sink drains, respectively. Epidemiological relationships amongst sinks were inferred on the basis of pairwise comparisons of ST profiles. Further research aimed at relating ST distribution patterns to environmental features encompassing sink location, utilization and microbial diversity is needed to improve the surveillance and management of opportunistic pathogens.

molecular biology↗