GEK1, a gene product of Arabidopsis thaliana involved in ethanol tolerance, is a d-aminoacyl-tRNA deacylase

GEK1, an Arabidopsis thaliana gene product, was recently identified through its involvement in ethanol tolerance. Later, this protein was shown to display 26% strict identity with archaeal d-Tyr-tRNA(Tyr) deacylases. To determine whether it actually possessed deacylase activity, the product of the GEK1 open reading frame was expressed in Escherichia coli from a multi-copy plasmid. Purified GEK1 protein contains two zinc ions and proves to be a broad-specific, markedly active d-aminoacyl-tRNA deacylase in vitro. Moreover, GEK1 expression is capable of functionally compensating in E. coli for the absence of endogeneous d-Tyr- tRNA(Tyr) deacylase. Possible connections between exposure of plants to ethanol/acetaldehyde and misaminoacylation of tRNA by d-amino acids are considered

Isolation and Characterization of Commensal Bifidobacteria Strains in Gut Microbiota of Neonates Born Preterm: A Prospective Longitudinal Study

Bifidobacterial population dynamics were investigated using a longitudinal analysis of dominant species isolated from feces of neonates born preterm (singletons (n = 10), pairs of twins (n = 11)) from birth up to 16 months of age. We performed quantification, isolation, and identification of the dominant bifidobacteria strains. The genetic relationship of the isolates was investigated via pulsed field gel electrophoresis (PFGE) genotyping, and PCR was used to screen the specific genetic marker tet genes. Additionally, all of the isolated strains were phenotypically characterized by their response to gastro-intestinal stresses and the MIC determination of tetracycline. In the same individual, our results showed a turnover of the bifidobacteria dominant population not only at species but also at strain levels. In addition, we found clonally related strains between twins. A minority of strains were tolerant to gastric (6%) and intestinal (16%) stresses. Thirteen percent of the strains were resistant to tetracycline. This work is original as it provides insights at the strain level of the early life in vivo dynamics of gut microbiota bifidobacteria in preterm neonates. It highlights the need to take into consideration the fluctuation of bifidobacteria populations that may occur for one individual

Etude de deux facteurs sigma secondaires de Lactococcus lactis

L expression des gènes codant les facteurs sigma ComX et SigX chez Lactococcus lactis varie pendant la croissance : le pic d expression de comX correspond à l entrée en phase stationnaire et celui de sigX se situe en phase exponentielle. Le but de ce travail est de comprendre le rôle de ces facteurs sigma chez L. lactis.ComX est homologue aux sigma induisant la transcription des gènes tardifs de compétence naturelle chez certains streptocoques. L. lactis n est pas répertorié comme naturellement compétent mais son génome comporte des gènes codant un système tardif potentiel. Deux types de ComX (ComXIL et ComXMG) ont été identifiés chez les lactocoques divergeant par des substitutions d acides aminés. La surexpression de comXIL engendre l induction de la transcription des gènes tardifs. Un motif conservé ( cin-box ) est retrouvé en amont de ces gènes et pourrait correspondre à la séquence reconnue par ComXIL pour initier la transcription. La cin-box s est révélée être très conservée chez les lactocoques, y compris dans les souches comportant le ComXMG. La purification de ComXIL, ComXMG et de l ARN polymérase a été entreprise pour étudier l affinité des ComX pour la cin-box et pour l ARN polymérase. Pour identifier le régulon contrôlé par SigX, deux approches sont menées en comparant deux conditions : surexpression ou non de sigX. La première approche était la protéomique. Cette étude n a pas révélé de cibles de SigX. La seconde approche est la transcriptomique. Les premiers résultats indiquent que SigX contrôlerait la transcription de gènes codant des protéines membranaires. Cette étude devrait aboutir à l identification du rôle de SigX chez L. lactis.The expression of the genes encoding the sigma factors ComX and SigX in Lactococcus lactis differs during growth : the expression peak of comX corresponds at the onset of the stationary phase and the one of sigX takes place in exponential phase. The aim of this work is to understand the role of these sigma factors in Lactococcus lactis.ComX is homologous to sigma inducing the transcription of the late genes of natural competence in several streptococci. L. lactis is not listed as a natural competent bacterium but its genome includes some genes encoding a potential late system. Two types of ComX (ComXIL and ComXMG) were identified in lactococci that diverge by amino-acid substitutions. The overeexpression of ComXIL allows the induction of the transcription of the late genes. A conserved motif ( cin-box ) is found upstream of these genes and could correspond to the sequence recognised by ComXIL to initiate the transcription. The cin-box revealed itself to be very conserved in lactococci, including in the strains with a ComX of type ComXMG. The purification of ComXIL, ComXMG and of the RNA polymerase has been undertaken to study the affinity of the ComX for the cin-box and for the RNA polymerase. To identify the regulon controlled by SigX, two approaches are used by comparing two conditions : overexpression or not of sigX. The first approach was the proteomic. This study did not reveal any targets of SigX. The second approach is the transcriptomic. The first results indicate that SigX may control the transcription of genes encoding membrane proteins. This study should lead to the identification of the role of SigX in L. lactis.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

La phagothérapie dans la lutte contre les bactéries multi-résistantes aux antibiotiques (des propriétés intéressantes qui peinent à s'imposer en France)

PARIS-BIUP (751062107) / SudocSudocFranceF

Établissement du microbiote

Certaines pathologies semblent avoir une origine développementale et, aujourd’hui, le microbiote apparaît comme un déterminant de l’état de santé de l’homme et son établissement, une étape importante chez le nouveau-né. Des variations dans sa constitution, incluant un déséquilibre (ou dysbiose), ont ainsi été associées à de nombreuses pathologies. De récents travaux suggèrent qu’une colonisation bactérienne de l’utérus, du liquide amniotique ou encore du placenta, des sites auparavant pensés stériles, existerait. Durant les phases de son développement, le foetus pourrait ainsi rencontrer des bactéries in utero. Elles contribueraient à l’établissement de son microbiote avant même l’accouchement et donc avant la rencontre avec les microorganismes des microbiotes vaginal, fécal et cutané, ceux-ci variant selon les modes d’accouchement (voie basse ou césarienne). Les premières études sur l’existence d’un microbiote in utero, qui se caractérise par une faible biomasse, sont cependant controversées

GEK1, une protéine d'Arabidopsis thaliana impliquée dans la tolérance à l'éthanol, est une D-aminoacyl-ARNt désacylase

National audienc

Direct Interaction of RNA Polymerase II and Mediator Required for Transcription in Vivo

International audienc

Evidence for contamination as the origin for bacteria found in human placenta rather than a microbiota.

Until recently the in utero environment of pregnant women was considered sterile. Recent high-sensitivity molecular techniques and high-throughput sequencing lead to some evidence for a low-biomass microbiome associated with the healthy placenta. Other studies failed to reveal evidence for a consistent presence of bacteria using either culture or molecular based techniques. Comparing conflicting "placental microbiome" studies is complicated by the use of varied and inconsistent protocols. Given this situation, we undertook an evaluation of the in utero environment sterility using several controlled methods, in the same study, to evaluate the presence or absence of bacteria and to explain contradictions present in the literature. Healthy pregnant women (n = 38) were recruited in three maternity wards. Placenta were collected after cesarean section with or without Alexis® and vaginal delivery births. For this study we sampled fetal membranes, umbilical cord and chorionic villi. Bacterial presence was analyzed using bacterial culture and qPCR on 34 fetal membranes, umbilical cord and chorionic villi samples. Shotgun metagenomics was performed on seven chorionic villi samples. We showed that the isolation of meaningful quantities of viable bacteria or bacterial DNA was possible only outside the placenta (fetal membranes and umbilical cords) highlighting the importance of sampling methods in studying the in utero environment. Bacterial communities described by metagenomics analysis were similar in chorionic villi samples and in negative controls and were dependent on the database chosen for the analysis. We conclude that the placenta does not harbor a specific, consistent and functional microbiota

Isolation and Characterization of Commensal Bifidobacteria Strains in Gut Microbiota of Neonates Born Preterm: A Prospective Longitudinal Study

Bifidobacterial population dynamics were investigated using a longitudinal analysis of dominant species isolated from feces of neonates born preterm (singletons (n = 10), pairs of twins (n = 11)) from birth up to 16 months of age. We performed quantification, isolation, and identification of the dominant bifidobacteria strains. The genetic relationship of the isolates was investigated via pulsed field gel electrophoresis (PFGE) genotyping, and PCR was used to screen the specific genetic marker tet genes. Additionally, all of the isolated strains were phenotypically characterized by their response to gastro-intestinal stresses and the MIC determination of tetracycline. In the same individual, our results showed a turnover of the bifidobacteria dominant population not only at species but also at strain levels. In addition, we found clonally related strains between twins. A minority of strains were tolerant to gastric (6%) and intestinal (16%) stresses. Thirteen percent of the strains were resistant to tetracycline. This work is original as it provides insights at the strain level of the early life in vivo dynamics of gut microbiota bifidobacteria in preterm neonates. It highlights the need to take into consideration the fluctuation of bifidobacteria populations that may occur for one individual

Redesigning the stereospecificity of tyrosyl-tRNA synthetase

International audienceD-Amino acids are largely excluded from protein synthesis, yet they are of great interest in biotechnology. Unnatural amino acids have been introduced into proteins using engineered aminoacyl-tRNA synthetases (aaRSs), and this strategy might be applicable to D-amino acids. Several aaRSs can aminoacylate their tRNA with a D-amino acid; of these, tyrosyl-tRNA synthetase (TyrRS) has the weakest stereospecificity. We use computational protein design to suggest active site mutations in Escherichia coli TyrRS that could increase its D-Tyr binding further, relative to L-Tyr. The mutations selected all modify one or more sidechain charges in the Tyr binding pocket. We test their effect by probing the aminoacyl-adenylation reaction through pyrophosphate exchange experiments. We also perform extensive alchemical free energy simulations to obtain L-Tyr/D-Tyr binding free energy differences. Agreement with experiment is good, validating the structural models and detailed thermodynamic predictions the simulations provide. The TyrRS stereospecificity proves hard to engineer through charge-altering mutations in the first and second coordination shells of the Tyr ammonium group. Of six mutants tested, two are active towards D-Tyr; one of these has an inverted stereospecificity, with a large preference for D-Tyr. However, its activity is low. Evidently, the TyrRS stereospecificity is robust towards charge rearrangements near the ligand. Future design may have to consider more distant and/or electrically neutral target mutations, and possibly design for binding of the transition state, whose structure however can only be modeled