The Complete Genome of Propionibacterium freudenreichii CIRM-BIA1T, a Hardy Actinobacterium with Food and Probiotic Applications

Background: Propionibacterium freudenreichii is essential as a ripening culture in Swiss-type cheeses and is also considered for its probiotic use [1]. This species exhibits slow growth, low nutritional requirements, and hardiness in many habitats. It belongs to the taxonomic group of dairy propionibacteria, in contrast to the cutaneous species P. acnes. The genome of the type strain, P. freudenreichii subsp. shermanii CIRM-BIA1 (CIP 103027T), was sequenced with an 11-fold coverage. Methodology/Principal Findings: The circular chromosome of 2.7 Mb of the CIRM-BIA1 strain has a GC-content of 67% and contains 22 different insertion sequences (3.5% of the genome in base pairs). Using a proteomic approach, 490 of the 2439 predicted proteins were confirmed. The annotation revealed the genetic basis for the hardiness of P. freudenreichii, as the bacterium possesses a complete enzymatic arsenal for de novo biosynthesis of aminoacids and vitamins (except panthotenate and biotin) as well as sequences involved in metabolism of various carbon sources, immunity against phages, duplicated chaperone genes and, interestingly, genes involved in the management of polyphosphate, glycogen and trehalose storage. The complete biosynthesis pathway for a bifidogenic compound is described, as well as a high number of surface proteins involved in interactions with the host and present in other probiotic bacteria. By comparative genomics, no pathogenicity factors found in P. acnes or in other pathogenic microbial species were identified in P. freudenreichii, which is consistent with the Generally Recognized As Safe and Qualified Presumption of Safety status of P. freudenreichii. Various pathways for formation of cheese flavor compounds were identified: the Wood-Werkman cycle for propionic acid formation, amino acid degradation pathways resulting in the formation of volatile branched chain fatty acids, and esterases involved in the formation of free fatty acids and esters. Conclusions/Significance: With the exception of its ability to degrade lactose, P. freudenreichii seems poorly adapted to dairy niches. This genome annotation opens up new prospects for the understanding of the P. freudenreichii probiotic activity

Human placental inositolphosphoglycans may be released by glycosylphosphatidylinositol-phospholipase D, both present in the syncytial brush border membrane investigation in normal and pre-eclamptic placentae

Pre-eclampsia is a disorder affecting 5 to 10% of all pregnancies. In the pre-eclamptic placenta, increased levels of an inositolphosphoglycan (IPG) second messengers have been reported. The objective of this thesis has been the study of IPGs in the placenta and their possible release by the glycosylphosphatidylinositol-phospholipase D (GPI-PLD) enzyme. The presence of IPGs was investigated in the placenta. Immunostaining on sections of chorionic villi revealed the presence of IPGs and confirmed the higher levels of IPGs in pre-eclampsia. IPGs of both sub-types A and P were extracted from microvilli preparations of both normal and pre-eclamptic placentae. After showing the presence of biologically active IPGs in microvilli preparation, the next step was to investigate the presence of glycosylphosphatidylinositol (GPI), the putative precursor molecule of IPGs. GPI was extracted from microvilli of normal placenta but unexpectedly not detected in pre-eclamptic samples. An elevated GPI-PLD activity could cause increased catabolism of GPI, and thus be responsible for both the absence of detectable GPI and the increased IPG levels found in pre-eclamptic samples. GPI-PLD hydrolysis activity was measured in microvilli preparations from both normal and pre-eclamptic placenta. Whereas GPI-PLD is described as a 100 kDa protein, a protein of 50 kDa was mainly detected. GPI-PLD mRNA was not detected in the placenta. We therefore propose a model in which placental GPI-PLD is taken up by the syncytiotrophoblast from the maternal blood and transferred to a lysosomal compartment where it is cleaved into an active 50 kDa protein before returning to the plasma membrane. Finally, the susceptibility of extracted placental GPI to hydrolysis by recombinant GPI-PLD has been studied. GPI-PLD was able to cleave GPI in vitro. To conclude, this work confirms the presence of IPGs in placenta and more specifically in microvilli and supports previous reports of increased IPG-P type in preeclampsia. The study of placental GPI and GPI-PLD throws light on the possible mechanism underlying the generation of IPGs in both normal and pre-eclamptic microvilli

Oxydation par le chlore et l'ozone de composés organiques à effets modulateurs endocriniens

L'objectif de ce travail a été de contribuer à une meilleure connaissance du devenir des composés perturbateurs endocriniens au cours de la filière de traitement des eaux. Pour cela, une étude de l'oxydation par le chlore et l'ozone (deux procédés de désinfection fréquemment employés comme étapes de potabilisation) de sept composés à effets modulateurs endocriniens (17b-estradiol, estriol, estrone, progestérone, 17a-éthinylestradiol, 4-n-nonylphénol et bisphénol A) a été menée. Dans un premier temps, une étude cinétique a été conduite à 20 +- 2 C et différents pH. Dans ces conditions, pour l'ensemble des composés phénoliques étudiés (17b-estradiol, estriol, estrone, 17a-éthinylestradiol, 4-n-nonylphénol et bisphénol A), quel que soit l'oxydant employé, une dégradation possible et plus ou moins rapide suivant le pH a été observée. Les réactions élémentaires d'oxydation et leur constante cinétique associée ont alors été déterminées. Pour la progestérone (dépourvue de cycle phénolique), aucune dégradation notable par le chlore n'a été relevée. Quel que soit le pH, une oxydation possible par l'ozone a, en revanche, été notée. Dans un second temps, pour deux des composés perturbateurs endocriniens étudiés (le bisphénol A et la progestérone), un travail d'identification des sous-produits d'ozonation a été entrepris. Par analyses chromatographiques (CLHP/UV, CLHP/SM, CLHP/SM/SM, CG/SM), pour chacun ces composés, de nombreux sous-produits d'ozonation ont été mis en évidence. Leur stabilité à l'ozone a alors été évaluée et les schémas réactionnels impliqués ont été discutés.The objective of this work was to improve the present knowledge about fate of endocrine disruptors during water treatment process. Chlorination and ozonation (two disinfection steps frequently applied) of seven endocrine disruptors (17b-estradiol, estriol, estrone, 17a-ethinylestradiol, 4-n-nonylphenol et bisphenol A) were then studied. In a first time, a kinetic study was performed at 20 +- 2 C and different pH levels. Under these conditions, all molecules with a phenolic group in their structure (17b-estradiol, estriol, estrone, 17a-ethinylestradiol, 4-n-nonylphenol et bisphenol A) were removed more or less rapidly as function of pH, irrespective of the studied oxidant. Elementary oxidation reactions and associated rate constants were then determined. Concerning progesterone (devoid of phenolic ring), no removal in presence of chlorine was shown. However, a possible oxidation by ozone was observed. In a second time, by-products ozonation of two studied endocrine disruptors (bisphenol A, progesterone) were studied. Numerous by-products were identified by chromatographic analyses (HPLC/UV, HPLC/MS, HPLC/MS/MS, GC/MS). Stability by-products in presence of ozone was then studied and reactional pathways were suggested.POITIERS-BU Sciences (861942102) / SudocSudocFranceF

Étude de la réactivité de deux composés pharmaceutiques, le paracétamol et la carbamazépine, au cours de la chloration

International audienc

TOWARDS A METABOLOMIC CHARACTERISATION OF THE GRAPEVINE RESPONSE TO FLAVESCENCE DORÉE INFECTION BY NMR AND LC-MS PROFILING

Flavescence dorée (FD) is a quarantine disease of grapevine, involving interactions between the plants, leafhopper vectors (phytophagous insect Scaphoideus titanus Ball, Cicadellidae family), and Flavescence dorée phytoplasma. This disease is a major threat to vineyard survival in different European grapegrowing areas. Differences in FD susceptibility between grapevine varieties exist, in terms of multiplication of the associated phytoplasma and its spread in the plant. Cabernet Sauvignon (CS) is highly susceptible to FD, whereas Merlot (M) is less susceptible. The objective of this project is to determine which primary and specialised metabolites are altered in grapevines following infection by FD phytoplasma under controlled conditions. Plants of CS and M were infected with FD phytoplasma on a single leaf via the natural insect vector S. titanus in a high containment greenhouse. Sampling took place 7-day, or 7-week post-inoculation. The leaves were harvested and immediately frozen in liquid N2 to quench metabolism. Phytoplasma titers were determined on fresh powder of each leaf. Metabolomic analyses were performed on a selection of the leaf sample extracts by 1 H-NMR, LC-LTQ/Orbitrap-HRMS, and LC-QqQ-MS. NMR spectra were acquired at 500 MHz and processed using NMRProcFlow (nmrprocflow.org). For both LC-MS, metabolites were separated by reverse-phase chromatography with a H2O/MeCN gradient. Untargeted experiments were performed on a UHPLC-ESI-LTQ/Orbitrap-MS with an ESI probe operated in positive mode and processed using MS Dial v4.7 8. Targeted experiments were performed on an LC-QqQ-MS system with a MRM method in positive or negative mode and the quantification by using calibration curves built with standards.The phytoplasma titre data, metabolite quantification data or metabolomic signatures of leaf samples were managed with ODAM tool

Transcriptional and Metabolic Adjustments in ADP-Glucose pyrophosphorylase -deficient bt2 Maize kernels

International audienceDuring the cloning of monogenic recessive mutations responsible for a defective kernel phenotype in a Mutator-induced Zea mays mutant collection, we isolated a new mutant allele in Brittle2 (Bt2), which codes for the small subunit of ADP-glucose pyrophosphorylase (AGPase), a key enzyme in starch synthesis. Reverse transcription-polymerase chain reaction experiments with gene-specific primers confirmed a predominant expression of Bt2 in endosperm, of Agpsemzm in embryo, and of Agpslzm in leaf, but also revealed considerable additional expression in various tissues for all three genes. Bt2a, the classical transcript coding for a cytoplasmic isoform, was almost exclusively expressed in the developing endosperm, whereas Bt2b, an alternative transcript coding for a plastidial isoform, was expressed in almost all tissues tested with a pattern very similar to that of Agpslzm. The phenotypic analysis showed that, at 30 d after pollination (DAP), mutant kernels were plumper than wild-type kernels, that the onset of kernel collapse took place between 31 and 35 DAP, and that the number of starch grains was greatly reduced in the mutant endosperm but not the mutant embryo. A comparative transcriptome analysis of wild-type and bt2-H2328 kernels at middevelopment (35 DAP) with the 18K GeneChip Maize Genome Array led to the conclusion that the lack of Bt2-encoded AGPase triggers large-scale changes on the transcriptional level that concern mainly genes involved in carbohydrate or amino acid metabolic pathways. Principal component analysis of 1H nuclear magnetic resonance metabolic profiles confirmed the impact of the bt2-H2328 mutation on these pathways and revealed that the bt2-H2328 mutation did not only affect the endosperm, but also the embryo at the metabolic level. These data suggest that, in the bt2-H2328 endosperms, regulatory networks are activated that redirect excess carbon into alternative biosynthetic pathways (amino acid synthesis) or into other tissues (embryo)