A sensitive bacterial-growth-based test reveals how intestinal Bacteroides meet their porphyrin requirement

Heme-PPIX-Screen detection of pure heme or in biological samples. (XLSX 20 kb

Identification of DNA Motifs Implicated in Maintenance of Bacterial Core Genomes by Predictive Modeling

Bacterial biodiversity at the species level, in terms of gene acquisition or loss, is so immense that it raises the question of how essential chromosomal regions are spared from uncontrolled rearrangements. Protection of the genome likely depends on specific DNA motifs that impose limits on the regions that undergo recombination. Although most such motifs remain unidentified, they are theoretically predictable based on their genomic distribution properties. We examined the distribution of the “crossover hotspot instigator,” or Chi, in Escherichia coli, and found that its exceptional distribution is restricted to the core genome common to three strains. We then formulated a set of criteria that were incorporated in a statistical model to search core genomes for motifs potentially involved in genome stability in other species. Our strategy led us to identify and biologically validate two distinct heptamers that possess Chi properties, one in Staphylococcus aureus, and the other in several streptococci. This strategy paves the way for wide-scale discovery of other important functional noncoding motifs that distinguish core genomes from the strain-variable regions

Navigating the garden of forking paths for data exclusions in fear conditioning research

In this report, we illustrate the considerable impact of researcher degrees of freedom with respect to exclusion of participants in paradimgs with a learning element. We illustrate this empirically through case examples from human fear conditioning research where the exclusion of ‘non-learners’ and ‘non-responders’ is common-despite a lack of consensus on how to define these groups. We illustrate the substantial heterogeneity in exclusion criteria based on a systematic literature search and highlight potential problems and pitfalls of different definitions through case examples based on re-analyses of existing data sets. Based on this, we propose a consensus on evidence-based rather than idiosyncratic criteria including clear guidelines on reporting details. Taken together, we illustrate how flexibility in data collection and analysis can be avoided, which will benefit the robustness and replicability of research findings and can be expected to be applicable to other fields of research that involve a learning element

Two Coregulated Efflux Transporters Modulate Intracellular Heme and Protoporphyrin IX Availability in Streptococcus agalactiae

Streptococcus agalactiae is a major neonatal pathogen whose infectious route involves septicemia. This pathogen does not synthesize heme, but scavenges it from blood to activate a respiration metabolism, which increases bacterial cell density and is required for full virulence. Factors that regulate heme pools in S. agalactiae are unknown. Here we report that one main strategy of heme and protoporphyrin IX (PPIX) homeostasis in S. agalactiae is based on a regulated system of efflux using two newly characterized operons, gbs1753 gbs1752 (called pefA pefB), and gbs1402 gbs1401 gbs1400 (called pefR pefC pefD), where pef stands for ‘porphyrin-regulated efflux’. In vitro and in vivo data show that PefR, a MarR-superfamily protein, is a repressor of both operons. Heme or PPIX both alleviate PefR-mediated repression. We show that bacteria inactivated for both Pef efflux systems display accrued sensitivity to these porphyrins, and give evidence that they accumulate intracellularly. The ΔpefR mutant, in which both pef operons are up-regulated, is defective for heme-dependent respiration, and attenuated for virulence. We conclude that this new efflux regulon controls intracellular heme and PPIX availability in S. agalactiae, and is needed for its capacity to undergo respiration metabolism, and to infect the host

Etude de la respiration chez Lactococcus lactis et implication des systèmes Redox dans la physiologie cellulaire

Lactococcus lactis a été étudié jusqu'à présent pour ses capacités de fermentation. Notre équipe a mis en évidence qu'en présence d'hème et d'oxygène, L. lactis est capable de respirer. La croissance par respiration s'accompagne d'une augmentation de la biomasse, d'une baisse de la production de lactate et d'une amélioration de la survie à long terme.Une étude par électrophorèse bidimensionnelle (E2D) menée sur des cultures en phase exponentielle montre que la plupart des protéines cytoplasmiques présentes en respiration le sont déjà en fermentation. Une seule protéine spécifique de la respiration a été détectée, un régulateur transcriptionnel régulant un transporteur putatif de lactate, en accord avec la consommation de lactate observée en respiration. Cette étude suggère l'existence d'autres régulateurs que ceux caractérisés jusqu'alors dans la nutrition azotée, ainsi que de régulateurs intervenant dans la consommation de lactate en respiration. Cette étude a également mis en évidence une diminution en respiration de l'oxydation d'une enzyme de la glycolyse, essentielle chez L. lactis, la glyceraldéhyde-3-phosphate déshydrogénase. Celle-ci est aussi moins oxydée dans un mutant trxB1, codant une thiorédoxine réductase. TrxB1 n'est pas essentielle chez L. lactis, mais son absence rend la cellule très sensible à la présence d'oxygène. Un mutant trxB1 est viable, même en l'absence de composés réducteurs pouvant pallier l'absence de thiorédoxine réductase. La physiologie de ce mutant a été analysée par E2D. Les résultats révèlent une plus forte expression d'enzymes anti-oxydantes et des changements dans le métabolisme des lipides, suggérant des dégâts membranaires.Lactococcus lactis has been extensively studied for his fermentative capacities. We demonstrated that in presence of heme and oxygen, L. lactis was able to undergo respiration. In this condition, growth and long term survival were improved and lactate production was lower than during fermentative growth.We used a proteomic approach to study differences between fermentative and respirative growth. Results showed that most of the proteins present in respirative growth are also present in fermentative growth. Only one protein was detected as specific for respiration, a transcriptional regulator involved in the expression of an operon encoding a putative lactate transporter, in agreement with lactate consumption specifically observed during respirative growth. This study suggest the existence of new unknown regulators involved in nitrogen metabolism and in lactate consumption. We also demonstrated that an essential enzyme of glycolysis, glyceraldehyde-3-phosphate dehydrogenase was less oxidised in respiration than in fermentation conditions.We studied the thioredoxin reductase TrxB1 of L. lactis by constructing a mutant in the corresponding gene. The mutant was viable even in the absence of dithiotreitol, suggesting that the thioredoxin system is not essential in L. lactis. However, this mutant was very sensitive to oxygen in the absence of dithiotreitol. Physiology of this mutant under fermentative aerobic growth was analysed by proteomics. Results showed increased expression of anti-oxidant enzymes and changes in lipid metabolism, suggesting membrane damages. Glyceraldehyde-3-phosphate dehydrogenase was surprisingly also less oxidised in this mutant.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

De l'analyse du génome à létude d'un nouveau facteur de virulence de la famille des internalines chez Enterococcus faecalis

ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF

Métabolisme respiratoire chez Lactococcus lactis dans un environnement oxydant et identification de composants de la chaîne respiratoire

LACTOCOCCUS LACTIS EST TRES UTILISEE DANS L'INDUSTRIE AGROALIMENTAIRE. A CETTE FIN ELLE A ETE PRINCIPALEMENT ETUDIEE POUR SES CAPACITES FERMENTAIRE. LE DOGME DES BACTERIES LACTIQUES EXCLUSIVEMENT FERMENTAIRE EST CADUQUE DEPUIS QU'IL A ETE MONTRE QUE L. LACTIS EST CAPABLE D'ADOPTER UN METABOLISME RESPIRATOIRE EN PRESENCE D'HEME EXOGENE ET D'OXYGENE. DANS CES CONDITIONS UN DOUBLEMENT DE LA BIOMASSE, UNE AUGMENTATION DU PH ET LA SURVIE LARGEMENT AUGMENTEE, SONT OBSERVEES PAR RAPPORT A UN METABOLISME FERMENTAIRE. MON PROJET DE THESE S'EST FOCALISE SUR LE METABOLISME RESPIRATOIRE DE L. LACTIS ET S'EST ARTICULE AUTOUR DE DEUX AXES : IDENTIFIER ET CARACTERISER LES COMPOSES DE LA CHAINE RESPIRATOIRE ET COMPRENDRE LES RAISONS DE LA MEILLEURE SURVIE. NOUS AVONS MIS EN EVIDENCE PAR MUTAGENESE CIBLEE LE ROLE DES MENAQUINONES DANS LE PROCESSUS DE TRANSFERT D'ELECTRONS A LA CYTOCHROME OXYDASE AINSI QUE SON IMPLICATION DANS LA PRODUCTION D'ION SUPEROXYDE ET DE REDUCTION DU FER EXTRACELLULAIRE. LES NADH DESHYDROGENASES NOXA ET NOXB SEMBLENT IMPLIQUEES EN PARTIE DANS CE PROCESSUS. LA CYTOCHROME BD OXYDASE DEJA IDENTIFIEE POUR SA FONCTION EN TANT QUE SEULE TERMINALE OXYDASE A ETE CONFIRMEE PAR NOS MUTANTS CYDA ET CYDB OBTENUS LORS DE LA MUTAGENESE ALEATOIRE. LA MISE EN PLACE DU METABOLISME RESPIRATOIRE PERMET A L. LACTIS DE SURVIVRE PLUS LONGTEMPS, NOTAMMENT GRACE A UNE DIMINUTION DE L'ENVIRONNEMENT OXYDANT CYTOPLASMIQUE ET L'AUGMENTATION DU PH. L'ENVIRONNEMENT CREE PAR L'ACTIVITE RESPIRATOIRE AUGMENTE EGALEMENT LA SURVIE DES BACTERIES FERMENTAIRES, PRESENTES DANS LE MEME MILIEU.LACTOCOCCUS LACTIS IS MAINLY USED IN THE FOOD INDUSTRY, AND HAS BEEN MAINLY STUDIED FOR ITS CAPACITIES TO FERMENT SUGAR SOURCES. RECENTLY OUR LABORATORY DISCOVERED THAT L. LACTIS ARE CAPABLE OF A RESPIRATION METABOLISM WHEN EXOGENOUS HEME AND OXYGEN ARE PRESENT IN THE MEDIUM. RESPIRATION GROWTH RESULTS IN IMPROVED BIOMASS, HIGHER PH AND A STRIKING INCREASED LONG TERM SURVIVAL COMPARED TO FERMENTATION METABOLISM.MY THESIS PROJECT WAS FOCUSED ON THE RESPIRATORY METABOLISM OF L. LACTIS, AND PRINCIPALLY ADDRESSED TWO OBJECTIVES : THE IDENTIFICATION OF RESPIRATORY CHAIN COMPONENTS, AND CLARIFICATION OF THE FACTORS THAT ASSOCIATE IMPROVED SURVIVAL WITH RESPIRATION GROWTH . WE SHOWED BY MUTAGENESIS THAT MENAQUINONES FUNCTION TO ASSURE ELECTRON TRANSFER TO CYTOCHROME OXIDASE. WE ALSO DEMONSTRATE A ROLE OF MENAQUINONES IN GENERATING SUPEROXYDE SPECIES, AND IN EXTRACELLULAR IRON REDUCTION. OUR RESULTS SUGGEST THAT NADH DEHYDROGENASE NOXA AND NOXB MAY BE ACTING AS ELECTRON DONORS FOR THE RESPIRATION CHAIN. THE ROLE OF CYTOCHROME BD OXIDASE, ALREADY KNOWN FOR ITS FUNCTION AS THE ONLY TERMINAL OXIDASE IN L. LACTIS, WAS CONFIRMED BY OUR MUTANTS CYDA AND CYDB OBTAINED BY A RANDOM MUTAGENESIS APPROACH. OUR RESULTS INDICATE THAT GREATER L. LACTIS SURVIVAL AFTER RESPIRATION METABOLISM (COMPARED TO AERATION GROWTH IN THE ABSENCE OF HÈME) IS ASSOCIATED WITH, AND LIKELY DUE TO A DECREASE IN CYTOPLASMIC OXIDATIVE STRESS AND AN INCREASE OF PH. THE ENVIRONMENT CREATED BY RESPIRATORY CHAIN ACTIVITY ALSO IMPROVE THE LONG TERM SURVIVAL OF FERMENTING BACTERIA PRESENT IN THE SAME MEDIUM.ORSAY-PARIS 11-BU Sciences (914712101) / SudocSudocFranceF