Rozprawy doktorskie na temat „Bacterial metabolic activity”

Biological wastewater treatment relies on the diverse and complex metabolic activities of bacteria to remove pollutants. Its success depends on the metabolic efficiency of the bacteria. Activated sludge models use parameters that attempt to depict bacterial growth and metabolic processes. However, current methods do not separate metabolic activity from growth and maintenance. As a result, activated sludge processes are misinterpreted or over-simplified. Alternative methods for gauging bacterial activity have been proposed and include the measurements of cellular derived compounds that relate specifically to energy cycling and include Nicotinamide Adenine Dinucleotide [NADH]. To date, NADH has been largely measured within activated sludge using commercial online fluorimeters with in situ probes. However, this current method provides a measure of the 'bulk' (raw) fluorescence within the system, resulting in difficulties when interpreting fluorescence data and poor sensitivity for detecting changes in intracellular [NADH]. This study has developed a more reliable method for estimating intracellular [NADH] and thus metabolic activity within activated sludge systems. Separating extracellular from intracellular [NADH] in samples was crucial because NADH was released and accumulates in the extracellular environment at a concentration of 200 ~M immediately following bacterial death or lysis. This concentration did not decline overtime. This not only caused high background fluorescence but also reduced the sensitivity of detection for changes in intracellular [NADH]. In particular, considerably higher [NADH] values to those from the extracellular suspensions were obtained following extraction of the intracellular material, suggesting that the cell membranes were not being penetrated by the excitable light source. Of the extraction procedures examined, filtration followed by extraction of the intracellular material with a hot Tris buffer was the most efficient and was recommended for accurate estimates of intracellular [NADH] in situ. In addition, standards were used to quantify NADH (moles per cell and/or unit volume) from unknown samples. The limits of detection were found to be 1.058 - 353 uM, whereas concentrations above 353 jAM self-quenched. Sample concentrations were always within these limits of detection. Hence, the sensitivity, reliability and experimental application of the original method was improved upon and able to be used for the direct measurement of microbial metabolic activity, something that has not been demonstrated before now. This study found that bacteria have between 106~ I 08 NADH molecules per cell depending on their metabolic state. A highly metabolically active bacterial cell had between 1O6~ tO7 NADH molecules, while a less active bacterial cell had between to7 -to8 NADH molecules. These measurements of metabolic activity were simultaneously monitored alongside other measures of bacterial growth, such as the incorporation of radiolabelled thymidine into DNA as a direct measure of DNA replication (new cell synthesis), the incorporation of radiolabelled leucine into protein as a direct measure of protein synthesis, oxygen uptake rates (OUR) as a direct measure of respiration, ATP as a measure of potential energy and dissolved organic carbon (DOC) as a measure of substrate assimilation. As OUR deceased, bacterial growth (using both the thymidine and leucine assays), specific [NADH] and specific [ATP] increased. High OUR and substrate oxidation rates simultaneous with low specific [NADH] indicated high rates of electron transport and thus efficient metabolic activity. Also, low OUR and substrate oxidation rates simultaneous with high specific [NADHI indicated inefficient rates of electron transport, therefore inhibiting oxidative phosphorylation (ATP production). A lack of oxygen as the terminal electron acceptor did not efficiently reoxidise NADH to NAD and resulted in an accumulation of NADH within the cell. Thus, a measure of low specific [NADHI was linked to the efficient rate of reoxidation of NADH to NAD* and reflects high metabolic efficiency. DNA and protein syntheses were coupled following substrate enrichment (glucose or acetate), indicating that bacteria were in balanced growth. However, DNA and protein syntheses became uncoupled once substrate was depleted, indicating unbalanced growth. An average Leu:TdR ratio of 7.4 was determined for activated sludge and was comparable to values published from marine systems. This ratio increased during log growth phase and decreased during stationary growth phases. Specific growth rates determined using the [3HITdR and [3H]Leu assay yielded values ranging from 2 - 10.5 d' and from 2.5 - 6 d1, respectively and were comparable to published values. Changes in OUR, NADH, ATE', DNA replication and protein synthesis were statistically ordinated using multidimensional scaling, and changes (in magnitude and direction) in bacterial metabolic activity were observed. Such methods enable the tracing of where bacteria divert their energies, such as to growth or maintenance and thus provide a greater understanding of bacterial behaviour in activated sludge. While studying anoxic and anaerobic conditions were beyond the scope of this work, the use of such methods to monitor bacterial metabolic activity under such conditions is warranted.

Biological wastewater treatment relies on the diverse and complex metabolic activities of bacteria to remove pollutants. Its success depends on the metabolic efficiency of the bacteria. Activated sludge models use parameters that attempt to depict bacterial growth and metabolic processes. However, current methods do not separate metabolic activity from growth and maintenance. As a result, activated sludge processes are misinterpreted or over-simplified. Alternative methods for gauging bacterial activity have been proposed and include the measurements of cellular derived compounds that relate specifically to energy cycling and include Nicotinamide Adenine Dinucleotide [NADH]. To date, NADH has been largely measured within activated sludge using commercial online fluorimeters with in situ probes. However, this current method provides a measure of the 'bulk' (raw) fluorescence within the system, resulting in difficulties when interpreting fluorescence data and poor sensitivity for detecting changes in intracellular [NADH]. This study has developed a more reliable method for estimating intracellular [NADH] and thus metabolic activity within activated sludge systems. Separating extracellular from intracellular [NADH] in samples was crucial because NADH was released and accumulates in the extracellular environment at a concentration of 200 ~M immediately following bacterial death or lysis. This concentration did not decline overtime. This not only caused high background fluorescence but also reduced the sensitivity of detection for changes in intracellular [NADH]. In particular, considerably higher [NADH] values to those from the extracellular suspensions were obtained following extraction of the intracellular material, suggesting that the cell membranes were not being penetrated by the excitable light source. Of the extraction procedures examined, filtration followed by extraction of the intracellular material with a hot Tris buffer was the most efficient and was recommended for accurate estimates of intracellular [NADH] in situ. In addition, standards were used to quantify NADH (moles per cell and/or unit volume) from unknown samples. The limits of detection were found to be 1.058 - 353 uM, whereas concentrations above 353 jAM self-quenched. Sample concentrations were always within these limits of detection. Hence, the sensitivity, reliability and experimental application of the original method was improved upon and able to be used for the direct measurement of microbial metabolic activity, something that has not been demonstrated before now. This study found that bacteria have between 106~ I 08 NADH molecules per cell depending on their metabolic state. A highly metabolically active bacterial cell had between 1O6~ tO7 NADH molecules, while a less active bacterial cell had between to7 -to8 NADH molecules. These measurements of metabolic activity were simultaneously monitored alongside other measures of bacterial growth, such as the incorporation of radiolabelled thymidine into DNA as a direct measure of DNA replication (new cell synthesis), the incorporation of radiolabelled leucine into protein as a direct measure of protein synthesis, oxygen uptake rates (OUR) as a direct measure of respiration, ATP as a measure of potential energy and dissolved organic carbon (DOC) as a measure of substrate assimilation. As OUR deceased, bacterial growth (using both the thymidine and leucine assays), specific [NADH] and specific [ATP] increased. High OUR and substrate oxidation rates simultaneous with low specific [NADH] indicated high rates of electron transport and thus efficient metabolic activity. Also, low OUR and substrate oxidation rates simultaneous with high specific [NADHI indicated inefficient rates of electron transport, therefore inhibiting oxidative phosphorylation (ATP production). A lack of oxygen as the terminal electron acceptor did not efficiently reoxidise NADH to NAD and resulted in an accumulation of NADH within the cell. Thus, a measure of low specific [NADHI was linked to the efficient rate of reoxidation of NADH to NAD* and reflects high metabolic efficiency. DNA and protein syntheses were coupled following substrate enrichment (glucose or acetate), indicating that bacteria were in balanced growth. However, DNA and protein syntheses became uncoupled once substrate was depleted, indicating unbalanced growth. An average Leu:TdR ratio of 7.4 was determined for activated sludge and was comparable to values published from marine systems. This ratio increased during log growth phase and decreased during stationary growth phases. Specific growth rates determined using the [3HITdR and [3H]Leu assay yielded values ranging from 2 - 10.5 d' and from 2.5 - 6 d1, respectively and were comparable to published values. Changes in OUR, NADH, ATE', DNA replication and protein synthesis were statistically ordinated using multidimensional scaling, and changes (in magnitude and direction) in bacterial metabolic activity were observed. Such methods enable the tracing of where bacteria divert their energies, such as to growth or maintenance and thus provide a greater understanding of bacterial behaviour in activated sludge. While studying anoxic and anaerobic conditions were beyond the scope of this work, the use of such methods to monitor bacterial metabolic activity under such conditions is warranted.
Thesis (PhD Doctorate)
Doctor of Philosophy (PhD)
School of Environmental Engineering
Full Text

Fluoroquinolones are a class of antibiotics used clinically to treat a wide array of bacterial infections. These therapeutics act by targeting a bacterial enzyme required for cell viability, bacterial type-II topoisomerases. Fluoroquinolones act by forming a ternary complex with bacterial type II topoisomerases and cleaved DNA; religation of DNA is subsequently blocked, therefore leading to bacterial cell death. In ternary complex the keto-acid moiety of the fluoroquinolone is complexed with a divalent magnesium ion, forming a drug-magnesium-water bridge to a serine and an aspartate (or glutamate) residue on helix-4 of the topoisomerase enzyme. A major issue with fluoroquinolones is the rise in bacterial resistance. Resistance arises through substitutions of the serine or aspartate/glutamate residue, therefore preventing formation of the magnesium-water bridge and dramatically diminishing the overall antibiotic activity of the fluoroquinolone. Quinazoline-2,4-diones are structurally similar to fluoroquinolones; diones also form a ternary complex similar to fluoroquinolones, however, these complexes are less active due to lack of a potent magnesium-water bridge interaction in helix-4. While quinazoline-2,4-diones are therefore less potent antibiotics, their non-reliance on the magnesium water bridge generally affords equipotent activity with wild-type and fluoroquinolone-resistant strains of bacteria. The first objective of this work was to probe the helix-4 interaction of the bacterial type-II topoisomerase by quinazoline-2,4-dione modification, specifically at the N3 and C4 positions of the quinazoline-2,4-dione scaffold to afford potentially new binding contacts. These modified quinazoline-2,4-diones will provide deeper understanding of the helix-4 interaction and potentially afford potent novel quinazoline-2,4-dione scaffolds, against both wild-type and resistant bacteria, for iterative drug design. Metabolism is one of the primary sources of detoxification, inactivation, and clearance of drugs from the body and is a critical consideration for all early stage therapeutic development. Clinically used fluoroquinolones, i.e. Moxifloxacin and Ciprofloxacin, historically are metabolically stable, and are not known to be metabolized by Phase I and/or Phase II drug metabolizing enzymes. However, major modifications to the Moxifloxacin and Ciprofloxacin scaffolds, due to the development of next generation antibiotics, may display different metabolic stability profiles. Moreover, metabolism of quinazoline-2,4-diones, developed for fluoroquinolone-resistant bacteria, is not extensively studied and may be subject to different metabolic liabilities that may render the quinazoline-2,4-dione an ineffective potential antibiotic. The second objective of this work was to determine the in vitro Phase I and Phase II metabolic stabilities of fluoroquinolone and quinazoline-2,4-dione scaffolds to determine any structural features that render the potential therapeutic a metabolic liability. The results from these two objectives have led to the discovery of a novel bacterial type-II topoisomerase catalytic inhibitor and the acquisition of initial metabolic stability data of fluoroquinolone and quinazoline-2,4-dione scaffolds. These findings further promote research into quinazoline-2,4-diones as bacterial topoisomerase targets, and provide metabolic considerations for both fluoroquinolone and quinazoline-2,4-dione therapeutic development, which is severely underrepresented in the field of quinolone antibiotics.

Thesis (Ph.D.)--Boston University PLEASE NOTE: Boston University Libraries did not receive an Authorization To Manage form for this thesis or dissertation. It is therefore not openly accessible, though it may be available by request. If you are the author or principal advisor of this work and would like to request open access for it, please contact us at open-help@bu.edu. Thank you.
The discovery of antibiotics was one of the most important medical breakthroughs of the twentieth century, having a broad impact on overall life expectancy and public health. Unfortunately, antibiotic discovery has slowed significantly in recent times and has failed to match the rising incidence of antibiotic-resistant pathogens. Gram-negative pathogens are a particularly troublesome threat, primarily because these bacteria possess an outer membrane that prevents many antibiotics from accessing their primary cellular targets. While the discovery of novel antibiotics could help to address these issues, alternative strategies, such as improving the activity of preexisting antibiotics, are also needed. Bactericidal antibiotics have recently been shown to share a common mechanism of cell death, despite having different primary, cellular targets. This shared mechanism involves the metabolic production of reactive oxygen species (ROS), which can damage proteins, lipids, and nucleic acids, and can ultimately result in bacterial cell death. The body of work described here shows that this common mechanism can be exploited to improve antibiotic activity, regardless of the antibiotic's primary mode of action. First, I will describe how bacterial metabolism can be predictably perturbed to increase endogenous ROS production, and that increasing endogenous ROS is sufficient to enhance bacterial sensitivity to treatments with ROS-generating biocides, antibiotics, and immune cell attack. I will then describe work indicating that an ancient antimicrobial agent, silver salts, can also increase endogenous ROS production and potentiate the activity of multiple antibiotic classes. Furthermore, I show that silver salts can increase the outer membrane permeability of a Gram-negative organism. This property is exploited to enable vancomycin, an antibiotic that is specific for Gram-positive bacteria, to work against a Gram-negative organism. Together, this body of work demonstrates that bacterial ROS metabolism can be exploited effectively to enhance. antibiotic activity, which ultimately could result in the discovery and development of novel antimicrobial agents.

Le sel joue un rôle essentiel dans la conservation des produits de charcuterie puisqu’il inhibe le développement bactérien. Or les recommandations nutritionnelles visent à réduire les teneurs en sel dans les denrées alimentaires. Les objectifs de ce projet de thèse étaient (1) de caractériser la communauté bactérienne des chipolatas et le phénomène d’altération sous l’effet d’une réduction de sel et (2) de corréler la dégradation organoleptique des produits aux modifications de la communauté bactérienne.Nous avons tout d’abord caractérisé l’altération des chipolatas par des mesures sensorielles et physico-chimiques. Les travaux démontrent que l’intensité de l’altération est plus importante quand la teneur en sel est réduite et plus particulièrement lorsque les chipolatas sont conditionnées sous atmosphère modifiée. L’altération est caractérisée par la production d’odeurs soufrées, aigres et rances, une diminution du pH des chipolatas et une augmentation de la production d’exsudat.Parallèlement, nous avons décrit la diversité bactérienne des chipolatas altérées par analyse des ARNr 16S bactériens et l’abondance des espèces bactériennes a été quantifiée par qPCR. Par ces méthodes, nous avons pu distinguer la population dominante de la population sous-dominante. La baisse de sel entraine un déséquilibre d’abondance entre ces deux populations et ce déséquilibre résulte non pas de l’augmentation des espèces dominantes mais d’une diminution d’abondance des espèces sous-dominantes.Pour comprendre le rôle respectif de ces populations bactériennes, nous avons analysé leurs activités métaboliques par approche RNAseq. Les travaux montrent une forte activité métabolique des espèces sous-dominantes. Lorsque le sel est en plus faible concentration, l’expression des gènes de Serratia spp. impliqués dans la fermentation du pyruvate pour produire de l’éthanol, du CO2 et de l’acétate est plus importante. La production d’acétate par cette espèce bactérienne peut être reliée aux défauts d’altération observés (baisse de pH, production d’exsudat, odeur aigre).Ces travaux démontrent que le sel impacte la communauté bactérienne des chipolatas (abondance et activités métaboliques) et que cette perturbation compromet la qualité organoleptique des produits
Salt content plays a key role in meat product preservation since it inhibits bacterial growth. However, dietary guidelines aim to reduce salt content in food. The objectives of this study were (1) to characterize the bacterial community of raw pork sausages and the spoilage phenomenon of these products under salt reduction conditions and (2) to correlate the organoleptic deterioration of the products to modifications in bacterial community.We first characterized the raw pork sausages spoilage by sensory and physicochemical analysis. The work demonstrates that spoilage intensity is greater under a reduced salt content, particularly when sausages are packaged under modified atmosphere. The spoilage is characterized by the production of sulfur, sour and rancid off-odors, a decrease in pH of the sausages and an increase of exudate production.At the same time, we described the bacterial diversity of spoiled sausages through 16S rRNA analysis. Abundance of bacterial species was quantified by qPCR. With these methods, we were able to distinguish the dominant population from the subdominant population. Reducing salt content causes an abundance imbalance between these two populations. This imbalance does not result from an increase of the dominant species; it results from a decrease in abundance of subdominant species.To understand the roles of these bacterial populations, we analyzed their metabolic activities by RNA-Seq approach. The works highlight a high metabolic activity of the subdominant species. When the salt concentration is lowest, the expression of Serratia sp. genes involved in the fermentation of pyruvate to produce ethanol, CO2 and acetate is most important. The acetate production may be connected to the spoilage defaults observed (decrease in pH, exudate production and sour off-odors).These studies reveal that salt reduction impacts the bacterial community of raw pork sausages (abundance and metabolic activities) and this disruption compromises the organoleptic quality of the products

Des experiences realisees sur grains stockes en cellules etanches a differentes activites d'eau ont permis de montrer qu'il est inutile de creer une atmosphere anoxique artificielle pour stabiliser les populations microbiennes naturelles. Une relation quantitative directe existe entre croissance, sporulation et quantite d'oxygene disponible dans l'ecosysteme. Aux tres faibles pressions partielles d'oxygene, la perte de viabilite des propagules fongiques est fonction de l'activite d'eau des grains. Les qualites technologiques des grains restent stables pour les activites d'eau inferieures a 0. 95. Seules les levures de stockage sont capables de se developper dans ces conditions gazeuses, des lors que l'activite d'eau est superieure ou egale a 0. 86. Le developpement particulier d'hyphopichia burtonii (boidin) en presence de traces d'oxygene et l'arret de sa croissance en anaerobiose stricte montrent que c'est une espece "microaerotolerante". En presence de traces d'oxygene, le dioxyde de carbone a un effet stabilisant sur les populations fongiques (aspergillus, penicillium). Cependant, le dioxyde de carbone employe seul ne saurait assurer la stabilite microbiologique des grains conserves en structures insuffisamment etanches

Les compartiments géologiques profonds suscitent un intérêt grandissant dans la communauté scientifique depuis les 50 dernières années. Néanmoins, ces écosystèmes demeurent largement méconnus du fait de leur difficulté d'accès. Le forage profond réalisé par l'ANDRA dans le Bassin parisien en 2008 a offert une opportunité unique de les étudier. Dans ce cadre, cette thèse avait deux objectifs majeurs ; i) caractériser, d'un point de vue microbiologique, quatre formations sédimentaires terrestres triasiques situées entre 1700 et 2000 m de profondeur et ii) étudier les effets combinés des paramètres de température, pression et salinité ainsi que de leur interaction sur l'activité métabolique de procaryotes anaérobies afin de mieux appréhender leur comportement au cours d'un enfouissement géologique.Malgré la recherche de microorganisme par la réalisation d'une gamme de milieux de culture diversifiée, ciblant préférentiellement les types trophiques fréquemment rencontrés en subsurface (méthanogènes, fermentaires, réducteurs de composés soufrés), aucun microorganisme viable et cultivable n'ait été isolé. En parallèle, une approche moléculaire complémentaire, composée (i) de l'étude comparative de l'efficacité de différentes méthodes d'extraction directe d'ADN et (ii) de l'analyse de la diversité bactérienne par la réalisation d'inventaires moléculaires, par DGGE (Denaturing Gel Gradient Electrophoresis) et clonage, a été réalisée sur le coeur des carottes de roches, conservées à pression atmosphérique ou sous pression, dans leurs états initiaux et post-incubation. L'exploration de ces formations sédimentaires profondes a indiqué la présence d'une très faible biomasse et d'une biodiversité microbienne pauvre principalement composée de membres aérobies et mésophiles appartenant au domaine Bacteria. Cette communauté bactérienne inattendue car a priori peu adaptée aux conditions régnant in-situ, également retrouvée dans divers écosystèmes de subsurface ainsi que dans des biotopes extrêmes, pourrait provenir en partie d'une paléo-recharge de l'aquifère du Trias par des eaux froides dérivées de la fonte des glaces formées lors de la dernière glaciation du Pléistocène.Le second objectif a été abordé à travers l'élaboration d'un plan factoriel complet dans le but d'identifier les effets des paramètres sur les activités microbiennes. Ainsi, les activités métaboliques de huit souches microbiennes halophiles et thermo-tolérantes ont été mesurées sous trente conditions distinctes de température (40, 55 et 70°C), pression (1, 90 et 180 bars) et salinité (13, 50, 110, 180 et 260 g.l-1). Toutes les souches originaires d'environnements profonds se sont révélées être au minimum piézo-tolérantes et capables de maintenir leur activité métabolique sous pressions hydrostatiques. Les métabolismes fermentaires (Thermovirga lienii et Halothermothrix orenii) et thiosulfato-réducteurs (Petrotoga mexicana et Thermosipho japonicus) se sont avérés particulièrement bien adaptées, d'un point de vue métabolique, aux hautes pressions, les plus hautes activités ayant été détectées sous pression. Certaines souches ont montré une résistance accrue aux hautes températures sous pression (Petrotoga mexicana). Toutefois une résistance variable à la salinité dans les différentes conditions de température et de pression a été observée pour chacune des souches, suggérant que certains mécanismes de résistance contre la pression osmotique seraient également efficaces pour lutter contre les températures et les pressions hydrostatiques élevées.Ce travail souligne que l'étude des écosystèmes terrestres profonds d'un point de vue microbiologique ne doit pas se restreindre à la recherche et à l'analyse de la diversité présente. L'étude des activités métaboliques de souches de subsurface en conditions profondes ouvre la voie à une meilleure compréhension des rôles joués par les communautés microbiennes en milieu extrême.

Ubiquitaires et très anciens, les communautés des tapis microbiens font preuve de capacités métaboliques et adaptatives très importantes. Situés en zone côtières, ces écosystèmes peuvent être soumis à des contaminations pétrolières. Dans ce contexte, cette étude vise d’une part à décrire la structure et le fonctionnement de tapis microbiens et d’autre part à comprendre l’impact d’une contamination pétrolière sur ces écosystèmes. Cette étude porte sur deux tapis microbiens de l’étang de Berre aux paramètres physico-Chimiques proches mais présentant des contaminations pétrolières contrastées. Le fonctionnement du système étant tributaire d’autres facteurs tels que la lumière et les saisons, les variations saisonnières et nycthémérales ont été prises en compte dans cette étude. Un accent particulier a également été porté sur le cycle du soufre de par son importance en milieu marin. Les résultats de cette étude mettent en évidence des structures de communautés différentes entre les deux tapis au niveau global, la séparation spatiale prévalant sur la séparation saisonnière. La fraction active de la communauté du site contaminé présente une évolution linéaire tandis que celle du site témoins suit pour sa part les variations saisonnières. Au niveau du site contaminé une augmentation de l’expression des gènes impliqués dans la dégradation des hydrocarbures couplée à une biodégradation des hydrocarbures suggère que le tapis contaminé est adapté à la contamination pétrolière. Malgré les différences de structures et d’activités de dégradation, des profils métaboliques très semblables sont cependant observables entre les deux tapis, avec des fonctions similaires laissant supposer une redondance fonctionnelle. Des variations saisonnières et nycthémérales ont également été observées avec notamment des Desulfobulbaceae plus abondantes au printemps et plus actives en journée. Des études culturales ont été réalisées en parallèle. Elles permettront d’appréhender de manière complémentaire la dynamique des communautés des sulfato-Réducteurs au sein du tapis et de mieux comprendre les variations mises en évidence dans cette étude
Ubiquist and very ancient, the microbial mats communities demonstrate very important metabolic and adaptive capacities. Located in the coastal area, these ecosystems may be subject to oil contamination. In this context, the aim of this study is on one hand to describe the structure and functioning of microbial mats and on the other to understand the impact of oil contamination on these ecosystems. This study focused on two microbial mats from the Berre lagoon with close physical chemical parameters but with contrasted hydrocarbon contamination levels. The functioning of the system is dependent on other factors such as light and seasons, diurnal and seasonal variations were taken into account in this study. Special emphasis was placed on the sulfur cycle due to its importance in the marine environments. The results of this study highlighted different communities’ structures at the global level between both mats, the spatial variation prevailed on seasonal variation. The active part of the community from the contaminated site shows a linear trend while that one of the uncontaminated site follows the seasonal variations. The contaminated site shows genes involved in hydrocarbon degradation more expressed coupled to a hydrocarbon biodegradation suggesting that the contaminated mat is adapted to the petroleum contamination. Despite these differences in the structure and the degradation capacities, very similar metabolic profiles are observed between the two mats with similar functions, suggesting functional redundancy. Seasonal and diurnal variation was also observed, the Desulfobulbaceae were particularly more abundant in spring and more active during the day. A complementary cultural approach will allow to better understanding the dynamics of sulfate-Reducers communities in the mat and comprehending these variations

Dans le but de preciser l'interet que presente la determination de la composition biochimique du materiel particulaire lacustre, pour l'etude de l'activite metabolique des microorganismes, la dynamique des populations phytoplanctoniques et les relations trophiques phyto-zooplanctoniques, une etude a court terme puis a long terme de la composition en acide gras des lipides phytoplanctoniques est menee. Ces acides gras sont ensuite utilises comme marqueurs organiques, pour suivre le transfert de matiere organique au sein du reseau trophique. Une demarche nouvelle est proposee par l'etude des relations trophiques phyto-zooplancton

Halymenia floresii, une Rhodophycée présente une surface remarquablement exempte d'épiphytes dans les conditions de l'Aquaculture MultiTrophique Intégrée (AMTI). Ce phénomène traduit la présence potentielle en surface de composés actifs allélopathiques. L'objectif de ce travail a été d'explorer les mécanismes de défense développés par H. floresii contre l'épibiose, de détecter et d'identifier les métabolites secondaires produits à la surface de l’algue et d'étudier les relations avec les bactéries épiphytes. Nous avons ainsi pu isoler la communauté épibactérienne de H. floresii cultivée dans des conditions contrôlées (AMTI) et non contrôlées (échantillons collectés in situ). Les épibactéries isolées ont été criblées in vitro pour analyser les signaux de détection de Quorum Sensing (QS). Les extraits produits en surface ont été analysés pour détecter toute interférence avec le Quorum Sensing. Les épibactéries pathogènes et non-pathogènes ont été différenciées par leur capacité à induire une maladie algale, le blanchiment. Vibrio owensii, ainsi que son signal C4-HSL QS, a été identifié comme pathogène opportuniste induisant un blanchiment. Les métabolites extraits de la surface et de cellules entières de H. floresii ont été analysés par LC-MS. Une base de données a été constituée à partir d’une analyse métabolomique non ciblée. Quarante et un métabolites actifs ont été identifiés, parmi lesquels les composés halogénés, des furanones et divers inhibiteurs étaient surreprésentés. Fait intéressant, les deux premières classes sont connues comme de puissants composés interférant avec le QS. La présence relativement élevée de métabolites allélopathiques à la surface de H. floresii soutient fortement l'hypothèse selon laquelle ils doivent être impliqués dans la protection de l'hôte. Des recherches supplémentaires seront nécessaires pour explorer l’ensemble des métabolites secondaires produits par H. floresii et leurs rôles chez l’algue
The surface of Halymenia floresii, a Mexican Rhodophyta, was observed to be remarkably free of epiphytes under Integrated MultiTrophic Aquaculture (IMTA) conditions. This suggests the presence of allelopathic active compounds released by this macroalgae. The aim of this work was to explore the defence mechanisms developed by H. floresii against surface epibiosis, to detect and identify the secondary metabolites produced at the surface of the algae, and to study its relation with surface associated bacteria. For the first time, we isolated the epibacterial community of H. floresii cultivated under controlled conditions (IMTA) and uncontrolled ones (beach-cast material collected in the area). The isolated epibacteria were screened in vitro to analyse Quorum Sensing (QS) signals, and others H. floresii surface extracts were assayed for any QS interference with them. We differentiated the epibacteria significant pathogens from the non- pathogens ones by their ability to induce bleaching, a well-known algal disease. Vibrio owensii was identified as an opportunistic pathogen inducing bleaching in H. floresii which was also associated to the presence of its C4-HSL QS signal. The surface and whole cell metabolites extracts from H. floresii specimens cultivated under controlled conditions were analysed by means of LC/MS. An untargeted metabolomic analysis of H. floresii was performed to provide a global metabolic profile as a first database. We identified ‘41’ active metabolites in H. floresii, among which halogenated compounds, furanones and various inhibitors were overrepresented. Interestingly, the first two classes are well known potent QS interfering compounds. The relatively higher occurrences of allelopathic metabolites at the surface of H. floresii strongly supports the hypothesis that they must be involved in the host protection. Further investigations are needed to explore the secondary metabolites of H. floresii et their role in the seaweed

Seuls les microorganismes et les plantes sont capables de produire la biotine. La voie de biosynthese de la biotine (vitamine h), bien connue chez les bacteries, n'a encore jamais ete etablie chez les vegetaux superieurs. Dans les differents compartiments subcellulaires (chloroplastes, mitochondries, cytosol et vacuoles) de feuilles de pois (pisum sativum l. ), la biotine libre est localisee dans le cytosol alors que la biotine liee aux proteines est presente dans les chloroplastes et les mitochondries. La biotine liee est associee a trois polypeptides de 34, 76 et 220 kda. Les polypeptides de 34 et 76 kda sont localises respectivement dans le stroma des chloroplastes et la matrice des mitochondries. Le polypeptide de 220 kda detecte dans l'extrait foliaire total n'a pu etre localise dans aucun compartiment subcellulaire. Seules deux activites carboxylases a biotine sont detectees, la 3-methylcrotonyl-coa carboxylase dans la matrice des mitochondries et l'acetyl-coa carboxylase dans le stroma des chloroplastes. Le polypeptide de 76 kda represente la sous-unite biotinylee de la 3-methylcrotonyl-coa carboxylase. Ses caracteristiques biochimiques et structurales sont analogues a celles etablies pour les enzymes animale et bacterienne. Dans les feuilles de pois, comme dans les graines de diverses plantes, l'acetyl-coa carboxylase est associee au polypeptide 220 kda. Plusieurs hypotheses ont ete proposees pour comprendre la nature de cette enzyme. L'etude de la biosynthese de la biotine dans les cellules de lavande demontre que chez les plantes cette voie est identique a celle decrite chez les bacteries: l'acide pimelique est le precurseur et les intermediaires sont le pimelyl-coa, le kapa, le dapa et la desthiobiotine. Un nouvel intermediaire thiol implique dans la conversion de la desthiobiotine en biotine est identifie: la mercaptodesthiobiotine (mdtb). L'acide actithiazique, inhibiteur de cette biosynthese chez les bacteries, inhibe la biosynthese de la biotine dans les cellules vegetales. Ce compose possede egalement une action herbicide sur des plantes monocotyledones et dicotyledones. Les trois premieres enzymes de la voie (pimelyl-coa synthetase, kapa synthase et dapa aminotransferase) sont presentes dans le cytosol des cellules foliaires de pois. Deux anticorps diriges contre la pimelyl-coa synthetase et la kapa synthase de bacillus. Sphricus reconnaissent deux proteines cytosoliques de pois et inhibent les activites enzymatiques correspondantes. En conclusion, ce travail a permis d'etablir la localisation et la biosynthese de la biotine dans les cellules vegetales. Il a mis en evidence un nouvel intermediaire de biosynthese. Dans l'avenir, nous envisageons de caracteriser l'ensemble de la voie de biosynthese chez les plantes. Ensuite, nous purifierons ces enzymes (clonage des adnc et surexpression des proteines) et nous rechercherons des inhibiteurs de cette biosynthese qui, comme l'acide actithiazique, pourraient posseder une activite herbicide potentielle

This thesis describes the development and application of a new quantitative and species-specific approach to measure the abundance and metabolic activity of selected populations of microorganisms in complex communities. The approach is based on a positive correlation between growth rate and RNA:DNA ratio, which was shown to exist in selected microorganisms able to degrade resin acids from pulp and paper mill effluent. Species included in the study belonged to the genera Pseudomonas, Sphingomonas, Zoogloea and Escherichia. Only the zoogloeal isolate did not exhibit a positive correlation between growth rate and RNA: DNA ratio during exponential growth, and neither were the trends in its RNA:DNA ratio similar to the trends in other species during batch growth. In other organisms, during batch growth, the ratio was low during lag phase, had a brief maximum during exponential growth and declined during decelerating and stationary phase. In no case was the ratio observed to remain high during the entire period of exponential growth. Slot-blot hybridization and competitive RT-PCR/PCR assays, targeting hypervariable regions of the 16S ribosomal RNA sequences, were used to measure the DNA and RNA concentrations in a species-specific manner. The hybridization assay proved to be not sensitive enough and burdened with high background when applied to activated sludge samples. The competitive RT-PCR/PCR assay had a much higher sensitivity (10 target copies per ml of activated sludge) and no background. Sphingomonas sp. DhA-33 and Pseudomonas abietaiphila BKME-9 were examined for their ability to survive and to contribute to the resin acid biodegradation in mixed communities. In both cases, DNA data showed survival of the strains, and the RNA:DNA ratio measured their metabolic activity over time, which varied with changes in resin acid concentration, aeration and pH. The quantitative competitive PCR/RT-PCR method contributes significantly to our ability to estimate growth or turnover rates of populations in complex environments when their population sizes appear to be stable. The method allows for the processing of large representative samples and, in conjunction with other molecular microbiological techniques, will contribute toward our understanding of microbial activities in situ.

Urban areas are important sources for river contamination with nitrogen and phosphorus compounds (nutrients). Their extensive amount in urban surface waters promote the proliferation of cyanobacterial harmful algal blooms (CyanoHABs) which could harm biodiversity and human health. Therefore, sequential sedimentation-biofiltration systems (SSBSs) were implemented to collect polluted water from urban rivers to reduce the nutrient load. Microbial communities play an important role in nutrient cycling in the environment, thus, the present study focused on the investigation of such communities in sediments from three SSBSs, two in Lodz city and one in Gniezno city (Poland). Two publications described how the differences in the system design, season, and physico-chemical parameters, influenced the functional metabolic activity of microbial communities (using community level physiological profile approach), and the abundance of nitrifying and denitrifying bacteria (using qPCR to estimate the gene abundance of amoA and nosZ, respectively). The results indicated that SSBS’s geochemical zones containing limestone presented the highest metabolic activity in summer, and the highest abundance of nitrifying bacteria in spring, while denitrifying zones containing brown coal were more abundant with denitrifying bacteria in summer. Moreover, 16S rRNA High throughput sequencing suggested that Commamonadaceae, Flavobacteriaceae, and Crenotrichaceae were the most representative families containing nitrogen-transforming bacteria, while the Rhodocyclaceae with polyphosphate accumulating bacteria. Finally, a third publication described the isolation and characterization of ten nitrogen-transforming bacterial strains from SSBSs sediments, from which two presented the most promising results in the removal of nitrogen compounds in controlled assays: Citrobacter fruendii Bzr02 and Pseudomonas mandelii Str21.
GuateFuturo Foundation, Guatemala, Guatemala city. The scholarship-loan program for young professionals for the specialization in master and doctoral programs abroad (https://guatefuturo.org/). Under the project code: PCB-2017-01. Faculty of Biology and Environmental Protection, University of Lodz. Specialized subsidy for activities related to the conduct of scientific research, development of work, and related tasks for the development of young scientists and participants of doctoral studies under the title of: Dynamika występowania bakterii zaangażowanych w przemiany biogenów w wybranych strefach miejskich systemów sedymentacyjno-biofiltracyjnych. Under the project code: B1711000001531.02. 2017. Faculty of Biology and Environmental Protection, University of Lodz. Specialized subsidy for activities related to the conduct of scientific research, development of work, and related tasks for the development of young scientists and participants of doctoral studies under the title of: Sezonowa i przestrzenna dynamika występowania bakterii zaangażowanych w przemiany biogenów w sekwencyjnych systemach sedymentacyjno-biofiltracyjnych. Under the code: B1811000001811.02. 2018. Faculty of Biology and Environmental Protection, University of Lodz. Specialized subsidy for activities related to the conduct of scientific research, development of work, and related tasks for the development of young scientists and participants of doctoral studies under the title of: Dynamika występowania oraz aktywność metaboliczna mikroorganizmów zaangażowanych w usuwanie biogenów w miejskich sekwencyjnych systemach sedymentacyjno-biofiltracyjnych. Under the code: B1911000002130.03. 2019. Cooperation with the European Regional Centre for Ecohydrology of the Polish Academy of Sciences (ERCE PAS)

Yes
In Escherichia coli, the biogenesis of both cytochrome bd-type quinol oxidases and periplasmic cytochromes requires the ATP-binding cassette-type cysteine/GSH transporter, CydDC. Recombinant CydDC was purified as a heterodimer and found to be an active ATPase both in soluble form with detergent and when reconstituted into a lipid environment. Two-dimensional crystals of CydDC were analyzed by electron cryomicroscopy, and the protein was shown to be made up of two non-identical domains corresponding to the putative CydD and CydC subunits, with dimensions characteristic of other ATP-binding cassette transporters. CydDC binds heme b. Detergent-solubilized CydDC appears to adopt at least two structural states, each associated with a characteristic level of bound heme. The purified protein in detergent showed a weak basal ATPase activity (approximately 100 nmol Pi/min/mg) that was stimulated ∼3-fold by various thiol compounds, suggesting that CydDC could act as a thiol transporter. The presence of heme (either intrinsic or added in the form of hemin) led to a further enhancement of thiol-stimulated ATPase activity, although a large excess of heme inhibited activity. Similar responses of the ATPase activity were observed with CydDC reconstituted into E. coli lipids. These results suggest that heme may have a regulatory role in CydDC-mediated transmembrane thiol transport.
This work was supported by Biotechnology and Biological Sciences Research Council grant BBS/B/14418 (Membrane Protein Structure Initiative).