Microbial decomposition of organic matter in aquatic environments plays an important role in natural fluxes of methane and carbon dioxide because the gases are end-products in microbial energy metabolism of organic matter. Microbial metabolism depends on the use of electron donors and electron acceptors in redox reactions that generate energy for growth and maintenance. Energy yields can be used to envisage specific patterns of microbial redox reactions and these predictions depend on the hypothesis that, in a specified environment, the metabolic reaction that yields most energy will dominate over any competing reactions. The energy yield hypothesis indicates a sequential order in electron acceptor use by microbes and also make it tempting to conclude that degradation rate of organic matter is different depending on available electron acceptors. The main purpose of this thesis was to study how the presences of different electron acceptors in freshwater sediments influence organic matter decomposition. Mineralization rates of organic matter under six different conditions regarding the electron acceptor availability were investigated in a river sediment sample from Stångån, Sweden, by measuring carbon dioxide and methane production using gas chromatography. This was done during a fixed time period, in vials containing a mixture of water, sediment, buffer solution and a dominating electron acceptor. Six different metabolic processes; aerobic respiration, denitrification, manganese reduction, iron reduction, sulphate reduction and methanogenesis were included. The overall result indicates similar mineralization rates in both oxic and anoxic treatments. The result also indicates that methane formation was present in the iron reduction and methanogenesis treatments and not evident in the oxic treatments. Sulphate reduction, denitrification and manganese reduction seems to inhibit methanogenesis, but the result also indicates that no significant total mineralization was apparent when NO3- and Mn(IV) were the dominating electron acceptors. The similarities between oxic and anoxic mineralization rates indicates that organic matter degradation rates are not dependent on available electron acceptors and that degradation rates of organic matter are independent of the thermodynamically based energy yield.

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No. of bitstreams: 2 Reprodução Não Autorizada.pdf: 47716 bytes, checksum: 0353d988c60b584cfc9978721c498a11 (MD5) license_rdf: 0 bytes, checksum: d41d8cd98f00b204e9800998ecf8427e (MD5) Previous issue date: 2017-11-03 / CNPq - Conselho Nacional de Desenvolvimento Científico e Tecnológico / Microorganisms are of fundamental importance for maintaining the equilibrium of living organisms and the chemical elements of our planet. They occur in all nature environments, including in foods. Several researches have shown that microorganisms found in the oral cavity and intestines have the ability to degrade components found in milk (lactose) and in some cereals (gluten). This work had as objectives, to isolate, purify and morphologically characterize cheeses and yoghurt bacteria commercialized in the city of Manaus, Amazonas, as well as to test their crude extracts capable of degrading lactose and gluten, aiming as an alternative to minimize or solve the problem of lactose intolerance and Celiac disease that affect part of the world population. Seventy-five bacteria were isolated from the 10 types of cheese and 10 types of yogurts used for the tests. All 75 bacteria showed high growth in the first 24 hours of evaluation in both culture media tested (gluten or lactose). Of the 75 bacteria tested, 61 presented halo formation of degradation in medium containing gluten. For the Gluten Degradation Index (GDI), of the 61 bacteria evaluated, 22 did not present GID during the test, 30 had GIDs considered low, 9 GIDs were considered medium and none had GID considered high. The carbon source of the culture medium (gluten or lactose) influenced the morphological characteristics of the colonies of the 20 selected bacteria as the best ones. In the Gram staining test, 16 bacteria were Gram Negative and four Gram positive. Of the 13 bacteria used in the genetic characterization, it was possible to identify only four at the species level. Four isolates are of the genus Pseudomonas, four of the genus Bacillus, two of the genus Stenotrophomonas, one of the genus Brevibacillus, one of the genus Achromobacter and one of the genus Burkholderia. In order to evaluate twelve bacteria selected from previous tests, laboratory experiments were performed in a liquid medium containing lactose or gluten as a source of carbon at pH of 2.3 (similar to that of the stomach) and 8.0 (similar to that of the intestines) at temperatures of 37 ° C and 55 ° C. The bacteria BLG01 (Pseudomonas sp.), BLG02 (Bacillus sp.), BLG06 (Pseudomonas sp.), BLG16 (Stenotrophomonas maltophilia), BLG25 (Stenotrophomonas sp.), BLG28), BLG38 (Bacillus sp.), BLG45 (Burkholderia sp.), BLG52 (Brevibacillus parabrevis), BLG56 (Achromobacter sp.) and BLG73 (Bacillus sp.). showed potential to be used as probiotics after future confirmation of non-pathogenicity or as suppliers of enzymes capable of degrading lactose and gluten. All bacteria showed sensitivity to acidity and alkalinity equivalent to those of the human stomach and intestines, indicating that this acidity/alkalinity bipolarity can be a defense mechanism of the human organism against the action of pathogenic or undesirable bacteria. For desirable bacteria to be used as probiotics, they need to be ingested at high concentrations, above 106 células.mL-1. From the 12 bacteria tested, those with the highest potential for use as probiotics, if not pathogenics, are BLG28 (Bacillus sp.), BLG38 (Bacillus sp.), BLG45 (Burkholderia sp.), BLG52 (Brevibacillus parabrevis), BLG56 (Achromobacter sp.) and BLG73 (Bacillus sp.), since they are part of genera with little possibility of pathogenicity and because they present positive growth in the first two hours at pH 2.3, similar to that of the stomach. However, they need to colonize and participate of the intestinal flora, and to help to the lactose and gluten metabolism ingested as food. To evaluate if the carbon source and the growth phase of the bacteria affect the production and quality of proteases capable of degrading gluten, experiments were carried out on the crude extracts of nine bacteria, BLG02, BLG25, BLG28, BLG33, BLG38, BLG45, BLG52, BLG56 and BLG73. The extracts were obtained from culture of these bacteria in two culture media (gluten and lactose as carbon sources) and collected with 6, 12 and 24 hours of incubation. Dilutions of the extracts were also tested for their ability to degrade gluten. All bacteria produced extracts with the ability to break down gluten. The source of carbon (lactose or gluten) has affected the ability of these bacteria to produce extracts capable of degrading gluten. The majority of them produced more proteases when growing in the medium with this proteic complex. The time of collection of bacterial extracts also influenced their ability to degrade gluten, with the 24 hour extracts from most of these bacteria showing the highest degradability when comparing with those produced with 6 and 12 hours. The highest percentage of gluten degradation was 87.3% using the extract obtained with 24 hours of growth of bacterium BLG56 (Achromobacter sp.) grown in culture medium containing gluten. Based on the degradability, collection times of bacterial extracts and culture media, it can be concluded that the nine bacteria present different proteases capable of degrading gluten. This ability to degrade gluten varied with their concentrations in the solution in different ways and this feature may serve as an additional test to differentiate one from the other. More detailed studies, such as the purification of the extracts components of these bacteria, are needed to evaluate each of these proteases individually to choose the best with biotechnological potential. / Os microrganismos são fundamentais para a manutenção do equilíbrio dos organismos vivos e dos elementos químicos do nosso planeta. Eles ocorrem em todos os ambientes da natureza, inclusive em alimentos. Diversas pesquisas têm mostrado que microrganismos encontrados na cavidade bucal e intestinos apresentam habilidade de degradar componentes encontrados no leite e em alguns cereais, podendo degradar a lactose e o glúten. Esse trabalho teve como objetivo, isolar, purificar e caracterizar morfologicamente bactérias de queijos e iogurtes comercializados no município de Manaus, Amazonas, bem como testar seus extratos brutos capazes de degradar lactose e glúten visando uma alternativa para minimizar ou solucionar o problema de intolerância à lactose e doença Celíaca que afetam parte da população mundial. Foram isoladas 75 bactérias dos 10 tipos de queijo e 10 tipos de iogurtes usados para os testes. Todas as 75 bactérias apresentaram elevado crescimento nas primeiras 24 horas de avaliação em ambos os meios de cultura testados (glúten ou lactose). Das 75 bactérias testadas, 61 apresentaram formação de halo de degradação em meio contendo glúten. Pelo Índice de Degradação de Glúten (IDG), das 61 bactérias avaliadas, 30 apresentaram baixos IDG’s, 9 IDG’s médios e nenhuma apresentou IDG alto. O uso do glúten ou lactose nos meios de cultura influenciou as características morfológicas das colônias das 20 bactérias selecionadas como as melhores. Das 13 bactérias usadas na caracterização genética, foi possível identificar somente quatro ao nível de espécie. Quatro isolados são do gênero Pseudomonas sp., quatro do gênero Bacillus sp., duas do gênero Stenotrophomonas sp., uma do gênero Brevibacillus sp., uma do gênero Achromobacter sp. e uma do gênero Burkholderia sp. Foram realizados também, experimentos de laboratório em meio líquido contendo lactose ou glúten como fonte de carbono em pH’s de 2,3 (semelhante ao do estômago) e 8,0 (semelhante ao dos intestinos) nas temperaturas de 37 °C e 55 °C. As bactérias BLG01 (Pseudomonas sp.), BLG02 (Bacillus sp.), BLG06 (Pseudomonas sp.), BLG16 (Stenotrophomonas maltophilia), BLG25 (Stenotrophomonas sp.), BLG28 (Bacillus sp.), BLG33 (Pseudomonas aeruginosa), BLG38 (Bacillus sp.), BLG45 (Burkholderia sp.), BLG52 (Brevibacillus parabrevis), BLG56 (Achromobacter sp.) e BLG73 (Bacillus sp.). mostraram potencial para serem usadas como probióticas, desde que não sejam patogênicas em testes futuros e/ou como supridoras de enzimas capazes de degradarem a lactose e o glúten. Todas mostraram sensibilidade à acidez e à alcalinidade equivalentes às do estômago e às dos intestinos humanos, indicando que essa bipolaridade acidez/alcalinidade pode ser um mecanismo de defesa do organismo humano contra a ação de bactérias patogênicas ou indesejáveis. Para que bactérias desejáveis sejam utilizadas como probióticas, elas precisam ser ingeridas em altas concentrações, acima de 106 células.mL-1. Das doze bactérias testadas, as com maiores potenciais de utilização como probióticas, desde que comprovadamente não sejam patogênicas, são as BLG28 (Bacillus sp.), BLG38 (Bacillus sp.), BLG45 (Burkholderia sp.), BLG52 (Brevibacillus parabrevis), BLG56 (Achromobacter sp.) e BLG73 (Bacillus sp.), por fazerem parte de gêneros com pouca possibilidade de patogenicidade e por apresentarem crescimento positivo nas duas primeiras horas em pH 2,3, semelhante ao do estômago. No entanto, elas precisam colonizar e fazer parte da flora intestinal, além de ajudarem no metabolismo da lactose e do glúten ingeridos como alimentos. Para avaliar se a fonte de carbono e a fase de crescimento das bactérias afeta a produção e qualidade de proteases capazes de degradar o glúten, foram realizados experimentos com os extratos brutos de nove dessas bactérias, BLG02, BLG25, BLG28, BLG33, BLG38, BLG45, BLG52, BLG56 e BLG73. Os extratos foram obtidos do cultivo dessas bactérias em dois meios de cultura (glúten e lactose como fontes de carbono) e coletados com 6, 12 e 24 horas de incubação. Diluições dos extratos também foram testadas quanto às suas capacidades de degradar o glúten. Todas as bactérias produziram extratos com capacidade de degradação do glúten. A fonte de carbono (lactose ou glúten) afetou a capacidade dessas bactérias em produzirem extratos capazes de degradarem o glúten. A maioria delas produziu mais proteases quando crescidas em meio contendo esse complexo proteico. O tempo de coleta dos extratos bacterianos também influenciou nas suas capacidades de degradarem o glúten, com os produzidos com 24 horas de crescimento sendo melhores para a maioria dessas bactérias quando comparados pelos produzidos com 6 e 12 horas. O maior percentual de degradação de glúten foi de 87,3 % usando o extrato obtido com 24 horas de crescimento da bactéria BLG56 (Achromobacter sp.) cultivada em meio de cultura contendo glúten. Com base na capacidade de degradação, nos tempos de coletas dos extratos bacterianos e meios de cultura, pode-se concluir que as nove bactérias apresentam diferentes proteases capazes de degradarem o glúten. Essa capacidade de degradarem o glúten variou com suas concentrações na solução de formas diferentes e essa característica pode servir como um teste a mais para diferenciar uma da outra. São necessários estudos mais detalhados, como o de purificação dos componentes dos extratos dessas bactérias, para avaliar cada uma dessas proteases individualmente, para escolher as de melhores potenciais biotecnológicos.

The amount of data obtained by barcoding of prokaryotic 16S rDNA from natural habitats is increasing exponentially. Thus, methods enabling us to extract useful information from these data are of increasing importance. In this thesis microbial communities from water, sludge and drilling dust were analyzed by 16S rDNA sequencing in three geologically well described sedimentary aquifers in Bohemian Massif. The main goal of this research was to establish how different analytical approaches can be useful in interpretation of groundwater biogeochemical processes. Three approaches were used: First, taxonomy and metabolic traits of the most abundant microorganisms were assessed. Second, ordination methods showing metabolic and taxonomic variability between communities were used. Last the analysis of phylogenetic dissimilarity using UniFrac metrics was performed. When analyzing individual localities separately, the shift in microbial community composition corresponds with the change of environmental conditions. The unconstrained ordination method based on the variability in metabolic traits indicated, that sludge samples are more informative than water samples when asking which electron donor is used in microbial communities. On the other hand, unconstrained ordination methods were useless when the...

Thesis: Ph. D., Joint Program in Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Department of Earth, Atmospheric, and Planetary Sciences; and the Woods Hole Oceanographic Institution), 2018. / Cataloged from PDF version of thesis. / Includes bibliographical references (pages 213-240). / Life is ubiquitous in the environment and an important mediator of Earth's carbon cycle, but quantifying the contribution of microbial biomass and its metabolic fluxes is difficult, especially in spatially and temporally-remote environments. Microbes leave behind an often scarce, unidentifiable, or nonspecific record on geologic timescales. This thesis develops and employs novel geochemical and genetic approaches to illuminate diagnostic signals of microbial metabolisms. Field studies, laboratory cultures, and computational models explain how methanogens produce unique nonequilibrium methane clumped isotopologue (1 3CH3D ) signals that do not correspond to growth temperature. Instead, [Delta]13CH3D values may be driven by enzymatic reactions common to all methanogens, the C-H bond inherited from substrate precursors including acetate and methanol, isotope exchange, or environmental processes such as methane oxidation. The phylogenetic relationship between substrate-specific methyl-corrinoid proteins provides insight into the evolutionary history of methylotrophic methanogenesis. The distribution of corrinoid proteins in methanogens and related bacteria suggests that these substrate-specific proteins evolved via a complex history of horizontal gene transfer (HGT), gene duplication, and loss. Furthermore, this work identifies a previously unrecognized HGT involving chitinases (ChiC/D) distributed between fungi and bacteria (~650 Ma). This HGT is used to tether fossil-calibrated ages from within fungi to bacterial lineages. Molecular clock analyses show that multiple clades of bacteria likely acquired chitinase homologs via HGT during the late Neoproterozoic into the early Paleozoic. These results also show that, following these HGT events, recipient terrestrial bacterial clades diversified ~400-500 Ma, consistent with established timescales of arthropod and plant terrestrialization. Divergence time estimates for bacterial lineages are broadly consistent with the dispersal of chitinase genes throughout the microbial world in direct response to the evolution and expansion of detrital-chitin producing groups including arthropods. These chitinases may aid in dating microbial lineages over geologic time and provide insight into an ecological shift from marine to terrestrial systems in the Proterozoic and Phanerozoic eons. Taken together, this thesis may be used to improve assessments of microbial activity in remote environments, and to enhance our understanding of the evolution of Earth's carbon cycle. / by Danielle S. Gruen / Ph. D.

Thesis (Ph. D.)--Joint Program in Oceanography/Applied Ocean Science and Engineering (Massachusetts Institute of Technology, Dept. of Biology; and the Woods Hole Oceanographic Institution), February 2013. / "February 2013." Cataloged from PDF version of thesis. / Includes bibliographical references. / Intact polar diacylglycerolipids (IP-DAGs) were used to study microbial dynamics in the surface ocean. IP-DAGs from surface ocean seawater were quantified using high performance liquid chromatography-mass spectrometry (HPLC-MS), after first developing a sensitive, high throughput molecular ion independent triple quadrupole MS method for quantification. Using this analytical technique I examined the distribution of the nine most abundant classes of IPDAGs across the Mediterranean, and found that phospholipids as a percent of total IP-DAGs correlated with phosphate concentration. Furthermore, phospholipids were a higher percent of total particulate phosphorus where phosphate was higher, ranging from 1-14%. Thus IP-DAGs can play not only a significant but also a dynamic role in defining planktonic nutrient needs and cellular C:N:P ratios in the environment. Additionally, microcosm incubations were amended with phosphate and ammonium, and in the course of several days this elicited a shift in the ratios of IP-DAGs. This study was the first to demonstrate the dynamic response of membrane lipid composition to changes in nutrients in a natural, mixed planktonic community, and indicated that the change in IP-DAG ratios in response to changing nutrients may be a useful indicator of microbial nutrient stress. In the surface waters of the western North Atlantic I used three experimental approaches to identify the microbial sources of the nine most abundant classes of IP-DAGs. Phytoplankton are the primary source of one class of sulfolipid, sulfoquinovosyldiacylglycerol, and one class of betaine lipid, diacylglyceryl-trimethyl-homoserine, while heterotrophic bacteria are the dominant source of the phospholipids phosphatidylglycerol and phosphatidylethanolamine. In regrowth experiments in the Sargasso Sea and the North Pacific I demonstrated that phospholipid specific production rate is representative of heterotrophic bacterial cell specific growth rate. I measured phospholipid specific production rate and bacterial production rate using uptake of 3H-leucine (³H-Leu) and 3H-thymidine (³H-TdR) across the North Atlantic, across the Mediterranean, and in the North Pacific subtropical gyre. I found that phospholipid specific production rates estimate heterotrophic bacterial cell specific growth rates that are on the order of 1 per day, an order of magnitude faster than cell specific growth rates suggested by uptake of ³H-Leu and ³H-TdR. / by Kimberly J. Popendorf. / Ph.D.

The loss of efficiency and performance of bioprocesses on scale-up is well known, but not fully understood. This work addresses this problem, by studying the effect of some fermentation gradients (pH, glucose and oxygen) at a larger scale in a bench-scale two compartment reactor (PFR + STR) using the cadaverine-producing recombinant bacterium, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT. The initial scale down strategy increased the magnitude of these gradients by only increasing the mean cell residence time in the plug flow reactor (τ_PFR). The cell growth and product related rate constants were compared as the τ_PFR was increased; differences were significant in some cases, but only up to 2 min residence time. For example, losses in cadaverine productivity when compared to the control fed-batch fermentation on average for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 42 % and 46 % respectively. This indicated that the increasing the τ_PFR alone does not necessarily increase the magnitude of fermentation gradients. The new scale-down strategy developed here, increased the magnitude of fermentation gradients by not only increasing the τ_PFR, but also considering the mean frequency at which the bacterial cells entered the PFR section (f_m). The f_m was kept constant by reducing the broth volume in the STR. Hence, the bacterial cells also spent shorter times in the well mixed STR, as the τ_PFR was increased (hypothesised as giving the bacterial cells less time to recover the non-ideal PFR section of the SDR). On adoption of this strategy cadaverine productivity decreases for the τ_PFR of 1 min, 2 min and 5 min were 25 %, 32 % and 53 % respectively. Thus, highlighting that loss in performance is most likely to occur as the magnitude of heterogeneity within the fermentation environment increases. However, Corynebacterium glutamicum DM1945 Δact3 Ptuf-ldcC_OPT did show some resilience in its biomass productivity. It was only marginally affected in the harshest of conditions simulated here.

Continental shelves located along eastern boundary currents occupy relatively small volumes of the world’s oceans, yet are responsible for a large proportion of global primary production. The Oregon coast is among these ecosystems. Recent analyses of dissolved oxygen at shallow depths in the water column has suggested increasing episodes of hypoxia and anoxia, events that are detrimental to larger macro-faunal species. Microbial communities, however, are metabolically diverse, capable of utilizing alternative electron donors and acceptors, and can withstand transient periods of low dissolved oxygen. Understanding the phylogenetic and metabolic diversity of microorganisms in these environments is important for assessing the impact hypoxic events have on local and global biogeochemistry. Several molecular ecology tools were used to answer questions about the distribution patterns and activities of microorganisms residing along the coast of Oregon in this dissertation. Ribosomal rRNA fingerprinting and sequence analyses of samples collected during 2007-2008 suggested that bacterial community structure was not substantially influenced by changes in dissolved oxygen. However, substantial depth dependent changes were observed, with samples collected in the bottom boundary layer (BBL) displaying significant differences from those collected in the surface layer. Phylogenetic analyses of bacterial rRNA genes revealed novel phylotypes associated with this area of the water column, including groups with close evolutionary relationships to putative or characterized sulfur oxidizing bacteria (SOB). Analysis of metagenomes and metatranscriptomes collected during 2009 suggested increasing abundances of chemolithoautrophic organisms and their activities in the BBL. Thaumarchaea displayed significant depth dependent increases during the summer, and were detected at maximal frequencies during periods of hypoxia, suggesting that nitrification maybe influenced by local changes in dissolved oxygen. Metagenomic analysis of samples collected from 2010 revealed substantial variability in the metabolic potential of the microbial communities from different water masses. Samples collected during the spring, prior to upwelling clustered independently of those collected during the summer, during a period of upwelling, and did not display any clear stratification. Samples collected during the summer did cluster based on depth, consistent with previous observations, and increases in the relative abundances of chemolithotrophic gene suites were observed in the BBL during stratified conditions, suggesting that the metabolic potential for these processes is a repeatable feature along the Oregon coast. Overall, these observations suggest that depth impacts microbial community diversity, metabolic potential, and transcriptional activity in shallow areas of the Northeast Pacific Ocean. The increase in lithotrophic genes and transcripts in the BBL suggests that this microbial community includes many organisms that are able to use inorganic electron donors for respiration. We speculate that the dissolved organic material in the BBL is semi-labile and not available for immediate oxidation, favoring the growth for microorganisms that are able to use alternative electron donors. / Graduation date: 2012

L'objet de cette thèse de bioinformatique est de mieux comprendre l’ensemble des systèmes de régulation génique chez les bactéries. La disponibilité de centaines de génomes complets chez les bactéries ouvre la voie aux approches de génomique comparative et donc à l’étude de l’évolution des réseaux transcriptionnels bactériens. Dans un premier temps, nous avons implémenté et validé plusieurs méthodes de prédiction d’opérons sur base des génomes bactériens séquencés. Suite à cette étude, nous avons décidé d’utiliser un algorithme qui se base simplement sur un seuil sur la distance intergénique, à savoir la distance en paires de bases entre deux gènes adjacents. Notre évaluation sur base d’opérons annotés chez Escherichia coli et Bacillus subtilis nous permet de définir un seuil optimal de 55pb pour lequel nous obtenons respectivement 78 et 79% de précision. Deuxièmement, l’identification des motifs de régulation transcriptionnelle, tels les sites de liaison des facteurs de transcription, donne des indications de l’organisation de la régulation. Nous avons développé une méthode de recherche d’empreintes phylogénétiques qui consiste à découvrir des paires de mots espacés (dyades) statistiquement sur-représentées en amont de gènes orthologues bactériens. Notre méthode est particulièrement adaptée à la recherche de motifs chez les bactéries puisqu’elle profite d’une part des centaines de génomes bactériens séquencés et d’autre part les facteurs de transcription bactériens présentent des domaines Hélice-Tour-Hélice qui reconnaissent spécifiquement des dyades. Une évaluation systématique sur 368 gènes de E.coli a permis d’évaluer les performances de notre méthode et de tester l’influence de plus de 40 combinaisons de paramètres concernant le niveau taxonomique, l’inférence d’opérons, le filtrage des dyades spécifiques de E.coli, le choix des modèles de fond pour le calcul du score de significativité, et enfin un seuil sur ce score. L’analyse détaillée pour un cas d’étude, l’autorégulation du facteur de transcription LexA, a montré que notre approche permet d’étudier l’évolution des sites d’auto-régulation dans plusieurs branches taxonomiques des bactéries. Nous avons ensuite appliqué la détection d’empreintes phylogénétiques à chaque gène de E.coli, et utilisé les motifs détectés comme significatifs afin de prédire les gènes co-régulés. Au centre de cette dernière stratégie, est définie une matrice de scores de significativité pour chaque mot détecté par gène chez l’organisme de référence. Plusieurs métriques ont été définies pour la comparaison de paires de profils de scores de sorte que des paires de gènes ayant des motifs détectés significativement en commun peuvent être regroupées. Ainsi, l’ensemble des nos méthodes nous permet de reconstruire des réseaux de co-régulation uniquement à partir de séquences génomiques, et nous ouvre la voie à l’étude de l’organisation et de l’évolution de la régulation transcriptionnelle pour des génomes dont on ne connaît rien.<p><p>The purpose of my thesis is to study the evolution of regulation within bacterial genomes by using a cross-genomic comparative approach. Nowadays, numerous genomes have been sequenced facilitating in silico analysis in order to detect groups of functionally related genes and to predict the mechanism of their relative regulation. In this project, we combined prediction of operons and regulons in order to reconstruct the transcriptional regulatory network for a bacterial genome. We have implemented three methods in order to predict operons from a bacterial genome and evaluated them on hundreds of annotated operons of Escherichia coli and Bacillus subtilis. It turns out that a simple distance-based threshold method gives good results with about 80% of accuracy. The principle of this method is to classify pairs of adjacent genes as “within operon” or “transcription unit border”, respectively, by using a threshold on their intergenic distance: two adjacent genes are predicted to be within an operon if their intergenic distance is smaller than 55bp. In the second part of my thesis, I evaluated the performances of a phylogenetic footprinting approach based on the detection of over-represented spaced motifs. This method is particularly suitable for (but not restricted to) Bacteria, since such motifs are typically bound by factors containing a Helix-Turn-Helix domain. We evaluated footprint discovery in 368 E.coli K12 genes with annotated sites, under 40 different combinations of parameters (taxonomical level, background model, organism-specific filtering, operon inference, significance threshold). Motifs are assessed both at the level of correctness and significance. The footprint discovery method proposed here shows excellent results with E. coli and can readily be extended to predict cis-acting regulatory signals and propose testable hypotheses in bacterial genomes for which nothing is known about regulation. Moreover, the predictive power of the strategy, and its capability to track the evolutionary divergence of cis-regulatory motifs was illustrated with the example of LexA auto-regulation, for which our predictions are remarkably consistent with the binding sites characterized in different taxonomical groups. A next challenge was to identify groups of co-regulated genes (regulons), by regrouping genes with similar motifs, in order to address the challenging domain of the evolution of transcriptional regulatory networks. We tested different metrics to detect putative pairs of co-regulated genes. The comparison between predicted and annotated co-regulation networks shows a high positive predictive value, since a good fraction of the predicted associations correspond to annotated co-regulations, and a low sensitivity, which may be due to the consequence of highly connected transcription factors (global regulator). A regulon-per-regulon analysis indeed shows that the sensitivity is very weak for these transcription factors, but can be quite good for specific transcription factors. The originality of this global strategy is to be able to infer a potential network from the sole analysis of genome sequences, and without any prior knowledge about the regulation in the considered organism. / Doctorat en Sciences / info:eu-repo/semantics/nonPublished

Indiana University-Purdue University Indianapolis (IUPUI) / Organic-rich substrates such as soil provide an excellent carbon source for bacteria. However, hydrocarbons such as those found in various ignitable liquids can also serve as a source of carbon to support bacterial growth. This is problematic for fire debris analysis as samples may be stored at room temperature for extended periods before they are analyzed due to case backlog. As a result, selective loss of key components due to bacterial metabolism can make identifying and classifying ignitable liquid residues by their chemical composition and boiling point range very difficult. The ultimate goal of this project is to preserve ignitable liquid residues against microbial degradation as efficiently and quickly as possible. Field and laboratory studies were conducted to monitor microbial degradation of gasoline and other ignitable liquids in soil samples. In addition to monitoring degradation in potting soil, as a worst case scenario, the effect of soil type and season were also studied. The effect of microbial action was also compared to the effect of weathering by evaporation (under nitrogen in the laboratory and by the passive headspace analysis of the glass fragments from the incendiary devices in the field studies). All studies showed that microbial degradation resulted in the significant loss of n-alkanes and lesser substituted alkylbenzenes predominantly and quickly, while more highly substituted alkanes and aromatics were not significantly affected. Additionally, the residential soil during the fall season showed the most significant loss of these compounds over the course of 30 days. To combat this problem, a chemical solution is to be immediately applied to the samples as they are collected. Various household and commercial products were tested for their efficacy at low concentrations to eliminate all living bacteria in the soil. Triclosan (2% (w/v) in NaOH) proved to be the most effective at preserving ignitable liquid residues for at least 30 days.