An increasing appreciation for the role of small RNA regulators prompted us to investigate the scope of RNA regulation in the bacterium, Streptomyces coelicolor. Our search revealed an antisense RNA that corresponds to the upstream region of four genes encoding cell wall cleavage enzymes (cell wall hydrolases), and a previously uncharacterized population of transfer RNA (tRNA) cleavage products. Further characterization of the 'tRNAs led to the discovery that S. coelicolor tRNAs are cleaved into 'tRNA halves' in a developmentally regulated fashion. All tRNAs seem to be susceptible to tRNA cleavage, although a bias was detected for tRNAs specifying highly used codons. To date, our work is the sole description of 'tRNA half production in a bacterium, and recent studies suggest that it is a widespread phenomenon among eukaryotic organisms. In a separate line of investigation, we noticed that a previous study had predicted that the genes associated with the antisense RNA are under the control of a riboswitch- a regulatory RNA element that directly controls gene expression in response to specific conditions. Our multifaceted characterization of this system began with the construction and phenotypic analyses of deletion mutant strains for several of the cell wall hydrolase-encoding genes. We demonstrate that S. coelicolor cell wall hydrolases are involved in germination, vegetative growth, and sporulation. Finally, we studied the potential for riboswitch regulation of one of the cell wall hydrolase-encoding genes, rpfA. RpfA is a resuscitation: Qromoting factor protein that is important for the revival of dormant bacteria, including the human pathogen and S. coelicolor relative - Mycobacterium tuberculosis. Our investigation uncovered evidence suggesting that the riboswitch region is involved in the regulation of rpfA, and we identified specific conditions under which it is repressed. This work represents a novel paradigm in the regulation of cell wall hydrolase expression. / Thesis / Doctor of Philosophy (PhD)

The growing problem of antibiotic resistance has prompted the need for new and novel antimicrobial therapies. The bacterial cell division pathway holds great promise for the development of novel broad-spectrum antibiotics as the majority of the proteins are essential for viability. The wealth of information regarding bacterial cell division has come from studies of the model organisms Escherichia coli and Bacillus subtilis. Although much has been elucidated regarding this pathway, the functions of many individual proteins remain unsolved. An important model organism for the investigation of cell division is Streptomyces coelicolor. The mycelial Streptomyces are sporulating, Gram-positive bacteria that grow in long branching networks of filamentous cells much like filamentous fungi. The normally essential process of cell division is dispensable for growth and viability of S. coelicolor. More interestingly, there are two different modes of cell division in this organism, one for vegetative growth and one is utilized for synchronous septation during sporulation. It is still unclear how developmental regulators control this switch, but advancements in fluorescence microscopy have shed some insight into the cell division process by allowing direct visualization of many cellular components and their dynamics. To better understand bacterial cell division and its regulation in S. coelicolor, three additional fluorescent proteins (FPs), including m.RFP, CyPet and YPet, have been established in this work. An m.RFP shuttle vector was constructed and the utility of m.RFP was tested by translationally fusing it to a tip-localizing protein, DiviVA. This work demonstrated that m.RFP is functional and an efficient marker for localized proteins. Also, established in this work is a two-colour fluorescence reporter system, which includes the fluorescent proteins CyPet and YPet that can be used to study co-localization and protein-protein interactions within cells. Future plans are to use co-localization of FP fusions and fluorescence resonance energy transfer (FRET) between CyPet and YPet to investigate the assembly of protein complexes within the cells, such as those involved in cell division. These studies will reveal critical information that is needed for the development of drugs that have novel mechanisms of action. / Thesis / Master of Science (MSc)

The Streptomyces are invaluable as a natural source of antibiotics and other bioactive compounds used in medicine and agriculture. S. coelicolor is the model streptomycete, and is studied for its complex secondary metabolism and multicellular life cycle. The subject of this work is bldB, a gene essential for development and antibiotic production in S. coelicolor, and one of its many homologues, located in the abaA antibiotic regulatory locus. The aim was to study the transcriptional regulation of bldB using a luminescent reporter, and investigate the role of each of the genes in the abaA cluster in regulation of antibiotic production, in order to understand the function and mechanism of action of bldB and its homologues. Individual deletion of each of the four genes in the abaA cluster resulted in varying effects on production of the antibiotic CDA. The bldB homologue, SCO0703, was shown to be a positive regulator of CDA, as the null mutant was severely defective in CDA production. It was found that bldB is expressed in most other bld developmental mutants, with the exception of bldD. There was no direct interaction observed between BldD and the bldB promoter, and possible mechanisms of indirect regulation are proposed. / Thesis / Master of Science (MSc)

Ce travail décrit l’analyse de la distribution des flux de carbones au sein de deux souches de Streptomyces coelicolor A3(2) : la souche sauvage nommée M145 et son mutant M1146 incapable de produire les antibiotiques actinorhodine, undecylprodigiosine, et l’antibiotique dépendant du calcium. Metabolite Balance Analysis et Isotopomer Balance Analysis sont mis en œuvre pour proposer un modèle de distribution des flux de carbones de S. coelicolor en phase exponentielle de croissance. Les souches M145 et M1146 sont cultivées dans un milieu minimum limitant en azote et leurs comportements métaboliques sont comparés. Dans la souche non productrice M1146, un taux de croissance plus élévé, un flux plus important dans la voie des pentoses phosphates, un flux plus faible au niveau du cycle de Krebs ainsi qu’une activité respiratoire plus faible sont mis en évidence. Cela traduit le coût énergétique important associé à la production d’actinorhodine par M145. De plus, ce travail propose un rôle important de la nicotinamide nucléotide transhydrogénase pour le maintien de l’homéostasie du NADPH lors de la production d’actinorhodine par M145. Comme il existe de bonnes corrélations entre les données expérimentales et celles issues de la modélisation au niveau des bilans carbones, des bilans de pouvoir réducteur et des échanges gazeux, il sera intéressant d’utiliser cette modélisation avec la technique de Flux Balance Analysis pour prédire les variations de la distribution des flux de carbones dans des mutants de S. coelicolor pour lesquels des gènes auraient été sur-exprimés ou délétés. / This work describes an analysis of carbon flux distribution in two strains of Streptomyces coelicolor A3(2), namely the wild type strain M145 and its derivative M1146 that is no longer able to produce the antibiotics actinorhodin, undecylprodigiosin and the calcium dependent antibiotic. Metabolite Balance Analysis and Isotopomer Balance Analysis were used to propose a model for carbon flux distribution in S. coelicolor during the exponential phase of growth. Strains M145 and M1146 were grown under nitrogen limitation in minimal medium and their metabolic behaviour were compared. In the non-producing strain M1146, a higher growth rate, a higher flux via the pentose phosphate pathway, a decreased flux through the TCA cycle and a decreased respiratory activity were evidenced. This highlighted the high energetic cost for actinorhodin production in M145. In this paper, we also propose a key role for the nicotinamide nucleotide transhydrogenase in NADPH homeostasis in M145 during actinorhodin production. As there are good correlations between experimental data and the model in terms of carbon balance, reducing power balance and gas exchanges, this model will be of great interest for Flux Balance Analysis to predict carbon-flux distribution changes in S. coelicolor strains in which gene are deleted or overexpressed.

Doctor en Ciencias de la Ingeniería, Mención Ingeniería Química y Biotecnología / Streptomyces leeuwenhoekii C34 es un actinomiceto aislado del desierto de Atacama, Chile, productor de los antibióticos chaxamicinas A a D y chaxalactinas A a C. El objetivo de este trabajo fue identificar los genes involucrados en la biosíntesis de chaxamicinas y chaxalactinas y producir chaxamicinas en un huésped heterólogo. Capítulo Uno detalla procedimientos de crecimiento y modificación genética de S. leeu- wenhoekii C34. La temperatura óptima para producción de chaxamicinas en medio líquido fue 30 °C, y la que permitió alcanzar altos niveles de esporulación en medio sólido fue 37 °C. S. leeuwenhoekii C34 fue sensible a tioestreptona, apramicina, higromicina B y kanamicina, mientras que no a ácido nalidíxico, antibióticos usados para seleccionar bac- terias genéticamente modificadas. Un vector sensible a temperatura (pGM1190) y otro con un sistema de integración del fago C31 (pSET152) fueron exitosamente transferi- dos a S. leeuwenhoekii C34 por conjugación con una cepa que no metila ADN, E. coli ET12567/pUZ8002. Suplementación del medio de conjugación con 120 mM MgCl2 ó 60 mM CaCl2, incrementó la frecuencia de conjugación de forma significativa, comparado con el control sin adición de sales. En este trabajo se demostró que S. leeuwenhoekii C34 incorpora ADN foráneo no metilado. Capítulo Dos describe la identificación y expresión heteróloga de los genes de biosín- tesis de chaxamicinas. El genoma de S. leeuwenhoekii C34 fue secuenciado de novo combinando tecnologías de secuenciación Illumina y PacBio. Un grupo de 27 genes (80,2 kb, locus 1.210.347 1.290.550), codifica enzimas para la biosíntesis de chaxam- icinas. pIJ12853 contenía un inserto del genoma de S. leeuwenhoekii C34 de 145 kb que incluye el segmento de 80,2 kb, el que fue transferido a una cepa que no produce chaxamicinas, S. coelicolor M1152, resultando en la producción de chaxamicinas A D, confirmando que los genes presentes en pIJ12853 codifican para la ruta de biosíntesis de chaxamicinas. Genes del huésped heterólogo podrían estar involucrados en biosíntesis y/o exporte de estas moléculas. La deleción del gen AHBA sintasa (cxmK) en S. leeu- wenhoekii C34, dio origen a la cepa M1653 que pierde su capacidad de producir chax- amicinas. Para demostrar que la interrupción en la producción de chaxamicinas fue sólo debido a su incapacidad de producir AHBA, un cultivo líquido de M1653 suplementado con AHBA comercial permitió restablecer la producción de chaxamicinas. Esto es una prueba más de que el los genes de biosíntesis de chaxamicinas fueron identificados. Capítulo Tres describe la identificación bioinformática de los genes de biosíntesis de chaxalactinas. Una secuencia de 80,7 kb (locus 7.146.903 7.227.608) contenía genes codificantes de 5 subunidades de una policétido sintasa, cuya arquitectura de dominios coincidía con la predicha para biosíntesis de chaxalactina A. Un gen que codifica una citocromo P450 sería responsable de la hidroxilación C-14 de chaxalactina A que da ori- gen a chaxalactina B. Dos genes codificantes de O-metiltransferasas estarían involucra- dos en una O-metilación C-13 de chaxalactina B, que da origen a chaxalactina C.

All living organisms must organize their genome so that it not only fits within the cell, but remains accessible for cellular processes. In bacteria, an arsenal of nucleoid-associated proteins contributes to chromosome condensation. A novel nucleoid-associated protein was recently discovered in actinobacteria, and is essential in Mycobacterium. It was classified as an integration host factor protein (IHF); however, it does not share sequence or structural homology with the well characterized Escherichia coli IHF. In this study, we characterize the structure and function of Streptomyces coelicolor IHF (sIHF). We have used a combination of biochemistry and structural biology to characterize the role of sIHF in DNA binding and DNA topology. We have solved crystal structures of sIHF bound to various double-stranded DNA substrates, and show that sIHF is able to contact DNA at multiple surfaces. Furthermore, sIHF inhibits the activity of TopA, impacting DNA topology in vitro. Our work demonstrates that sIHF is a novel nucleoid-associated protein with key roles in condensing DNA. We believe that sIHF performs its function by differentially using multiple nucleic-acid binding surfaces. Further characterization is required to confirm this hypothesis in vivo. Given that the Mycobacterium homolog of sIHF (mIHF) is essential, our studies lay the foundation to explore novel drug targets for Mycobacterium tuberculosis and Mycobacterium leprae. / Thesis / Master of Science (MSc) / Unconstrained, the bacterial genome exceeds the size of the cell by 1 000- 10 000 times; thus, compacting it into a condensed structure, known as the nucleoid, is essential for life. An arsenal of nucleoid-associated proteins contributes to this process. In this study, we characterize the structure and function of a novel nucleoid–associated protein from the soil dwelling organism Streptomyces coelicolor. We used a combination of genetics, biochemistry, and structural biology to characterize the role of this protein in DNA binding and nucleoid organization. Since this protein is also found in important human pathogens, this work lays the foundation to explore the use of nucleoid-associated proteins as antimicrobial drug targets.

Il y a une dizaine d'années, de nouvelles flavoenzymes nommées hydroxylases flavine-dépendantes à deux composants ont été identifiées chez certains microorganismes. Le rôle physiologique de ces enzymes est maintenant bien connu. Elles sont impliquées dans les processus de biosynthèse et de biodégradation d'une multitude de molécules organiques. Ces hydroxylases sont composées de deux enzymes distinctes. La première est une flavine réductase qui catalyse la formation de flavine réduite nécessaire au fonctionnement de la seconde enzyme, une monooxygénase flavine-dépendante. Au début de notre projet, le mécanisme enzymatique de ces nouvelles hydroxylases était encore inconnu. Pour comprendre le détail de leur fonctionnement, nous avons choisi d'étudier le système ActVA-ActVB, un nouveau membre de la famille des hydroxylases flavine-dépendantes impliqué dans la dernière étape de biosynthèse de l'actinorhodine, un antibiotique naturel synthétisé par Streptomyces coelicolor. La caractérisation préalable de ActVB avait permis de montrer que cette enzyme était une NADH:FMN oxydoréductase capable de catalyser la réduction du FMN par le NADH selon un mécanisme de type séquentiel ordonné. Nos résultats ont permis d'identifier ActVA-Orf5, une monooxygénase flavine-dépendante capable d'utiliser la flavine réduite fournie par ActVB pour catalyser l'hydroxylation du précurseur de l'actinorhodine, la DHK. Le mécanisme de transfert de flavine entre les deux protéines a été étudié. Pour cela, les constantes de dissociation du FMNox et FMNred vis-à-vis de ActVA et ActVB ont été déterminées. Nos donnés montrent clairement qu'à l'état réduit, la flavine est bien plus affine pour la monooxygénase ActVA que pour la réductase ActVB alors qu'à l'état oxydé, elle possède une meilleure affinité pour la réductase que pour la monooxygénase. Cette différence d'affinité permet d'orienter le transfert de flavine d'une protéine à l'autre sans nécessiter d'interaction entre les deux protéines. Nous avons montré de plus que ActVA avait la capacité de stabiliser un intermédiaire activé de l'oxygène, une espèce électrophile nommée C(4a)-hydroperoxyflavine, au sein de son site actif. Cet intermédiaire réagit très rapidement avec la DHK nucléophile pour former son analogue hydroxylé : la DHK-OH. En accord avec ce mécanisme, il semble que le pouvoir nucléophile du substrat est très important pour cette réaction car seule la forme réduite à deux électrons de DHK (hydroquinone) est hydroxylée. D'autre part, ActVA ne semble pas être très spécifique car elle parvient également à catalyser l'hydroxylation de l'énantiomère de la DHK, la NNM-A et de l'analogue lactonique de la NNM-A, la NNM-D. Finalement le système ActVA-ActVB n'a pas la capacité de dimériser la DHK-OH pour former l'actinorhodine et l'enzyme intervenant dans la dernière étape de cette biosynthèse reste à identifier.

The main topic of this diploma thesis is ARE (resistance) proteins from the ABC-F family of the second class of ABC proteins. ARE proteins confer resistance to antibiotics that bind to a large ribosomal subunit and therefore inhibit proteosynthesis. One of the ARE proteins is the Lmr (C) protein, which is part of the linkomycin biosynthesis cluster of Streptomyces lincolnensis, and according to new results, Lmr (C) does not have to be just resistant protein but may have also regulatory function. We decided to study Sco0636, the closest homologue to Lmr (C) in Streptomyces coelicolor, which is a model organism in the study of secondary metabolism. Thanks to the production of color pigments, it is possible to monitor the effect of ARE proteins on secondary metabolism directly on the plates. I prepared the deletion mutant and the strain with constitutive expression of sco0636, and observed the effect on the phenotype. I followed the production of a blue asset and set a minimum inhibitory concentration to selected antibiotics, which bind to the ribosome. I have found that Sco0636 gives high resistance to tiamulin and so it has been named TiaA. The deletion of gene sco0636 accelerated production of actinorodine, and constitutive expression of this gene slowed down production. Keywords: ABC proteins,...

The main topic of this diploma thesis is ARE (resistance) proteins from the ABC-F family of the second class of ABC proteins. ARE proteins confer resistance to antibiotics that bind to a large ribosomal subunit and therefore inhibit proteosynthesis. One of the ARE proteins is the Lmr (C) protein, which is part of the linkomycin biosynthesis cluster of Streptomyces lincolnensis, and according to new results, Lmr (C) does not have to be just resistant protein but may have also regulatory function. We decided to study Sco0636, the closest homologue to Lmr (C) in Streptomyces coelicolor, which is a model organism in the study of secondary metabolism. Thanks to the production of color pigments, it is possible to monitor the effect of ARE proteins on secondary metabolism directly on the plates. I prepared the deletion mutant and the strain with constitutive expression of sco0636, and observed the effect on the phenotype. I followed the production of a blue asset and set a minimum inhibitory concentration to selected antibiotics, which bind to the ribosome. I have found that Sco0636 gives high resistance to tiamulin and so it has been named TiaA. The deletion of gene sco0636 accelerated production of actinorodine, and constitutive expression of this gene slowed down production. Keywords: ABC proteins,...

Les modèles de Markov d’ordre 2 (HMM2) sont des modèles stochastiques qui ont démontré leur efficacité dans l’exploration de séquences génomiques. Cette thèse explore l’intérêt de modèles de différents types (M1M2, M2M2, M2M0) ainsi que leur couplage à des méthodes combinatoires pour segmenter les génomes bactériens sans connaissances a priori du contenu génétique. Ces approches ont été appliquées à deux modèles bactériens afin d’en valider la robustesse : Streptomyces coelicolor et Streptococcus thermophilus. Ces espèces bactériennes présentent des caractéristiques génomiques très distinctes (composition, taille du génome) en lien avec leur écosystème spécifique : le sol pour les S. coelicolor et le milieu lait pour S. thermophilus / Second-order Hidden Markov Models (HMM2) are stochastic processes with a high efficiency in exploring bacterial genome sequences. Different types of HMM2 (M1M2, M2M2, M2M0) combined to combinatorial methods were developed in a new approach to discriminate genomic regions without a priori knowledge on their genetic content. This approach was applied on two bacterial models in order to validate its achievements: Streptomyces coelicolor and Streptococcus thermophilus. These bacterial species exhibit distinct genomic traits (base composition, global genome size) in relation with their ecological niche: soil for S. coelicolor and dairy products for S. thermophilus. In S. coelicolor, a first HMM2 architecture allowed the detection of short discrete DNA heterogeneities (5-16 nucleotides in size), mostly localized in intergenic regions. The application of the method on a biologically known gene set, the SigR regulon (involved in oxidative stress response), proved the efficiency in identifying bacterial promoters. S. coelicolor shows a complex regulatory network (up to 12% of the genes may be involved in gene regulation) with more than 60 sigma factors, involved in initiation of transcription. A classification method coupled to a searching algorithm (i.e. R’MES) was developed to automatically extract the box1-spacer-box2 composite DNA motifs, structure corresponding to the typical bacterial promoter -35/-10 boxes. Among the 814 DNA motifs described for the whole S. coelicolor genome, those of sigma factors (B, WhiG) could be retrieved from the crude data. We could show that this method could be generalized by applying it successfully in a preliminary attempt to the genome of Bacillus subtilis