Functional and structural genomics of amino acid metabolism in Streptomyces coelicolor

An investigation of amino acid metabolism in Streptomyces coelicolor, including the anabolism of tryptophan, histidine, the branched-chain amino acids and proline, as well as the catabolism of the latter, is reported. The experiments reported herein were conceptually conceived within a functional genomics framework. For this purpose the complete genome sequence of S. coelicolor was systematically exploited. Moreover, the current knowledge on the physiology of Streptomyces was taken onboard, as well as the prevailing and emerging notions on the evolution of proteins and metabolic pathways. Some of the results obtained using S. coelicolor as a model organism were expanded to other actinomycetes, such as Mycobacterium tuberculosis. This was aided by a comparative genomics analysis of the actinomycetes whose genomes have been sequenced. The theoretical principles that give support to this thesis are introduced in Chapter 1. This study was greatly facilitated by the development of a novel PCRtargeting mutagenesis method of which details can be found in Chapter VII. The discovery of a common isomerase for tryptophan and histidine biosynthesis is reported in Chapter II. This discovery arose from efforts aimed at reconstructing the tryptophan biosynthetic pathway of S. coelicolor, since the genome sequence project of this organism failed to identifiy a trpF gene coding for the enzyme phosphoribosyl anthranilate isomerase. The solution of this functional genomics discrepancy led to the discovery of a putative (~a)8-barrel enzyme, termed PriA, whose preliminary functional and structural characterisation is reported in Chapter III. The evolutionary implications of the discovery of PriA are discussed within Chapters III and N. A comparative genomics analysis of actinomycetes centred on the priA gene is presented in the latter Chapter, supporting the notion that this novel protein is spread across the high (0 + C) content Gram-positive organisms. Indeed, it was predicted that a priA orthologue accounts for the lack of a trpF gene from the genome of M tuberculosis, a hypothesis that proved to be correct. Finally, evidence to support the notion that the histidine and tryptophan biosynthetic pathways co-evolved is presented. In contrast to the isomerisation catalysed by PriA, in which an enzyme is shared by two amino acid biosynthetic pathways, several paralogous enzymes with the potential to account for the first step of tryptophan biosynthesis from chorismate were found on the genome of S. coelicolor. These chorismate-utilising enzymes are investigated in Chapter V. Mutational analysis of some of this paralogues is reported and it is anticipated that the analysis and results reported therein will serve to direct future experiments aimed at identifying the trpE paralogue encoding the enzyme anthranilate synthase. Chapter VI reports on the identification of the proC gene involved in the last step of proline biosynthesis in S. coelicolor. The pyrroline-5-carboxylate reductase activity of the enzyme encoded by the putative proC gene was extensively characterised, with particular emphasis on the interaction between primary and secondary metabolism. Furthermore, mutational analysis of proC suggested that paralogues of this gene are present on the genome of this organism, since its deletion did not lead to an auxotrophic phenotype. Investigation of this observation showed that two paralogous enzymes encoded by i1vC-like genes, involved in biosynthesis of the branched-chain amino acids, are capable of compensating for the lack of proC. This is the first example of a physiological link between the biosynthesis of proline and the branched-chain amino acids. To sum up, the results reported in this thesis represent an advancement towards understanding the physiology of S. coelicolor as a model actinomycete, within a functional and structural genomics framework. They also offer evidence on the evolutionary principles that lead to the appearance of novel proteins and metabolic pathways in bacteria.EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Functional and structural genomics of amino acid metabolism in Streptomyces coelicolor

An investigation of amino acid metabolism in Streptomyces coelicolor, including the anabolism of tryptophan, histidine, the branched-chain amino acids and proline, as well as the catabolism of the latter, is reported. The experiments reported herein were conceptually conceived within a functional genomics framework. For this purpose the complete genome sequence of S. coelicolor was systematically exploited. Moreover, the current knowledge on the physiology of Streptomyces was taken onboard, as well as the prevailing and emerging notions on the evolution of proteins and metabolic pathways. Some of the results obtained using S. coelicolor as a model organism were expanded to other actinomycetes, such as Mycobacterium tuberculosis. This was aided by a comparative genomics analysis of the actinomycetes whose genomes have been sequenced. The theoretical principles that give support to this thesis are introduced in Chapter 1. This study was greatly facilitated by the development of a novel PCRtargeting mutagenesis method of which details can be found in Chapter VII. The discovery of a common isomerase for tryptophan and histidine biosynthesis is reported in Chapter II. This discovery arose from efforts aimed at reconstructing the tryptophan biosynthetic pathway of S. coelicolor, since the genome sequence project of this organism failed to identifiy a trpF gene coding for the enzyme phosphoribosyl anthranilate isomerase. The solution of this functional genomics discrepancy led to the discovery of a putative (~a)8-barrel enzyme, termed PriA, whose preliminary functional and structural characterisation is reported in Chapter III. The evolutionary implications of the discovery of PriA are discussed within Chapters III and N. A comparative genomics analysis of actinomycetes centred on the priA gene is presented in the latter Chapter, supporting the notion that this novel protein is spread across the high (0 + C) content Gram-positive organisms. Indeed, it was predicted that a priA orthologue accounts for the lack of a trpF gene from the genome of M tuberculosis, a hypothesis that proved to be correct. Finally, evidence to support the notion that the histidine and tryptophan biosynthetic pathways co-evolved is presented. In contrast to the isomerisation catalysed by PriA, in which an enzyme is shared by two amino acid biosynthetic pathways, several paralogous enzymes with the potential to account for the first step of tryptophan biosynthesis from chorismate were found on the genome of S. coelicolor. These chorismate-utilising enzymes are investigated in Chapter V. Mutational analysis of some of this paralogues is reported and it is anticipated that the analysis and results reported therein will serve to direct future experiments aimed at identifying the trpE paralogue encoding the enzyme anthranilate synthase. Chapter VI reports on the identification of the proC gene involved in the last step of proline biosynthesis in S. coelicolor. The pyrroline-5-carboxylate reductase activity of the enzyme encoded by the putative proC gene was extensively characterised, with particular emphasis on the interaction between primary and secondary metabolism. Furthermore, mutational analysis of proC suggested that paralogues of this gene are present on the genome of this organism, since its deletion did not lead to an auxotrophic phenotype. Investigation of this observation showed that two paralogous enzymes encoded by i1vC-like genes, involved in biosynthesis of the branched-chain amino acids, are capable of compensating for the lack of proC. This is the first example of a physiological link between the biosynthesis of proline and the branched-chain amino acids. To sum up, the results reported in this thesis represent an advancement towards understanding the physiology of S. coelicolor as a model actinomycete, within a functional and structural genomics framework. They also offer evidence on the evolutionary principles that lead to the appearance of novel proteins and metabolic pathways in bacteria

Molecular annotation of ketol-acid reductoisomerases fromStreptomycesreveals a novel amino acid biosynthesis interlock mediated by enzyme promiscuity

The 6-phosphogluconate dehydrogenase superfamily oxidize and reduce a wide range of substrates, making their functional annotation challenging. Ketolacid reductoisomerase (KARI), encoded by the ilvC gene in branched-chain amino acids biosynthesis, is a promiscuous reductase enzyme within this superfamily. Here, we obtain steady-state enzyme kinetic parameters for 10 IlvC homologues from the genera Streptomyces and Corynebacterium, upon eight selected chemically diverse substrates, including some not normally recognized by enzymes of this superfamily. This biochemical data suggested a Streptomyces biosynthetic interlock between proline and the branched-chain amino acids, mediated by enzyme substrate promiscuity, which was confirmed via mutagenesis and complementation analyses of the proC, ilvC1 and ilvC2 genes in Streptomyces coelicolor. Moreover, both ilvC orthologues and paralogues were analysed, such that the relationship between gene duplication and functional diversification could be explored. The KARI paralogues present in S. coelicolor and Streptomyces lividans, despite their conserved high sequence identity (97%), were shown to be more promiscuous, suggesting a recent functional diversification. In contrast, the KARI paralogue from Streptomyces viridifaciens showed selectivity towards the synthesis of valine precursors, explaining its recruitment within the biosynthetic gene cluster of valanimycin. These results allowed us to assess substrate promiscuity indices as a tool to annotate new molecular functions with metabolic implications

Whole genome sequencing reveals widespread distribution of typhoidal toxin genes and VirB/D4 plasmids in bovine-associated nontyphoidal Salmonella

Aedes aegypti is the primary urban mosquito vector of viruses causing dengue, Zika and chikungunya fevers –for which vaccines and efective pharmaceuticals are still lacking. Current strategies to suppress arbovirus outbreaks include removal of larval-breeding sites and insecticide treatment of larval and adult populations. Insecticidal control of Ae. aegypti is challenging, due to a recent rapid global increase in knockdown-resistance (kdr) to pyrethroid insecticides. Widespread, heavy use of pyrethroid spacesprays has created an immense selection pressure for kdr, which is primarily under the control of the voltage-gated sodium channel gene (vgsc). To date, eleven replacements in vgsc have been discovered, published and shown to be associated with pyrethroid resistance to varying degrees. In Mexico, F1,534C and V1,016I have co-evolved in the last 16 years across Ae. aegypti populations. Recently, a novel replacement V410L was identifed in Brazil and its efect on vgsc was confrmed by electrophysiology. Herein, we screened V410L in 25 Ae. aegypti historical collections from Mexico, the frst heterozygote appeared in 2002 and frequencies have increased in the last 16 years alongside V1,016I and F1,534C. Knowledge of the specifc vgsc replacements and their interaction to confer resistance is essential to predict and to develop strategies for resistance management

Evolution of substrate specificity in a retained enzyme driven by gene loss.

The connection between gene loss and the functional adaptation of retained proteins is still poorly understood. We apply phylogenomics and metabolic modeling to detect bacterial species that are evolving by gene loss, with the finding that Actinomycetaceae genomes from human cavities are undergoing sizable reductions, including loss of L-histidine and L-tryptophan biosynthesis. We observe that the dual-substrate phosphoribosyl isomerase A or priA gene, at which these pathways converge, appears to coevolve with the occurrence of trp and his genes. Characterization of a dozen PriA homologs shows that these enzymes adapt from bifunctionality in the largest genomes, to a monofunctional, yet not necessarily specialized, inefficient form in genomes undergoing reduction. These functional changes are accomplished via mutations, which result from relaxation of purifying selection, in residues structurally mapped after sequence and X-ray structural analyses. Our results show how gene loss can drive the evolution of substrate specificity from retained enzymes

Evolution of substrate specificity in a recipient's enzyme following horizontal gene transfer

Despite the prominent role of horizontal gene transfer (HGT) in shaping bacterial metabolism, little is known about the impact of HGT on the evolution of enzyme function. Specifically, what is the influence of a recently acquired gene on the function of an existing gene? For example, certain members of the genus Corynebacterium have horizontally acquired a whole L-tryptophan biosynthetic operon, whereas in certain closely related actinobacteria, for example, Mycobacterium, the trpF gene is missing. In Mycobacterium, the function of the trpF gene is performed by a dual-substrate (βα)8 phosphoribosyl isomerase (priA gene) also involved in L-histidine (hisA gene) biosynthesis. We investigated the effect of a HGT-acquired TrpF enzyme upon PriA’s substrate specificity in Corynebacterium through comparative genomics and phylogenetic reconstructions. After comprehensive in vivo and enzyme kinetic analyses of selected PriA homologs, a novel (βα)8 isomerase subfamily with a specialized function in L-histidine biosynthesis, termed subHisA, was confirmed. X-ray crystallography was used to reveal active-site mutations in subHisA important for narrowing of substrate specificity, which when mutated to the naturally occurring amino acid in PriA led to gain of function. Moreover, in silico molecular dynamic analyses demonstrated that the narrowing of substrate specificity of subHisA is concomitant with loss of ancestral protein conformational states. Our results show the importance of HGT in shaping enzyme evolution and metabolism

Reflexión Política. Volumen 7 No. 14 de 2005

En el debate electoral venidero se va a practicar en Colombia la reforma política que en parte fue expedida por el Congreso tres años atrás (acto legislativo No. 1 de 2003), y la que zanjó la Corte Constitucional recientemente echando la base de un cambio indudablemente importante, al aprobar la reelección inmediata del Presidente de la República. Así se practica en no pocos Estados que ponen de presente su madurez democrática. Cabe esperar, a juzgar por las tentativas conocidas, que en lo futuro tengamos también reelección de alcaldes municipales y de gobernadores seccionales, proyecto que aún carece de suficiente opinión favorable.In the upcoming electoral debate, the political reform that was partially issued by Congress three years ago (legislative act No. 1 of 2003) and which was recently settled by the Constitutional Court will be practiced in Colombia, laying the foundation for an undoubtedly change. important, by approving the immediate re-election of the President of the Republic. This is how it is practiced in not a few States that show their democratic maturity. It is to be expected, judging by the known attempts, that in the future we will also have re-election of municipal mayors and sectional governors, a project that still lacks sufficient favorable opinion

Adaptación a la docencia online de las prácticas preclínicas de Cirugía Bucal I

Los cambios en la docencia debido a la pandemia por la COVID-19 ha llevado a la reducción de la presencialidad y a un aumento de la docencia online. Por ello se diseñó este proyecto, cuya finalidad fue la adaptación a la docencia online de las prácticas preclínicas de Cirugía Bucal I. Para el desarrollo de este trabajo se realizaron las rúbricas de evaluación de cada módulo de la asignatura, se elaboraron videos y documentación para subir al campus virtual antes de la realización de la práctica. Finalmente, se elaboraron cuestionarios de evaluación de la satisfacción de los estudiantes y profesorado con esta metodología. Los resultados mostraron una elevada satisfacción de ambos grupos, considerándola una herramienta de utilidad en el aprendizaje y de implementación en cursos posteriores

7th Drug hypersensitivity meeting: part two

No abstract availabl