All members of the genus Xanthomonas are considered to be plant pathogenic, with specific pathovars infecting several high value agricultural crops. One of these pathovars, X. campestris pv. zeae (as this is only a proposed name it will further on be referred to as Xanthomonas BLSD) the causal agent of bacterial leaf steak of maize, has established itself as a widespread significant maize pathogen within South Africa. Insufficient information about the present distribution of the pathogen is available. The main aim of the study was thus to isolate and characterise the pathogen using molecular methods. Results demonstrated that the causal agent of bacterial leaf streak disease (Xanthomonas BLSD: potentially X. campestris pv. zeae) was widely distributed within the major maize cultivation regions of South Africa. Most of the isolates collected originated from the Highveld maize production provinces (North West, Free State, Gauteng and Mpumalanga provinces) as well as from irrigated maize fields in the Northern Cape province. The XgumD gene marker was used to determine if the isolates belonged to the genus Xanthomonas. The gumD gene fragment is located within the gumB-gumM region of the operon and is conserved among Xanthomonas species. This gene fragment is partially responsible for xanthan production. This marker was amplified from all isolates and a selected number were sequenced. The marker was only able to confirm that the causal agent was a member of the genus Xanthomonas. PCR methods were used for the characterisation of the isolates. This included PCR and sequencing of ribosomal RNA- gyraseB and gumD genes. A fingerprinting method BOX-PCR was also employed. Good quality DNA of sufficient quantities was obtained from the various isolates. Amplification produced no non-specific amplification products. This resulted in good quality sequences that could be analysed using bioinformatics tools. Phylogenetic analyses of the ribosomal RNA and gyraseB genes could not detect differences amongst the 47 Xanthomonas BLSD isolates. However, these genes were able to distinguish between the type strain of these isolates and various Xanthomonas species and pathovars. From all three neighbour joining trees the Xanthomonas BLSD isolates had close association with X. axonopodis pv. vasculorum strain ATCC 35938. For the 16S rRNA gene there exists no sequence differences between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. A single nucleotide difference was observed between Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938 for the 23S rRNA gene. The gyraseB gene detected a total of six nucleotide variations between these two Xanthomonas species. For all of the phylogenetic trees there was no clustering of Xanthomonas BLSD with X. campestris pathovars. Genetic profiling (via BOX-PCR) based on present/absent analysis revealed no variations amongst the Xanthomonas BLSD isolates. All isolates shared an identical pattern produced by 12 distinct PCR products. This profiling technique did differentiate between the isolates of Xanthomonas BLSD and X. axonopodis pv. vasculorum strain ATCC 35938. Their profiles shared common bands, but differed in the number and overall pattern of the bands. These results suggest two main conclusions: (i) Xanthomonas BLSD has a clonal origin with geographical separation not impacting genetic variation. The fact that all the isolates appear to be clonal may imply that when resistant maize cultivars are developed these should be resistant to all isolates of the pathovar irrespective of their geographical origin. This is a suggestion that will have to be corroborated using more isolates and additional genetic fingerprinting techniques (ii) the Xanthomonas BLSD isolates from this study may not belong to X. campestris. Further studies using other markers should be conducted to determine the real identity of Xanthomonas BLSD. / MSc Environmental Sciences, North-West University, Potchefstroom Campus, 2015

This thesis covers a wide variety of projects within the domain of computational structural biology. Structural biology is concerned with the molecular structure of proteins and nucleic acids, and the relationship between structure and biological function. We used molecular modeling and simulation, a purely computational approach, to study DNA-linked molecular nanowires. We developed a computational tool that allows potential designs to be screened for viability, and then we used molecular dynamics (MD) simulations to test their stability. As an example of using molecular modeling to create experimentally testable hypotheses, we were able to suggest a new design based on pyrrylene vinylene monomers. In another project, we combined experiments and molecular modeling to gain insight into factors that influence the kinetic binding dynamics of fibrin "knob" peptides and complementary "holes." Molecular dynamics simulations provided helpful information about potential peptide structural conformations and intrachain interactions that may influence binding properties. The remaining projects discussed in this thesis all deal with RNA structure. The underlying approach for these studies is a recently developed chemical probing technology called 2'-hydroxyl acylation analyzed by primer extension (SHAPE). One study focuses on ribosomal RNA, specifically the 23S rRNA from T. thermophilus. We used SHAPE experiments to show that Domain III of the T. thermophilus 23S rRNA is an independently folding domain. This first required the development of our own data processing program for generating quantitative and interpretable data from our SHAPE experiments, due to limitations of existing programs and modifications to the experimental protocol. In another study, we used SHAPE chemistry to study the in vitro transcript of the RNA genome of satellite tobacco mosaic virus (STMV). This involved incorporating the SHAPE data into a secondary structure prediction program. The SHAPE-directed secondary structure of the STMV RNA was highly extended and considerably different from that proposed for the RNA in the intact virion. Finally, analyzing SHAPE data requires navigating a complex data processing pipeline. We review some of the various ways of running a SHAPE experiment, and how this affects the approach to data analysis.

La plupart des molécules d’ARN doivent se replier en structure tertiaire complexe afin d’accomplir leurs fonctions biologiques. Cependant, les déterminants d’une chaîne de polynucléotides qui sont nécessaires à son repliement et à ses interactions avec d’autres éléments sont essentiellement inconnus. L’établissement des relations structure-fonction dans les grandes molécules d’ARN passe inévitablement par l’analyse de chaque élément de leur structure de façon individuelle et en contexte avec d’autres éléments. À l’image d’une construction d’immeuble, une structure d’ARN est composée d’unités répétitives assemblées de façon spécifique. Les motifs récurrents d’ARN sont des arrangements de nucléotides retrouvés à différents endroits d’une structure tertiaire et possèdent des conformations identiques ou très similaires. Ainsi, une des étapes nécessaires à la compréhension de la structure et de la fonction des molécules d’ARN consiste à identifier de façon systématique les motifs récurrents et d’en effectuer une analyse comparative afin d’établir la séquence consensus. L’analyse de tous les cas d’empaquetage de doubles hélices dans la structure du ribosome a permis l’identification d’un nouvel arrangement nommé motif d’empaquetage le long du sillon (AGPM) (along-groove packing motif). Ce motif est retrouvé à 14 endroits dans la structure du ribosome de même qu’entre l’ARN ribosomique 23S et les molécules d’ARN de transfert liées aux sites ribosomaux P et E. Le motif se forme par l’empaquetage de deux doubles hélices via leur sillon mineur. Le squelette sucre-phosphate d’une hélice voyage le long du sillon mineur de l’autre hélice et vice versa. Dans chacune des hélices, la région de contact comprend quatre paires de bases. L’empaquetage le plus serré est retrouvé au centre de l’arrangement où l’on retrouve souvent une paire de bases GU dans une hélice interagissant avec une paire de bases Watson-Crick (WC) dans l’autre hélice. Même si la présence des paires de bases centrales GU versus WC au centre du motif augmente sa stabilité, d’autres alternatives existent pour différents représentants du motif. L’analyse comparative de trois librairies combinatoires de gènes d’AGPM, où les paires de bases centrales ont été variées de manière complètement aléatoire, a montré que le contexte structural influence l’étendue de la variabilité des séquences de nucléotides formant les paires de bases centrales. Le fait que l’identité des paires de bases centrales puisse varier suggérait la présence d’autres déterminants responsables au maintien de l’intégrité du motif. L’analyse de tous les contacts entre les hélices a révélé qu’en dehors du centre du motif, les interactions entre les squelettes sucre-phosphate s’effectuent via trois contacts ribose-ribose. Pour chacun de ces contacts, les riboses des nucléotides qui interagissent ensemble doivent adopter des positions particulières afin d’éviter qu’ils entrent en collision. Nous montrons que la position de ces riboses est modulée par des conformations spécifiques des paires de bases auxquelles ils appartiennent. Finalement, un autre motif récurrent identifié à l’intérieur même de la structure de trois cas d’AGPM a été nommé « adenosine-wedge ». Son analyse a révélé que ce dernier est lui-même composé d’un autre arrangement, nommé motif triangle-NAG (NAG-triangle). Nous montrons que le motif « adenosine-wedge » représente un arrangement complexe d’ARN composé de quatre éléments répétitifs, c’est-à-dire des motifs AGPM, « hook-turn », « A-minor » et triangle-NAG. Ceci illustre clairement l’arrangement hiérarchique des structures d’ARN qui peut aussi être observé pour d’autres motifs d’ARN. D’un point de vue plus global, mes résultats enrichissent notre compréhension générale du rôle des différents types d’interactions tertiaires dans la formation des molécules d’ARN complexes. / Most RNA molecules have to adopt a complex tertiary structure to accomplish their biological functions. However, the important determinants of a polynucleotide chain that are required for its proper folding and its interactions with other elements are essentially unknown. The establishment of structure-function relationships in large RNA molecules goes inevitably through the analysis of each element of their structure separately and in context with other elements. Like a building, an RNA structure is built of repetitive pieces that are glued together in a specific way. These repetitive elements, instead of being bricks, are recurrent motifs. Recurrent RNA motifs are arrangements of nucleotides found in different parts of a tertiary structure and have identical or very similar conformations. Thus, a necessary step toward the understanding of RNA structure and function consists in the systematic identification of recurrent motifs, followed by their comparative analysis and establishment of their sequence consensus. The analysis of all instances of helical packing within the ribosome structure led to the identification of a new structural arrangement, named the along-groove packing motif (AGPM), which is found in 14 places of the ribosome structure as well as between the 23S ribosomal RNA and the transfer RNA molecules bound to the P and E sites. The motif is formed by the packing of two double helices via their minor grooves. The sugar-phosphate backbone of one helix goes along the minor groove of the other helix and vice versa. In each helix, the contact region includes four base pairs. The closest packing occurs in the center where one can often see a GU base pair packed against a WC base pair. While the presence of the central base pairs GU versus WC in the core of the motif enhances its stability, other alternatives are also present among available structures of the motif. A comparative analysis of three different combinatorial gene libraries of AGPM, in which the central base pairs were fully randomized, shows that the structural context influences the scope of nucleotide sequence variability of the central base pairs. The fact that the identity of the central base pairs can vary suggested that there are other determinants responsible of the motif’s integrity. Analysis of all other inter-helix contacts has shown that outside the center of the motif the interactions between backbones are made via three ribose-ribose contacts. Within each of these contacts, the riboses of the nucleotides that are in touch adopt particular positions in order to provide for collision-free interactions between them. We show that the position of these riboses is modulated by the specific base pair conformation in which it belongs. Finally, another recurrent arrangement that occurs within the structure of three cases of AGPM was identified and called the adenosine-wedge. Analysis has shown that the latter motif is itself composed of a smaller arrangement, called the NAG-triangle motif. We show that the adenosine-wedge motif represents a complex RNA arrangement composed of four repetitive elements, AGPM, the hook-turn, the A-minor and the NAG-triangle, which clearly illustrates the hierarchical organisation of the structure that could also occur in other RNA motifs as well. Altogether, my results enrich our general understanding of the role of different types of tertiary interactions in the formation of large RNA molecules.

A corrosão metálica é um problema que afeta a economia mundial, sendo responsável pelo aumento dos custos de geração, transmissão e distribuição de energia elétrica e pode ser influenciada por atividades microbianas, podendo levar a aceleração ou inibição do processo. O presente trabalho teve por objetivo isolar e identificar as bactérias associadas a processos de corrosão em torres de transmissão de energia, bem como avaliar a resistência destas bactérias a diferentes metais pesados buscando selecionar micro-organismos com potencial para biorremediação. A identificação taxonômica dos isolados associados a elementos metálicos foi efetuada por análise filogenética das sequências parciais do gene RNA ribossomal 16S, resultando em 101 bactérias distribuídas em oito gêneros: Lysinibacillus, Serratia, Stenotrophomonas, Pseudomonas, Exiguobacterium, Chryseobacterium, Bacillus e Acinetobacter. Todos os gêneros foram resistentes a mais de um metal, sendo que o metal que menos afetou o crescimento dos isolados foi o cromo e o que mais afetou foi o mercúrio. / Metallic corrosion is a problem that affects the world economy, being responsible for the increased costs of generating, transmission and distribution of electricity and can be influenced by microbial activities, leading to inhibition or acceleration of the process. This study aimed to isolate and identify bacteria associated with corrosion processes in metallic structures of power transmission towers, as well as evaluating the resistance of these bacteria to different heavy metals, in order to select microorganisms with potential for biorremediation. The taxonomic identification of the isolates of bacteria associated with metallic elements was performed by phylogenetic analysis of partial sequences of 16S ribosomal RNA gene, resulting in 101 bacteria distributed in eight genera: Lysinibacillus, Serratia, Stenotrophomonas, Pseudomonas, Exiguobacterium, Chryseobacterium, Bacillus and Acinetobacter. All genera were resistant to more than one metal. Chromium and mercury were the metals that least and most affected the growth of isolates, respectively.

La plupart des molécules d’ARN doivent se replier en structure tertiaire complexe afin d’accomplir leurs fonctions biologiques. Cependant, les déterminants d’une chaîne de polynucléotides qui sont nécessaires à son repliement et à ses interactions avec d’autres éléments sont essentiellement inconnus. L’établissement des relations structure-fonction dans les grandes molécules d’ARN passe inévitablement par l’analyse de chaque élément de leur structure de façon individuelle et en contexte avec d’autres éléments. À l’image d’une construction d’immeuble, une structure d’ARN est composée d’unités répétitives assemblées de façon spécifique. Les motifs récurrents d’ARN sont des arrangements de nucléotides retrouvés à différents endroits d’une structure tertiaire et possèdent des conformations identiques ou très similaires. Ainsi, une des étapes nécessaires à la compréhension de la structure et de la fonction des molécules d’ARN consiste à identifier de façon systématique les motifs récurrents et d’en effectuer une analyse comparative afin d’établir la séquence consensus. L’analyse de tous les cas d’empaquetage de doubles hélices dans la structure du ribosome a permis l’identification d’un nouvel arrangement nommé motif d’empaquetage le long du sillon (AGPM) (along-groove packing motif). Ce motif est retrouvé à 14 endroits dans la structure du ribosome de même qu’entre l’ARN ribosomique 23S et les molécules d’ARN de transfert liées aux sites ribosomaux P et E. Le motif se forme par l’empaquetage de deux doubles hélices via leur sillon mineur. Le squelette sucre-phosphate d’une hélice voyage le long du sillon mineur de l’autre hélice et vice versa. Dans chacune des hélices, la région de contact comprend quatre paires de bases. L’empaquetage le plus serré est retrouvé au centre de l’arrangement où l’on retrouve souvent une paire de bases GU dans une hélice interagissant avec une paire de bases Watson-Crick (WC) dans l’autre hélice. Même si la présence des paires de bases centrales GU versus WC au centre du motif augmente sa stabilité, d’autres alternatives existent pour différents représentants du motif. L’analyse comparative de trois librairies combinatoires de gènes d’AGPM, où les paires de bases centrales ont été variées de manière complètement aléatoire, a montré que le contexte structural influence l’étendue de la variabilité des séquences de nucléotides formant les paires de bases centrales. Le fait que l’identité des paires de bases centrales puisse varier suggérait la présence d’autres déterminants responsables au maintien de l’intégrité du motif. L’analyse de tous les contacts entre les hélices a révélé qu’en dehors du centre du motif, les interactions entre les squelettes sucre-phosphate s’effectuent via trois contacts ribose-ribose. Pour chacun de ces contacts, les riboses des nucléotides qui interagissent ensemble doivent adopter des positions particulières afin d’éviter qu’ils entrent en collision. Nous montrons que la position de ces riboses est modulée par des conformations spécifiques des paires de bases auxquelles ils appartiennent. Finalement, un autre motif récurrent identifié à l’intérieur même de la structure de trois cas d’AGPM a été nommé « adenosine-wedge ». Son analyse a révélé que ce dernier est lui-même composé d’un autre arrangement, nommé motif triangle-NAG (NAG-triangle). Nous montrons que le motif « adenosine-wedge » représente un arrangement complexe d’ARN composé de quatre éléments répétitifs, c’est-à-dire des motifs AGPM, « hook-turn », « A-minor » et triangle-NAG. Ceci illustre clairement l’arrangement hiérarchique des structures d’ARN qui peut aussi être observé pour d’autres motifs d’ARN. D’un point de vue plus global, mes résultats enrichissent notre compréhension générale du rôle des différents types d’interactions tertiaires dans la formation des molécules d’ARN complexes. / Most RNA molecules have to adopt a complex tertiary structure to accomplish their biological functions. However, the important determinants of a polynucleotide chain that are required for its proper folding and its interactions with other elements are essentially unknown. The establishment of structure-function relationships in large RNA molecules goes inevitably through the analysis of each element of their structure separately and in context with other elements. Like a building, an RNA structure is built of repetitive pieces that are glued together in a specific way. These repetitive elements, instead of being bricks, are recurrent motifs. Recurrent RNA motifs are arrangements of nucleotides found in different parts of a tertiary structure and have identical or very similar conformations. Thus, a necessary step toward the understanding of RNA structure and function consists in the systematic identification of recurrent motifs, followed by their comparative analysis and establishment of their sequence consensus. The analysis of all instances of helical packing within the ribosome structure led to the identification of a new structural arrangement, named the along-groove packing motif (AGPM), which is found in 14 places of the ribosome structure as well as between the 23S ribosomal RNA and the transfer RNA molecules bound to the P and E sites. The motif is formed by the packing of two double helices via their minor grooves. The sugar-phosphate backbone of one helix goes along the minor groove of the other helix and vice versa. In each helix, the contact region includes four base pairs. The closest packing occurs in the center where one can often see a GU base pair packed against a WC base pair. While the presence of the central base pairs GU versus WC in the core of the motif enhances its stability, other alternatives are also present among available structures of the motif. A comparative analysis of three different combinatorial gene libraries of AGPM, in which the central base pairs were fully randomized, shows that the structural context influences the scope of nucleotide sequence variability of the central base pairs. The fact that the identity of the central base pairs can vary suggested that there are other determinants responsible of the motif’s integrity. Analysis of all other inter-helix contacts has shown that outside the center of the motif the interactions between backbones are made via three ribose-ribose contacts. Within each of these contacts, the riboses of the nucleotides that are in touch adopt particular positions in order to provide for collision-free interactions between them. We show that the position of these riboses is modulated by the specific base pair conformation in which it belongs. Finally, another recurrent arrangement that occurs within the structure of three cases of AGPM was identified and called the adenosine-wedge. Analysis has shown that the latter motif is itself composed of a smaller arrangement, called the NAG-triangle motif. We show that the adenosine-wedge motif represents a complex RNA arrangement composed of four repetitive elements, AGPM, the hook-turn, the A-minor and the NAG-triangle, which clearly illustrates the hierarchical organisation of the structure that could also occur in other RNA motifs as well. Altogether, my results enrich our general understanding of the role of different types of tertiary interactions in the formation of large RNA molecules.

A corrosão metálica é um problema que afeta a economia mundial, sendo responsável pelo aumento dos custos de geração, transmissão e distribuição de energia elétrica e pode ser influenciada por atividades microbianas, podendo levar a aceleração ou inibição do processo. O presente trabalho teve por objetivo isolar e identificar as bactérias associadas a processos de corrosão em torres de transmissão de energia, bem como avaliar a resistência destas bactérias a diferentes metais pesados buscando selecionar micro-organismos com potencial para biorremediação. A identificação taxonômica dos isolados associados a elementos metálicos foi efetuada por análise filogenética das sequências parciais do gene RNA ribossomal 16S, resultando em 101 bactérias distribuídas em oito gêneros: Lysinibacillus, Serratia, Stenotrophomonas, Pseudomonas, Exiguobacterium, Chryseobacterium, Bacillus e Acinetobacter. Todos os gêneros foram resistentes a mais de um metal, sendo que o metal que menos afetou o crescimento dos isolados foi o cromo e o que mais afetou foi o mercúrio. / Metallic corrosion is a problem that affects the world economy, being responsible for the increased costs of generating, transmission and distribution of electricity and can be influenced by microbial activities, leading to inhibition or acceleration of the process. This study aimed to isolate and identify bacteria associated with corrosion processes in metallic structures of power transmission towers, as well as evaluating the resistance of these bacteria to different heavy metals, in order to select microorganisms with potential for biorremediation. The taxonomic identification of the isolates of bacteria associated with metallic elements was performed by phylogenetic analysis of partial sequences of 16S ribosomal RNA gene, resulting in 101 bacteria distributed in eight genera: Lysinibacillus, Serratia, Stenotrophomonas, Pseudomonas, Exiguobacterium, Chryseobacterium, Bacillus and Acinetobacter. All genera were resistant to more than one metal. Chromium and mercury were the metals that least and most affected the growth of isolates, respectively.

CSIR / Translation initiation is a rate limiting step during protein biosynthesis. Initiation occurs by formation of an initiation complex comprising 30S subunit of ribosome, mRNA, initiator tRNA, and initiation factors. The initiator tRNA has a specialized function of binding to ribosomal P site whereas all the other tRNAs are selected in the ribosomal A site. The presence of a highly conserved 3 consecutive G-C base pairs in the anticodon stem of the initiator tRNA has been shown to be responsible for its P-site targeting. The exact molecular mechanism involved in the P-site targeting of the initiator tRNA is still unclear and focus of our study. Using genetic methods, we obtained mutant E. coli strains where initiator tRNA mutants lacking the characteristic 3-GC base pairs can also initiate translation. One such mutant strain, A30, was selected for this study. Using standard molecular genetic tools, the mutation was mapped and identified to be a mutation in a transcription remodeling factor, RapA (A511V). RapA is a transcription recycling factor and it displaces S1 when it performs its transcription recycling activity. We found this mutation to cause an increase in the S1-depleted ribosomes leading to decreased fidelity of translation initiation as the mutant RapA inefficiently displaces S1 from RNA polymerase complex. The mutation in the RapA was also found to cause changes in the transcriptome which leads to downregulation of major genes important for methionine and purine metabolism. Using mass spectrometric analysis, we identified deficiencies of methionine and adenine in the strain carrying mutant RapA. Our lab had previously reported that methionine and S-adenosyl methionine deficiency cause deficiency of methylations in ribosome which in turn decreases the fidelity of protein synthesis initiation. We used strains deleted for two newly identified methyltransferases, namely RlmH and RlmI, for our study and these strains also showed decreased fidelity of initiation. RlmH and RlmI methylate 1915 and 1962 positions of 23S rRNA respectively. We found that deletion of these methyltransferases also caused defects in ribosome biogenesis and compromised activity of ribosome recycling factor. We constructed phylogenetic trees of the initiator tRNA from 158 species which distinctly assembled into three domains of life. We also constructed trees using the minihelix or the whole sequence of species specific tRNAs, and iterated our analysis on 50 eubacterial species. We identified tRNAPro, tRNAGlu, or tRNAThr (but surprisingly not elongator tRNAMet) as probable ancestors of tRNAi. We then determined the factors imposing selection of methionine as the initiating amino acid. Overall frequency of occurrence of methionine, whose metabolic cost of synthesis is the highest among all amino acids, remains almost unchanged across the three domains of life. Our results indicate that methionine selection, as the initiating amino acid was possibly a consequence of the evolution of one-carbon metabolism, which plays an important role in regulating translation initiation. In conclusion, the current study reveals the importance of methylations in ribosome biogenesis and fidelity of translation initiation. It also strongly suggests a co-evolution of the metabolism and translation apparatus giving adaptive advantage to the cells where presence of methionine in the environment can be a signal to initiate translation with methionine initiator tRNA.

Orientador: Fabiana Fantinatti-Garboggini / Dissertação (mestrado) - Universidade Estadual de Campinas, Instituto de Biologia / Made available in DSpace on 2018-08-18T12:39:46Z (GMT). No. of bitstreams: 1 Mantovani_CristinaKampus_M.pdf: 1137189 bytes, checksum: 2db8a522e2d39a0ae80a5e302d4d79e8 (MD5) Previous issue date: 2011 / Resumo: Nas últimas décadas um grande número de compostos de interesse biotecnológico, como por exemplo, citotoxinas, agentes antifúngicos, antimicrobianos, antivirais e anticancerígenos têm sido isolados de esponjas marinhas. Entretanto, estudos comprovam que, em muitos casos, os compostos ativos desses animais são oriundos de micro-organismos associados, que podem compor até 60% do volume tecidual das esponjas. A presente proposta teve por objetivo a caracterização taxonômica da diversidade de bactérias cultiváveis associadas às esponjas coletadas no litoral norte do estado de São Paulo, Brasil, e a avaliação da atividade antimicrobiana a partir de extratos orgânicos brutos dessas bactérias. Um total de 86 bactérias foi recuperado das esponjas Axinella corrugata, Dragmacidon reticulata, Chelonaplysilla erecta e Petromica citrina utilizando diferentes meios de cultivo. A diversidade das bactérias foi caracterizada utilizando dados de morfologia, ARDRA (Amplified Ribossomal Restriction Analysis) e sequenciamento do gene RNA ribossomal 16S, cuja análise permitiu a identificação de membros pertencentes aos filos Proteobacteria, Actinobacteria, Bacteroidetes e Firmicutes num total de 15 gêneros distintos. O gênero Pseudovibrio foi o único presente em todas as esponjas amostradas, e os gêneros Bacillus, Ruegeria, Vibrio, Staphylococcus e Erythrobacter estavam presentes em mais de uma esponja. A esponja Dragmacidon reticulata apresentou a maior diversidade bacteriana, englobando oito diferentes gêneros, dentre eles, um representate do gênero Cyclobacterium, o qual até onde se sabe, foi isolado pela primeira vez de uma esponja marinha. O gênero Bacillus esteve presente em três esponjas, mas na Petromica citrina, endêmica do Brasil, o gênero ficou representado em 74% dos isolados obtidos. Este estudo foi o primerio relato sobre a diversidade de bactérias cultiváveis da esponja Petromica critrina. Todos os isolados foram avaliados quanto à presença ou ausência dos fragmentos dos genes PKS (Polyketide Synthases) e NRPS (Non Ribossomal Peptide Synthetases), visando à investigação do potencial biotecnológico das bactérias, e mais da metade delas apresentaram pelo menos um dos genes estudados. Uma triagem da atividade antimicrobiana utilizando o método da difusão em bloco de ágar demonstrou que 21 isolados foram promissores para produção de antimicrobianos. Destes isolados foram obtidos os extratos orgâncios brutos, os quais foram testados quanto à determinação da concentração inibitória mínima contra oito micro-organismos indicadores. Um total de 13 extratos orgânicos brutos, em sua maioria respresentantes do gênero Bacillus, demonstraram ação contra o micro-organismo Bacillus subtilis ATCC 6051 e um deles demonstrou ação contra o micro-organismo Escherichia coli ATCC 11775. A numerosa inibição de estirpes de Bacillus por outros Bacillus sugere que a atividade possa ser gerada por bacteriocinas, polipeptídeos produzidos pela via ribossomal que atuam na inibição de crescimento de grupos próximos de micro-organismos. Sua possível função no meio ambiente é prover vantagem seletiva através da eliminação de um competidor relativamente próximo. Ainda, um representante do gênero Exiguobacterium apresentou atividade antimicrobiana contra B. subtilis, resultado este não descrito até o presente na literatura / Abstract: In recent decades a large number of compounds of biotechnological interest, such as cytotoxins, antifungal, antimicrobial, antiviral and anticancer substances have been isolated from marine sponges, however, studies show that, in many cases, the active compounds are actually produced by associated microorganisms, which can comprise up to 60% of the volume of sponge tissue. This proposal aimed to characterize the taxonomic diversity of culturable bacteria associated with sponges collected in the northern coast of São Paulo, Brazil, and to evaluate the antimicrobial activity from crude organic extracts of these bacteria. A total of 86 bacteria were recovered from sponges the Axinella corrugata, Dragmacidon reticulata, Petromica citrina and Chelonaplysilla erecta using different culture media. The diversity of bacteria was characterized using data from morphology, ARDRA (Amplified Ribossomal Restriction Analysis) and sequencing of 16S ribosomal RNA gene, whose analysis allowed the identification of members belonging to the phyla Proteobacteria, Actinobacteria, Bacteroidetes and Firmicutes, in a total of 15 distinct genera. The genus Pseudovibrio was the only one present in all sponges sampled, and the genera Bacillus, Ruegeria, Vibrio, Staphylococcus and Erythrobacter were present in more than one sponge sampled. The sponge Dragmacidon reticulata showed the highest bacterial diversity, encompassing eight different genera, among which the genus Cyclobacterium, which, as far as is known, was first isolated from a marine sponge. The genus Bacillus was present in three sponges, but in Petromica citrina, endemic to Brazil, the genus accounted for 74% of the isolates. This study was the first report on the diversity of culturable bacteria from the sponge Petromica critrina. All isolates were evaluated for the presence or absence of NRPS (non ribossomal peptide synthetases) and PKS (polyketide synthase) genes in order to investigate the biotechnological potential of bacteria, and over half of the isolates had at least one of these genes. A screening of antimicrobial activity using the diffusion agar disk method showed that 21 isolates were promising for the production of antibiotics. Crude organic extracts from these isolates were produced and tested against eight indicator microorganisms to determine the minimum inhibitory concentration (MIC). A total of 13 crude organic extracts, most of the genus Bacillus, showed inhibitory activity against the microorganism Bacillus subtilis ATCC 6051, and one of them showed activity against the microorganism Escherichia coli ATCC 11775. The large inhibition of Bacillus strains to other Bacillus strains suggests that the activity can be generated by bacteriocins produced through ribosomal polypeptides that inhibit close groups of microorganisms. Its possible role in the environment is to provide a selective advantage by eliminating a relatively close competitor. Still, a representative of the genus Exiguobacterium showed antimicrobial activity against B. subtilis, which was not described in the literature up to date / Mestrado / Microbiologia / Mestre em Genética e Biologia Molecular

Delta subunit of bacterial RNA pol and its role in regulation of gene expression in B. subtilis. In this work I focus on regulation of eubacterial gene expression. First, I describe recent knowledge about a key stage of gene expression - transcription, focusing on regulation of trancription iniciation via small effector molecules (guanosine tetraphosphate, initiating nucleoside triphosphate) that are important for the regulation of ribosomal RNA. Second, in the experimental part of my work, I focus on the role of the _ protein, a subunit of RNA polymarase in gram positive bacteria, in transcription iniciation and its effects on regulation of RNA polymerase by the concentration of initiating nucleoside triphosphates.

Teleost fishes (Teleostei) encompass more than half of the extant vertebrate biodiversity. Their genomes display considerable plasticity and flexibility, going hand in hand with polyploidization events and repetitive DNA dynamics. Teleosts also display a striking diversity in mechanisms of sex determination and differentiation. The aim of the present thesis was to study the mechanisms underlying the karyotype and sex chromosome differentiation through cytogenetic mapping of repetitive DNA (by fluorescence in situ hybridization, FISH) in selected representatives of the African annual killifishes of the genus Nothobranchius (Teleostei: Nothobranchiidae). Nothobranchius spp. evolved a unique adaptation to freshwater temporary water pools whose existence is limited to periods of rainy season in African savannahs. Recent diversification, allopatric speciation in non-overlapping generations and small-sized populations together with known cytogenetics collectively suggest fast dynamics of chromosomal changes in Nothobranchius killifishes. The study took advantage of the availability of i) more populations of several closely related species for analysis and ii) data on specific repetitive DNA composition in selected Nothobranchius genomes as revealed by RepeatExplorer analysis. My work showed considerable...