Thesis advisor: Steven D. Bruner / Thesis advisor: Larry W. McLaughlin / Nonribosomal peptides are therapeutically important natural products produced through pathways that utilize large multimodular enzymes, termed nonribosomal peptide synthetases (NRPSs). Central to the assembly line methodology, the monomer building blocks and the growing polymer chain are covalently linked to dedicated peptidyl carrier protein domains as phosphopantetheinyl thioesters. Although structures of multidomain NRPS fragments have been solved recently, the active conformation of the carrier domains with their attached phosphopantetheinyl arms has not been determined. Significant conformational changes in carrier domains are likely to occur as the domains shuttle peptidyl phosphopantetheinyl thioesters between the active sites of the partner domains. This thesis focuses on the application of the synthetic isosteric non-hydrolyzable CoA analogs to manipulate carrier domain geometry of NRPS assemblies through. The synthetic conjugates are designed to deliver an inhibitor moiety to a domain of interest. Using this strategy, various complexes have been designed to direct the phosphopantetheinyl arm to active sites of adenylation domains and thioesterase domains in catalytically relevant conformations. The structurally restrained multidomain NRPS assemblies are useful for elucidating the complex structure and mechanism of NRPSs. An X-ray crystal structure of a peptidyl carrier-thioesterase NRPS didomain fragment from enterobactin synthetase has been solved with a phosphopantetheinyl analog which forms a cross-link between the two domains. This structure provides, for the first time, detailed insights into the phosphopantetheinyl arm interaction with an NRPS partner domain, as well as an active confirmation of a mutidomain NRPS in the holo-form. In addition, the hydrolytically stable CoA analogs have been successfully used as probes in the structural and mechanistic study of a CoA-utilizing enzyme DpgC, a unique cofactor-independent dioxygenase involved in vancomycin biosynthesis. / Thesis (PhD) — Boston College, 2009. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

No description available.

Strain MS14, exhibiting antifungal activity, was classified to belong to Burkholderia contaminans. Occidiofungin produced by strain MS14 is an octapeptide dedicated to a broad range of antifungal activities of the bacterium. The 58.2-kb genomic fragment containing 18 open reading frames (ORFs), named occidiofungin (occ) gene cluster, is required for occidiofungin production. Putative proteins encoded by five nonribosomal peptide synthetase genes (occA – occE) of the gene cluster were predicted to contain the catalytic modules responsible for the biosynthesis of occidiofungin. Transcription of all the ORFs identified in the region except ORF1 and ORF16 was regulated by both ambR1 and ambR2, the LuxR-type regulatory genes located at the left border of the cluster. The functional ambR1 gene was essential for transcription of ambR2, and constitutive expression of ambR2 did not restore the phenotype of the mutant MS14GG44(ambR1::nptII). Sequence analysis revealed that the occ gene cluster shared high similarity (99% nucleotide coverage and 91% identity) to an uncharacterized DNA region of B. ambifaria strain AMMD. The gene cluster was not found in other Burkholderia strains available in GenBank (nucleotide coverage < 24%). Analysis of G+C composition and prediction using “IslandPick” indicate that the occ gene cluster has possibly been horizontally transferred between bacteria. In addition, the absence of the gene cluster in clinical strains of Burkholderia indicates that occidiofungin is not required for potential human pathogenesis. The findings have provided insights into the development of antifungal medicines and agricultural fungicides based on occidiofungin.

O espaço intergênico (IGS) juntamente com suas subunidades flanqueadoras (cpcB) e (cpcA) do operon do ficocianina foi usado para identificar linhagens de cianobactérias. Dentro do domínio Bacteria somente as cianobactérias possuem o operon da ficocianina e a região cpcBA-IGS é suficientemente variável para diferenciar linhagens desses microrganismos. No presente estudo 25 linhagens de cianobactérias isoladas de diversos locais brasileiros foram caracterizadas usando a seqüência cpcBA-IGS. DNA genômico foi extraído das ordens Chroococcales (oito linhagens), Oscillatoriales (duas linhagens), Nostocales (onze linhagens) e Stigonematales (quatro linhagens). Os oligonucleotídeos iniciadores PC&#946;F/PC&#945;R, específicos para a seqüência cpcBA-IGS, foram usados para amplificar fragmentos de DNA de aproximadamente 685 pb. Os produtos da PCR foram clonados, seqüenciados e as seqüências foram comparadas pela análise BLAST. Todas as seqüências de Microcystis e também as seqüências de Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 e Gloeotrichia UFV-B2 mostraram identidades com seqüências do GenBank. Entretanto, nenhuma identidade foi encontrada para as seqüências restantes. As relações filogenéticas das seqüências de cpcBA-IGS foram investigadas junto com outras seqüências de cianobactéria do Genbank usando a análise “Neighbour Joining". A topologia da árvore foi congruente com outras árvores de cianobactérias, com exceção de todas as seqüências sem identidades no GenBank, as quais formaram um agrupamento separado. Os dados das seqüências de cpcBA-IGS analisadas confirmam que as cianobactéria heterocitadas formam um grupo monofilético. Estudos anteriores realizados com linhagens de cianobactéria mostraram que estes microrganismos são uma fonte rica de produtos naturais. No presente estudo conduzido com 59 linhagens de cianobactérias, sendo a maioria isolada de ambientes brasileiros, isto foi confirmado. Para alcançar esse objetivo, dois conjuntos de iniciadores degenerados foram usados para produzir seqüências amplificadas por PCR das sintetases de peptídeos não-ribossômicos (NRPSs), e de sintases policetídeos (PKSs) modulares, as quais são enzimas multifuncionais envolvidas na produção de produtos naturais. O sistema híbrido NRPS/PKS também foi amplificado por PCR usando uma combinação de iniciadores de NRPS e de PKS. Essa abordagem molecular mostrou a presença de genes de NRPS e de PKS em 93% e 81% linhagens de cianobactérias, respectivamente. Genes de NRPS/PKS foram encontrados em 87% das cianobactérias examinadas. Numa tentativa de atribuir funções a oito fragmentos de PKS identificados por PCR, estas seqüências foram clonadas, seqüenciadas e analisadas filogeneticamente. As seqüências de PKSs da Microcystis aeruginosa NPCD1 e Fischerella CENA62 mostraram correlação com a síntese de sideróforo e de microcistina, respectivamente. Todas as 59 linhagens foram analisadas para a produção do microcistinas e 20 linhagens apresentaram resultados positivos. Para a maioria das linhagens potencialmente produtoras de microcistinas os produtos de PCR esperados de NRPS, PKS e NRPS/PKS foram amplificados. A produção de sideróforos foi testada em 28 linhagens e somente cinco produziram resultados positivos. Em três linhagens produtoras de sideróforos todos os três sistemas moleculares analisados estavam presentes. Estes resultados serão altamente valiosos na exploração futura de cada peptídeo dessas cianobactérias e para a elucidação da bioatividade de tais produtos naturais. / The intergenic spacer (IGS) together with its flanking subunits &#61538; (cpcB) and &#61537; (cpcA) of the phycocyanin operon has been used to identify cyanobacterial strains. Within the Bacteria domain only cyanobacteria present phycocyanin operon and the cpcBA-IGS region is variable enough to differentiate strains of these microorganisms. In the present study 25 cyanobacterial strains isolated from several Brazilian locations were characterized using the cpcBA-IGS sequence. Genomic DNA was extracted from the orders Chroococcales (eight strains), Oscillatoriales (two strains), Nostocales (eleven strains) and Stigonematales (four strains). The primers PC&#946;F/PC&#945;R targeting the cpcBA-IGS sequence were used to amplify DNA fragments of approximately 685 bp. The PCR products were cloned, sequenced and the sequences were compared by BLAST analysis. All Microcystis sequences and also sequences from Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 and Gloeotrichia UFV-B2 showed identities with sequences from GenBank. However, no identities were found for the remaining sequences. Phylogenetic relationships of the cpcBA-IGS sequences were investigated together with other cyanobacterial sequences from Genbank using the Neighbour Joining analysis. The tree topology was congruent with previous cyanobacterial trees, except for all sequences with no identities in the GenBank, which formed a separated cluster. The cpcBA-IGS sequences analysis data confirm that heterocyte-forming cyanobacteria are a monophyletic group. Previous studies carried out with cyanobacterial strains showed that these microorganisms are a rich source of natural products. This has been confirmed in the present study conducted with 59 cyanobacterial strains, with the majority of them isolated from Brazilian environment. To reach this goal, two sets of degenerate primers were used to generate PCR amplification sequences of nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), which are multifunctional enzymes implicated in natural products production. Also, NRPS/PKS hybrid system was PCR amplified by using a combination of NRPS and PKS primers. This molecular approach revealed the presence of NRPS and PKS genes in 93% and 81% cyanobacterial strains, respectively. NRPS/PKS genes were found in 87% of cyanobacteria examined. In an attempt to attribute functions to eight PCR identified PKS fragments, these sequences were cloned, sequenced and phylogenetically analyzed. PKSs sequences of Microcystis aeruginosa NPCD1 and Fischerella CENA62 showed correlation with the synthesis of siderophore and microcystin, respectively. All 59 strains were analyzed for microcystin production and 20 strains presented positive results. For the majority of potentially producing-microcystin strains expected PCR products of NRPS, PKS and NRPS/PKS were amplified. The siderophores production was tested in 28 strains and only five gave positive results. In three producing-siderophore strains all three molecular systems analyzed were present. These results will be highly valuable for further exploring each of these cyanobacterial peptides and for elucidating the bioactivity of such natural products.

Heterocycles play an important role in many biological processes and are widespread among natural products. Oxazole-containing natural products possess a broad range of bioactivities and are of great interest in the pharmaceutical and agrochemical industries. Herein, the biosynthetic routes to the oxazole-containing phthoxazolins and the bis(benzoxaozle) AJI9561, were investigated. Phthoxazolins A-D are a group of oxazole trienes produced by a polyketide synthase-nonribosomal peptide synthetase (PKS-NRPS) pathway in Streptomyces sp. KO-7888 and Streptomyces sp. OM-5714. The phthoxazolin pathway was used as a model to study 5-oxazole and primary amide formation in PKS-NRPS pathways. An unusually large gene cluster for phthoxazolin biosynthesis was identified from the complete genome sequence of the producer strains and various gene deletions were performed to define the minimal gene cluster. PhoxP was proposed to encode an ATP-dependent cyclodehydratase for 5-oxazole formation on an enzyme-bound N-formylglycylacyl-intermediate, and its deletion abolished phthoxazolin production. In vitro reconstitution of the early steps of phthoxazolin biosynthesis was attempted to validate the role of PhoxP, but was unsuccessful. Furthermore, Orf3515, a putative flavin-dependent monooxygenase coded by a remote gene, was proposed to hydroxylate glycine-extended polyketide-peptide chain(s) at the α-position to yield phthoxazolins with the primary amide moiety. On the other hand, an in vitro approach was employed to establish the enzymatic logic of the biosynthesis of AJI9561, a bis(benzoxazole) antibiotic isolated from Streptomyces sp. AJ9561. The AJI9561 pathway was reconstituted using the precursors 3-hydroxyanthranilic acid and 6-methylsalicylic acid and five purified enzymes previously identified from the pathway as key enzymes for benzoxazole formation, including two adenylation enzymes for precursor activation, an acyl carrier protein (ACP), a 3-oxoacyl-ACP synthase and an amidohydrolase-like cyclase. Intermediates and shunt products isolated from enzymatic reactions containing different enzyme and precursor combinations were assessed for their competence for various steps of AJI9561 biosynthesis. Further bioinformatic analysis and in silico modelling of the amidohydrolase-like cyclase shed light on the oxazole cyclisation that represents a novel catalytic function of the amidohydrolase superfamily.

O espaço intergênico (IGS) juntamente com suas subunidades flanqueadoras (cpcB) e (cpcA) do operon do ficocianina foi usado para identificar linhagens de cianobactérias. Dentro do domínio Bacteria somente as cianobactérias possuem o operon da ficocianina e a região cpcBA-IGS é suficientemente variável para diferenciar linhagens desses microrganismos. No presente estudo 25 linhagens de cianobactérias isoladas de diversos locais brasileiros foram caracterizadas usando a seqüência cpcBA-IGS. DNA genômico foi extraído das ordens Chroococcales (oito linhagens), Oscillatoriales (duas linhagens), Nostocales (onze linhagens) e Stigonematales (quatro linhagens). Os oligonucleotídeos iniciadores PC&#946;F/PC&#945;R, específicos para a seqüência cpcBA-IGS, foram usados para amplificar fragmentos de DNA de aproximadamente 685 pb. Os produtos da PCR foram clonados, seqüenciados e as seqüências foram comparadas pela análise BLAST. Todas as seqüências de Microcystis e também as seqüências de Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 e Gloeotrichia UFV-B2 mostraram identidades com seqüências do GenBank. Entretanto, nenhuma identidade foi encontrada para as seqüências restantes. As relações filogenéticas das seqüências de cpcBA-IGS foram investigadas junto com outras seqüências de cianobactéria do Genbank usando a análise “Neighbour Joining”. A topologia da árvore foi congruente com outras árvores de cianobactérias, com exceção de todas as seqüências sem identidades no GenBank, as quais formaram um agrupamento separado. Os dados das seqüências de cpcBA-IGS analisadas confirmam que as cianobactéria heterocitadas formam um grupo monofilético. Estudos anteriores realizados com linhagens de cianobactéria mostraram que estes microrganismos são uma fonte rica de produtos naturais. No presente estudo conduzido com 59 linhagens de cianobactérias, sendo a maioria isolada de ambientes brasileiros, isto foi confirmado. Para alcançar esse objetivo, dois conjuntos de iniciadores degenerados foram usados para produzir seqüências amplificadas por PCR das sintetases de peptídeos não-ribossômicos (NRPSs), e de sintases policetídeos (PKSs) modulares, as quais são enzimas multifuncionais envolvidas na produção de produtos naturais. O sistema híbrido NRPS/PKS também foi amplificado por PCR usando uma combinação de iniciadores de NRPS e de PKS. Essa abordagem molecular mostrou a presença de genes de NRPS e de PKS em 93% e 81% linhagens de cianobactérias, respectivamente. Genes de NRPS/PKS foram encontrados em 87% das cianobactérias examinadas. Numa tentativa de atribuir funções a oito fragmentos de PKS identificados por PCR, estas seqüências foram clonadas, seqüenciadas e analisadas filogeneticamente. As seqüências de PKSs da Microcystis aeruginosa NPCD1 e Fischerella CENA62 mostraram correlação com a síntese de sideróforo e de microcistina, respectivamente. Todas as 59 linhagens foram analisadas para a produção do microcistinas e 20 linhagens apresentaram resultados positivos. Para a maioria das linhagens potencialmente produtoras de microcistinas os produtos de PCR esperados de NRPS, PKS e NRPS/PKS foram amplificados. A produção de sideróforos foi testada em 28 linhagens e somente cinco produziram resultados positivos. Em três linhagens produtoras de sideróforos todos os três sistemas moleculares analisados estavam presentes. Estes resultados serão altamente valiosos na exploração futura de cada peptídeo dessas cianobactérias e para a elucidação da bioatividade de tais produtos naturais. / The intergenic spacer (IGS) together with its flanking subunits &#61538; (cpcB) and &#61537; (cpcA) of the phycocyanin operon has been used to identify cyanobacterial strains. Within the Bacteria domain only cyanobacteria present phycocyanin operon and the cpcBA-IGS region is variable enough to differentiate strains of these microorganisms. In the present study 25 cyanobacterial strains isolated from several Brazilian locations were characterized using the cpcBA-IGS sequence. Genomic DNA was extracted from the orders Chroococcales (eight strains), Oscillatoriales (two strains), Nostocales (eleven strains) and Stigonematales (four strains). The primers PC&#946;F/PC&#945;R targeting the cpcBA-IGS sequence were used to amplify DNA fragments of approximately 685 bp. The PCR products were cloned, sequenced and the sequences were compared by BLAST analysis. All Microcystis sequences and also sequences from Radiocystis fernadoi SPC736, Planktothrix mougeotii SPC788, Geitlerinema splendidum SPC923, Microchaete investiens CENA64 and Gloeotrichia UFV-B2 showed identities with sequences from GenBank. However, no identities were found for the remaining sequences. Phylogenetic relationships of the cpcBA-IGS sequences were investigated together with other cyanobacterial sequences from Genbank using the Neighbour Joining analysis. The tree topology was congruent with previous cyanobacterial trees, except for all sequences with no identities in the GenBank, which formed a separated cluster. The cpcBA-IGS sequences analysis data confirm that heterocyte-forming cyanobacteria are a monophyletic group. Previous studies carried out with cyanobacterial strains showed that these microorganisms are a rich source of natural products. This has been confirmed in the present study conducted with 59 cyanobacterial strains, with the majority of them isolated from Brazilian environment. To reach this goal, two sets of degenerate primers were used to generate PCR amplification sequences of nonribosomal peptide synthetases (NRPSs) and modular polyketide synthases (PKSs), which are multifunctional enzymes implicated in natural products production. Also, NRPS/PKS hybrid system was PCR amplified by using a combination of NRPS and PKS primers. This molecular approach revealed the presence of NRPS and PKS genes in 93% and 81% cyanobacterial strains, respectively. NRPS/PKS genes were found in 87% of cyanobacteria examined. In an attempt to attribute functions to eight PCR identified PKS fragments, these sequences were cloned, sequenced and phylogenetically analyzed. PKSs sequences of Microcystis aeruginosa NPCD1 and Fischerella CENA62 showed correlation with the synthesis of siderophore and microcystin, respectively. All 59 strains were analyzed for microcystin production and 20 strains presented positive results. For the majority of potentially producing-microcystin strains expected PCR products of NRPS, PKS and NRPS/PKS were amplified. The siderophores production was tested in 28 strains and only five gave positive results. In three producing-siderophore strains all three molecular systems analyzed were present. These results will be highly valuable for further exploring each of these cyanobacterial peptides and for elucidating the bioactivity of such natural products.

Alternaria brassicicola is a necrotrophic pathogen that causes black spot disease on virtually all cultivated Brassicas, A. brassicicola is renowned for its ability to prodigiously produce secondary metabolites. To test the hypothesis that secondary metabolites produced by A. brassicicola contribute to pathogenicity, we identified seven nonribosomal peptide synthetases (NPSs) and 10 polyketide synthases (PKSs) in the A. brassicicola genome. The phenotype resulting from knockout mutations of each PKS and NPS gene was investigated with an emphasis on discovery of fungal virulence factors. A highly efficient gene disruption method using a short linear double stranded DNA construct with minimal elements was developed, optimized, and used to functionally disrupt all NPS and PKS genes in A. brassicicola. Three NPS and two PKS genes, and one NPS-like gene appeared to be virulence factors based upon reduced lesion development of each mutant on inoculated green cabbage and Arabidopsis compared with the wild-type strain. Furthermore some of the KO mutants exhibited developmental phenotypic changes in pigmentation and conidiogenesis. To further characterize the roles of several genes of interest in A. brassicicola development and pathogenesis, the genes AbNPS2, AbPKS9, and NPS-like tmpL were selected for in-depth functional analysis. We provide substantial evidence that the AbNPS2-associated metabolite is involved in conidial cell wall construction, possibly as an anchor connecting two cell wall layers. We also characterized a biosynthetic gene cluster harboring the AbPKS9 gene and demonstrated that this cluster is responsible for the biosynthesis of depudecin, an inhibitor of histone deacetylases and a minor virulence factor. Finally, we demonstrated that a NPS-like protein named TmpL is involved in a filamentous fungi-specific mechanism for regulating levels of intracellular reactive oxygen species during conidiation and pathogenesis in both plant and animal pathogenic fungi. Collectively our results indicate that small molecule nonribosomal peptides and polyketides in A. brassicicola play diverse, but also fundamental, roles in fungal development and pathogenesis. / Ph. D.

Os microrganismos atraem considerável atenção por serem uma fonte de compostos biotecnológicos e farmacêuticos. Diversos produtos naturais peptídicos produzidos por fungos e bactérias são sintetizados por grandes enzimas, conhecidas como peptídeo sintetase não ribossômica (NRPS) e policetídeo sintase (PKS). A bioprospecção dos microrganismos isolados do solo de Terra Preta Antropogênica (TPA) da Amazônia Ocidental é de grande importância para o conhecimento deste bioma tropical. Este estudo correlacionou a presença de sideróforos e de compostos antimicrobianos produzidos pelos microrganismos isolados de TPA e dos solos adjacentes com a presença dos genes que codificam para NRPS e PKS. Linhagens bacterianas foram isoladas das amostras do solo coletadas de 10, 20 e 40 cm de profundidade. Os isolados foram cultivados em meio líquido específico por 2 dias a 28oC. Um total de 143 isolados foi testado para a atividade de sideróforo e para isso, as linhagens foram inoculadas em um meio com baixa concentração de ferro (MM9) contendo o complexo cromoazurol S-Fe3. Do total, 72 isolados apresentaram reação positiva para a produção de sideróforo. O DNA genômico dos isolados foi extraído e a amplificação por PCR foi realizada usando iniciadores específicos para NRPS e PKS. Os resultados mostraram que quinze isolados apresentaram o gene que codifica para NRPS, vinte isolados para PKS e somente dez isolados apresentaram ambos os genes. A presença de genes de NRPS e PKS em 31% dos isolados testados sugere que a produção dos sideróforos possa ocorrer pela via não ribossomal. Dois isolados foram selecionados para estudos de identificação e caracterização dos compostos. O isolado TP11 foi identificado como Pseudomonas putida através de seqüenciamento do 16S rRNA e apresentou resultado negativo para hidroxamato e catecol, sugerindo que o tipo de sideróforo não possui nenhum destes grupos funcionais. O isolado TP16 foi identificado como Pseudomonas putida e apresentou produção de sideróforo do tipo catecol e hidroxamato, sugerindo a produção de mais de um sideróforo. Além disso, esta linhagem produziu um composto antimicrobiano, com atividade de sideróforo identificado por espectrometria de massas como pseudomonina com massa molar de 330 Da. / Microorganisms have attracted considerable attention as a source for biotechnological and pharmaceutical agents. Several peptidic natural products synthesized by fungi and bacteria are assembled by large enzymes, referred as nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). Bioprospection of microorganisms isolated from Anthropological Dark Earth soil of Brazilian Occidental Amazon is of great importance to the knowledge of this tropical biome. This study aimed to correlate the presence of siderophores and antimicrobial compounds produced by microorganisms isolated from Dark Earth and adjacent soils of Brazilian Amazon with the presence of genes encoding NRPS and PKS. Bacterial strains were isolated from soil samples collected at 10, 20 and 40 cm depth. The isolates were grown in specific liquid medium for 2 d at 28oC. A total of 143 isolates were screened for siderophore activity and for this, bacterial strains were inoculated on plates containing an iron-limited medium (MM9) amended with a chromeazurol S-Fe3 complex. From the total, seventy-two isolates showed positive reaction for siderophore production. Genomic DNA of the isolates was extracted and PCR amplification was carried out using specific primers for NRPS and PKS. The results showed that fifteen isolates presented NRPS, twenty isolates presented PKS and only ten isolates showed both genes. The presence of NRPS and PKS genes in 31% of the isolates tested suggests that production of siderophores may occur by a nonribosomal pathway. Two isolates were selected for further studies. Isolate TP11 was identified as Pseudomonas putida by 16S rDNA sequencing analysis and was negative for hydroxamate and catechol, suggesting that the siderophore type has no hydroxamate- or catechol-type functional groups. The isolate TP16 was identified as Pseudomonas putida and showed the production of catechol and hydroxamate siderophore-type, suggesting the production of more than one siderophore. In addition, this strain produced an antimicrobial compound, with siderophore activity identified through mass spectrometry as pseudomonine with a molar mass of 330 Da.

Os microrganismos atraem considerável atenção por serem uma fonte de compostos biotecnológicos e farmacêuticos. Diversos produtos naturais peptídicos produzidos por fungos e bactérias são sintetizados por grandes enzimas, conhecidas como peptídeo sintetase não ribossômica (NRPS) e policetídeo sintase (PKS). A bioprospecção dos microrganismos isolados do solo de Terra Preta Antropogênica (TPA) da Amazônia Ocidental é de grande importância para o conhecimento deste bioma tropical. Este estudo correlacionou a presença de sideróforos e de compostos antimicrobianos produzidos pelos microrganismos isolados de TPA e dos solos adjacentes com a presença dos genes que codificam para NRPS e PKS. Linhagens bacterianas foram isoladas das amostras do solo coletadas de 10, 20 e 40 cm de profundidade. Os isolados foram cultivados em meio líquido específico por 2 dias a 28oC. Um total de 143 isolados foi testado para a atividade de sideróforo e para isso, as linhagens foram inoculadas em um meio com baixa concentração de ferro (MM9) contendo o complexo cromoazurol S-Fe3. Do total, 72 isolados apresentaram reação positiva para a produção de sideróforo. O DNA genômico dos isolados foi extraído e a amplificação por PCR foi realizada usando iniciadores específicos para NRPS e PKS. Os resultados mostraram que quinze isolados apresentaram o gene que codifica para NRPS, vinte isolados para PKS e somente dez isolados apresentaram ambos os genes. A presença de genes de NRPS e PKS em 31% dos isolados testados sugere que a produção dos sideróforos possa ocorrer pela via não ribossomal. Dois isolados foram selecionados para estudos de identificação e caracterização dos compostos. O isolado TP11 foi identificado como Pseudomonas putida através de seqüenciamento do 16S rRNA e apresentou resultado negativo para hidroxamato e catecol, sugerindo que o tipo de sideróforo não possui nenhum destes grupos funcionais. O isolado TP16 foi identificado como Pseudomonas putida e apresentou produção de sideróforo do tipo catecol e hidroxamato, sugerindo a produção de mais de um sideróforo. Além disso, esta linhagem produziu um composto antimicrobiano, com atividade de sideróforo identificado por espectrometria de massas como pseudomonina com massa molar de 330 Da. / Microorganisms have attracted considerable attention as a source for biotechnological and pharmaceutical agents. Several peptidic natural products synthesized by fungi and bacteria are assembled by large enzymes, referred as nonribosomal peptide synthetase (NRPS) and polyketide synthase (PKS). Bioprospection of microorganisms isolated from Anthropological Dark Earth soil of Brazilian Occidental Amazon is of great importance to the knowledge of this tropical biome. This study aimed to correlate the presence of siderophores and antimicrobial compounds produced by microorganisms isolated from Dark Earth and adjacent soils of Brazilian Amazon with the presence of genes encoding NRPS and PKS. Bacterial strains were isolated from soil samples collected at 10, 20 and 40 cm depth. The isolates were grown in specific liquid medium for 2 d at 28oC. A total of 143 isolates were screened for siderophore activity and for this, bacterial strains were inoculated on plates containing an iron-limited medium (MM9) amended with a chromeazurol S-Fe3 complex. From the total, seventy-two isolates showed positive reaction for siderophore production. Genomic DNA of the isolates was extracted and PCR amplification was carried out using specific primers for NRPS and PKS. The results showed that fifteen isolates presented NRPS, twenty isolates presented PKS and only ten isolates showed both genes. The presence of NRPS and PKS genes in 31% of the isolates tested suggests that production of siderophores may occur by a nonribosomal pathway. Two isolates were selected for further studies. Isolate TP11 was identified as Pseudomonas putida by 16S rDNA sequencing analysis and was negative for hydroxamate and catechol, suggesting that the siderophore type has no hydroxamate- or catechol-type functional groups. The isolate TP16 was identified as Pseudomonas putida and showed the production of catechol and hydroxamate siderophore-type, suggesting the production of more than one siderophore. In addition, this strain produced an antimicrobial compound, with siderophore activity identified through mass spectrometry as pseudomonine with a molar mass of 330 Da.

La colistine, antibiotique appartenant à la famille des polymyxines, est un polypeptide cyclique, cationique, ciblant les membranes bactériennes. Elle est produite par Paenibacillus polymyxa via des complexes multi-enzymatiques appelés Non-Ribosomal Peptides Synthétases (NRPS). Dans le cas de la mucoviscidose, et malgré des effets secondaires importants, la colistine est utilisée comme ultime recours pour lutter contre les bactéries Gram-négatives multirésistantes responsables d’infections pulmonaires dont Pseudomonas aeruginosa. Jusqu’ici les systèmes génétiques à l’origine de la production de la colistine étaient peu connus. Au cours de cette étude, nous avons caractérisé par LC-MS haute résolution des molécules antimicrobiennes, dont des colistines, produites par un nouveau Paenibacillus. Afin d’identifier et de cloner le cluster de gène responsable de la production de ces antibiotiques, une banque d’ADN génomique a été construite et criblée par homologie de séquence avec des systèmes de production déjà connus. Ce criblage a permis de sélectionner quatre clones d’intérêt. L’étude in silico de leurs séquences a permis d’identifier les différents modules d’un nouveau cluster NRPS qui serait à l’origine de la synthèse de variants de la colistine. À terme, cette découverte pourrait permettre de mieux contrôler la production de la colistine et d’obtenir des composés plus actifs et/ou présentant des effets secondaires amoindris. En parallèle à ce premier travail, nous avons également recherché la présence de nouvelles NRPS chez une centaine de micro-organismes issus d’une station d’étude environnementale du laboratoire (vasière intertidale). Ce travail a permis de découvrir des nouvelles séquences et d’isoler un nouveau micro-organisme producteur d’antibiotique(s). / Colistin is a cationic cyclic polypeptide antibiotic belonging to the polymyxin family and targeting bacterial membranes. It is produced by Paenibacillus polymyxa through a Nonribosomal Peptide Synthetase (NRPS) mechanism. In the context of cystic fibrosis (CF), colistin is used for the treatment of lung infections caused by multiresistant Gram-negative bacteria including Pseudomonas aeruginosa. Unfortunately, this molecule is also known for its strong side effects. So far, genetic systems controlling the production of polymyxins were little known. In this study we characterized by High-resolution LC-MS the antimicrobial molecules, including colistins, of a new Paenibacillus. A genomic library of this strain was constructed and screened to identify genes involved in the production of these antibiotics. A degenerated PCR screening was performed and allowed to select four clones in the genomic library. In silico study allowed to identify a new NRPS gene cluster responsible for the biosynthesis of colistin variants. In the future, this work might allow the harnessing of the production of colistin derived structures, more active and/or showing fewer side effects. In parallel, a second investigation was performed in order to find new NRPS genes in a collection of one hundred intertidal mudflat bacterial isolates. This work has allowed the identification of new sequences and the characterization of a new antimicrobial producing strain.