Some specialist microbes can deploy a range of mechanisms to cause disease on one or more host plant species. To identify entirely new classes of pathogenicity and virulence factors, a bioinformatics-reverse genetics approach has been applied to a plant pathogen where near complete genomic sequence information was available. A genomic region was identified on chromosome 1 of the important cereal pathogen Fusarium graminearum that contains a significant grouping of homologues of known virulence genes. Targeted deletion of these genes revealed a role for the neutral trehalase (NTH1) and protein kinase A regulatory subunit (PKAR) genes in the rate of disease symptom spread by F. graminearum, in addition to the previously reported SNF1 Ser/Thr protein kinase and STE7 MAP kinase kinase genes. Subsequent investigation of further genes at this locality revealed the presence of a gene, here named Fusarium graminearum Contributor to Virulence 1 (FCV1), which represent a novel class of gene required for a full rate of symptom spread. Targeted deletion of FCV1 led to a reduced rate of disease development by F. graminearum on wheat ears and Arabidopsis floral tissue, but did not affect trichothecene mycotoxin production. The fcv1 deletion mutant also exhibits altered hyphal growth, reduced asexual sporulation and altered sensitivity to oxidative and osmotic stress. In the complemented strain, wild-type traits were completely or partially restored. This micro-region of < 40 kb containing these five important genes represents a novel type of gene cluster containing genes required for a full rate of disease development. This micro-region is located in a genomic region of low recombination, is highly conserved in three other Fusarium species, but is less conserved in other plant pathogenic species. The micro-region is not defined by a distinct GC content or coordinated gene expression patterns, nor is it flanked by highly repetitive sequences. This micro-region is distinct from the previously identified fungal and bacterial virulence gene clusters and the clustered biosynthesis-associated genes required to synthesis metabolites which contribute to pathogenicity. This method for novel disease development-contributing gene identification is applicable to any sequenced pathogen species.

This PhD thesis presents results of a research focussed on the evolution of phototrophy in the bacterial order Rhodobacterales with a special regard to its haloalkaliphilic representatives. The photoheterotrophic bacterium Rhodobaca barguzinensis alga05 was used as an organism of choice. Its phylogeny, genome organization, and metabolic potential was characterized. The main result of the thesis is that phototrophy is a genuine trait among the haloalkaliphilic representatives of the Rhodobacter-Rhodobaca group inside the Rhodobacterales clade.

Nodularia spumigena é uma espécie cianobacteriana conhecida como produtora da hepatotoxina nodularina. Essa cianotoxina é uma potente e irreversível inibidora de proteínas fosfatases da família serina/treonina (PP1 e PP2A) de células eucarióticas e é uma promotora tumoral e suspeita carcinogéna. Além da nodularina, a N. spumigena também é produtora de outros peptídeos não ribossômicos, tais como espumiginas, aeruginosinas e anabaenopeptinas. O primeiro relato de N. spumigena formadora de florações no Brasil ocorreu em 2011 em tanques de produção de camarões no Rio Grande, RS, e estimulou o interesse na obtenção de informações sobre o seu genoma e potencial biossíntético. Dessa forma, a objetivo deste estudo foi avaliar os aspectos genômicos e funcionais da linhagem Nodularia spumigena CENA596 isolada de um tanque de produção de camarões de Rio Grande. Para isso, uma cultura da linhagem N. spumigena CENA596 foi submetida a um tratamento com hipoclorito de sódio (2%) para eliminação de contaminantes e o DNA extraído das células tratadas foi sequenciado na plataforma MiSeq e analisado com ferramentas genômicas. O sequenciamento e a montagem do seu genoma originaram 291 sequências contíguas com percentual GC de 41,19 e tamanho total de 5.189.679 pb. A análise filogenética baseada na sequência do gene que codifica o 16S rRNA agrupou a linhagem CENA596 com outras de N. spumigena da Austrália e América do Norte. Na árvore filogenômica construída com as sequências concatenadas de 31 proteínas, a linhagem brasileira CENA596 agrupou-se com valor de reamostragem de 100% com a N. spumigena CCY9414 originária do mar Báltico. As análises comparativas entre os genomas dessas duas linhagens indicaram um grande número de genes compartilhados, os quais estão relacionados principalmente ao metabolismo primário das células. Por outro lado, foram encontrados genes específicos para cada uma delas que estão envolvidos em respostas celulares a estresses oxidativos, patógenos e antibióticos. A mineração do genoma da N. spumigena CENA596 revelou 13 agrupamentos gênicos hipoteticamente relacionados à síntese de metabólitos secundários, a maioria dos quais mostrou similaridade significativa com agrupamentos conhecidos. As análises químicas confirmaram a produção de duas variantes de nodularina, espumigina, namalida, aeruginosina e aminoácidos tipo micosporina, e uma variante de geosmina. A linhagem brasileira N. spumigena CENA596 mostrou-se capaz de produzir uma variedade significante de moléculas bioativas e seu genoma revelou-se ser consideravelmente conservado em relação ao genoma da linhagem CCY9414, a qual é conhecida por causar grandes florações tóxicas no Mar Báltico / Nodularia spumigena is a cyanobacterial species known as a producer of the hepatotoxin nodularin. This cyanotoxin is a potent and irreversible inhibitor of eukaryotic cell serine/threonine protein phosphatases (PP1 and PP2A) and is a tumor promoter and suspected carcinogen. In addition to nodularin, N. spumigena is also produces other non-ribosomal peptides, such as spumigins, aeruginosines and anabaenopeptins. The first report of bloom-forming N. spumigena in Brazil occurred in 2011 in shrimp production ponds, Rio Grande, RS, and stimulated interest in obtaining information on its genome and biosynthetic potential. Thus, the objective of this study was to evaluate the genomic and functional aspects of the strain N. spumigena CENA596 isolated from a shrimp production pond of the Rio Grande. For this, a culture of the strain N. spumigena CENA596 was submitted to a treatment with sodium hypochlorite (2%) to eliminate contaminants and the DNA extracted from treated cells was sequenced in a platform MiSeq and analyzed with genomic tools. Genome sequencing and assembly resulted in 291 contiguous sequences with GC percentage of 41.19 and total size of 5,187,679 bp. Phylogenetic analysis based on the gene sequence encoding the 16S rRNA grouped the strain CENA596 with other N. spumigena from Australia and North America. In the phylogenomic tree constructed with the concatenated sequences of 31 proteins, the Brazilian strain CENA596 grouped with a bootstrap value of 100% with the N. spumigena CCY9414 originating from the Baltic sea. Comparative analyses between the genomes of these two strains indicated a large number of shared genes, which are mainly related to the primary metabolism of the cells. Otherwise, genes specific for each of the two strains were identified as involved in cellular responses to oxidative stress, pathogens and antibiotics. Genome mining revealed 13 gene clusters hypothetically related to the synthesis of secondary metabolites, most of which showed significant similarity to known clusters. Chemical analyses confirmed the production of two variants of nodularin, spumigin, namalide, aeruginosin and mycosporine-like amino acid, and one variant of geosmin. The Brazilian strain N. spumigena CENA596 was able to produce a significant variety of bioactive molecules and its genome revealed to be considerably conserved in relation to the genome of the strain CCY9414, which is known to cause large toxic blooms in the Baltic Sea

Streptomyces clavuligerus produces industrially important secondary metabolites such as cephamycin C (a &beta / -lactam antibiotic) and clavulanic acid (a potent &beta / -lactamase inhibitor). Cephamycin C is active against penicillin-resistant bacteria due to presence of methoxyl group in C-7 position of cephalosporin nucleus. Clavulanic acid is prescribed in combination with &beta / -lactams for treatment of various bacterial infections. Cephamycin C and clavulanic acid gene clusters form &beta / -lactam supercluster in S. clavuligerus genome. CcaR (Cephamycin C-Clavulanic Acid Regulator), encoded by ccaR, located in cephamycin C gene cluster, is a positive regulator of &beta / -lactam supercluster. Previous studies on cephamycin C gene cluster have used different techniques, such as S1 nuclease (Paradkar et al., 1994), Northern blot (Perez-Llarena et al., 1997), and Western blot (Alexander and Jensen, 1998) to determine expression of cephamycin C genes at mRNA level and to identify their functions at protein level, and they have studied on different parts of the cluster. Hence, a comprehensive study is needed to understand molecular mechanisms of pathway-specific regulation of cephamycin C production by S. clavuligerus. In this study, time-dependent expression levels of cephamycin C gene cluster in a ccaR-disrupted mutant and ccaR-overexpressed recombinant strain of S. clavuligerus as compared to those in the wild strain were analysed by RT-PCR and qRT-PCR. In addition, DNA-binding sequences of CcaR on cephamycin C gene cluster were examined by EMSA. The effect of ccaR disruption and overexpression on cephamycin C and clavulanic acid yields were determined by bioassay and HPLC. Three polycistronic and two monocistronic transcripts were obtained by RT-PCR. CcaR regulation showed its effect on mostly ccaR, lat, cmcI, cefD, blp and cefF expression levels. qRT-PCR data was supported by EMSA showing CcaR binding to lat, cefD&ndash / cmcI and ccaR promoters. ccaR overexpression from multi-copy recombinant plasmid resulted in significant increase in cephamycin C and clavulanic acid yields, making the respective recombinant strain as an attractive industrial strain. qRT-PCR data presented herein constitute the first that reveal the effect of CcaR activator on the expression of cephamycin C genes in a time-dependent manner.

Natural products continue to be a major chemical lead matter for drug discovery due to their diverse chemical structures and bioactivities. Clinically significant natural products include anti-cancer and anti-infective compounds and while many more of these compounds show promising bioactivity, their clinical relevance is often limited by toxicity or poor solubility. Combinatorial biosynthesis can be employed to modify existing chemical scaffolds towards reducing these limitations. To fully take advantage of these biochemical tools, it is important to understand the biosynthesis and mechanism of action of the molecules. Saccharides in glycosylated natural products provide specific interactions with cellular targets and are often crucial for a compound’s bioactivity. Genetic engineering of sugar pathways can modify glycosylation patterns leading to the diversification of natural products. Saquayamycins (SQN) H and I are cytotoxic angucycline antibiotics containing five deoxyhexoses including the rare amino sugar rednose. Elucidating the biosynthetic pathway of rednose could add to the arsenal of combinatorial biosynthesis tools for drug development. Our research goal of investigating the rednose biosynthetic pathway was pursued through two specific aims: the identification of the Streptomyces sp. KY 40-1 gene cluster involved in the biosynthesis of SQN H and I (sqn) (specific aim 1), and the validation of the proposed L-rednose biosynthetic pathway up to the glycosyl transfer through enzymatic synthesis of NDP-3,6-dideoxy-L-idosamine (specific aim 2). The sqn gene cluster revealed deoxysugar biosynthetic genes that could be used to alter glycosylation patterns to generate novel compounds while the enzymatic synthesis afforded novel genetic engineering tools to generate novel TDP-deoxysugars that could be used to diversify compounds such as aminoglycosides to circumvent resistance mechanisms. The first step to generate TDP-glucosamine enzymatically was accomplished, however later steps were unsuccessful. The aureolic acid mithramycin (MTM) was recently tested in clinical trials for Ewing sarcoma following the discovery of MTM as a potent inhibitor of the oncogenic transcription factor EWS-FLI1 present only in Ewing sarcoma cells It is understood that MTM binds the minor groove of G/C rich DNA as an Mg2+-coordinated dimer disrupting transcription of proto-oncogenes; however, the DNA recognition rules were not completely understood, making further interrogation of MTM’s DNA binding preferences necessary. This research goal of further understanding the mechanism of action for MTM was approached through two specific aims: the investigation of the dimerization of MTM (specific aim 3), and the investigation of MTM’s DNA binding preferences (specific aim 4). This work established that MTM and its biosynthetic precursor premithramycin B (PreMTM B), and several MTM analogues with modified 3-side chains: mithramycin SDK (MTM SDK), mithramycin SA tryptophan (MTM SA-Trp), and mithramycin SA alanine (MTM SA-Ala) dimerize even in the absence of DNA under physiologically relevant conditions. The study also demonstrated that modification of the 3-side chain modulates DNA binding affinity of MTM analogues, established a minimum MTM binding site on DNA, and revealed MTM DNA recognition is driven by direct (sequence) and not indirect (conformation) readout laying the foundation for subsequent research based on the interaction between MTM, DNA, and the oncogenic transcription factor EWS-FLI1 in the rational design of new MTM analogues for the treatment of Ewing sarcoma.

Nodularia spumigena é uma espécie cianobacteriana conhecida como produtora da hepatotoxina nodularina. Essa cianotoxina é uma potente e irreversível inibidora de proteínas fosfatases da família serina/treonina (PP1 e PP2A) de células eucarióticas e é uma promotora tumoral e suspeita carcinogéna. Além da nodularina, a N. spumigena também é produtora de outros peptídeos não ribossômicos, tais como espumiginas, aeruginosinas e anabaenopeptinas. O primeiro relato de N. spumigena formadora de florações no Brasil ocorreu em 2011 em tanques de produção de camarões no Rio Grande, RS, e estimulou o interesse na obtenção de informações sobre o seu genoma e potencial biossíntético. Dessa forma, a objetivo deste estudo foi avaliar os aspectos genômicos e funcionais da linhagem Nodularia spumigena CENA596 isolada de um tanque de produção de camarões de Rio Grande. Para isso, uma cultura da linhagem N. spumigena CENA596 foi submetida a um tratamento com hipoclorito de sódio (2%) para eliminação de contaminantes e o DNA extraído das células tratadas foi sequenciado na plataforma MiSeq e analisado com ferramentas genômicas. O sequenciamento e a montagem do seu genoma originaram 291 sequências contíguas com percentual GC de 41,19 e tamanho total de 5.189.679 pb. A análise filogenética baseada na sequência do gene que codifica o 16S rRNA agrupou a linhagem CENA596 com outras de N. spumigena da Austrália e América do Norte. Na árvore filogenômica construída com as sequências concatenadas de 31 proteínas, a linhagem brasileira CENA596 agrupou-se com valor de reamostragem de 100% com a N. spumigena CCY9414 originária do mar Báltico. As análises comparativas entre os genomas dessas duas linhagens indicaram um grande número de genes compartilhados, os quais estão relacionados principalmente ao metabolismo primário das células. Por outro lado, foram encontrados genes específicos para cada uma delas que estão envolvidos em respostas celulares a estresses oxidativos, patógenos e antibióticos. A mineração do genoma da N. spumigena CENA596 revelou 13 agrupamentos gênicos hipoteticamente relacionados à síntese de metabólitos secundários, a maioria dos quais mostrou similaridade significativa com agrupamentos conhecidos. As análises químicas confirmaram a produção de duas variantes de nodularina, espumigina, namalida, aeruginosina e aminoácidos tipo micosporina, e uma variante de geosmina. A linhagem brasileira N. spumigena CENA596 mostrou-se capaz de produzir uma variedade significante de moléculas bioativas e seu genoma revelou-se ser consideravelmente conservado em relação ao genoma da linhagem CCY9414, a qual é conhecida por causar grandes florações tóxicas no Mar Báltico / Nodularia spumigena is a cyanobacterial species known as a producer of the hepatotoxin nodularin. This cyanotoxin is a potent and irreversible inhibitor of eukaryotic cell serine/threonine protein phosphatases (PP1 and PP2A) and is a tumor promoter and suspected carcinogen. In addition to nodularin, N. spumigena is also produces other non-ribosomal peptides, such as spumigins, aeruginosines and anabaenopeptins. The first report of bloom-forming N. spumigena in Brazil occurred in 2011 in shrimp production ponds, Rio Grande, RS, and stimulated interest in obtaining information on its genome and biosynthetic potential. Thus, the objective of this study was to evaluate the genomic and functional aspects of the strain N. spumigena CENA596 isolated from a shrimp production pond of the Rio Grande. For this, a culture of the strain N. spumigena CENA596 was submitted to a treatment with sodium hypochlorite (2%) to eliminate contaminants and the DNA extracted from treated cells was sequenced in a platform MiSeq and analyzed with genomic tools. Genome sequencing and assembly resulted in 291 contiguous sequences with GC percentage of 41.19 and total size of 5,187,679 bp. Phylogenetic analysis based on the gene sequence encoding the 16S rRNA grouped the strain CENA596 with other N. spumigena from Australia and North America. In the phylogenomic tree constructed with the concatenated sequences of 31 proteins, the Brazilian strain CENA596 grouped with a bootstrap value of 100% with the N. spumigena CCY9414 originating from the Baltic sea. Comparative analyses between the genomes of these two strains indicated a large number of shared genes, which are mainly related to the primary metabolism of the cells. Otherwise, genes specific for each of the two strains were identified as involved in cellular responses to oxidative stress, pathogens and antibiotics. Genome mining revealed 13 gene clusters hypothetically related to the synthesis of secondary metabolites, most of which showed significant similarity to known clusters. Chemical analyses confirmed the production of two variants of nodularin, spumigin, namalide, aeruginosin and mycosporine-like amino acid, and one variant of geosmin. The Brazilian strain N. spumigena CENA596 was able to produce a significant variety of bioactive molecules and its genome revealed to be considerably conserved in relation to the genome of the strain CCY9414, which is known to cause large toxic blooms in the Baltic Sea

As the spread of antibiotic resistance threatens our ability to treat infections, avoiding the return of a pre-antibiotic era urgently requires the discovery of novel antibiotics. Actinomycetes, a family of bacteria commonly isolated from soil, are a proven source of clinically useful antibiotics. However, easily identifiable metabolites have been exhausted and the rediscovery of common antibiotics thwarts searches for rarer molecules. Sequencing of actinomycete genomes reveals that they contain far more biosynthetic gene clusters with the potential to encode antibiotics than whose products can be readily observed in the laboratory. The work presented in this thesis revolves around developing approaches to mine these previously inaccessible metabolites as a source of new antibiotics. First, I describe how inactivation of biosynthetic gene clusters for common antibiotics can uncover rare antibiotics otherwise masked in these strains. By applying CRISPR-Cas9 to knockout genes encoding nuisance antibiotics, I develop a simple strategy to reveal the hidden biosynthetic potential of actinomycete strains that can be used to discover rare or novel antibiotics. Second, I describe the use of the evolutionary history of biosynthetic gene clusters to prioritize divergent members of an antibiotic family, the glycopeptide antibiotics, that are likely to possess new biological activities. Using these predictions, I uncover a novel functional class of glycopeptide antibiotics that blocks the action of autolysins, essential peptidoglycan hydrolases required for remodelling the cell wall during growth. Finally, I apply target-directed genome mining, which makes use of target duplication as a predicted resistance mechanism within an antibiotic’s biosynthetic gene cluster. Using this approach, I discover the association of a family of gene clusters with the housekeeping protease ClpP and characterize the produced metabolite’s effect on ClpP function. These three research projects mine previously inaccessible chemical matter from a proven source of antibiotics, actinomycetes. The techniques and antibiotics described are required now more than ever to develop life-saving antibiotics capable of combatting multidrug-resistant pathogens. / Dissertation / Doctor of Philosophy (PhD) / Antibiotics are essential for treating life-threatening infections, but the rise of antibiotic resistance renders them ineffective. To treat these drug-resistant infections, new antibiotics that work in new ways are required. A family of bacteria commonly isolated from soil called Actinomycetes produce most antibiotics we use today, but it has become increasingly difficult to find new antibiotics from this source. My work describes three techniques that can be applied to actinomycetes to help overcome the challenges associated with antibiotic discovery. Specifically, these techniques guide discovery efforts by making use of regions in actinomycete genomes called biosynthetic gene clusters that often encode antibiotics. In doing so, I describe ways to uncover rare antibiotics from actinomycete strains that produce common and uninteresting antibiotics, use antibiotic family trees to discover antibiotics that work in new ways, and apply antibiotic resistance to identify biosynthetic gene clusters likely to act on a certain bacterial target.

Phytophthora root and stem rot is the most destructive soybean soil-borne disease worldwide and can be managed using soybean cultivars with genes conferring resistance to <i>Phytophthora sojae</i>. Here we show that soybean <i>Rps11</i> is an ~27-kb nucleotide-binding site leucine-rich repeat (NLR) gene that confers broad-spectrum resistance to the pathogen. This giant gene is located in a genomic region containing 12 unusually large NLR genes of a single origin and was formed by rounds of intergenic/intragenic unequal recombination that involves the promoter regions and the LRR regions. Comparison of the genomic region in the Rps11 donor line with its corresponding regions in 29 diverse soybean genomes revealed drastic regional diversification including NLR copy number variation ranging from 5 to 23, and absence of allelic copy of <i>Rps11</i> in all 29 genomes. This study highlights innovative evolution and complexity of an NLR cluster and enables precise selection of <i>Rps11</i> for cultivar improvement.

No description available.

No description available.