Thesis advisor: Marc Snapper / Thesis advisor: Steven Bruner / Peptide natural products have diverse, elaborate scaffolds and are important leads in the development of new drugs. A complete understanding of the natural biosynthetic pathways of these compounds can improve chemical syntheses and boost bioengineering efforts. There are two classes of peptide natural products: ribosomal and nonribosomal peptides. Ribosomally produced and posttranslationally modified peptides (RiPPs) are produced by the ribosome using the 20 canonical amino acids and undergo extensive tailoring to yield the active natural products. Nonribosomal peptides (NRPs) are assembled through an enzyme dependent system and can incorporate over 500 different amino and acyl building blocks to impart complexity. These peptides can also undergo additional tailoring to further modify the core peptide. The microviridins are a class of RiPPs that are modified by two ATP dependent ligases to create a total of three macrocyclic bonds. We have solved the three dimensional protein structures of each of these ligases to establish the mechanism of substrate recognition and cyclization. Vancomycin is a NRP that contains five nonproteinogenic aromatic amino acids that are necessary for biological activity. One of these amino acids is derived from a polyketide pathway and undergoes a four-electron oxidation by a cofactor independent dioxygenase, DpgC. We have solved the structure of this enzyme and have established a radical mechanism. We have investigated this mechanism using synthetic probes and mutagenesis. We have examined O₂ binding using molecular dynamics and mutagenesis. NRPs are synthesized by the multidomain, modular nonribosomal peptide synthetases (NRPSs) in an enzyme templated, ATP-dependent manner. We have synthesized domain specific probes to study the structures and mechanisms of these pathways. Our continued work will provide the insight necessary to manipulate these pathways to provide biologically active compounds. / Thesis (PhD) — Boston College, 2013. / Submitted to: Boston College. Graduate School of Arts and Sciences. / Discipline: Chemistry.

L’ilot génomique pks de Escherichia coli et d’autres Enterobacteriaceae code des synthases depolycétides et de peptides non ribosomaux qui permettent l’assemblage d’un composé hybride polycétidepeptidenon ribosomal putative. Ce composé nommé colibactine induit des cassures double-brin de l’ADN descellules eucaryotes.La machinerie enzymatique codée par l’ilot pks comporte une protéine essentielle ClbP, atypique dansce type d’ilot. Nous avons montré que ClbP possède une partie N-terminale catalytique et périplasmique, et unepartie C-terminale associée à la membrane cytoplasmique. La structure cristalline de ClbP et des expériences demutagenèse ont révélé un site actif à sérine et des caractéristiques structurales originales, qui sont compatiblesavec une activité peptidase, confirmée par des analyses biochimiques. Dix homologues de ClbP ont été identifiésin silico dans des ilots génomiques de synthases de peptides non ribosomaux d’espèces bactériennes proches etéloignées. Tous les homologues testés ont présenté une promiscuité fonctionnelle avec ClbP. ClbP est donc leprototype d’une nouvelle sous-famille de peptidases, qui sont probablement impliquées dans la maturation decomposés peptidiques non ribosomaux.Par ailleurs, nous avons réalisé deux études épidémiologiques sur la prévalence de l’ilot pks dansl’espèce E. coli dans deux contextes physiopathologiques, l’urosepsis et les cancers coliques et rectaux. L’ilotpks était significativement associé aux souches issues d’urosepsis comparé à des souches commensales, et auxsouches issues de biopsies de tumeurs coliques comparé à des souches commensales ou issues de biopsies detumeurs rectales, de diverticuloses et de lésions iléales de maladie de Crohn. / The pks genomic island of Escherichia coli and other Enterobacteriaceae encodes polyketide andnonribosomal peptide synthases that build a putative hybrid PK-NRP compound. This compound designatedColibactin induces DNA double-strand breaks in eukaryotic cells.The pks-encoded enzymatic machinery comprises an essential protein ClbP, atypical for this type ofgenomic islands. We report that ClbP harbors a catalytic and periplasmic N-terminal part, and a C-terminal partassociated to the cytoplasmic membrane. ClbP crystal structure and mutagenesis experiments revealed a serineactivesite and original structural features, which are compatible with peptidase activity confirmed bybiochemical assays. Ten ClbP homologs were identified in silico in NRPS-encoding genomic islands of closeand distant-related bacterial species. All tested ClbP homologs showed functional promiscuity with ClbP. ClbPis therefore a prototype of a new subfamily of peptidases, which are probably involved for the maturation ofNRP compounds.Furthermore, we undertook two epidemiological studies on the prevalence of pks island in E. coli in twopathophysiology contexts; urosepsis and colorectal cancers. The pks island was significantly associated withurosepsis strains compared to commensal strains, and strains isolated from biopsies of colon tumors comparedwith commensal strains or strains isolated from biopsies of rectal tumors, diverticulosis and ileal lesions ofCrohn disease.

The increasing spectrum of multidrug-resistant bacteria has caused a major global public health concern, necessitating the discovery of novel antimicrobial agents. Additionally, recent advancements in the use of microbial cells for the scalable production of industrial enzymes has encouraged the screening of new environments for efficient microbial cell factories. The unique ecological niche of the Red Sea points to the promising metabolic and biosynthetic potential of its microbial system. Here, ten sequenced Bacilli strains, that are isolated from microbial mat and mangrove mud samples from the Red Sea, were evaluated for their use as platforms for protein production and biosynthesis of bioactive compounds. Two of the species (B.paralicheniformis Bac48 and B. litoralis Bac94) were found to secrete twice as much protein as Bacillus subtilis 168, and B. litoralis Bac94 had complete Tat and Sec protein secretion systems. Additionally, four Red Sea Species (B. paralicheniformis Bac48, Virgibacillus sp. Bac330, B. vallismortis Bac111, B. amyloliquefaciens Bac57) showed capabilities for genetic transformation and possessed competence genes. More specifically, the distinctive biosynthetic potential evident in the genomes of B. paralicheniformis Bac48 and B. paralicheniformis Bac84 was assessed and compared to nine available complete genomes of B. licheniformis and three genomes of B. paralicheniformis. A uniquely-structured trans-acyltransferase (trans-AT) polyketide synthase/nonribosomal peptide synthetase (PKS/NRPS) cluster in strains of this species was identified in the genome of B. paralicheniformis 48. In total, the two B. paralicheniformis Red Sea strains were found to be more enriched in modular clusters compared to B. licheniformis strains and B. paralicheniformis strains from other environments. These findings provided more insights into the potential of B. paralicheniformis 48 as a microbial cell factory and encouraged further focus on the strain’s metabolism at the system level. Accordingly, a draft metabolic model for B. paralicheniformis Bac48 (iPARA1056) was reconstructed, refined, and validated using growth rate and growth phenotypes under different substrates, generated using high-throughput Phenotype Microarray technology. The presented studies indicate that several of the isolated strains represent promising chassis for the development of cell factories for enzyme production and also point to the richness of their genomes with specific modules of secondary metabolism that have likely evolved in Red Sea Bacilli due to environmental adaptation.

Microbial natural products have been a source for new drugs for many decades and are unrivaled in their capacity to generate not only future therapeutic agents, but also providing key agents for agricultural and industrial use. LC-MS/MS based metabolomic tools and technologies have been developed that can rapidly dereplicate nonribosomal peptides and statistically identify related congeners in an automated nontargeted process from complex natural product extracts with nanogram sensitivity. This data-base search approach is designed to handle linear, cyclic and cyclic-branched nonribosomal peptides from proteinogenic and nonproteinogenic amino acids without genomic data or traditional bioactivity directed fractionation. Chemometric work-flows combined with a comprehensive metabolomic guided discovery strategy were used to profile the chemical space of a diverse collection of understudied fungal endophytes from fruiting plants. This approach allowed for the prioritization of unique isolates and for the focused discovery, isolation and characterization of distinct outlier metabolites by LC-SPE, 1D and 2D NMR, HRMS and single crystal X-ray analysis. These metabolomic tools and strategies have led to the discovery and characterization of 35 new and over 40 known natural products, many of which are biologically active. This thesis with enabling metabolomic tools and novel discoveries has demonstrated the utility of these analytical methodologies as an effective strategy for the untargeted discovery of new natural products from bacteria and endophytic fungi. / Thesis / Doctor of Philosophy (PhD)

A presença de antibióticos no ecossistema representa um sério risco à saúde humana e animal em virtude do desenvolvimento crescente de resistência bacteriana. Uma vez que a maioria dos antibióticos é persistente à degradação biológica, os processos oxidativos avançados são apontados como uma das tecnologias mais efetivas para decompor esses compostos em águas residuárias. A bacitracina zíncica (Bc-Zn) é um potente antibiótico constituído por uma mistura complexa de peptídeos não-biodegradáveis, conjugados ao zinco. Apesar de ser um antibiótico amplamente consumido na medicina humana e animal, é preocupante a escassez de estudos que investigam sua degradação e destino ambiental. O presente trabalho analisou a degradação da Bc-Zn através dos processos de fotólise direta e UV/H2O2 em diferentes condições de radiação UVC e concentração inicial de H2O2. Os parâmetros cinéticos rendimento quântico da fotólise, constantes cinéticas de pseudo-primeira ordem e constante cinéticas de segunda ordem foram satisfatoriamente estimados pela modelagem do sistema fotoquímico experimental. Os resultados revelaram que a fotólise direta permitiu degradar todos os congêneres da mistura de Bc-Zn nas maiores doses de radiação UVC empregadas. No entanto, não houve remoção de TOC após 120 minutos de irradiação. A adição de H2O2 acelerou substancialmente a fotodegradação do antibiótico, apresentando constantes cinéticas de pseudo-primeira ordem uma ordem de grandeza superiores às obtidas por fotólise direta. Além disso, remoção considerável de até 71% do TOC foi alcançada. A análise estatística demonstrou que a radiação UV foi um fator bem mais significativo para a fotodegradação da Bc-Zn em relação à concentração inicial de H2O2, sendo as melhores condições do processo alcançadas para a maior taxa específica de emissão de fótons (1,11×10-5 Einstein L-1 s-1). Ensaios biológicos com soluções tratadas por fotólise direta e UV/H2O2 indicaram remoção completa da atividade antimicrobiana residual, ainda que os produtos da fotodegradação tenham se mostrado não-biodegradáveis. Análises de toxicidade indicaram que o metal zinco presente no antibiótico é responsável pela a toxicidade no micro-organismo-teste Vibrio fischeri. Estudos adicionais devem ser realizados para identificar os sub-produtos formados, bem como para investigar a degradação da Bc-Zn em efluentes industriais reais. / The presence of antibiotics in ecosystems represents a serious risk to human and animal health, caused by the increase in bacterial resistance. Since most antibiotics resist to biological degradation, advanced oxidation processes are pointed out as the most effective technologies for degrading these compounds in wastewater. Zinc bacitracin (Bc-Zn) is a potent antibiotic with a complex mixture of non-biodegradable peptides conjugated to zinc. Despite being a widely used antibiotic in human and animal medicine, the scarcity of studies dealing with its degradation and environmental fate is a matter of concern. In this work, Bc-Zn degradation by direct photolysis and the UV/H2O2 process was investigated for different UVC radiation conditions and initial H2O2 concentrations. Kinetic parameters, namely the photolysis quantum yield, pseudo-first order kinetic constants and second-order kinetic constants, were satisfactorily estimated from experimental data by modeling the photochemical system. The results showed that all the congeners of the Bc-Zn mixture were photolyzed at the highest UVC doses applied, while no TOC removal was observed after 120 minutes of irradiation. The addition of H2O2 substantially accelerated Bc-Zn photodegradation, with pseudo-first order kinetic constants of one order of magnitude higher than those observed under direct photolysis. In addition, a remarkable removal of up to 71% of TOC was achieved. Statistical analyses showed that UV radiation had a much more important effect on Bc-Zn photodegradation in comparison with initial H2O2 concentration, with the best process conditions achieved for the highest specific photon emission rate (1.11×10-5 Einstein L-1 s-1). Biological assays carried out with the solutions treated by direct photolysis and UV/H2O2 revealed no residual antimicrobial activity, though photodegradation products remained non-biodegradable. In addition, toxicity analyses indicated that the zinc metal present in the antibiotic is responsible for the toxic effect on the test microorganism Vibrio fischeri. Finally, further studies should be performed to identify the by-products formed and to investigate Bc-Zn degradation in real industrial wastewater.

A presença de antibióticos no ecossistema representa um sério risco à saúde humana e animal em virtude do desenvolvimento crescente de resistência bacteriana. Uma vez que a maioria dos antibióticos é persistente à degradação biológica, os processos oxidativos avançados são apontados como uma das tecnologias mais efetivas para decompor esses compostos em águas residuárias. A bacitracina zíncica (Bc-Zn) é um potente antibiótico constituído por uma mistura complexa de peptídeos não-biodegradáveis, conjugados ao zinco. Apesar de ser um antibiótico amplamente consumido na medicina humana e animal, é preocupante a escassez de estudos que investigam sua degradação e destino ambiental. O presente trabalho analisou a degradação da Bc-Zn através dos processos de fotólise direta e UV/H2O2 em diferentes condições de radiação UVC e concentração inicial de H2O2. Os parâmetros cinéticos rendimento quântico da fotólise, constantes cinéticas de pseudo-primeira ordem e constante cinéticas de segunda ordem foram satisfatoriamente estimados pela modelagem do sistema fotoquímico experimental. Os resultados revelaram que a fotólise direta permitiu degradar todos os congêneres da mistura de Bc-Zn nas maiores doses de radiação UVC empregadas. No entanto, não houve remoção de TOC após 120 minutos de irradiação. A adição de H2O2 acelerou substancialmente a fotodegradação do antibiótico, apresentando constantes cinéticas de pseudo-primeira ordem uma ordem de grandeza superiores às obtidas por fotólise direta. Além disso, remoção considerável de até 71% do TOC foi alcançada. A análise estatística demonstrou que a radiação UV foi um fator bem mais significativo para a fotodegradação da Bc-Zn em relação à concentração inicial de H2O2, sendo as melhores condições do processo alcançadas para a maior taxa específica de emissão de fótons (1,11×10-5 Einstein L-1 s-1). Ensaios biológicos com soluções tratadas por fotólise direta e UV/H2O2 indicaram remoção completa da atividade antimicrobiana residual, ainda que os produtos da fotodegradação tenham se mostrado não-biodegradáveis. Análises de toxicidade indicaram que o metal zinco presente no antibiótico é responsável pela a toxicidade no micro-organismo-teste Vibrio fischeri. Estudos adicionais devem ser realizados para identificar os sub-produtos formados, bem como para investigar a degradação da Bc-Zn em efluentes industriais reais. / The presence of antibiotics in ecosystems represents a serious risk to human and animal health, caused by the increase in bacterial resistance. Since most antibiotics resist to biological degradation, advanced oxidation processes are pointed out as the most effective technologies for degrading these compounds in wastewater. Zinc bacitracin (Bc-Zn) is a potent antibiotic with a complex mixture of non-biodegradable peptides conjugated to zinc. Despite being a widely used antibiotic in human and animal medicine, the scarcity of studies dealing with its degradation and environmental fate is a matter of concern. In this work, Bc-Zn degradation by direct photolysis and the UV/H2O2 process was investigated for different UVC radiation conditions and initial H2O2 concentrations. Kinetic parameters, namely the photolysis quantum yield, pseudo-first order kinetic constants and second-order kinetic constants, were satisfactorily estimated from experimental data by modeling the photochemical system. The results showed that all the congeners of the Bc-Zn mixture were photolyzed at the highest UVC doses applied, while no TOC removal was observed after 120 minutes of irradiation. The addition of H2O2 substantially accelerated Bc-Zn photodegradation, with pseudo-first order kinetic constants of one order of magnitude higher than those observed under direct photolysis. In addition, a remarkable removal of up to 71% of TOC was achieved. Statistical analyses showed that UV radiation had a much more important effect on Bc-Zn photodegradation in comparison with initial H2O2 concentration, with the best process conditions achieved for the highest specific photon emission rate (1.11×10-5 Einstein L-1 s-1). Biological assays carried out with the solutions treated by direct photolysis and UV/H2O2 revealed no residual antimicrobial activity, though photodegradation products remained non-biodegradable. In addition, toxicity analyses indicated that the zinc metal present in the antibiotic is responsible for the toxic effect on the test microorganism Vibrio fischeri. Finally, further studies should be performed to identify the by-products formed and to investigate Bc-Zn degradation in real industrial wastewater.