Regulatory Characteristics of Bacillus pumilus Protease Promoters

© 2017, Springer Science+Business Media New York.Expression of extracellular protease genes of Bacilli is subject to regulation by many positive and negative regulators. Here we analyzed 5′ regulatory regions of genes encoding proteolytic proteases AprBp, GseBp, and MprBp from Bacillus pumilus strain 3–19. Gfp fusion constructs with upstream genomic regions of different lengths were created for all three genes to identify their natural promoters (regulatory regions). Our results suggest that the aprBp gene, encoding the major subtilisin-like protease, has the most extensive promoter region of approximately 445 bp, while the minor protease genes encoding glutamyl endopeptidase (gseBp) and metalloproteinase (mprBp) are preceded by promoters of 150 and 250 bp in length, respectively. Promoter analysis of PaprBp-gfpmu3 and PgseBp-gfpmu3 reporter fusion constructs in degU and spo0A mutants indicates a positive regulatory effect of DegU and Spo0A on protease expression, while the disruption of abrB, sinR, and scoC repressor genes did not significantly affect promoter activities of all protease genes. On the other hand, the expression of PaprBp-gfpmu3 and PgseBp-gfpmu3 reporters increased 1.6- and 3.0-fold, respectively, in sigD-deficient cells, indicating that the prevention of motility gene expression promotes protease expression. Our results indicate that all examined regulators regulated serine proteases production in B. subtilis

Evolutionary Analysis of the Bacillus subtilis Genome Reveals New Genes Involved in Sporulation

Bacilli can form dormant, highly resistant, and metabolically inactive spores to cope with extreme environmental challenges. In this study, we examined the evolutionary age of Bacillus subtilis sporulation genes using the approach known as genomic phylostratigraphy. We found that B. subtilis sporulation genes cluster in several groups that emerged at distant evolutionary time-points, suggesting that the sporulation process underwent several stages of expansion. Next, we asked whether such evolutionary stratification of the genome could be used to predict involvement in sporulation of presently uncharacterized genes (y-genes). We individually inactivated a representative sample of uncharacterized genes that arose during the same evolutionary periods as the known sporulation genes and tested the resulting strains for sporulation phenotypes. Sporulation was significantly affected in 16 out of 37 (43%) tested strains. In addition to expanding the knowledge base on B. subtilis sporulation, our findings suggest that evolutionary age could be used to help with genome mining

Caractérisation du transfert d'ICEBs1 en fonction de la formation de biofilm

La résistance aux antibiotiques est un problème pour le milieu clinique et agricole générant de plus en plus d’inquiétude à l’échelle mondiale. Ce problème est exacerbé par le manque de développement de nouvelles classes d’antibiotiques au cours des trois dernières décennies. Les résistances aux antibiotiques sont principalement acquises par des éléments génétiques mobiles. Parmi ces éléments, on retrouve les éléments intégratifs et conjugatifs (ICEs). Un autre aspect intervenant lors d’infections bactériennes est la formation de biofilm. En effet, une majorité des infections chroniques sont causées par des bactéries retrouvées sous forme de biofilm. Il a déjà été démontré que le biofilm était considéré comme un environnement favorisant le transfert de plasmides conjugatifs, mais son impact réel n’a jamais été quantifié. Par ailleurs, le transfert des ICEs en biofilm n’a jamais été caractérisé et la manière dont le biofilm aurait un effet sur le transfert conjugatif n’a jamais été élucidée. Dans cette étude, pour répondre à ces questions, nous avons utilisé le microorganisme modèle Bacillus subtilis, une rhizobactérie à Gram positif. B. subtilis forme un biofilm robuste en plus d’être l’hôte d’un ICE nommé ICEBs1. Nous avons pu démontrer que la formation de biofilm amenait environ 100 à 10 000 fois plus de transfert d’ICEBs1, que ce soit en milieu solide ou liquide. De plus, l’activation du transfert d’ICEBs1 et de la formation de biofilm arrivent simultanément dans le temps. Nous avons également pu démontrer que l’augmentation du transfert d’ICEBs1 était due à un meilleur contact cellule-cellule, médié par la production de matrice extracellulaire. Cette production de matrice extracellulaire doit provenir de la bactérie réceptrice afin d’avoir un transfert optimal d’ICEBs1. De plus, nous avons montré qu’ICEBs1 était capable d’infiltrer un biofilm déjà établi. En somme, ces travaux nous permettent de mieux comprendre le transfert d’ICEs en présence de biofilms. Comme nos travaux démontrent un transfert élevé d’ICEs en présence de biofilms, les connaissances acquises vont aussi être applicables à l’étude de différents ICEs chez les bactéries à Gram positif. En effet, ces ICEs sont historiquement connus comme ayant des taux de transferts très faibles comparativement à ceux retrouvés chez les bactéries à Gram négatif

Neurotoxin regulation and the temperature stress response in Clostridium botulinum

Clostridium botulinum is a dangerous foodborne pathogen that forms highly resistant endospores and the extremely potent botulinum neurotoxin. Whereas endospores enable the survival and transmission of the organism in many harsh environments, the botulinum neurotoxin blocks neurotransmission and causes the severe and potentially lethal disease botulism in humans and animals. Both traits play an important role in the life of this pathogen and temporally overlap in C. botulinum batch cultures, suggesting common regulation. However, the co-regulation of sporulation and neurotoxin synthesis and the significance of both traits during stress conditions have not been examined in detail. This study focused on the role of the master regulator of sporulation Spo0A in neurotoxin synthesis in Group II C. botulinum type E strains, which lack the well-known neurotoxin gene regulator BotR. Furthermore, the role of the two traits during heat stress in Group I C. botulinum ATCC 3502 was investigated. Group II C. botulinum strains represent the main hazard in minimally-processed anaerobically-packaged foods relying on cold storage, as Group II strains are able to grow and produce toxin at temperatures as low as 3 °C. Cold tolerance mechanisms are scarcely known in psychrotrophic Group II C. botulinum. Studying the mechanisms required for adaption and growth at low temperatures is crucial to counter the safety hazards caused by this dangerous pathogen. The role of a two-component signal transduction system in the cold tolerance of Group II C. botulinum type E was investigated. A better understanding of neurotoxin gene regulation and mechanisms contributing to cold tolerance might enable the development of measures to reduce the risk of botulism outbreaks. The sporulation transcription factor Spo0A was shown to control the initiation of sporulation and neurotoxin synthesis in C. botulinum type E Beluga. The non-sporulating spo0A mutants produced drastically less neurotoxin than the wild-type strain, and in vitro binding assays showed that Spo0A binds to a putative Spo0A-binding box (CTTCGAA) upstream of the neurotoxin gene operon, suggesting the direct activation of neurotoxin synthesis by Spo0A. The sequence and location of the putative Spo0A-binding box is conserved among C. botulinum type E strains, and analysis of spo0A mutations in two more type E strains (K3 and 11/1-1) affirmed the important role of Spo0A in neurotoxin type E synthesis. Spo0A is the first neurotoxin regulator reported in C. botulinum type E strains that lack the neurotoxin gene activating alternative sigma factor BotR. However, co-regulation of sporulation and neurotoxin synthesis is probably not limited to type E strains. Analysis of heat shocked continuously growing C. botulinum type A ATCC 3502 cultures revealed simultaneous downregulation of both traits in response to heat stress, which was affirmed by decreased toxin synthesis and abolished sporulation in batch cultures incubated at 45 °C compared to cultures incubated at 39 °C. This suggests that both traits might be co-regulated in C. botulinum type A, possibly also via Spo0A, which was significantly downregulated after heat shock, whereas the expression of genes encoding the known neurotoxin gene transcription activators BotR and CodY was unaffected or even upregulated during the heat shock response. While heat stress had a negative effect on sporulation and neurotoxin synthesis, the expression of genes related to motility was induced after heat shock. This suggests that motility is the preferred choice when facing elevated temperatures, probably to search for environments with less harmful temperatures. In order to grow and produce neurotoxin at cold temperatures, bacteria have to sense low temperatures and adjust their metabolism and structure for efficient growth in cold environments. We identified the first two-component signal transduction system (TCS) induced during the cold-shock response and needed for efficient growth at low temperatures in psychrotrophic C. botulinum type E. Expression of the TCS genes clo3403 (encoding a histidine kinase for sensing) and clo3404 (encoding a DNA regulator for responding) was increased after cold shock and prolonged compared to the expression pattern observed at the optimal growth temperature, suggesting that the TCS CLO3403/CLO3404 is needed for cold adaptation. Furthermore, inactivation of the TCS genes clo3403 and clo3404 resulted in impaired growth with significantly reduced maximum growth rates at low temperatures but not at the optimum temperature compared to wild-type growth. The important role of the TCS CLO3403/CLO3404 for cold tolerance in C. botulinum type E was confirmed by successful complementation of the mutations. In summary, this study demonstrated that sporulation and neurotoxin synthesis are co-regulated via the master regulator of sporulation Spo0A in C. botulinum type E and that heat stress has a negative effect on both traits in C. botulinum ATCC 3502, which also suggests common regulation in type A strains. Sporulation-dependent neurotoxin synthesis might play a central role in the life of this dangerous pathogen and represents a key intervention point for control. Finally, we identified a TCS (CLO3403/CLO3404) important for cold adaptation in psychrotrophic C. botulinum type E, which represents a major hazard in anaerobically-packaged chilled foods.Clostridium botulinum on vaarallinen ruokamyrkytysbakteeri, joka muodostaa kestäviä itiöitä ja tuottaa voimakkainta luonnollista myrkkyä, botulinumneurotoksiinia. Itiöiden avulla bakteeri selviytyy epäsuotuisissa olosuhteissa ja päätyy elintarvikkeisiin tai elimistöön. Neurotoksiini nieltynä tai elimistössä muodostuessaan estää hermoimpulssin aiheuttaen ihmisille ja eläimille henkeä uhkaavan velttohalvauksen, botulismin. C. botulinumia esiintyy yleisesti elintarvikkeiden raaka-aineissa ja lämmönkestävät itiöt selviävät useimmista nykyaikaisista elintarvikkeiden prosesseista. Osa C. botulinum -kannoista kasvaa ja tuottaa toksiinia jopa jääkaappilämpötiloissa. Nämä kannat muodostavat merkittävän terveysriskin pakatuissa kylmäsäilytetyissä elintarvikkeissa. Itiömuodostus ja toksiinituotanto ovat keskeisiä elementtejä C. botulinumin biologiassa ja epidemiologiassa. Mekanismit, joilla bakteeri säätelee itiömuodostusta ja toksiinituotantoa, tunnetaan huonosti. On myös epäselvää, miten erilaiset ympäristöolosuhteet vaikuttavat C. botulinumin itiöitymiseen ja toksiinituotantoon ja miten bakteeri sopeutuu erilaisiin olosuhteisiin esimerkiksi elintarvikkeiden valmistuksen ja säilytyksen aikana. Väitöskirjatyössä osoitettiin, että itiöivillä bakteereilla itiöitymisen pääsäätelijänä yleisesti tunnettu Spo0A-proteiini säätelee myös C. botulinumin neurotoksiinituotantoa. Spo0A on ensimmäinen neurotoksiinituotannon säätelytekijä, joka on kuvattu E-tyypin toksiinia tuottavilla C. botulinum –kannoilla. Näitä kantoja esiintyy yleisesti Suomen vesistöissä ja niiden kaloissa, ja ne aiheuttavat botulismiriskiä ihmiselle tyhjiöpakattujen lämminsavukalatuotteiden välityksellä. Lisäksi väitöskirjatyössä löydettiin kaksoiskomponenttijärjestelmiin kuuluva säätelytekijäpari, jonka avulla bakteeri todennäköisesti aistii ympäristön lämpötilaa ja sopeutuu kasvamaan ja tuottamaan toksiinia ja itiöitä kylmässä. Korkeaan lämpötilaan bakteerin havaittiin sopeutuvan aktivoimalla liikkumista edesauttavia tekijöitä ja vähentämällä itiöitymistä ja neurotoksiinituotantoa. C. botulinumin kasvua, sopeutumista ja toksiinituotantoa säätelevien solutason mekanismien ja ympäristötekijöiden selvittäminen avaa uusia mahdollisuuksia hallita bakteerin aiheuttamia terveysriskejä elintarvikkeissa ja elimistössä

Evaluation and method development for the biosynthesis of microbial lipopeptides by bacillus species

Microbial lipopeptides are secondary metabolites produced by bacteria and single-celled microorganisms. They consist of a cyclic or linear peptide chain linked to a lipid residue. Due to their high-foaming biosurfactant properties, they have various industrial applications such as in detergents, food emulsifiers, bioremediation, and enhanced oil recovery. Additionally, they possess other functional properties such as antifungal activity, making them an environmentally friendly alternative to synthetic fertilizers and fungicides. Bacillus species produce cyclic lipopeptides known for their potent antifungal activity, which makes them a potential source of bio-fungicides in agriculture. However, the production titer of wild-type Bacillus species does not meet industrial needs. Thereby, genetic modification of producer strains and bioprocess engineering can help increase the production of lipopeptides. Nevertheless, the regulation and basis of biosynthesis for Bacillus lipopeptides are still not completely understood, and ongoing research aims to enhance their production. In general, three main lipopeptide families, including surfactins, iturins, and fengycins are produced by different Bacillus species. Among these, surfactin as the strong biosurfactant is the most extensively studied lipopeptide produced by Bacillus species. The focus of this doctoral thesis was mainly to evaluate the biosynthesis of iturin and fengycin families, which are strong antimicrobial lipopeptides produced by Bacillus subtilis and Bacillus velezensis. This involved developing strains through genetic engineering and enhancing the lipopeptide titer by evaluating the cultivation medium. Initially, the entire genome of the bacteria used in this thesis was examined in terms of lipopeptide biosynthesis, and the structure and yield of the different produced lipopeptides were analyzed. Regarding the lipopeptide producer derivatives of the domesticated laboratory model strain B. subtilis 168 and B. subtilis 3NA, a spore deficient strain appropriate for bioreactor cultivation, surfactin is the lipopeptide with the highest yield, while plipastatin which is a member of fengycin family, is produced in lower quantities. In the present thesis, the biosynthesis of plipastatin by B. subtilis BMV9 as the lipopeptide producer derivative of strain 3NA was evaluated. The study aimed to convert BMV9 to a constitutive plipastatin mono-producer strain. In this sense, overexpressing plipastatin biosynthesis operon using the stronger constitutive Pveg promoter led to a five-fold increase in plipastatin production. Interestingly, it was observed that deletion of srfAA-AD operon in BMV9 and the constructed constitutive plipastatin producer strain has not improved plipastatin production. Therefore, it can be stated that presumably the biosynthesis of plipastatin may be positively influenced in a post-transcriptional manner by the surfactin synthetase or some of its subunits. However, the regulatory mechanism behind this effect remained unknown and requires further research. Another attempt to enhance the plipastatin biosynthesis in strain BMV9 was repairing the degQ expression. One main genome characterization of strains with B. subtilis 168 and 3NA background is that the pleiotropic degQ gene expression, which is known to have a positive effect on plipastatin biosynthesis, is silenced due to a mutation in the promoter area. However, while repair of degQ expression in BMV9 increased the plipastatin production, combination of both repaired degQ expression and promoter exchange (Ppps::Pveg) has not significantly increased the plipastatin yield. To further evaluate the impact of degQ expression on surfactin and plipastatin biosynthesis, two strains of B. subtilis were selected: JABs24, a lipopeptide producer derived from the 168 strain, and DSM10T, the wild-type strain expressing native degQ. The findings demonstrated that surfactin biosynthesis is negatively affected by DegQ-associated DegU regulation, while increased plipastatin biosynthesis is achieved in the presence of native degQ expression. In addition to production of lipopeptides, the DegU regulatory system also plays a role in the formation of secretory proteases. A comparison of extracellular protease activities between JABs24 and DSM10T showed that degQ expression led to DSM10T having five times higher protease activity than JABs24. Interestingly, production of extracellular proteases has not affected the stability of both plipastatin and surfactin during cultivation, suggesting that lipopeptides are less targeted by extracellular proteases. The identification of proficient wild-type strains is critical to the advancement of bio-fungicide in agriculture. Therefore, the subsequent approach of this thesis centered on the production of microbial lipopeptide by wild-type B. velezensis strains. Here, the lipopeptide productivity and antifungal ability of B. velezensis UTB96 was higher than B. velezensis FZB42, as a well-established strain for biocontrol of plant pathogens in agriculture. Furthermore, addition of certain amino acids stimulated lipopeptide production, and using a bioreactor system resulted in enhancement of lipopeptide production, especially iturin A by UTB96. Overall, the doctoral thesis evaluates the biosynthesis of antimicrobial lipopeptides produced by B. subtilis and B. velezensis. The study involves genetic engineering such as promoter exchange, deletion of genes involved in competing biosynthetic pathways and cultivation medium development with amino acid supplementation to enhance the lipopeptide titer. The thesis also identifies B. velezensis UTB96 as a promising candidate for further research to be used as a wild-type antifungal agent in agriculture.Mikrobielle Lipopeptide sind Sekundärmetabolite, die von Bakterien und einzelligen Mikroorganismen produziert werden. Sie bestehen aus einer zyklischen oder linearen Peptidkette, die an einen Lipidrest gebunden ist. Aufgrund der stark schäumenden Eigenschaften von Biotensiden, werden diese in der Industrie in verschiedenen Bereichen eingesetzt, wie z. B. in Reinigungsmitteln, Lebensmittele-mulgatoren, bei der Bioremediation und der verbesserten Ölgewinnung. Darüber hinaus besitzen sie weitere funktionelle Eigenschaften, wie z. B. eine antimykotische Wirkung, was sie zu einer sicheren und umweltfreundlichen Alternative zu synthetischen Düngemitteln und Pestiziden macht. Bacillus‐Arten produzieren zyklische Lipopeptide, die für ihre starke antimykotische Wirkung gegen Phytopathogene bekannt sind, was sie zu einer potenziellen Quelle für Bio‐Fungizide in der Landwirtschaft macht. Der Produktionstiter von Wildtyp‐Bacillus‐Arten entspricht jedoch nicht dem industriellen Bedarf. Durch genetische Veränderung und Bioverfahrenstechnik kann die Produktion von Lipopeptiden gesteigert werden. Dennoch sind die Regulierung und die Grundlagen der Biosynthese von Bacillus‐Lipopeptiden noch nicht vollständig geklärt, und die laufende Forschung zielt auf die Optimierung ihrer Produktion ab. Es ist bekannt, dass Bacillus‐Arten drei Hauptlipopeptidfamilien produzieren, darunter Surfactin, Iturin und Fengycin. Unter diesen ist Surfactin das am besten untersuchte Biotensid. Im Mittelpunkt dieser Doktorarbeit stand die Untersuchung der Biosynthese der Iturin- und Fengycin‐Familien. Diese stark antimikrobiellen Lipopeptide werden von Bacillus subtilis und Bacillus velezensis produziert. Dazu wurden gentechnisch modifizierte Stämme entwickelt und der Lipopeptid‐Titer durch Verbesserung des Kultivierungsmediums erhöht. Zunächst wurde das gesamte Genom der in dieser Arbeit verwendeten Bakterien im Hinblick auf die Lipopeptidbiosynthese untersucht, und die Struktur und Ausbeute der verschiedenen produzierten Lipopeptide wurden analysiert. Bei den lipopeptidproduzierenden Derivaten des domestizierten Labormodellstammes B. subtilis 168 und B. subtilis 3NA, einem für die Bioreaktorkultivierung geeigneten nicht sporulierenden Stamm, ist Surfactin das Lipopeptid mit der höchsten Ausbeute. Im Gegensatz dazu wird Plipastatin, ein Mitglied der Fengycinfamilie, in geringeren Mengen produziert. In der vorliegenden Arbeit wurde die Biosynthese von Plipastatin durch B. subtilis BMV9, dem Lipopeptidproduzenten‐Derivat des Stammes 3NA, untersucht. Der Regulationsmechanismus, der diesem Effekt zugrunde liegt, ist jedoch noch unerforscht und bedarf weiterer Forschung. Ein weiterer Versuch, die Plipastatin‐ Biosynthese im Stamm BMV9 zu verbessern, war die Reparatur der degQ‐Expression. Eine wesentliche Genomcharakterisierung von Stämmen mit B. subtilis 168 und 3NA-Hintergrund besteht darin, dass die pleiotrope degQ‐Genexpression, von der bekannt ist, dass sie sich positiv auf die Plipastatinbiosynthese auswirkt, aufgrund einer Mutation im Bereich des Promotors inaktiv ist. Während jedoch die Reparatur der degQ‐Expression in BMV9 die Plipastatinproduktion erhöhte, konnte die Kombination aus reparierter degQ-Expression und Promotoraustausch (Ppps::Pveg) die Plipastatinausbeute nicht signifikant steigern. Um die Auswirkungen der degQ‐Expression auf die Surfactin‐ und Plipastatin‐Biosynthese weiter zu untersuchen, wurden zwei B. subtilis‐Stämme ausgewählt: JABs24, ein von Stamm 168 abgeleiteter Lipopeptidproduzent, und DSM10T, der Wildtyp‐Stamm, der natives degQ exprimiert. Die Ergebnisse zeigten, dass die Surfactin‐Biosynthese durch die DegQ‐assoziierte DegU‐Regulierung negativ beeinflusst wird, während die Plipastatin‐ Biosynthese bei Vorhandensein einer nativen DegQ‐Expression gesteigert wird. Neben der Produktion von Lipopeptiden spielt das DegU‐Regulationssystem ebenfalls eine Rolle bei der Bildung von sekretorischen Proteasen. Interessanterweise hat die Produktion von extrazellulären Proteasen die Stabilität von Plipastatin und Surfactin während der Kultivierung nicht beeinträchtigt, was darauf hindeutet, dass Lipopeptide kaum von extrazellulären Proteasen angegriffen werden. Die Identifizierung tauglicher Wildtyp‐Stämme ist für die Weiterentwicklung von Biofungiziden in der Landwirtschaft von entscheidender Bedeutung. Daher konzentrierte sich der weitere Ansatz dieser Arbeit auf die Produktion von mikrobiellen Lipopeptiden durch Wildtyp‐B. velezensis‐Stämme. Dabei waren die Lipopeptidproduktivität und die antimykotische Fähigkeit von B. velezensis UTB96 höher als die von B. velezensis FZB42, einem etablierten Stamm zur Biokontrolle von Pflanzenpathogenen in der Landwirtschaft. Außerdem stimulierte die Zugabe bestimmter Aminosäuren die Lipopeptidproduktion, und die Verwendung eines Bioreaktorsystems führte zu einer Steigerung der Lipopeptidproduktion, insbesondere von Iturin A durch UTB96. Zusammenfassend wird in der Dissertation die Biosynthese antimikrobieller Lipopeptide untersucht, die von B. subtilis und B. velezensis produziert werden. Die Studie umfasst gentechnische Eingriffe wie den Austausch von Promotoren, die Deletion von Genen, die an konkurrierenden Biosynthesewegen beteiligt sind, und die Entwicklung von Kultivierungsmedien mit Aminosäuresupplementierung zur Steigerung des Lipopeptid‐ Titers. In der Dissertation wird B. velezensis UTB96 als vielversprechender Kandidat für weitere Forschungen identifiziert, um als Wildtyp‐Antimykotikum in der Landwirtschaft eingesetzt zu werden

Complex Organization and Dynamic Regulation of the pks Gene Cluster in Bacillus subtilis

The pks genes are the largest antibiotic- encoding gene cluster in Bacillus subtilis and encode the Pks enzymatic complex that produces bacillaene. Bacillaene plays important roles in the fitness of B. subtilis during competition with other bacterial species. In this dissertation, I investigate the regulatory mechanisms used by B. subtilis to control the expression of the pks genes and the production of bacillaene. First, I focus on understanding the transcriptional regulatory network that coordinates the activation of the pks genes. My results indicate that multiple transcriptional regulators, in particular the stationary phase regulators Spo0A and CodY, coordinate the control of the pks gene activation. Also, cells dedicated to the formation of biofilms and spores but not motility induce the expression of the pks genes. I discuss these findings in light of their roles during bacterial competition. I also identified multiple regulatory elements along the pks genes. Promoters upstream of pksB, pksC and pksS are active during vegetative growth while a promoter upstream of pksG is active only during spore formation. The activity of the pksG promoter is exclusive to the nascent spores and not the mother cells. In addition to promoters, a cis-regulatory element at the intergenic region of pksC and pksD promotes readthrough of transcription terminators along the pks genes. Finally, I focus on the function of PksA, previously presumed to regulate the pks genes. I have found that PksA is not involved in the control of the pks gene expression. Instead, PksA negatively regulates the expression of ymcC. My data suggests that YmcC is not involved in bacillaene production but, consistent with structural prediction, I have found that YmcC is a membrane protein produced during sporulation. I hypothesize the function of YmcC during spore maturation or germination and propose experiments to elucidate this role. In general, this dissertation contributes to the understanding of pks gene regulation and its implications in the competitive fitness of B. subtilis. This work also provides a model for the activation of Type I trans-AT PKSs encoded in gene clusters with similar organization to the pks genes