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

Bacterial Competition Reveals Differential Regulation of the pks Genes by Bacillus subtilis

Bacillus subtilis is adaptable to many environments in part due to its ability to produce a broad range of bioactive compounds. One such compound, bacillaene, is a linear polyketide/nonribosomal peptide. The pks genes encode the enzymatic megacomplex that synthesizes bacillaene. The majority of pks genes appear to be organized as a giant operon (>74 kb from pksC-pksR). In previous work (P. D. Straight, M. A. Fischbach, C. T. Walsh, D. Z. Rudner, and R. Kolter, Proc. Natl. Acad. Sci. U. S. A. 104:305–310, 2007, doi:10.1073/pnas.0609073103), a deletion of the pks operon in B. subtilis was found to induce prodiginine production by Streptomyces coelicolor. Here, colonies of wild-type B. subtilis formed a spreading population that induced prodiginine production from Streptomyces lividans, suggesting differential regulation of pks genes and, as a result, bacillaene. While the parent colony showed widespread induction of pks expression among cells in the population, we found the spreading cells uniformly and transiently repressed the expression of the pks genes. To identify regulators that control pks genes, we first determined the pattern of pks gene expression in liquid culture. We next identified mutations in regulatory genes that disrupted the wild-type pattern of pks gene expression. We found that expression of the pks genes requires the master regulator of development, Spo0A, through its repression of AbrB and the stationary-phase regulator, CodY. Deletions of degU, comA, and scoC had moderate effects, disrupting the timing and level of pks gene expression. The observed patterns of expression suggest that complex regulation of bacillaene and other antibiotics optimizes competitive fitness for 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

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

In vivo and in vitro studies of the lrpC-topB operon of Bacillus subtilis

Tesis doctoral inédita. Universidad Autónoma de Madrid, Facultad de Ciencias, Departamento de Biología Molecular. Fecha de lectura: 16-11-201

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