Diss. (sammanfattning) Uppsala : Uppsala universitet, 2006. / Härtill 4 uppsatser.

Thesis (MTech (Chemical Engineering))--Cape Peninsula University of Technology, 2009 / The main purpose of this study was to quantify the growth and oxygen mass transfer kinetic parameters of the filamentous bacterium Streptomyces coelicolor, immobilised on the external surface of a ceramic membrane in a continuously operated pressurised Membrane Gradostat Bioreactor (MGR). One of the most important and critical parameters required when studying biofilms, are the growth kinetics, as they can be utilised to model both the mass transfer and biological reactions occurring within the biofilm. Single fibre MGR's (SFMGR) were operated using a pneumatic system to supply humidified pressurised air to the extra capillary space (ECS) and pressurised growth medium to the lumen of the ceramic membrane. Two growth media; a complex growth medium, ISP2, and a defined growth medium, were tested and supplied to the lumen of the ceramic membrane in the dead-end mode

𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘮𝘺𝘤𝘦𝘴 initiate a complex developmental program during their 5-day life cycle, consisting of aerial mycelium formation and antibiotic production. Several developmental genes are involved in regulating these events, one of which is 𝘣𝘭𝘥𝘉. I have complemented a 𝘣𝘭𝘥𝘉 null mutant, which demonstrated the loss of aerial mycelium formation and antibiotic production, restoring both characteristics. This demonstrated that 𝘣𝘭𝘥𝘉 is essential for 𝘚. 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳 morphogenesis but not viability. Using a bacterial two hybrid system devised, I screened the 𝘚. 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳 genome using BldB as "bait" for binding partners of BldB. The two most compelling candidates were bbpl, a homologue of UspA in 𝘌. 𝘤𝘰𝘭𝘪, and bbp2, a homologue of SrmR in 𝘚. 𝘢𝘮𝘣𝘰𝘧𝘢𝘤𝘪𝘦𝘯𝘴. Furthermore, I have investigated these interactions biochemically by affinity chromatography to further elucidate the details involved in these interactions. Preliminary results showed three protein bands obtained at approximately 68kDa, 55kDa and 35kDa, respectively. / Thesis / Master of Science (MS)

The RamC protein is required for the production of spore-forming cells called aerial hyphae in colonies of 𝘚𝘵𝘳𝘦𝘱𝘵𝘰𝘮𝘺𝘤𝘦𝘴 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳. RamC can be detected during the period between 24 and 48 hours following spore germination however there is a dramatic drop in RamC levels thereafter. This could be explained either by the existence of an active means of RamC removal or by the fact that at later time points in the 𝘚. 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳 lifecycle non-RamC producing cells vastly outnumber RamC-producing cells. characterized a large number of 𝘣𝘭𝘥 mutants and found that most of them do not produce RamC. In the majority of the 𝘣𝘭𝘥 mutants that do produce RamC, we observed the same pattern of accumulation and loss during colony growth as in wildtype colonies. Furthermore, we identified a small number of mutants that produced RamC such that it persisted at detectable levels for a longer duration or only appeared after a substantial delay relative to the wildtype. None of these RamC-producing 𝘣𝘭𝘥 mutants was complemented by plasmids containing a cloned 𝘣𝘭𝘥𝘔, 𝘣𝘭𝘥𝘕 or 𝘳𝘢𝘮𝘙 gene, mutations in which also cause persistent or delayed RamC production. These results suggest either that there is more than one differentiated cell type within the substrate mycelium or that 𝘚. 𝘤𝘰𝘦𝘭𝘪𝘤𝘰𝘭𝘰𝘳 colonies actively rid themselves of RamC once the protein's biological function has passed. / Thesis / Master of Science (MS)

Earlier investigations indicated that the genes involved in the biosynthesis of tryptophan from anthranilic acid were located in at least two separate regions on the chromosome of Streptomyces coelicolor A3(2). Tryptophan-requiring mutants capable of utilizing indole mapped counterclockwise of hisC9 between hisC9 and proAl. Trp mutants unable to use indole in place of tryptophan mapped clockwise of hisC9 between hisC9 and ammA5. Other genetic markers, located between proAl and hisC9, include leuAl, rif'B37 and thiC2. Twenty indole-utilizing Trp mutants were mapped relative to leuAl, rifB37 and thiC2. All but two recently isolated mutants, Trp-e8 and e22, mapped between rifB37 and thiC2. Genetic and enzymatic analyses of Trp-e8 and e22 indicate that these mutants map between hisC9 and ammA5, fail to complement each other in vivo and lack indoleglycerol phosphate synthase. Two mutants, Trp-e14 and 8, mapping between hisC9 and ammA5 were assayed for their tryptophan enzymes. Trp-el4 lacks tryptophan synthase B activity. Tryptophan synthase A activity was missing and tryptophan synthase B activity was relatively weak in crude extracts of Trp-8. Anthranilate-PRPP phosphoribosyltransferase activity was not detected in crude extracts of two mutants, Trp-e6 and e10, mapping between rifB37 and thiC2. / Master of Science

Since microorganisms normally co-exist with other species in nature, they have developed complex metabolic and physiological responses as a result of such inter-species interactions. Biotic elicitation mimics the inter-species interactions in nature by introducing cell extracts, parts of cell wall, or dead cells into the culture of another species thus resulting in complex metabolic responses in the elicited microorganisms. In this thesis we report the exploitation of the interspecies interaction in order to enhance the antibiotic production by the model organism Streptomyces coelicolor. It produces four known antibiotics: actinorhodin, undecylprodigioisn, methylenomycin and the calcium dependent antibiotic. We investigated the production of actinorhodin and undecylprodigiosin only because of the lack of quantitative analytical methods for the other two. The pure cultures of S. coelicolor in a defined medium produce higher concentrations of actinorhodin compared with undecylprodigiosin. However, undecylprodigiosin is more important due to its antitumor activities. We introduced live and dead cells of E. coli, Bacillus subtilis and Staphylococcus aureus, separately, to the S. coelicolor culture. Investigations were performed on Petri dishes, shake flasks and 2 L bioreactors. The suitable amount of each elicitor bacterium was first determined based on its ability to grow in the S. coelicolor medium so that it did not overtake the growth of S. coelicolor. Growth of S. coelicolor and glucose consumption of the elicited cultures were studied and compared with those in the pure culture. Our results revealed an alteration in the antibiotic production pattern by S. coelicolor, such that undecylprodigiosin production was significantly enhanced and actinorhodin decreased. The maximum enhancement occurred in the culture elicited with the live cells of E. coli with an increase of 3.5-fold, whereas the minimum was with elicitation using S. aureus cells (2.1-fold increase). Also, a considerable suppression in the production of actinorhodin was observed upon elicitation with live cells of E. coli or S. aureus. Furthermore, another positive outcome of the elicitation was the earlier onset of undecylprodigiosin production by 24-35h compared to the pure culture of S. coelicolor. Moreover, this study showed that the dead cells of B. subtilis and S. aureus had the same elicitation effects as the live cells, contrary to the heat-killed cells of E. coli that had no such effect. Some optimisation experiments on the amount and the timing of the elicitation were performed and the optimal conditions were chosen that would increase undecylprodigiosin production. Elicitation in the bioreactor resulted in as much as six-fold increase in the production of undecylprodigiosin compared with the pure culture and approximately double that obtained in the shake flasks. The antimicrobial activities of the extracted actinorhodin and undecylprodigiosin on the elicitor bacteria were tested in agar diffusion tests. Undecylprodigiosin always inhibited the growth of the elicitor bacteria whereas actinorhodin was less effective. In addition, our results indicated that the interaction between S. coelicolor and E. coli was mediated via a molecule present in the E. coli culture, while no such evidence was found in the case of interaction with B. subtilis or S. aureus. The results showed that the elicitation with the cells of B. subtilis and S. aureus was not due to peptidoglycan or N-acetyl glucoseamine which is the constituents of the cell wall that may have been released by lyses during the culture process. Such inter-species interactions may form the basis of new strategies in the search for novel antibiotics and other bioactive compounds. They can also be used to increase the productivity of existing processes for antibiotics as it was found in this work.

The pleiotropic effect of WD-40 domain containing proteins on cellular differentiation and production of secondary metabolites in Streptomyces coelicolor WD-40 domains, also known as beta-transducin repeats, are highly conserved repeating amino acid units, which are found in a wide variety of eukaryotic proteins that have a range of different functions. In the late 1990s, the first WD-40 containing proteins were identified in prokaryotes, however the knowledge about their function is scarce. Streptomyces coelicolor is a gram-positive bacterium with complicated morphological and physiological differentiation in the course of its life cycle. The genome of Streptomyces coelicolor encodes 6 potential genes encoding proteins with WD-repeat motifs. To determine the function of two of these WD-40 genes (wdpB and wdpC), the deletion replacement mutants in both genes were prepared. Both mutants exhibited medium-dependent phenotypes, which are markedly evident on modified R3 plates. Phenotypic studies revealed that deletion of wdpB gene resulted in substantial reduction of aerial hyphae formation and reduced production of undecylprodigiosin. In addition, the hyphae of ΔwdpB mutant were unusually branched and showed the signs of precocious lysis. Delayed spore-containing hyphae were irregularly septated....

No description available.

Magíster en Ciencias de la Ingeniería, Mención Química. Ingeniera Civil en Biotecnología / El estudios de Streptomyces, bacterias Gram positivas del filo Actinobacteria y cosmopolitas (encontradas en diferentes tipos de ambientes), ha llevado al descubrimiento de un gran número de metabolitos secundarios con potencial terapéutico. Uno de los nuevos tipos de moléculas producidas por estas bacterias son los lazo péptidos. Estos se caracterizan por formar un lazo dentro de su estructura y por presentar actividad antimicrobiana, citotóxica contra diferentes líneas celulares de cáncer y actividad inhibitoria de la replicación de ciertos virus, entre otras. Nuevos estudios han hallado cepas de Streptomyces en ambientes extremos. Una de ellas es la cepa Streptomyces sp. HST28 aislada desde el Salar de Huasco en el norte chileno. Análisis de actividad realizados a este organismo arrojaron potencial antibiótico, antifúngico y citotóxico, además su genoma fue secuenciado dando paso a la realización de minería de genomas sobre su secuencia. El presente trabajo se enfocó en la identificación de los lazo péptidos presentes en Streptomyces sp. HST28 y su caracterización. Para esto se utilizó la estrategia de minería de genomas ubicando los clústers de genes de estos péptidos, luego estos clústers fueron reconstruidos de manera bioinformática. Como segunda etapa se realizó la expresión heteróloga de cada lazo péptido empleando como huésped Streptomyces coelicolor M1152 y M1154. Una vez clonados se detectó el péptido a través de espectrometría de masa y se evaluó su la actividad. Paralelamente al estudio de laboratorio se realizó una investigación in silico de cada lazo péptido creando modelos por homología de secuencia y estructurales, usando como plantilla o molde lazo péptidos ya descritos y caracterizados estructuralmente, buscando similitudes en campos electrostáticos, distribuciones de hidrofobicidad y espaciales. Se encontraron cuatro clústers de genes de lazo péptidos en el genoma de Streptomyces sp. HST28, nombrados como LP1, LP2, LP3 y LP4. Tres de estos lazo péptidos fueron clasificados como clase II y uno de clase I (LP1). La organización de sus clústers es acorde a lo encontrado en bibliografía a excepción del de LP3. Las secuencias codificantes para los cuatro péptidos se clonaron en el vector pIJ10257, que posee el promotor constitutivo ermE*. Solo LP1 y LP3 fueron clonados exitosamente en S. colelicolor M1152, el producto predicho para LP1 no fue encontrado en los clones analizados por espectrometría de masa. En la investigación in silico se logró modelar los cuatro lazo péptidos y se realizó una comparación preliminar con otras estructuras, deduciendo que es posible implementar esta metodología para tener una aproximación del comportamiento de estas moléculas.

Streptomyces coelicolor produces fatty acids for both primary metabolism and for production of the components of natural products such as undecylprodiginine. Primary metabolism makes the longer and predominantly branched-chain fatty acids, while undecylprodiginine utilizes shorter and almost exclusively straight chain fatty acids. The first step in fatty acid biosynthetic process is catalyzed by FabH (β-ketoacyl synthase III), which catalyzes a decarboxylative condensation of an acyl-CoA primer with malonyl-acyl carrier protein (ACP). The resulting 3-ketoacyl-ACP product is reduced by NADPH-dependent FabG into 3-hydroxyacyl-ACP, which is dehydrated by FabA to form enoyl-ACP. The NADH-dependent FabI (InhA) completes the cycle. Subsequent rounds of elongations in the pathways are catalyzed by the condensing enzyme FabF. For undecylprodiginine biosynthesis in S. coelicolor, homologues of the condensing enzymes (FabH and FabF) and the ACP (FabC) are encoded by redP, redR and redQ respectively in the red gene cluster. The genes encoding 3-ketoacyl-ACP reductase (FabG), 3-hydroxyacyl-ACP dehydratase (FabA), and enoyl-ACP reductase (FabI), are putatively shared between fatty acid and undecylprodigine biosynthesis, since the corresponding genes are not present within the red gene cluster of S. coelicolor. RedP is proposed to initiate biosynthesis of undecylprodiginine alkane chain by condensing an acetyl-CoA with a malonyl-RedQ, in contrast to FabH which process a broad range of acyl-CoA with malonyl-FabC. The 3-keto group of the resulting 3-ketoacyl-RedQ is then reduced to provide butyryl-RedQ, presumably by the type II FAS enzymes FabG, FabA and FabI. These enzymes would not differentiate between straight and branched-chain substrates, and have equal preference for FabC and RedQ ACPs. RedR would then catalyze four subsequent elongation steps with malonyl-RedQ, with appropriate 3-keto group processing after each step. The proposed role and substrate specificities of condensing enzymes RedP and FabH have not been investigated in S. coelicolor. The genes encoding FabG, FabA, and FabI have not been characterized in Streptomyces. Analysis of the S. coelicolor genome sequence has revealed the presence of one fabI gene (SCO1814, encoding an enoyl-ACP reductase), and three likely fabG genes (SCO1815, SCO1345, and SCO1346, encoding β-ketoacyl-ACP reductase). In the current study the substrates specificities of both RedP and FabH were determined from assays using pairings of two acyl-CoA substrates (acetyl-CoA and isobutyryl-CoA) and two malonyl-ACP substrates (malonyl-RedQ and malonyl-FabC) (FabC is a dedicated ACP for fatty acid biosynthesis and RedQ for undecylprodiginine biosynthesis in S. coelicolor). For RedP, activity was only observed with a pairing of acetyl-CoA and malonyl-RedQ. No activity was observed with isobutyryl-CoA consistent with the proposed role for RedP and the observation that acetyl CoA-derived prodiginines predominate in S. coelicolor. Malonyl-FabC is not a substrate for RedP, indicating that ACP specificity is one of the factors that permit a separation between prodiginine and fatty acid biosynthetic processes. In contrast to RedP, FabH was active with all pairings but demonstrated the greatest catalytic efficiency with isobutyryl-CoA using malonyl-FabC. Lower catalytic efficiency was observed using an acetyl-CoA and malonyl-FabC pairing consistent with the observation that in streptomycetes, a broad mixture of fatty acids are biosynthesized, with those derived from branched chain acyl-CoA starter units predominating. Diminished but demonstrable FabH activity was also observed using malonyl-RedQ, with the same preference for isobutyryl-CoA over acetyl-CoA, completing biochemical and genetic evidence that in the absence of RedP this enzyme can also play a role in prodiginine biosynthesis, producing branched alkyl chain prodiginines. The identification and characterization of both enzymes FabG and FabI was also carried out. A series of straight and branched-chain β-ketoacyl and enoyl substrates tethered to either NAC or ACP were synthesized and used to elucidate the functional role and substrate specificity of these enzymes. Kinetic analysis demonstrates that of the three S. coelicolor enzymes, SCO1815 and SCO1345 have NADPH-dependent β-ketoacyl-reductase activity, in contrast to SCO1346, which has NADH-dependent β-ketoacyl-reductase activity. Spectrophotometric assays revealed that all three FabGs are capable of utilizing both straight and branched-chain β-ketoacyl-NAC substrates. These results are consistent with FabGs role in fatty acid and undecylprodiginine biosynthesis, wherein it processes branched-chain for primary metabolism as well as straight-chain products for undecylprodiginine biosynthesis. LC/MS assays demonstrate that these FabG enzymes do not discriminate between primary metabolism ACP (FabC) and secondary metabolism ACP (RedQ) (except for SCO1345, which does not have any activity with RedQ). This relaxed substrate specificity allows these enzymes to process 3-ketoacyl-FabC substrates for fatty acid biosynthesis as well as 3-ketoacyl-RedQ substrates for undecylprodiginine biosynthesis. Similar to FabG, spectrophotometric and LC/MS assays were also carried out to elucidate the functional role and substrate specificity of S. coelicolor FabI. The kinetic analyses demonstrate that SCO1814 has NADH-dependent enoyl-ACP reductase activity. Spectrophotometric and LC/MS assays demonstrated that FabI does not differentiate between straight and branched-chain substrates, and has equal preference for FabC and RedQ ACPs. These observations provide experimental support for the hypothesis that these enzymes are shared and process the intermediates in the elongation cycle of both fatty acid and undecylprodiginine biosynthesis. In summary, these studies have demonstrated the activity of enzymes RedP, FabH, FabG and FabI (InhA) previously uncharacterized in S. coelicolor and clarified their role in fatty acid and undecylprodiginine biosynthesis.