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1
Patterson, Kathryn Grace. "Gene regulation in the lac operon." Thesis, Montana State University, 2009. http://etd.lib.montana.edu/etd/2009/patterson/PattersonK0809.pdf.
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The lac operon, a jointly controlled series of genes in the bacteria E. coli, has been studied extensively since the 1940's. The lac operon genes are transcribed and then translated into proteins necessary for transport and digestion of lactose. The operon is activated in the presence of lactose after glucose, the preferred carbon source, has been expended. In this thesis, we introduce a biophysical model using the Shea-Ackers framework for modeling promoter dynamics. The model spans two scales: the inputs are biophysical parameters of molecular interactions and the result is a level of gene expression - a macroscopic behavior of the cell. We include all experimentally suggested control mechanisms into the model, even though the experimental evidence is stronger for some of these mechanisms than others. We compare our model to experimental data and explore the individual contribution of the proposed mechanisms by removing them one by one and testing the reduced model's fit to the data. Finally, we find a minimal model which faithfully represents the available data, yet includes only the minimal number of control mechanisms.
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Lazarus, Linda Ruth. "The role of FIS in tyrT transcriptional regulation." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259766.
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Vichivanives, Padungsri. "Transcriptional regulation of the Rhodobacter Capsulatus CO? fixation operons /." The Ohio State University, 2001. http://rave.ohiolink.edu/etdc/view?acc_num=osu1488205318510482.
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Nold, Niklas. "Untersuchungen zur Regulation des sol-Operons in Clostridium acetobutylicum." [S.l. : s.n.], 2008. http://nbn-resolving.de/urn:nbn:de:bsz:289-vts-65443.
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Clarke, Simon Richard. "Regulation of the bla operon in Staphylococcus aureus." Thesis, University of Oxford, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.326077.
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Min, Kyung-Tai. "Regulation of the spoIIA operon in Bacillus subtilis." Thesis, University of Oxford, 1993. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.333440.
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Low, Yuen Li. "Metal regulation of the E. faecalis efaCBA operon." Thesis, University of Bath, 2002. https://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.760823.
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Dole, Sudhanshu. "Multiple level regulation of the Escherichia coli bgl operon." [S.l. : s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963645978.
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Davies, Ian J. "Transcriptional regulation of the qua operon of Escherichia coli." Thesis, University of Southampton, 1989. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.314696.
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Yuen, Hiu-fung. "A study of the catabolite repression of the dehalogenase IVa gene of Burkholderia cepacia MBA4." Click to view the E-thesis via HKUTO, 2004. http://sunzi.lib.hku.hk/hkuto/record/B30682113.
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Stephen, Robert John. "A study of the proU operon of Salmonella typhimurium." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.318587.
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Gould, Phillip Spencer. "Regulation and role of the three chaperonin operons of Rhizobium leguminosarum." Thesis, University of Birmingham, 2002. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.273921.
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Coleman, Struan Howard. "The regulation of the rpmB,G operon of Escherichia coli." Thesis, University of Oxford, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.260156.
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Shevtsov, Mikhail Borisovich. "Structural studies of the trp operon regulation in Bacillus subtilis." Thesis, University of York, 2009. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.507488.
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Yuen, Hiu-fung, and 阮曉峰. "A study of the catabolite repression of the dehalogenase IVa gene of Burkholderia cepacia MBA4." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2004. http://hub.hku.hk/bib/B30682113.
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Steven, Blaire. "Regulation and expression of the mdh-sucCDAB operon of Sinorhizobium meliloti." Thesis, McGill University, 2003. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=80880.
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The genes encoding malate dehydrogenase (mdh), succinyl-CoA synthetase (sucCD), and subunits of 2-oxoglutarate dehydrogenase (sucAB) constitute an operon in the order mdh-sucCDAB in Sinorhizobium meliloti. Regulation of the operon was studied using beta-galactosidase gene fusions. Expression of the operon was assayed in response to the carbon source provided, and over the growth of the culture. A promoter upstream of the mdh gene was identified, and although the promoter was active in S. meliloti it was not expressed in Escherichia coli. It was demonstrated that the role of 2-oxoglutarate dehydrogenase (OGD) is minimal in symbiosis, as nodules with no OGD activity formed nodules able to fix nitrogen. Alfalfa plants inoculated with strains of S. meliloti carrying extra-chromosomal copies of the mdh gene did not show any increase in shoot dry weight compared to plants inoculated with the wild-type strain.
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Norman, Richard Albert. "Signal transduction in the regulation of operon expression in Pseudomonas aeruginosa." Thesis, University College London (University of London), 1998. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.298366.
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Wang, Baomin. "Studies on the Regulation of the Assimilatory Nitrate Reductase Operon in Azotobacter vinelandii." Diss., The University of Arizona, 2009. http://hdl.handle.net/10150/195085.
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Azotobacter vinelandii is a free-living diazotroph. This bacterium fixes atmospheric nitrogen in different environments using three genetically distinct nitrogenases. A. vinelandii is also capable of utilizing nitrate and nitrite from the environment. Nitrate is reduced sequentially into nitrite and ammonia. The assimilatory nitrate reductase and nitrite reductase are encoded by the nasAB operon. Previous genetic studies identified a number of factors that influence nasAB expression. However, the molecular mechanisms controlling the expression of nasAB are unclear.The current study was initiated to characterize the region preceding the nasAB operon which was previously implicated in its regulation and to further study the molecular mechanisms of nasAB regulation. The results confirm that nasAB is subject to multiple layers of regulation. The operon is under the control of an NtrC-dependent promoter; nitrate/nitrite induction occurs at the post-transcriptional level via antitermination within the nasAB leader region; and nitrate/nitrite induction is mediated by NasS/NasT, a sensor-antiterminator two-component regulatory system.Together, these data suggest a model for the regulation of the assimilatory nitrate reductase operon in A. vinelandii.
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Hillebrand, Annette. "Transkriptionsfaktor- und wachstumsabhängige Regulation der sieben verschiedenen ribosomalen RNA-Operons in Escherichia coli." [S.l.] : [s.n.], 2001. http://deposit.ddb.de/cgi-bin/dokserv?idn=963523325.
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Tover, Andres. "Regulation of transcription of the phenol degradation pheBB operon in pseudomonas putida /." Online version, 2005. http://dspace.utlib.ee/dspace/bitstream/10062/1343/5/tover.pdf.
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Poon, Karen Kuen Huan. "Regulation of Bacillus subtilis glucitol operon, characterization of a transcription activator, GutR." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 2000. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0018/NQ54804.pdf.
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Tesfa-Selase, Fisehaye. "Regulation of the gua operon of Escherichia coli by the DnaA protein." Thesis, University of Southampton, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.385344.
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Botha, Jeanine. "The regulation and function of the ESAT-6 gene cluster operons of Mycobacterium tuberculosis." Thesis, Link to the online version, 2006. http://hdl.handle.net/10019/505.
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Neubert, Miranda J., Elizabeth A. Dahlmann, Andrew Ambrose, and Michael D. L. Johnson. "Copper Chaperone CupA and Zinc Control CopY Regulation of the Pneumococcal cop Operon." AMER SOC MICROBIOLOGY, 2017. http://hdl.handle.net/10150/626457.
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Any metal in excess can be toxic; therefore, metal homeostasis is critical to bacterial survival. Bacteria have developed specialized metal import and export systems for this purpose. For broadly toxic metals such as copper, bacteria have evolved only export systems. The copper export system (cop operon) usually consists of the operon repressor, the copper chaperone, and the copper exporter. In Streptococcus pneumoniae, the causative agent of pneumonia, otitis media, sepsis, and meningitis, little is known about operon regulation. This is partly due to the S. pneumoniae repressor, CopY, and copper chaperone, CupA, sharing limited homology to proteins of putative related function and confirmed established systems. In this study, we examined CopY metal crosstalk, CopY interactions with CupA, and how CupA can control the oxidation state of copper. We found that CopY bound zinc and increased the DNA-binding affinity of CopY by roughly an order of magnitude over that of the apo form of CopY. Once copper displaced zinc in CopY, resulting in operon activation, CupA chelated copper from CopY. After copper was acquired from CopY or other sources, if needed, CupA facilitated the reduction of Cu2+ to Cu1+, which is the exported copper state. Taken together, these data show novel mechanisms for copper processing in S. pneumoniae. IMPORTANCE As mechanisms of copper toxicity are emerging, bacterial processing of intracellular copper, specifically inside Streptococcus pneumoniae, remains unclear. In this study, we investigated two proteins encoded by the copper export operon: the repressor, CopY, and the copper chaperone, CupA. Zinc suppressed transcription of the copper export operon by increasing the affinity of CopY for DNA. Furthermore, CupA was able to chelate copper from CopY not bound to DNA and reduce it from Cu2+ to Cu1+. This reduced copper state is essential for bacterial copper export via CopA. In view of the fact that innate immune cells use copper to kill pathogenic bacteria, understanding the mechanisms of copper export could expose new small-molecule therapeutic targets that could work synergistically with copper against pathogenic bacteria.
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Tagliabue, L. "THE SUBTLE BIOFILM REGULATION IN ESCHERICHIA COLI: CSGD AND THE YDDV-DOS OPERON." Doctoral thesis, Università degli Studi di Milano, 2010. http://hdl.handle.net/2434/160739.
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In this PhD thesis work I investigated the expression modulation of the major adhesion factors in Escherichia coli; in particular I focused on the role of GGDEF and EAL proteins, on their modulation in E. coli biofilm formation in response to environmental signals and on regulation of curli fibers, cellulose and poly-N-acetylglucosamine (PNAG), the most important biofilm determinants in E. coli.
E. coli is an Enterobacterium, normally living inside the mammalian gut, at temperature of 37° C and in relatively nutrient-rich environment. Once outside the host, bacteria usually face much lower temperatures (< 30°C) and a nutrient-limiting environment. The biofilm determinants studied in this thesis are all expressed in response to environmental conditions such as low temperature, low osmolarity and starvation, suggesting that E. coli bacteria switch to a biofilm mode of growth as part of their adaptation to the natural environment. In response to reduction in growth rates, E. coli seems to canalize its energy consumption into production of extracellular features such as curli or exopolysaccharides. Biofilms can be thus considered as a “resistance form” of growth able to withstand stress conditions more efficiently than cells living in a planktonic mode of growth. The CsgD protein is the master regulator of E. coli biofilm formation. It is a transcriptional factor necessary for curli genes transcription and, through the AdrA protein, for cellulose biosynthesis. Gene regulation by CsgD is tightly connected to production and sensing of cyclic di-GMP, a bacterial second messenger involved in various cellular processes, including biosynthesis of extracellular polysaccharides (Simm et al., 2004), biofilm formation (Hickman et al., 2005), and virulence (Pratt et al., 2007; Tischler and Camilli, 2005), as well as morphological and physiological differentiation (Paul et al., 2004). The CsgD-dependent adrA gene, involved in cellulose biosynthesis (Zogaj et al., 2001), encodes a cyclic di-GMP synthase (Simm et al., 2004). CsgD can also activate yoaD, whose gene product is a cyclic di-GMP phosphodiesterase, suggesting that CsgD is directly involved in feedback regulation of cyclic di-GMP intracellular levels and of cellulose biosynthesis (Brombacher et al., 2006). CsgD is also able to activate the iraP gene: IraP acts as a stabilization factor for the σs protein, an alternative sigma factor of RNA polymerase which directs transcription of genes involved in adaptation to slow growth and to cellular stresses. Here I showed that CsgD transcription activation of the iraP gene does result in a significant increase of σs intracellular concentration by positively affecting σs protein stability, thus leading to altered expression of σs-dependent genes. CsgD-mediated increase of σs cellular concentrations via the iraP gene would trigger an autoactivation loop leading to an increased production of CsgD-dependent adhesion determinants such as curli fibers and cellulose. This autoregulatory circuitry might be further fueled by σs-dependent induction of genes encoding di-guanylate cyclases, i.e., proteins able to synthesize the second messenger di-cyclic- GMP, which, in turn, can positively affect csg gene expression (Kader et al., 2006; Weber et al., 2002). The yddV-dos operon is the most expressed among c-di-GMP-related genes showing dependence on σs (Weber et al., 2006; Sommerfeldt et al., 2009). It encodes, respectively, a protein with DGC activity and a PDE that can degrade c-di- GMP to pGpG. Both Dos and YddV are heme-binding oxygen sensors, and interact to form a stable protein complex (Tuckerman et al., 2009). Although it has been reported that YddV overexpression can stimulate biofilm formation (Mendez-Ortiz et al., 2006), the targets of yddV-dependent biofilm induction had not yet been identified. Here I showed that YddV acts modulating curli and PNAG expression. Control of curli production by yddV-dos takes place at the level of transcription regulation of the csgBAC operon, encoding curli structural subunits, and is mediated by the DGC and PDE activities of YddV and Dos. In contrast, the YddV–Dos protein complex does not strongly influence csgDEFG expression, nor does it affect the expression of the CsgD-dependent adrA gene, encoding a positive effector for cellulose biosynthesis. Regarding PNAG production, we showed that YddV is able to prevent degradation of pgaABCD transcript in the MG1655csrA background, thus suggesting that a DGC might regulate gene expression by affecting mRNA stability in E. coli. YddV regulation of pgaABCD operon in a wild type contest is still controversial: pgaABCD genes are expressed at low levels in MG1655 (the standard laboratory strain of E. coli) and their mRNA half-life is lower than two minutes regardless of the growth conditions tested; thus, possible effects of yddV inactivation on destabilization of the pga transcript are not easy to evaluate in the wt contest. In the last part of my thesis I tried to characterize a biofilm-forming mutant of E. coli, able to express pgaABCD genes at high levels. Even if initial data suggested that a mutation in the csrA gene could be responsible for pga mRNA stabilization in this mutant, actual the mutation leading to the adhesive phenotype and to PNAG production is outside the csrA gene and is still unknown. Moreover my data suggest a connection between pga expression and iron regulation in E. coli strains: it is conceivable that pgaABCD expression and consequent biofilm formation and the adherent phenotype depends on concerted production of different determinants, whose expression is also affected by iron concentration. Thus, my research highlighted that biofilm production is the result of coordinated expression of different adhesion determinants, whose regulation is complex and not fully understood. In particular, the precise extent and the molecular mechanism of c-di-GMP adhesion factors regulation remains to be largely identified and represents an exciting challenge for future research in the biofilm field.
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Bailey, Marc J. A. "HlyT, a transcriptional regulator of the Escherichai coli hemolysin operon." Thesis, University of Cambridge, 1992. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.259542.
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Deng, Liyu, and 鄧麗瑜. "Exploration of the transcription factors that regulate the expression of the haloacid operon in Burkholderia caribensis MBA4." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2014. http://hdl.handle.net/10722/208618.
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Bacterial dehalogenase is a key enzyme involved in bioremediation of halogenated organic compounds. A dehalogenase, Deh4a, was isolated from the Gram-negative bacterium Burkholderia caribensis MBA4, which can utilize haloacetic acids as carbon source. The haloacid operon in MBA4 was identified and characterized. It is composed of the structural genes forDeh4a and a transporter Deh4p. Transcription of this operon is negatively regulated, but the mechanism and the relevant regulator are still poorly understood. In this study, magnetic DNA affinity chromatography and Tn5transposon mutagenesis were employed to explore the regulatory factors that affected the expression of this haloacid operon.
A process that uses lysates from glycolate-grown cells, magnetic DNA affinity chromatography and LC-MS/MS has identified a TetR family transcriptional regulator, TetR8620, which binds to the promoter region of deh4a. Disruption of the TetR8620 gene in mutant Ins8620 abolished the formation of a slow migrating complex in electrophoretic mobility shift assay (EMSA) using lysates from glycolate-grown cells. Moreover, expressions of deh4a were enhanced in bothglycolate- and MCA- grown Ins8620. The addition of recombinant histidine-tagged TetR8620 to lysates of Ins8620 resumed the formation of a retardation complex, but different from that using purified His-tagged TetR8620.This suggested that TetR8620 is responsible for formation of retardation complexes, and an additional protein might be involved. To investigate other putative factors that interact with TetR8620, purified His-tagged TetR8620 was immobilized with Ni-NTA agarose and used for isolation of interacting proteins. Chemical cross-linking of the purified fraction with BS3established that TetR8620 interacts with a proteinof30 kDa. Separation of the cross-linked complex in SDS-PAGE gel also showed that a protein with similar MW was specifically pulled down. These results suggest that TetR8620 was interacting with a ~30 kDa protein. Protein identification using mass spectrometry assay proposed that this protein is probably a universal stress protein UspA encoding by peg.3485 or acetyl-glutamate kinase (EC 2.7.2.8) encoding by peg.714 in MBA4.
Tn5transposon mutagenesis was also employed to explore the factors that regulate the haloacid operon ofMBA4. A derivative of MBA4, MK06, which contains a kanamycin resistant gene (kan) with a deh4apromoter was constructed. Kanamycin resistancy of this derivative was MCA inducible. Transposon mutagenesis was conducted on this derivative, and Tn-containing mutants were isolated as tetracycline resistant colonies on pyruvate plates. These colonies were further selected on their resistance tokanamycin in pyruvate plates. Gene peg.6589 encoding a putative transcriptional regulator, DehR1, was disrupted by Tn insertion. While the production of dehalogenase was still MCA-inducible, this mutant has partially relieved the repression of the haloacid operon in media containing pyruvate. Moreover, constitutive production of DehR1 in MBA4 decreased the transcript levels of deh4ain medium containing pyruvate or MCA.
This study has identified two transcription factors, TetR8620 and DehR1, which regulate the expression of Deh4a negatively. TetR8620 is a DNA-binding protein that interacts with the deh4apromoter. Results from this study imply that the regulation of the haloacid operon in MBA4 is likely to be under the control of multiple factors.published_or_final_version
Biological Sciences
Doctoral
Doctor of Philosophy
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Allen, Darin. "Transcriptional regulation of the dlt operon in Enterococcus faecalis and further characterization of a dlta mutant." Thesis, Manhattan, Kan. : Kansas State University, 2008. http://hdl.handle.net/2097/783.
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Grainger, David Christopher. "Transcriptional regulation in the Escherichia Coli melibiose operon : interactions of the MeIR activator protein." Thesis, University of Birmingham, 2003. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.410284.
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The Escherichia coli melibiose (meý operon encodes proteins that facilitate the metabolism of melibiose. Expression of the mel operon is controlled by a single promoter (the meIAB promoter) and is co-dependent on two transcription activators, MeIR and the cyclic AMP receptor protein (CRP). MeIR is a member of the AraC family of transcription regulators and its activity is controlled by melibiose. CRP is a global regulator controlled by the second messenger, cyclic AMP, whose level is controlled by the availability of glucose. Thus, the co-dependence of the meIAB promoter on two activators couples induction to two physiological signals, the presence of melibiose and the absence of glucose. in the absence of melibiose, MeIR binds to three DNA target sites upstream of the melAB transcription start site. In the presence of melibiose, MeIR is able to bind to a fourth site, which has a low affinity for MeIR and overlaps the -35 hexamer of the melAB promoter. The four MeIR binding sites are organised as two pairs that are separated from each other by a binding site for CRP. Binding of CRP to this site requires co-operative interactions with adjacently bound MeR. These interactions stabilise the binding of both MeIR and CRP to their DNA target sites. In this work it is shown that MeIR binds to each pair of sites at the melAB promoter as a direct repeat. This defines the face of MeIR that is able to interact with adjacently bound CRP and, for MeIR bound close to the -35 hexamer, the a7O subunit of RNA polymerase. The MeIR-e interface has been characterised using a combination of genetics, biochemistry and structural modelling. Amino acid side chains in MeIR and RNA polymeraseth at interact during transcription activation haveb eeni dentified. Finally, simplified derivatives of the melAB promoter, which carry an improved pair of promoter proximal MeIR binding sites, have been used to study the interaction between MeIR and the C-terminal domain of the (x subunit of RNA polymerase. My data show that subunits of MeIR and (X protein can bind to the DNA co-operatively and that this co-operative binding is sufficient to stimulate transcription initiation by RNA polymerase lacking the entire (X C-terminal domain.
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Lee, Ka Man. "Regulation of expression of the type IV B pili-encoding operon of salmonella typhi /." View Abstract or Full-Text, 2003. http://library.ust.hk/cgi/db/thesis.pl?BICH%202003%20LEE.
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Thesis (M. Phil.)--Hong Kong University of Science and Technology, 2003.Includes bibliographical references (leaves 110-127). Also available in electronic version. Access restricted to campus users.
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Gliese, Nicole. "Untersuchungen zur Regulation des pqq-Operons und anderer Komponenten des Ethanol-oxidierenden Systems von Pseudomonas aeruginosa." Berlin mbv, Mensch-und-Buch-Verl, 2009. http://d-nb.info/998052116/04.
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Vélez, Acevedo Rosuany. "Analysis of the Regulation of the Transferrin Iron Acquisition System in Neisseria gonorrhoeae." VCU Scholars Compass, 2009. http://scholarscompass.vcu.edu/etd/1955.
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The neisserial transferrin binding proteins (Tbps) comprise a bipartite system for iron acquisition from human transferrin. TbpA is a TonB-dependent outer membrane protein that forms the pore for iron internalization. TbpB is a surface-exposed lipoprotein that makes the iron-uptake process more efficient. Previous studies have shown that the genes encoding these proteins are arranged in a bicistronic operon, with the tbpB gene located upstream of tbpA. The operon is under the control of the ferric uptake regulator (Fur) protein. However, promoter elements necessary for the regulation of the operon have not been experimentally defined. In this study, putative regulatory motifs were confirmed by mutagenesis. Further examination of the sequence upstream of these promoter/operator motifs led to the identification of two direct repeats. We hypothesized that these repeats may be involved in further regulation of the operon. Insertional mutagenesis of the repeats resulted in altered transcript and protein levels. These results confirmed that the region upstream of the operon serves as an extended regulatory region. A comprehensive investigation of the expression of the operon in response to different environmental stimuli that gonococci might encounter upon infection was also conducted. Changes in osmolarity, carbon source, cAMP availability, and H2O2 stress did not alter expression of the operon at the transcript or protein levels. However, low oxygen levels resulted in decreased tbpBA transcript and protein. These results are biologically relevant, and provide new insights into the use of the transferrin binding proteins as vaccine candidates. Lastly, the role of G4 DNA sequences identified in the vicinity of the tbpBA operon was investigated. We hypothesized that G4 DNA structures could be involved in the regulation of the operon. Results presented here indicate that interference with these sequences appears to have no effect on expression of the operon. However, identification of potential G4-forming sequences in the non-coding regions upstream and downstream of the operon suggests their importance, perhaps in mediating recombination which could lead to increased antigenic diversity.
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Jones, Melissa Kolsch. "Regulation of phase variation and deletion mutation in the Vibrio vulnificus group 1 CPS operon." [Gainesville, Fla.] : University of Florida, 2006. http://purl.fcla.edu/fcla/etd/UFE0013839.
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Quinlivan-Repasi, Vanessa H. "Transcriptional Regulation of the Acetone Carboxylase Operon via Two-Component Signal Transduction in Helicobacter pylori." W&M ScholarWorks, 2012. https://scholarworks.wm.edu/etd/1539626927.
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Col, Bekir. "Regulation of Fructose 1,6-bisphosphatase II (GlpX) Gene Expression in Escherichia coli." Diss., Virginia Tech, 2004. http://hdl.handle.net/10919/11282.
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The glpX gene of Escherichia coli encodes fructose 1,6-bisphosphatase II (FBPase II), an enzyme that would appear to be redundant with FBPase I, encoded by fbp. However, glpX mutants have no apparent phenotype, while fbp mutants are unable to grow on gluconeogenic substrates as sole carbon sources, suggesting that GlpX function is insufficient for growth of fbp mutants under these conditions. To gain insight into the physiological functions of the FBPases, regulation of glpX expression was investigated. It was found that glpX is transcribed as part of a complex glpFKX operon containing promoters upstream of glpF, glpK and glpX (PglpF, PglpK, PglpX, respectively). Transcription start sites of PglpX were found at -24 and -41 relative to the ATG translation initiation site using primer extension analysis. Unlike PglpF, these newly found promoters were not subject to regulation by GlpR or cAMP-CRP. Cra (Catabolite Repressor/Activator) positively regulated expression from PglpK and PglpX by increasing transcription approximately 2 fold. Western analysis using GlpX polyclonal antibodies revealed that GlpX levels were higher in cultures grown on glycerol compared with levels in maltose- or glucose-grown cultures (glycerol>maltose>glucose). Various strains and growth conditions were used to show that GlpX levels are regulated by GlpR, suggesting that PglpF can give rise to expression of glpX. GlpX protein was present in a strain containing a polar insertion in glpK, indicating that PglpX can also give rise to expression of glpX. Strains deficient in FBPase I or CsrA (carbon starvation regulator) did not reveal any difference in GlpX levels with respect to the wild type. All of these data indicate that glpX expression is achieved by its own promoter as well as the operon promoter, PglpF. Finally, the results show that the delta-fbp phenotype is not due to the absence of GlpX.Ph. D.
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Ucci, Amanda Piovesan. "Participação da ORF XAC3629 na regulação do operon de resistência a cobre copAB em Xanthomonas citri subsp. citri /." Araraquara : [s.n.], 2009. http://hdl.handle.net/11449/88003.
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Orientador: Maria Célia BertoliniBanca: Jesus Aparecido Ferro
Banca: Julio Cezar Franco de Oliveira
Resumo: Xanthomonas citri subsp. citri (Xcc) é uma bactéria Gram-negativa responsável pelo o cancro cítrico. A doença causada por este fitopatógeno constitui numa das mais graves doenças da citricultura brasileira, cujas medidas de controle capazes de eliminar completamente a doença são ineficientes até o momento. O sequenciamento e anotação do genoma de Xcc juntamente com estudos relacionados à genômica funcional nesta bactéria realizados em nosso laboratório mostraram a existência, na bactéria, de alguns genes (copA e copB) que codificam proteínas envolvidas com o mecanismo de resistência a cobre (CopA e CopB). Os genes que codificam ambas proteínas estão organizados em um operon, cuja transcrição é induzida por cobre e é específica ao metal. Componentes a base de cobre são comumente usados na agricultura como agentes microbicidas a fim de reduzir a severidade desta e também de outras doenças nas plantas. Contudo, a eficácia do cobre está sendo reduzida pelo aparecimento de linhagens de bactérias resistentes a este metal, portanto, estudos visando a compreensão dos mecanismos moleculares envolvidos na resistência a cobre em Xcc são de grande importância. Uma pequena ORF (XAC3629) que codifica uma proteína hipotética de 152 aminoácidos e que se localiza upstream aos genes copAB foi identificada. Neste trabalho foi estudado o papel da ORF na regulação do operon copAB. Linhagens mutantes contendo a ORF XAC3629 interrompida por um transposon (XAC3629::Tn), previamente construídas no laboratório, foram avaliadas em relação ao crescimento em meio contendo diferentes concentrações de cobre. Estas linhagens mostraram perda da resistência quando o metal foi adicionado ao meio, mesmo em baixas concentrações (0,25 mM), sugerindo seu envolvimento na regulação do operon de resistência a cobre copAB. Células da linhagem selvagem ... (Resumo completo, clicar acesso eletrônico abaixo)
Abstract: Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium that causes citrus canker, one of the most serious diseases in Brazilian crops, and the ways to eliminate it are not efficient. After sequencing and annotation of the Xcc genome our laboratory started to perform functional genomic studies in Xcc. Previous studies identified two genes (copA and copB) that encode proteins involved in the copper resistance mechanism (CopA and CopB). The genes are organized in an operon whose transcription is induced and specific to copper. Copper compounds are commonly used in agriculture in order to control plant diseases, however the copper effectiveness is being reduced due to the emergence of copper resistant bacteria strains. Therefore, studies related to copper resistance mechanism in Xcc are very relevant. A short ORF (XAC3629) which encodes an 152 amino acids hypothetical protein was identified upstream the operon copAB. In this work the role of the ORF in the operon regulation was studied. Mutant strains containing the ORF XAC3629 interrupetd by a transposon (XAC3629::Tn) were previously constructed in our laboratory and in this work their growth were evaluated in a medium containing different copper concentration. The mutant strains were unable to grow when the metal was added to the medium, even at low concentration (0.25 mM) suggesting that the ORF XAC3629 may have a role in regulating the operon copAB. Wild type Xcc cells are able to grow up to 1 mM copper concentration. Attempts to construct an ORF XAC3629 knocked out strain were performed but this mutant strain was not obtained. Two XAC3629::Tn mutants strains were transformed with a plasmid construction containing the ORF XAC3629 entire sequence plus its flanking regions. The results did not show functional complementation (recovery of the copper resistance when cells were grown in medium containing ... (Complete abstract click electronic access below)
Mestre
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Weissenborn, Deborah Louise. "Regulation of the glpFK operon of Escherichia coli K-12 and characterization of it gene products." Diss., Virginia Tech, 1990. http://hdl.handle.net/10919/39937.
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Bröms, Jeanette. "Type III secretion- the various functions of the translocon operon in bacterial pathogenesis." Doctoral thesis, Umeå University, Molecular Biology, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-331.
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In order to establish colonisation of a human host, pathogenic Yersinia use a type III protein secretion system to directly intoxicate host immune cells. Activation of this system requires target cell contact and is a highly regulated process. Both the intoxication and regulation events depend on the lcrGVHyopBD translocon operon, which is highly conserved in many bacterial pathogens. In this study, the role of individual operon members was analysed and functional domains identified by using the highly homologous pcrGVHpopBD operon of P. aeruginosa as a comparative tool.
Yersinia spp. and P. aeruginosa were shown to form translocation pores of a similar size that promoted equally efficient protein delivery. A strong dependency on interactions between native translocator(s) in protein delivery was revealed, suggesting that each pathogen has delicately fine-tuned this process to suit its own infection niche. In particular, the C-terminus of YopD was shown to possess functional specificity for effector delivery in Yersinia that could not be conferred by the comparable region in homologous PopD. Moreover, a role for LcrV and PcrV in substrate recognition during the protein delivery process was excluded.
The N-terminus of LcrH was recognized as a unique regulatory domain, mediating formation of LcrH-YscY regulatory complexes in Yersinia, while equivalent complexes with analogous proteins were not formed in P. aeruginosa. These results compliment the idea that a negative regulatory pathway involving LcrH, YopD, LcrQ and YscY is unique to Yersinia.
Finally, PcrH was identified as a new member of the translocator class of chaperones, being essential for assembly of a functional PopB/PopD mediated translocon in P. aeruginosa. However, in contrast to the other members of this family, PcrH was dispensable for type III regulation. Moreover, both LcrH and PcrH were shown to possess tetratricopeptide repeats crucial for their chaperone function. One tetratricopeptide repeat mutant in LcrH was even isolated that failed to secrete both YopB and YopD substrates, even though stability was maintained. This demonstrates for the first time that LcrH has a role in substrate secretion in addition to its critical role in promoting substrate stability.
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Hammar, Petter. "lac of Time : Transcription Factor Kinetics in Living Cells." Doctoral thesis, Uppsala universitet, Beräknings- och systembiologi, 2013. http://urn.kb.se/resolve?urn=urn:nbn:se:uu:diva-198814.
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Gene regulation mediated by transcription factors (TFs) is essential for all organisms. The functionality of TFs can largely be described by the fraction of time they occupy their regulatory binding sites on the chromosome. DNA-binding proteins have been shown to find their targets through facilitated diffusion in vitro. In its simplest form this means that the protein combines a random 3D search in the cytoplasm with 1D sliding along DNA. This has been proposed to speed up target location. It is difficult to mimic the in vivo conditions for gene regulation in biochemistry experiments; i.e. the ionic strength, chromosomal structure, and the presence of other DNA-binding macromolecules. In this thesis single molecule imaging assays for live cell measurements were developed to study the kinetics of the Escherichia coli transcription factor LacI. The low copy number LacI, in fusion with a fluorescent protein (Venus) is detected as a localized near-diffraction limited spot when being DNA-bound for longer than the exposure time. An allosteric inducer is used to control binding and release. Using this method we can measure the time it takes for LacI to bind to different operator sequences. We then extend the assay and show that LacI slides in to and out from the operator site, and that it is obstructed by another DNA-binding protein positioned next to its target. We present a new model where LacI redundantly passes over the operator many times before binding. By combining experiments with molecular dynamics simulations we can characterize the details of non-specific DNA-binding. In particular, we validate long-standing assumptions that the non-specific association is diffusion-controlled. In addition it is seen that the non-specifically bound protein diffuses along DNA in a helical path. Using microfluidics we design a chase assay to measure in vivo dissociation rates for the LacI-Venus dimer. Based on the comparison of these rates with association rates and equilibrium binding data we suggest that there might be a short time following TF dissociation when transcription initiation is silenced. This implies that the fraction of time the operator is occupied is not enough to describe the regulatory range of the promoter.
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Jonsson, Maria. "Contribution of the outer surface proteins of Borrelia burgdorferi s.l. to the pathogenesis of Lyme disease." Doctoral thesis, Umeå universitet, Mikrobiologi, 1994. http://urn.kb.se/resolve?urn=urn:nbn:se:umu:diva-100569.
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Borrelia burgdorferi s. l. is a spirochete which causes the multisystemic disorder Lyme disease. As the borreliae lack toxin production, the pathogenicity is thought to involve, at least in part, molecules from the outer surface. Most Lyme disease Borrelia strains express two major outer surface lipoproteins, OspA (31 kD) and OspB (34 kD), on their surface. However, some strains lack the expression of OspA and OspB, but express a smaller 21 to 25 kD OspC protein instead. This thesis focuses on the importance of these proteins in the pathogenesis of Lyme disease. Biochemical and immunochemical studies of the OspA and OspB proteins from strains of various geographic origins show considerable differences in the apparent molecular weights and in their reactivities to monoclonal antibodies. The cloning and sequencing of the ospAB opérons from strains of different origins has demonstrated that the heterogeneity is found also at the DNA level Comparison of the ospAB sequences allows the classification of the strains into three types, which coincide with the recent species designations, B. burgdorferi sensu stricto, B. afzelii and B. garinii The genes are located on a linear plasmid about 50 kb in size, and are cotranscribed as a single message. The expression of the osp operon in different strains was studied by Western blot and Northern blot analysis. The ospAB operon of strains expressing varying amounts of the Osp proteins was cloned and sequenced. The DNA sequence was found to be >99% identical. The regulation appears to be primarily at the transcriptional level. In patients who have received incomplete treatment, B. burgdorferi have been isolated several years after the onset of the disease. As mentioned above, the ospAB loci of different strains show considerable heterogeneity, and it has been speculated that the spirochetes evade the host’s immune system by antigenic variation of the Osp proteins. In a mouse model system it was shown that no variation of the osp genes occurs over the course of an infection, and that other escape mechanisms must be used. The OspB proteins in particular have been shown to be very heterogeneous in different isolates. The MAb 84C recognizes a wide variety of B. burgdorferi strains, and the binding epitope was mapped to a conserved region in the carboxyl terminus of the OspB protein with putative structural and/or functional importance. It is well known that antibodies can kill bacteria in the presence of complement and phagocytes. Some antibodies seem to have a bactericidal effect by themselves. H6831 is a monoclonal antibody recognizing the OspB protein of some B. burgdorferi strains. The bactericidal action of univalent FAb fragments from H6831 was further characterized, and the binding epitope was mapped to a very heterogeneous region of the carboxyl end of the OspB protein.Diss. (sammanfattning) Umeå : Umeå universitet, 1994, härtill 5 uppsatser
digitalisering@umu
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Ucci, Amanda Piovesan [UNESP]. "Participação da ORF XAC3629 na regulação do operon de resistência a cobre copAB em Xanthomonas citri subsp. citri." Universidade Estadual Paulista (UNESP), 2009. http://hdl.handle.net/11449/88003.
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Xanthomonas citri subsp. citri (Xcc) é uma bactéria Gram-negativa responsável pelo o cancro cítrico. A doença causada por este fitopatógeno constitui numa das mais graves doenças da citricultura brasileira, cujas medidas de controle capazes de eliminar completamente a doença são ineficientes até o momento. O sequenciamento e anotação do genoma de Xcc juntamente com estudos relacionados à genômica funcional nesta bactéria realizados em nosso laboratório mostraram a existência, na bactéria, de alguns genes (copA e copB) que codificam proteínas envolvidas com o mecanismo de resistência a cobre (CopA e CopB). Os genes que codificam ambas proteínas estão organizados em um operon, cuja transcrição é induzida por cobre e é específica ao metal. Componentes a base de cobre são comumente usados na agricultura como agentes microbicidas a fim de reduzir a severidade desta e também de outras doenças nas plantas. Contudo, a eficácia do cobre está sendo reduzida pelo aparecimento de linhagens de bactérias resistentes a este metal, portanto, estudos visando a compreensão dos mecanismos moleculares envolvidos na resistência a cobre em Xcc são de grande importância. Uma pequena ORF (XAC3629) que codifica uma proteína hipotética de 152 aminoácidos e que se localiza upstream aos genes copAB foi identificada. Neste trabalho foi estudado o papel da ORF na regulação do operon copAB. Linhagens mutantes contendo a ORF XAC3629 interrompida por um transposon (XAC3629
Xanthomonas citri subsp. citri (Xcc) is a Gram-negative bacterium that causes citrus canker, one of the most serious diseases in Brazilian crops, and the ways to eliminate it are not efficient. After sequencing and annotation of the Xcc genome our laboratory started to perform functional genomic studies in Xcc. Previous studies identified two genes (copA and copB) that encode proteins involved in the copper resistance mechanism (CopA and CopB). The genes are organized in an operon whose transcription is induced and specific to copper. Copper compounds are commonly used in agriculture in order to control plant diseases, however the copper effectiveness is being reduced due to the emergence of copper resistant bacteria strains. Therefore, studies related to copper resistance mechanism in Xcc are very relevant. A short ORF (XAC3629) which encodes an 152 amino acids hypothetical protein was identified upstream the operon copAB. In this work the role of the ORF in the operon regulation was studied. Mutant strains containing the ORF XAC3629 interrupetd by a transposon (XAC3629
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Pinto, Cecilia de Agrela. "The two-component system of a novel copper resistant operon of Marinobacter hydrocarbonoclasticus." Master's thesis, Faculdade de Ciências e Tecnologia, 2012. http://hdl.handle.net/10362/10062.
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Dissertation for the Master’s Degree in Structural and Functional BiochemistryThe majority of bacterial heavy metal resistance systems are regulated by twocomponent signal transduction systems. Stimuli from the environment interact with the histidine kinase, which in turn activates the response regulator by phosphorylation. The effector domain of the response regulator then binds to DNA, eliciting the specific response. Analysis of the Marinobacter hydrocarbonoclasticus genome revealed the presence of genes, copXAB, that code for proteins associated with copper response. The biochemical characterization of the two-component signal transduction system, copSR, is of interest due to the vital role it plays in the regulation of expression of the copXAB operon. The genes that encode for the CopR and CopS_C (cytosolic sensor domain of CopS) proteins were heterologously expressed in E.coli and expression was optimized for the production of soluble protein using LB medium. Due to solubility problems, the genes that code for these proteins were cloned as hexahistidine or glutathione S-transferase fusion proteins. CopR and its domains were optimally expressed at 16°C for 16 and 3 h after induction, respectively, whilst CopS_C was expressed at 37°C during 3 h after induction. Proteins were purified using different chromatographic strategies, most of them using affinity chromatography. The yields of pure protein per liter of growth culture obtained after complete purification from the soluble cellular extract were: 0.14 to 0.23 mg/L for CopR; 0.42 mg/L, CopR_NHis6; for the CopR_CHis6 it was 0.16 mg/L and 4.2 mg/L of CopS_C. The molecular mass of each protein was determined by gel filtration, 31 kDa for CopR, 17.5 kDa for CopR_NHis6, 15.1 kDa for CopR_CHis6 and 38.2 kDa for CopS_C. In the case of CopS_C there is the possibility that a dimer is formed, which should be evaluated. From the evaluation of disulfide bonds, using SDS PAGE and PAGE gels, all proteins or protein domains appeared to be monomers when in the presence of β-MEtOH. Circular dichroism evaluated the state of folding of the CopS_C and CopR proteins, which were shown to be folded in which the α-helix structures predominate. A model structure for CopR was also determined which agrees with this analysis. However, in the case of the CopR domains, the data obtained merely indicate folding, due to the low concentrations of the proteins. Phosphorylation and electrophoresis mobility shift assays of the CopR protein were, for the most part, inconclusive. However, in the absence of BSA, formation of the CopR:DNA complex in a gel filtration column is observed, though requires additional evaluation.
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Morante, Estela Ynés Valencia. "Estudo do sistema BlaR/Blal e de dois operons codificando sistemas de efluxo RND em Caulobacter crescentus." Universidade de São Paulo, 2012. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-04062012-105252/.
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O presente trabalho tem como objetivo caracterizar três agrupamentos de genes da alfa proteobactéria Caulobacter crescentus envolvidos na resposta a metais e antibióticos. Analisamos o agrupamento composto pelos genes CC1637-CC1640, que contém um sistema BlaR/BlaI de transdução de sinal, e realizamos uma análise comparativa de dois sistemas de efluxo da família RND composto pelos genes CC2720-CC2725 e CC2388-CC2390 que estariam envolvidos na resposta a metais cádmio e zinco. Mutantes simples e duplos com deleção em fase foram obtidos, e o estudo da atividade promotora foi realizado através de ensaio de b-galactosidase utilizando gene repórter lacZ . Ensaios de RT-PCR e atividade b-galactosidase mostraram que o gene CC1638 provavelmente não possui promotor próprio e que os genes CC1637- CC1640 podem consituir um operon. A atividade promotora do gene CC1640 não responde a H2O2, Cd2+ e Zn2+, mas a linhagem DCC1640 apresentou baixa viabilidade na presença de Cd2+. As linhagens DCC1637 e DCC1638 mostraram sensibilidade a t-butil-hidroperóxido. A linhagem DCC1640 mostrou-se sensível aos antibióticos CTX e PPT, e ensaios de b-galactosidase em placa e em meio liquido mostraram indução da expressão pelos antibióticos CTX e CFE. Observamos uma auto-regulação do operon pela proteína codificada pelo gene CC1640 (BlaI), confirmado por ensaios de EMSA. A presença de BlaR inibe a ligação da proteína BlaI ao promotor, sugerindo que ambas BlaI/BlaR regulem em conjunto o promotor do gene CC1640. Análise in silico do consenso TTACGNNCGTAA localizado no promotor de CC1640 identificou esta sequência na região promotora de outros genes. A análise da expressão relativa sugere que os genes CC1568, CC1230 e CC2661 são regulados pela proteína BlaI, sugerindo que BlaI regula a expressão de outros genes possivelmente envolvidos na resposta a antibióticos b-lactâmicos. Ensaios de atividade b-galactosidase da região intergênica CC2720-CC2721 mostraram que esta não possui atividade promotora, e análise por RT-PCR confirmou que os genes CC2720-CC2721 são co-transcritos e que fazem parte do operon CC2720-CC2725. A expressão do operon mostrou indução significativa na presença de Cd2+, moderada indução na presença de Zn2+ e Co2+, e pouca indução na presença de Ni2+. A expressão do operon CC2388-CC2390 é altamente induzida na presença de níquel e cobalto, não é induzida por cádmio e moderadamente induzida por zinco. A linhagem DCC2724 não é sensível a zinco, cobalto ou níquel. A linhagem DCC2390 é sensível a cobalto, pouco sensível a níquel e não sensível a zinco, e ambas as linhagens foram sensíveis a cádmio. A obtenção do duplo mutante, assim como sua complementação, foram realizadas, e os resultados sugerem que se trata de dois sistemas de efluxo com diferentes respostas a metal.The aim of this work is to characterize three clusters of genes from the alpha proteobacterium Caulobacter crescentus involved in metal and antibiotics response. We analyzed the cluster comprising genes CC1637-CC1640, which contains a BlaR/BlaI signal transduction system, and we performed a comparative analysis with two RND efflux systems consisting on genes CC2720-CC2725 and CC2388-CC2390, which are probably involved in cadmium and zinc response. Mutant strains for one or two of these genes were obtained, and the study of promoter activity was performed by b-galactosidase activity assays using lacZ as reporter gene. RT-PCR and b-galactosidase activity assays revealed that the CC1638 gene probably does not possess an exclusive promoter, and that the genes CC1637-CC1640 may constitute an operon. The promoter of CC1640 does not respond to H2O2, Cd2+ and Zn2+, but the DCC1640 strain presented low viability in the presence of Cd2+. The DCC1637 and DCC1638 strains showed sensitivity to t-butyl-hydroperoxide. The DCC1640 strain showed sensitivity to the antibiotics CTX and PPT, and b-galactosidase activity assays performed both on plates and liquid medium showed induction of the expression by the presence of antibiotics CTX and CFE. We observed auto-regulation of the operon by the protein encoded by the CC1640 gene (BlaI), which was confirmed by EMSA assays. The presence of BlaR inhibits the binding of the BlaI protein to the promoter, suggesting that both BlaI/BlaR regulate the CC1640 gene promoter. In silico analyses for the TTACGNNCGTAA consensus, located on CC1640 promoter, identified this sequence in promoter regions of other genes. Relative expression analyses indicate that the genes CC1568, CC1230 and CC2661 are regulated by the BlaI protein, suggesting that BlaI regulates the expression of other genes, possibly involved in b-lactamic antibiotics response. b-galactosidase activity assays of the intergenic region CC2720-CC2721 showed that it does not possess promoter activity, and a RT-PCR analysis confirmed that the genes CC2720-CC2721 are co-transcribed and belong to the CC2720-CC2725 operon. The expression of the operon showed significant induction in the presence of Cd2+, moderate induction in the presence of Zn2+ and Co2+, and a slight induction in the presence of Ni2+. The expression of the CC2388-CC2390 operon is highly induced in the presence of nickel and cobalt, not induced by cadmium and moderately induced by zinc. The DCC2724 strain is not sensitive to zinc, cobalt or nickel. The DCC2390 strain is sensitive to cobalt, slightly sensitive to nickel and not sensitive to zinc, and both strains are sensitive to cadmium. A double mutant was constructed, as well as a complemented strain, and results suggest that these are two efflux systems with distinct metal responses.
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Wienecke, Sarah Verfasser], and Dieter [Akademischer Betreuer] [Jahn. "Untersuchungen zur XylR-vermittelten Regulation des Xylose-Operons und phänotypischen Heterogenität in Bacillus megaterium / Sarah Wienecke ; Betreuer: Dieter Jahn." Braunschweig : Technische Universität Braunschweig, 2017. http://d-nb.info/1175817759/34.
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Chen, Chaw-Yuan. "Regulation, Evolution, and Properties of the ato Qperon and its Gene Products in Escherichia coli." Thesis, University of North Texas, 1993. https://digital.library.unt.edu/ark:/67531/metadc277592/.
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The regulation of short chain fatty acid metabolism has been examined. Metabolism of acetoacetate, and short chain fatty acids such as butyrate and valerate, is predicated upon the expression of genes of the ato operon. Acetoacetate induces expression of a CoA transferase (encoded by the atoDA genes) and expression of a thiolase (encoded by the atoB gene). Metabolism of saturated short chain fatty acids requires the activities of the transferase and thiolase and enzymes of 6-oxidation as well. Spontaneous mutant strains were isolated that were either constitutive or that were inducible by valerate or butyrate instead of acetoacetate.
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Toukoki, Chadia. "MTSR is a Dual Regulator that Controls Virulence Genes and Metabolic Functions in Addition to Metal Homeostasis in Group A Streptococcus." Digital Archive @ GSU, 2009. http://digitalarchive.gsu.edu/biology_diss/66.
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Group A Streptococcus (GAS) is a common pathogen of the human skin and mucosal surfaces and is capable of producing a variety of diseases. This dissertation investigates the function of a metalloregulator named MtsR in GAS physiology and disease process. An mtsR mutant was constructed and analyzed. Consistent with MtsR role in iron uptake regulation, the mtsR mutant accumulates more iron (80 ± 22.5%) than the wild type strain. Inactivation of mtsR results in constitutive transcription of the sia (Streptococcal Iron Acquisition) operon, which is negatively regulated by iron in the parent strain. We identified the promoter that controls the expression of the sia operon (Pshr) and used it as a model to study MtsR interaction with DNA. Electrophoretic mobility gel shift assays (EMSAs) demonstrated that MtsR binds to the shr upstream region specifically and in an iron and manganese dependent manner. DNase I footprint analysis revealed that MtsR protects a 69 bp segment in Pshr that includes 2 inverted repeats, overlapping the core promoter elements. A global transcriptional analysis determined that MtsR modulates the expression of 64 genes, of which 44 were upregulated and 20 were downregulated in the mtsR mutant. MtsR controls genes with diverse functions including immune evasion, colonization, dissemination, metal homeostasis, nucleic acid and amino acid metabolism, and protein stability. MtsR functions as a dual regulator as it binds to the promoters of the repressed genes ska, aroE, and nrdF.2, as well as the upstream region of the positively regulated genes mga, emm49, and pyrF. A 16 bp MtsR-binding consensus region was identified in all of the promoters that are directly regulated by MtsR. In conclusion, we have demonstrated that MtsR is a global regulator in GAS that controls the expression of vital virulence factors and genes involved in metal transport, virulence and metabolic pathways.
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Kim, Jean. "Alternative regulation of the alginate algD operon by an activated AlgB in nonmucoid Pseudomonas aeruginosa is dependent on Sigma 54." VCU Scholars Compass, 2010. http://scholarscompass.vcu.edu/etd/108.
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Alginate overproduction by Pseudomonas aeruginosa, which causes a mucoid phenotype, is a major virulence factor associated with chronic pulmonary infections in cystic fibrosis patients. Expression of the algD operon for alginate biosynthesis requires three major regulators in association with the ECF sigma factor, σ22, in mucoid strains that are typically defective in anti-sigma factor, MucA. One such algD regulator is AlgB, a member of the NtrC family of two-component systems, which typically utilize σ54. However, neither σ54 nor the cognate sensor kinase (KinB) of AlgB are required for algD expression in such mucoid strains. I hypothesized that KinB-phosphorylated AlgB must play some role in gene regulation, and so I sought to construct a constitutively active AlgB that simulated kinase-phosphorylation. I took a predictive approach and genetically introduced substitutions in AlgB that had been shown to activate DctD, a close homologue of AlgB in Rhizobium (52). When one such substitution, AlgBE125K, was transferred to a nonmucoid P. aeruginosa PAO ΔalgB-kinB (JK159) strain, alginate overproduction was observed. Interestingly, introduction of an algT mutation to remove σ22 did not block alginate production induced by AlgBE125K; although, it did stimulate the production of alginate in the presence of AlgBwt in trans to similar levels induced by the constitutive mutant. In contrast, introduction of an rpoN mutation showed that alginate production mediated by AlgBwt and AlgBE125K was σ54 dependent. The increase in expression of alginate by AlgBwt in the presence of σ54 and the absence of σ22 suggested a competition between the sigma factors for binding to PalgD. Biochemical assays were conducted to assess the constitutive property of AlgBE125K. For the ATPase assay, an equivalent amount of ATP hydrolysis was observed between the mutant and the wild type AlgB proteins. Slight differences seen for the EMSA data suggested possible higher order complex formation for AlgBE125K compared to AlgBwt. Collectively, these results suggested that in wild-type (MucA+) P. aeruginosa, expression of the algD operon is dependent on the phosphorylation of AlgB by KinB in a typical two-component fashion that is triggered by some as yet unknown environmental stimulus.
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Kühne, Caroline Verfasser], and Petra [Akademischer Betreuer] [Dersch. "Gene regulation dynamics of the flagellar master regulatory operon flhDC in Salmonella enterica serovar Typhimurium / Caroline Kühne ; Betreuer: Petra Dersch." Braunschweig : Technische Universität Braunschweig, 2017. http://d-nb.info/1175817678/34.
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Munevar, Nicolas Federico Villamil. "Análise do metabolismo de polifosfato e do operon pst em Pseudomonas aeruginosa." Universidade de São Paulo, 2015. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-10122015-101302/.
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O operon pst de P. aeruginosa codifica um transportador de fosfato de alta afinida-de e também a proteína PhoU que, em conjunto, atuam como repressores da ex-pressão do regulon Pho dessa espécie. A atividade de PhoU está também associada ao metabolismo de polifosfato (poliP), dado que mutantes phoU nulos apresentam um vasto acúmulo do biopolímero. Ensaios de β-galactosidase mostraram uma alteração na expressão dos genes ppk e ppx, envolvidos no metabolismo de poliP, no mutante phoU. Observou-se que na cepa selvagem, a transcrição de ppk e de ppx não responde às limitações de Pi ou de nitrogênio, sendo esses genes altamente expressos em condições normais de crescimento. Além disso, determinou-se que ppk é co-transcrito com o gene hemB, os quais formam, portanto, um operon. O operon pst também foi analisado. Foi identificado por ensaios de northern blot o transcrito do primeiro gene do operon, pstS, que codifica uma proteína periplasmática. Também, foi identificado um promotor imediatamente a montante de phoU, o gene mais distal do operon, que permitiria sua expressão em condições normais do crescimento bacteriano. Por fim, determinou-se por ensaios de EMSA que as duas sequências consenso Pho box presentes no operon pst são completamente funcionais.The pst operon in P. aeruginosa encodes a high-affinity phosphate transporter and the PhoU protein, which together act as repressors of Pho regulon of this species. The PhoU activity is also related with polyphosphate (polyP) metabolism, since phoU null mutants have a large accumulation of the biopolymer. β-galactosidase assays allowed to confirm a change in the expression of ppk and ppx genes, in-volved in PolyP metabolism, in the phoU mutant. It was also evidenced that in the wild type strain, the ppk and ppx transcription does not respond to Pi or nitrogen starvation, and that these genes are highly expressed under conditions of normal growth. In addition, it was determined that ppk is co-transcribed with hemB, a gene involved in the synthesis of porphyrins, and they constitute therefore an operon. The pst operon was also examined. Was identified by northern blot the transcript of the first gene in the operon, pstS, which encodes a periplasmic protein. Also, a promoter was identified immediately upstream of phoU, the most distal gene in the operon, allowing its expression in normal conditions of bacterial growth. Finally, it was determined by EMSA that the two consensus sequences Pho box present in the pst operon are fully functional.
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Barbosa, Rosicler Lazaro. "Analise funcional e estrutural da proteina BigR de Xylella fastidiosa envolvida na regulação do operon Xf0768-0764." [s.n.], 2008. http://repositorio.unicamp.br/jspui/handle/REPOSIP/316599.
Full textAbstract:
Orientador: Celso Eduardo BenedettiTese (doutorado) - Universidade Estadual de Campinas, Instituto de Biologia
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Resumo: O gene XF0767 de Xylella fastidiosa está localizado num operon composto por mais quatro genes classificados como proteínas hipotéticas (XF0768-XF0767-XF0766-XF0765-XF0764). Este operon está conservado em uma série de bactérias associadas a plantas, incluindo Agrobacterium tumefaciens, Mezorhizobium loti e Sinorhizobium meliloti. A região upstream ao códon de iniciação deste operon possui seqüências canônicas correspondentes a sítios -35 e ¿10 de regiões promotoras reguladas por fatores sigma70. O objetivo deste trabalho foi caracterizar o sistema de regulação do operon pela proteína XF0767, nomeada BigR (biofilm growth-associated repressor), visando à compreensão deste sistema e sua importância para a bactéria. A proteína BigR se liga na seqüência repetida invertida (9-4-9) localizada na região ¿10 do promotor do operon XF0768-0764, denominada BigRbox. BigR inibe a atividade do operon reprimindo a expressão do gene repórter GFP em Escherichia coli, Agrobacterium tumefaciens e Xylella fastidiosa. Mutações no BigRbox dos promotores de Xylella e Agrobacterium afetam a ligação do repressor e abole a transcrição do gene repórter. Esses dados indicam, portanto, que BigR compete com a RNA polimerase pelo mesmo sítio de ligação ao DNA, revelando assim o mecanismo de regulação do operon. BigR apresenta similaridade com fatores transcricionais de bactérias contendo domínio HTH (helix-turn-helix) de ligação ao DNA. Apesar de BigR ter sido inicialmente classificada como uma proteína da família SmtB/ArsR, as quais regulam operons relacionados à resistência a metais, nossos dados indicam que BigR não atua como sensor de metal. A adição de cádmio, ferro e cobre na mistura de ligação causam uma aparente dissociação do complexo DNA/BigR, entretanto, estudos in vivo na presença de metais não evidenciaram nenhuma alteração na expressão do gene repórter. Mutantes deficientes em BigR também não apresentam diferença de crescimento na presença de metais. O gene XF0768 codifica uma proteína nomeada BLH (beta-lactamase-like hydrolase) por pertencer à superfamília das metalo-beta-lactamases. Dada a presença de BigR e BLH no operon de Xylella e Agrobacterium, a atividade do operon foi analisada em diferentes condições como crescimento das células repórter in planta, co-cultivo com bactérias endofíticas e deficiência nutricional. Entretanto, uma maior atividade do operon em Xylella e Agrobacterium foi detectada apenas na condição de biofilme. Células de Agrobacterium aderidas em raiz de tabaco também apresentaram maior expressão do gene repórter quando comparadas às células em suspensão. Mutantes de Agrobacterium deficientes em BigR (bigR-) apresentam maior expressão do gene repórter, confirmando que BigR age como o repressor transcricional do operon. A quantificação da formação de biofilme das células mutante e selvagem revelou uma maior densidade de biofilme no mutante bigR- em superfície de vidro e também maior quantidade de células aderidas em raiz de tabaco, indicando que o operon pode estar envolvido com o processo de adesão ou desenvolvimento do biofilme bacteriano. Do ponto de vista estrutural, foi mostrado que a proteína BigR truncada no N-terminal (?BigR) encontra-se estruturada na forma de trímero, diferindo do observado para as proteínas com domínio HTH que em geral formam dímeros e monômeros. BigR é estável termicamente, começando a perder estrutura à 74oC, mas retendo um sinal residual de a-hélice até 90oC. Tratamentos com altas concentrações de (NH4) SO 2 4 interferiram na ligação da proteína ao DNA alvo e no conteúdo de estrutura secundária sem alterar, contudo, sua estabilidade térmica. A purificação e cristalização da proteína ?BigR resultou na coleta de alguns conjuntos de dados da proteína nativa e derivada, através de soaking ou marcada com SeMet. Apesar dos dados obtidos serem de boa qualidade, até o momento, não foi possível a resolução da estrutura cristalográfica desta proteína
Abstract: The XF0767 gene from Xylella fastidiosa is located in an operon composed by a set of genes (XF0768-XF0767-XF0766-XF0765-XF0764) of unknown function. This operon is conserved in a number of plant-associated bacteria including Agrobacterium tumefaciens, Mezorhizobium loti e Sinorhizobium meliloti. The DNA region upstream of the operon has canonical sequences corresponding to ¿35 and ¿10 elements found in sigma70-regulated promoters. The aim of this work was to elucidate the biological function of the protein encoded by XF0767, named BigR (biofilm growth-associated repressor), as a transcriptional regulator and its importance to the bacteria. BigR binds to an inverted repeat sequence (9-4-9) located in the ¿10 region of the XF0768-0764 operon promoter. This sequence was named BigRbox. BigR repressed transcription of its own operon upon binding to the BigRbox in Xylella fastidiosa and Agrobacterium tumefaciens. Mutations in the BigRbox significantly affected the repressor binding and abolished transcription of the reporter gene in both bacteria, indicating that BigR compete with the RNA polymerase for the same promoter site. BigR is similar to HTH transcriptional factors of the SmtB/ArsR family, which control tolerance and detoxification of heavy metals in prokaryotes. Despite the similarities, BigR does not appear to function as a metal sensor, as initially predicted. Although binding of BigR to its target DNA was diminished in the presence of cadmium, copper and iron, operon regulation in response to metals was not demonstrated in vivo. In addition, Agrobacterium mutants deficient in BigR did not show changes in growth rates in the presence of metals. BLH is an unusual beta-lactamase-like hydrolase coded by the XF0768 gene. To gain insights into the possible function of the operon, the activity of reporter cells was observed in the presence of different compounds and conditions including in planta growth, effect of endophytic competition and nutrient deficiency. Significantly, an increased operon activity was observed in Xylella and Agrobacterium biofilms. Agrobacterium cells attached to tobacco roots also showed high levels of reporter gene expression in comparison to cells in suspension. A. tumefaciens mutants deficient in the BigR showed constitutive expression of the operon, confirming that BigR acts as a transcriptional repressor. Biofilm quantification showed increased biofilm formation in glass surfaces as well as in tobacco roots, indicating that the operon may play a role in cell adherence or biofilm development. Structurally, the truncated BigR protein (?BigR) is a trimmer in solution, as opposed to most HTH regulators known, which are usually found as dimmers or monomers. BigR is stable at high temperatures (74oC); however, treatments with high concentrations of ammonium sulfate interfere with the protein secondary structure and its DNA binding capacity, without affecting its thermal stability. Good quality X-ray diffraction data were collected for the native and derivative ?bigR protein; however, structure resolution was not possible probably due to problems with the crystals
Doutorado
Genetica de Microorganismos
Doutor em Genetica e Biologia Molecular