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1
Brauer, Matthew Jonas. "Geometry and genetics of microbial adaptation /." Full text (PDF) from UMI/Dissertation Abstracts International, 2000. http://wwwlib.umi.com/cr/utexas/fullcit?p3004221.
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Hu, Yiguo. "Identification of Key Signaling Molecules with Therapeutic Potential for Ph+ Leukemia." Fogler Library, University of Maine, 2007. http://www.library.umaine.edu/theses/pdf/HuY2007.pdf.
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Lundin, Cecilia. "Homologous recombination at replication forks in mammalian cells /." Stockholm : Institutionen för genetik, mikrobiologi och toxikologi, Univ, 2004. http://urn.kb.se/resolve?urn=urn:nbn:se:su:diva-207.
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Imamura, Kely Braga [UNESP]. "Caracterização funcional de um fator de transcrição hipotético no fungo Neurospora crassa." Universidade Estadual Paulista (UNESP), 2015. http://hdl.handle.net/11449/135929.
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000857370.pdf: 3326185 bytes, checksum: 3f22efb9b253d3c96ffa4aefe4751ed0 (MD5)<br>O fungo Neurospora crassa tem sido amplamente utilizado como organismo modelo para o estudo de alguns aspectos da biologia em eucariotos. O sequenciamento de seu genoma permitiu analisar funcionalmente diversos fatores de transcrição e, portanto, atribuir função a proteínas anotadas como hipotéticas. Neste estudo, está sendo investigado o papel funcional do produto de ORF NCU01629, um fator de transcrição que pertence à família zinc-finger sem homólogos funcionais nos bancos de dados de fungos filamentosos. Análises de interação DNA-proteína in vitro foram previamente realizadas por pesquisadores colaboradores, permitindo a identificação do seu motif de ligação ao DNA, bem como os genes provavelmente regulados por este fator de transcrição. A partir destes dados, estes genes foram classificados pelo FunCat. Os resultados revelaram o envolvimento do fator de transcrição em eventos celulares relacionados ao estresse oxidativo, bem como morte celular, entre outros processos celulares. Análises do crescimento radial do fungo foram realizadas em placas de Petri contendo agentes indutores de diferentes tipos de estresse, tais como osmótico, térmico e oxidativo. A linhagem mutante mostrou crescimento semelhante à linhagem selvagem, em condições de estresse osmótico (NaCl 0,1-1,5M e sorbitol 1-1,5M), pH (4,2 e 7,8) e térmico (45ºC). Entretanto, o crescimento da linhagem mutante foi influenciado quando a linhagem foi exposta a diferentes agentes indutores de EROs, como o paraquat (10 μM), menadiona (50 μM), H2O2 (2 mM) e farnesol (10 μM). A linhagem mutante mostrou crescimento radial reduzido, quando comparado à linhagem selvagem no tratamento com diferentes concentrações de paraquat e farnesol e aumento da resistência quando expostos a H2O2 e menadiona. A expressão de genes relacionados a EROs (cat-1, cat-2, cat-3, gst-1, gst-2, sod e nox) e genes apoptóticos...<br>The fungus Neurospora crassa has been widely used as a model organism for the study of some aspects of biology in eukaryotes. The sequencing of its genome has enabled functionally analyze various transcription factors and therefore, assign function to hypothetical proteins. In this study, we investigated the functional role of the ORF NCU01629 product, a transcription factor that belongs to the zinc-finger protein family without functional homologues in fungi database. In vitro analysis of DNA-protein interaction, allowed the identification of its DNA binding motif and, as a consequence, the most likely genes regulated by this transcription factor. The genes were classified by FunCat. The results revealed the involvement of the transcription factor in multiple cellular processes including the response to oxidative stress and cell death. Analyses of radial growth were performed in Petri dishes containing agents that induce different types of stress such as osmotic, thermal and oxidative. The knockout strain showed similar growth to the wild type strain when exposed to osmotic (NaCl 0,1-1,5M and sorbitol 1-1,5M), pH (4.2 and 7.8) and heat (45°C) stresses. However, growth of the knockout strain was influenced when the strain was exposed to different ROS inducing agents, such as paraquat (10 μM), menadione (50 μM), H2O2 (2mM) and farnesol (10 μM). The knockout strain showed reduced radial growth, compared to the wild-type strain, when exposed to different concentrations of paraquat and farnesol and increased resistance to H2O2 and menadione. The expression of genes related to ROS (cat-1, cat-2, cat-3, gst-1, gst-2, sod, and nox) and apoptotic genes (bax, metascaspases, and p53) were analyzed by RT-qPCR. The results showed that the transcription factor is involved in the regulation of the oxidative stress response, controlling the expression of all genes. The gene encoding glutathione-S-transferase...
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Meng, Da. "Bioinformatics tools for evaluating microbial relationships." Pullman, Wash. : Washington State University, 2009. http://www.dissertations.wsu.edu/Dissertations/Spring2009/d_meng_042209.pdf.
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Thesis (Ph. D.)--Washington State University, May 2009.<br>Title from PDF title page (viewed on June 8, 2009). "School of Electrical Engineering and Computer Science." Includes bibliographical references.
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Robinson, Andrea Keryn. "Microbial zinc metallothioneins : function of SmtA and species distribution." Thesis, University of Newcastle Upon Tyne, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.366622.
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Lolle, Susan Janne. "Expression of killer preprotoxin cDNA in Saccharomyces cerevisiae : functional analysis of the N-terminal leader domain." Thesis, McGill University, 1987. http://digitool.Library.McGill.CA:80/R/?func=dbin-jump-full&object_id=75435.
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Expression of cDNA clones of the M1 double-stranded RNA killer preprotoxin coding region in Saccharomyces cerevisiae successfully directed the synthesis of secreted active toxin. Transformants harbouring these expression plasmids also displayed a K1 specific immunity phenotype. Immunoprecipitation of intracellular proteins with antitoxin antiserum showed that these transformants synthesize a 42kd glycosylated preprotoxin precursor. Two smaller unglycosylated immunoreactive species could also be resolved. These toxin precursor species were characterized by using secretory-defective hosts, by comparative electrophoretic mobilities, and by tunicamycin susceptibility. Such studies indicate that these protein species represent intermediates generated by signal cleavage of the preprotoxin and its subsequent glycosylation and provide evidence that these events occur post-translationally. Mutational analysis of the 44 amino acid preprotoxin N-terminal leader indicated that it is functionally bipartite, consisting of an N-terminal signal sequence and a C-terminal pro-sequence. Deletion of the leader perturbed but did not eliminate secretion of toxin.
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Simmons, Susan. "The microbial ecology of acidic environments." Thesis, University of Warwick, 2001. http://wrap.warwick.ac.uk/58964/.
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The microflora of two acidic environments was investigated using analysis of 16S rDNA amplified by the polymerase chain reaction (PCR) from environmental DNA. These environments had different chemical characteristics from most of the acidic environments studied by others. The first sample site, a coal spoil (Birch Coppice, Warwickshire), might have been expected to produce niches enriched in humic matter. The second, comprising geothermal vents on the Island of Vulcano, was unusual for natural acidic environments since it was saline. Three vent regions of different temperatures (30°C, 45°C and 80°C) were examined. Prior to the 16S rDNA analysis of the sites, a brief investigation into selection of a suitable method of DNA extraction was carried out. A bead-beating method and a chemical lysis/freeze-thaw method were compared. With regard to clone types found via each method, there was little qualitative difference. DNA was extracted from the two sites and 16S rRNA genes were amplified by PCR. PCR products were ligated and competent E. coli cells were transformed to produce clone libraries. Restriction fragment length polymorphisms (RFLPs) were examined and representatives of each RFLP type were sequenced and analysed with reference to RNA gene sequence data bases. The coal spoil clone library was dominated by sequences related to those from uncultured actinobacteria, particularly those found previously in peat bogs and various soils. Representatives of some well-known acidophiles were also found (e.g. Leptospirillum species). The clone bank from the saline, geothermal site DNA comprised sequences from acidophiles capable of growth at the respective temperatures of different samples. The lowest temperature samples produced sequences from a novel Acidithiobacillus species and also indicated a novel species probably related to Thiobacillus prosperus (which was isolated previously from Vulcano). A high temperature sample gave sequences from archaeal acidophiles, Acidianus brierleyi and, previously isolated from Vulcano, Acidianus infernus and Thermoplasma volcanium. Where the clone banks revealed the presence of novel organisms, attempts were made to isolate and characterise them. The novel actinobacteria did not appear to grow in laboratory enrichment cultures. The novel Acidithiobacillus species and two novel Thiobacillus prosperus-like species were characterised.
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Tsang, J. S. H. "The physiology and genetics of bacterial dehalogenases." Thesis, University of Kent, 1987. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.380588.
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Melendrez, Melanie Crystal. "Population genetics of Synehococcus species inhabiting the Mushroom Spring microbial mat, Yellowstone National Park." Thesis, Montana State University, 2010. http://etd.lib.montana.edu/etd/2010/melendrez/MelendrezM0510.pdf.
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The species concept in microbiology is under considerable debate. Some scientists believe that traditional approaches are adequate, while others search for more natural concepts. The ecotype concept (ecological species concept) was evaluated in this work. Two temperature sites of a well-studied microbial mat system in Yellowstone National Park were investigated. Previous molecular analyses with 16S rRNA and the adjacent internal transcribed spacer (ITS) suggested the dominance of two putative ecotypes (PEs) of cyanobacteria in these sites, Synechococcus genotypes A and B'. Higher resolution molecular approaches were developed to address the hypotheses that (i) there are more Synechococcus PEs than those discerned by 16S rRNA/ITS sequence variation, (ii) these PEs exhibit distinct ecological distribution patterns and (iii) recombination has been less important than mutation in shaping the evolution of these Synechococcus populations. Analysis of single protein-encoding loci revealed more sample-specific PEs than previously detected, but didn't account for recombination. Bacterial artificial chromosome (BAC) libraries were constructed to sample multiple loci near 16S rRNA genes for multi-locus sequence analyses (MLSA). Analysis of BAC clone end sequences revealed that 16S rRNA regions of the genomes of Synechococcus A- and B'-like populations have undergone rearrangement. Multiple BAC loci were analyzed using two population genetics algorithms; Evolutionary Simulation (ES) and eBURST. ES of concatenated MLSA sequences, but not eBURST analysis, suggested a much greater number of PEs than were detected by 16S rRNA and ITS and provided stronger evidence of sample-specificity. Recombination, suggested by phylogenetic incongruency among loci, multiple recombination tests and polymorphism patterns, appears to have been more frequent than mutation, but not to have erased ecotype structure. Many PEs predicted by ES contained a dominant variant surrounded by rare variants. eBURST predicted some clonal complexes with the same dominant variant, but different rare variants. ES appears to miss phylogenetically distant variants that differ at one locus, whereas eBURST appears to miss phylogenetically similar variants that differ at >1 locus. True ecotype populations in nature may contain both types of variants, but this must be evaluated by examining the distribution of all variants relative to environmental gradients.
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Al-Ani, Bahjat S. "Studies on extracellular protein gene expression in Staphylococcus aureus." Thesis, University of Nottingham, 1990. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.277372.
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Miller, Philip F. P. "Involvement of Ca2+ in the regulation of apical growth and branching in the pathogenic fungus Aspergillus fumigatus." Thesis, University of Aberdeen, 1993. http://digitool.abdn.ac.uk/R?func=search-advanced-go&find_code1=WSN&request1=AAIU053094.
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Calcium has been shown to play important roles in the control of tip growth in a wide range of cell types. This thesis sets out to test the effects of exogenous Ca2+ on various morphological events during the life cycle of the human pathogenic fungus Aspergillus fumigatus. The Ca2+ chelating agent EGTA induced marked morphological effects on colonies of A.fumigatus. That is radial growth rate decreased and branching frequency increased with increasing concentration of EGTA. Ion substitution experiments indicated that these effects were due to the chelation of Ca2+ and not Mg2+, Zn 2+, Cu2+ or Fe2+. However, Mn2+ was able to substitute for Ca2+. Calcium ions were required for germ tube emergence but not for the preceding period of spherical growth in spores of A.fumigatus. That is the rate of spherical growth was independent of exogenous [Ca2+ ] but germ tube emergence was retarded and spores became more swollen as exogenous Ca2+ decreased. The effect of applied electrical fields on spores of A.fumigatus was investigated. Germ tubes were anodotropic and polarisation increased with increasing field strength. The galvanotropic response was dependent on Ca2+ and polarisation decreased with exogenous [Ca2+ ]. The effect of exogenous [Ca2+] on growth kinetics of colonies of A.fumigatus growing on solid medium was investigated. When colonies were incubated on medium buffered at pCa 4-8 (10-4-10 -8 M) hyphal growth unit length and mean hyphal extension rate decreased linearly with the log of Ca2+ concentration. In contrast the specific growth rate remained constant over the range pCa 4-7 and was only reduced when colonies were incubated in medium buffered at pCa 8. This suggests that exogenous Ca2+ acts on processes that govern apical growth and branching but do not affect growth per se. The effect of a broad range of Ca2+-channel blockers and calmodulin antagonists on the growth and morphology of colonies of A.fumigatus were also investigated.
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Imamura, Kely Braga. "Caracterização funcional de um fator de transcrição hipotético no fungo Neurospora crassa /." Araraquara, 2015. http://hdl.handle.net/11449/135929.
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Orientador: Maria Célia Bertolini<br>Co-orientador: Rodrigo Duarte Gonçalves<br>Banca: Iran Malavazi<br>Banca: Márcia Eliana da Silva Ferreira<br>Resumo: O fungo Neurospora crassa tem sido amplamente utilizado como organismo modelo para o estudo de alguns aspectos da biologia em eucariotos. O sequenciamento de seu genoma permitiu analisar funcionalmente diversos fatores de transcrição e, portanto, atribuir função a proteínas anotadas como hipotéticas. Neste estudo, está sendo investigado o papel funcional do produto de ORF NCU01629, um fator de transcrição que pertence à família zinc-finger sem homólogos funcionais nos bancos de dados de fungos filamentosos. Análises de interação DNA-proteína in vitro foram previamente realizadas por pesquisadores colaboradores, permitindo a identificação do seu motif de ligação ao DNA, bem como os genes provavelmente regulados por este fator de transcrição. A partir destes dados, estes genes foram classificados pelo FunCat. Os resultados revelaram o envolvimento do fator de transcrição em eventos celulares relacionados ao estresse oxidativo, bem como morte celular, entre outros processos celulares. Análises do crescimento radial do fungo foram realizadas em placas de Petri contendo agentes indutores de diferentes tipos de estresse, tais como osmótico, térmico e oxidativo. A linhagem mutante mostrou crescimento semelhante à linhagem selvagem, em condições de estresse osmótico (NaCl 0,1-1,5M e sorbitol 1-1,5M), pH (4,2 e 7,8) e térmico (45ºC). Entretanto, o crescimento da linhagem mutante foi influenciado quando a linhagem foi exposta a diferentes agentes indutores de EROs, como o paraquat (10 μM), menadiona (50 μM), H2O2 (2 mM) e farnesol (10 μM). A linhagem mutante mostrou crescimento radial reduzido, quando comparado à linhagem selvagem no tratamento com diferentes concentrações de paraquat e farnesol e aumento da resistência quando expostos a H2O2 e menadiona. A expressão de genes relacionados a EROs (cat-1, cat-2, cat-3, gst-1, gst-2, sod e nox) e genes apoptóticos...<br>Abstract: The fungus Neurospora crassa has been widely used as a model organism for the study of some aspects of biology in eukaryotes. The sequencing of its genome has enabled functionally analyze various transcription factors and therefore, assign function to hypothetical proteins. In this study, we investigated the functional role of the ORF NCU01629 product, a transcription factor that belongs to the zinc-finger protein family without functional homologues in fungi database. In vitro analysis of DNA-protein interaction, allowed the identification of its DNA binding motif and, as a consequence, the most likely genes regulated by this transcription factor. The genes were classified by FunCat. The results revealed the involvement of the transcription factor in multiple cellular processes including the response to oxidative stress and cell death. Analyses of radial growth were performed in Petri dishes containing agents that induce different types of stress such as osmotic, thermal and oxidative. The knockout strain showed similar growth to the wild type strain when exposed to osmotic (NaCl 0,1-1,5M and sorbitol 1-1,5M), pH (4.2 and 7.8) and heat (45°C) stresses. However, growth of the knockout strain was influenced when the strain was exposed to different ROS inducing agents, such as paraquat (10 μM), menadione (50 μM), H2O2 (2mM) and farnesol (10 μM). The knockout strain showed reduced radial growth, compared to the wild-type strain, when exposed to different concentrations of paraquat and farnesol and increased resistance to H2O2 and menadione. The expression of genes related to ROS (cat-1, cat-2, cat-3, gst-1, gst-2, sod, and nox) and apoptotic genes (bax, metascaspases, and p53) were analyzed by RT-qPCR. The results showed that the transcription factor is involved in the regulation of the oxidative stress response, controlling the expression of all genes. The gene encoding glutathione-S-transferase...<br>Mestre
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Andrade, Fontes Carlos Mendes Godinho de. "Characterization of a microbial xylanase and its expression in mammalian cells." Thesis, University of Newcastle Upon Tyne, 1995. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.294720.
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Xia, Yu, and 夏雨. "Exploring microbial structure and carbohydrate metabolism of thermophilic anaerobic cellulose-degrading consortia by metagenomics based on next generation sequencing." Thesis, The University of Hong Kong (Pokfulam, Hong Kong), 2013. http://hdl.handle.net/10722/195961.
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The pressing need for clean renewable energy sources has aroused worldwide research interest on the exploration of biofuels produced from lignocellulosic feedstock (e.g. forestry or agricultural residues and municipal wastes). The general absence of cost-effective method to overcome the recalcitrant nature of cellulosic biomass is the major challenge for the industrialization of this so-called second-generation biofuel. With the purpose to enhance our understanding of the fundamental mechanism of thermophilic microbial cellulose conversion process, we used culture-independent metagenomic analysis based on Next Generation Sequencing to explore the physiological ecology of thermophilic cellulolytic microbial community and more importantly to discover metabolic potentials.
During the enrichment of thermophilic cellulolytic consortium, noticeable effects of co-substrate and pH was observed and subsequently investigated. Based on the community structure revealed by 16S rRNA gene sequencing at various pH values, we concluded that keeping pH higher than 6.0 was crucial to maintain effective cellulose conversion because the growth of Thermoanaerobacterium over other more efficient cellulolytic populations could be practically avoided.
Given in mind that uncharacterized microbial populations may possess critical enzymatic components that are essential for the breakdown of cellulosic feedstock, gene-centric metagenomic pipeline was developed to discover genes that are functionally beneficial for thermophilic cellulose hydrolysis. Aside from that, metagenomic gene mining based on functional prediction using HMM (Hidden Markov Model) showed higher positive ratio in identifying novel carbohydrate-active genes than that of functional screening. Without cultivation, near complete genomes of the major thermophilic cellulose degraders were recovered from the metagenome by a gene binning pipeline combining tetranucleotide frequency based primary k-means clustering and subsequent scaffolding with paired-end relationship between two reads (sequences).
Furthermore, by quantifying the transcriptional activities of various carbohydrate-active genes in the metatranscriptome of the enriched thermophilic cellulose-degrading consortium, we disclosed significance of enzymes of GH09 and GH48 which had been underestimated by previous metagenomic studies. Eventually, metagenomic survey of various sludge samples collected at specific operational conditions helped to confirm the metabolism potential of thermophilic sludge in cellulose up taking by possessing more enzymes of GH05 and GH04 families.<br>published_or_final_version<br>Civil Engineering<br>Doctoral<br>Doctor of Philosophy
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Carlton, Timothy M., and n/a. "Iron and microevolution in Mesorhizobia." University of Otago. Department of Microbiology & Immunology, 2006. http://adt.otago.ac.nz./public/adt-NZDU20070215.154441.
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Genome plasticity in soil bacteria is predicted to be evolutionarily advantageous, allowing bacteria to sample genetic variation for adaptation to local soil ecology. In the field population of mesorhizobia where the symbiosis island (ICEMlSym[R7A]; an I̲ntegrative C̲onjugative E̲lement) was first identified, individual members were found to have significant chromosomal variation downstream of the phe-tRNA gene or phe-tRNA integrated ICEMlSym[R7A]. However, the nature of this genetic variation and whether it contributed to the adaptation of the indigenous mesorhizobia to their field environment were unknown.
This work focused on a nodule isolate, Mesorhizobium sp. strain R88B, a member of the indigenous mesorhizobial population that received ICEMlSym[R7A] from strain R7A. The region downstream of ICEMlSym[R7A] was sequenced, revealing three distinct regions of non-conserved DNA, totalling 34.5 kb. Integrated directly downstream of ICEMlSym[R7A] was IMEMlAdh[R88B], a 24.3-kb novel I̲ntegrative M̲obilisable E̲lement. Using a PCR-based assay, it was shown that the IMEMlAdh[R88B] integrase could excise not only IMEMlAdh[R88B], but also a dual-IMEMlAdh[R88B]/ICEMlSym[R7A] hybrid, indicating the potential mobility of IMEMlAdh[R88B], and a likely evolutionary intermediate of a novel ICE. However, a functional role for MadA, (a putative adhesin and the sole adaptive trait encoded on IMEMlAdh[R88B]) was not discovered. Southern hybridisations with the mesorhizobial population provided evidence for the existence of a novel family of IMEs in the mesorhizobia, which, by diversifying their internal sequences, provide allele-specific variation to the population.
The two other regions downstream of IMEMlAdh[R88B] possessed no obvious mobile genetic element structures, and only the region adjacent to the core-chromosome encoded ORFs with putative functions. Mutation of two of these ORFs, fhuD1 and fhuB1, identified their function as two of the four components of a ferrichrome ABC-uptake (Fhu) system. Using genetic screens, the remaining components of this transporter were mapped to two separate loci. Thus, the functional transporter in R88B was a composite of at least two independently-acquired Fhu systems. The genetic screens also revealed that ferrichrome utilisation was dependent on a TonB energy-transduction system encoded downstream of the Fhu ATPase gene, fhuC.
Expression studies on the three fhu loci demonstrated that, despite their separate acquisition, their expression was coordinately up-regulated in response to low-iron conditions. Bioinformatics on the predicted promoter regions of the fhu genes identified the binding site of the rhizobial Fur analogue, RirA, which is likely to be responsible for this expression profile.
Southern hybridisations of DNA isolated from members of the mesorhizobial population revealed the three fhu loci were not conserved in the mesorhizobial population. The presence of FhuA was the best predictive marker for the trait. It is proposed that multiple rounds of acquisitions and recombinations, both illegitimate and legitimate, formed this transporter, with the constant need for iron offset by the negative selection pressure of FhuA being a target for phage. None of the Fhu-specific genes was present in the sequenced M. loti strain MAFF303099 though flanking sequences were, further emphasizing the role of genome microevolution in forming the Fhu phenotype.
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Mazzon, Ricardo Ruiz. "Estudo de genes de Caulobacter crescentus importantes para a sobrevivência em baixas temperaturas." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-20032012-162702/.
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Caulobacter crescentus sobrevive em baixas temperaturas e mostrou ser um organismo psicrotolerante e de alta resistência ao congelamento, característica resultante de múltiplos fatores. C. crescentus possui quatro genes codificantes para CSPs, sendo cspA e cspB induzidos em baixas temperaturas e cspB, cspC e cspD em fase estacionária. A ausência de cspA e cspB ou cspA e cspC confere grande deficiência de crescimento em baixas temperaturas. cspA e cspB não são autorregulados e são regulados pós-transcricionalmente via estabilização de seu mRNA e traducionalmente após o choque-frio. A expressão de cspB é influênciada por CspC a 30 graus e durante o choque-frio, e por CspC, SpdR e SpoT durante a fase estacionária. A ausência de CspC ou CspC e CspD compromete a adaptação à fase estacionária promovendo alterações morfológicas. Nenhuma das CSPs de C. crescentus é capaz de reverter o fenótipo de E. coli BX04 por expressão heteróloga, embora todas possuam atividade antiterminadora que, nestas proteínas, não depende dos mesmos aminoácidos que CspE de E. coli.<br>Characterization of Caulobacter crescentus cold response was performed. This bacterium showed to be psicrotolerant and have remarkable freezing resistance, which may be a result of multiple traits. C. crescentus has four CSP encoding genes, being cspA and cspB cold-induced and cspB, cspC and cspD stationary phase-induced. The absence of cspA and cspB or cspA and cspC led to growth deficiency under low temperature incubation. cspA and cspB are not self-regulated and are post-transcriptionally and translationally regulated during cold-shock. The cspB gene expression is affected by CspC at exponential growth phase and by CspC, SpdR and SpoT at stationary phase. The absence of CspC, or CspC and CspD, affects stationary phase fitness of this organism, also promoting morphological alterations. None of the C. crescentus CSPs were able to restore the phenotype of E. coli BX04 strain by heterologous expression. Although all of them have shown to be transcription antiterminators, this ability is not dependent on the same critical aminoacids displayed by CspE from E. coli.
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McCallum, Mark Edward. "Genetic studies of xanthomonas maltophilia." Diss., Georgia Institute of Technology, 1995. http://hdl.handle.net/1853/25214.
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White, Patricia McGuire. "Genetic studies of P̲s̲e̲u̲d̲o̲m̲o̲n̲a̲s̲ m̲a̲lt̲o̲p̲h̲i̲l̲i̲a̲." Diss., Georgia Institute of Technology, 1987. http://hdl.handle.net/1853/31068.
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Scanferlato, Vjera Sostarec. "Environment risk assessment for toxic chemicals and genetically-engineered microorganisms : a microcosm approach /." Diss., This resource online, 1990. http://scholar.lib.vt.edu/theses/available/etd-07282008-135357/.
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Kirke, David F. "Protein-nucleic acid interactions regulating bacterial quorum sensing." Thesis, University of Nottingham, 2000. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364668.
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Benjamin, Ashlee. "Genetic elements of microbes : a comprehensive and integrated genomic database application /." Online version of thesis, 2009. http://hdl.handle.net/1850/10833.
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Massini, Karen Cristina. "Bioprospecção de genes biossintéticos de policetídeos em DNA metagenômico de solo de Mata Atlântica." Universidade de São Paulo, 2009. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-29012010-111747/.
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A Mata Atlântica brasileira é apontada como um dos mais importantes refúgios da biodiversidade em todo o planeta. Este bioma é extremamente importante sob o aspecto da riqueza de espécies vegetais e animais na sua composição e interações, porém ainda pouco conhecido e explorado sob o ponto de vista microbiológico. Um grama de solo pode conter cerca de 10 bilhões de micro-organismos de diferentes espécies. A maioria dos micro-organismos presentes nos solos não é de fácil cultivo em laboratório (somente 0.1-10% são recuperados), sendo necessária à utilização de novas técnicas para superar este problema. Muitos micro-organismos presentes no solo tem grande importância biotecnológica por produzirem compostos bioativos. O filo Actinobacteria é abundante em solos e de grande importância econômica, tendo em vista que a maioria dos antibióticos comercializados é produzido por membros deste grupo. Porém a biodiversidade microbiana da Mata Atlântica, bem como, o seu potencial biotecnológico não tem sido plenamente estudado. Poucos trabalhos mostram produtos do metabolismo microbiano com potencial em uso em indústrias e mostram menos ainda antimicrobianos produzidos por isolados bacterianos desta região. Dentro deste contexto, o presente trabalho buscou em duas alternativas metodológicas como a técnica independente de cultivo, o metagenoma, verificar a presença de genes de uma importante via biossintética os policetídeos sintases e com a técnica dependente de cultivo, selecionar prováveis bactérias produtoras de composto bioativos. O metagenoma propõe fazer uma análise do DNA total de amostras do solo, visando conhecer a informação gênica destes compostos na complexa diversidade microbiana. Desta forma, várias abordagens foram empregadas para conseguir um DNA de alto peso molecular e de qualidade suficiente para construir bibliotecas metagenômicas, e procurar nestas, genes das vias de sínteses dos policetídeos (PKS) tipo I e tipo II, que ficam agrupados em clusters que variam de tamanho entre 20 a 100 kb. Otimizamos um método de extração de DNA do solo e conseguimos obter um DNA de aproximadamente 50kb, que foi amplificado por PCR utilizando primers para regiões conservadas dos genes policetídeos sintases tipo I e II (acetosintase ) de Actinomicetos. Os fragmentos obtidos, PKS I e PKS II, com tamanho entre 600pb a 700pb, foram clonados, construído-se duas bibliotecas metagenômicas (KSI e KS II). Os clones foram sequênciados e analisados em uma árvore filogenética. A análise filogenética de genes policetídeos tipo I demonstrou similaridade com estes genes de diversas divisões de bactérias, revelando a presença de prováveis genes novos não apenas relacionados a via de PKSI, como também aos genes de PKSI híbridos com peptídeos não ribossomais. Em complemento a filogenia de policetídeos tipo II apresentou uma similaridade com genes de Actinobacteria, formando um grupo que também está relacionados a presença de prováveis genes novos de importantes famílias de antibióticos. Através do cultivo utilizando meio seletivo para o crescimento de bactérias não cultivadas, foi possível isolar sete bactérias que possuem atividade antibacteriana e/ou antifúngica.<br>The Brazilian Atlantic Forest is considered as one of the most important reservoir of biodiversity in the planet. This biome is extremely important for its richness of plant and animal species but although with their composition and interactions poorly known and unexplored from a microbiological perspective. One gram of soil can contain near 10 billion microorganisms of different species. The majority of the soil microorganisms is not cultivable in laboratory (only 0.1-10% are recovered), being necessary to use new techniques to overcome this problem. Many of the soil microorganisms are biotechnologically important for the production of bioactive compounds. The Actinobacteria phylum is abundant in soil and important economically due to the capacity of synthesize many antibiotics. Nevertheless, the Atlantic Forest microbial biodiversity has not been properly study. Few works show microbial metabolic products with potential use in industries and, still less, antimicrobials isolated from this biome. The present work searched two new methodological alternatives: one culture independent, the metagenome, to verify the presence of polyketide synthases biosynthetic genes; and the second, the culture dependent, to select potential bacteria producers of bioactive compounds. The metagenome intend the total DNA analysis of a sample, focusing in to know the genetic information of the complex microbial diversity. Several approaches were used in order to obtain DNA of high molecular weight and quality toconstruct metagenomic libraries and search for polyketide synthases (PKS) genes types I and II, that usually are organized in clusters of 30 to 100 kb. A DNA extraction method was optimized obtaining DNA of approximately 50 kb, and used for the detection of PKS gens by PCR approaches using primers based in polyketide synthases type I and II (ketosynthase ) conserved regions of Actinomycetes. The PKS I and PKSII amplicons (600-700 bp) were cloned and two metagenoic libraries were obtained (KS I and KS II). The clones were sequenced and analyzed in a phylogenetic tree. Phylogenetic analysis of PKS I genes reveled high similarities with genes of several divisions of bacterias pointing the presence of provable new genes related with the synthesis of polyketides produzrd by PKS I and hybrid PKS with non ribosomal peptides (NRPs). Polyketide type I genes showed similarity with Streptomyces and uncultered bacteria. The analysis of polyketide II genes showed high similarity with genes of Actinobacteria gruped in two main groups, one of them with possible new genes related with the production of important antibiotics. Using selective medium for uncultered bacteria, seven isolates were obtained being studied taxonomically and tested for the production of secondary metabolites with antibacterial and antifungal activities.
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Ryan-Kewley, Angela E. "Microbial ecology of Propionibacterium acnes in patients undergoing treatment with isotretinoin." Thesis, University of Lincoln, 2011. http://eprints.lincoln.ac.uk/6073/.
Full textAbstract:
Following failure to respond to antibiotic therapy, 56 patients (mean age 22yrs; range 15-46yrs) with recorded active Acne vulgaris were treated with a standard course of isotretinoin (1mg/kg body weight for 16 weeks). This study investigated the recovery and analysis of skin surface organisms from several skin sites at the start of treatment, 8 weeks into treatment, at the end of the course and 1 month post treatment. The number of anaerobic isolates - presumptive Propionibacterium acnes and also aerobic isolates recovered by appropriate media from each of three specific sites per patient were compared with age-matched controls of healthy volunteers. Patients and controls exhibited similar total numbers of organisms on the cheek, nares and toe web before treatment. Following treatment with isotretinoin the majority of patients had a significant reduction in bacterial numbers (1-2 orders of magnitude) on the cheek. Numbers in the nares and toe web did not show this reduction. Isotretinoin has no antibacterial activity in vitro or in vivo so the observed reduction would strongly suggest that the effect is mediated through alteration of the skin nutritional micro-environment. The clinic-based patients demonstrated high levels of isolates with antibiotic resistance from the outset; the resistance status of each patients’ skin microbiota was determined before, during and after treatment and the dynamics of the population from these sites was investigated. Previous studies of the nature of skin organisms and of the complex multifactorial mechanisms that lead to the eruption of acne have shown that P. acnes play a significant but as yet poorly explained role in the pathogenesis of the disease although it is traditionally the target organism for anti-acne therapy. The clinical samples were cultured using conventional methods to confirm the presence of P. acnes and staphylococci. Novel modifications of PCR amplification methodology were developed to enhance the detection of specific organisms. New protocols for comprehensive and detailed RAPD-PCR analysis of whole cell PCR were developed and employed to profile recovered isolates from individual patients before and after clinical treatment. RAPD-PCR proved to be a useful tool to survey changing bacterial profiles within the cohort. Isolates from some patients were highly similar to each other at the outset, but displayed increased diversity by the end of therapy. Interestingly, other studies have linked clonal populations of bacteria with high pathogenicity. Some patients appear to have acquired a strain which was not evident at their original sampling, but was present in other patients attending the same clinic. P. acnes persist in the environment and may be found on many inanimate objects, from where they could become a source for transmission. As there is some evidence of acquisition of strains from the environment, this is of considerable significance to the management of dermatology clinics throughout the country.
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Isherwood, Karen Elizabeth. "Quorum sensing in Yersinia pestis." Thesis, University of Nottingham, 2001. http://ethos.bl.uk/OrderDetails.do?uin=uk.bl.ethos.364667.
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Escoda, Assens Lídia. "Applications of next-generation sequencing in conservation genomics: kinship analysis and dispersal patterns." Doctoral thesis, Universitat de Barcelona, 2018. http://hdl.handle.net/10803/586083.
Full textAbstract:
Knowledge of the genealogical relationships among individuals of a population and their dispersal patterns are essential to many studies of endangered species, especially those with small and fragmented populations. The main objective of this doctoral thesis is to use genomic data obtained with next-generation sequencing techniques to infer contemporary dispersal patterns of species from relatedness networks, to construct pedigrees from kinship categories, and to quantify the effect of anthropogenic and geographic barriers on the dispersal of individuals, using as a model the Pyrenean desman (Galemys pyrenaicus), a small semi-aquatic mammal endemic to the Iberian Peninsula.
First, the contact zone between two lineages of the Pyrenean desman in the Iberian Range (La Rioja) was studied using SNPs obtained from ddRAD (double-digest restriction associated DNA) genomic libraries. According to the genomic tree, the principal component analysis, and the population structure analyses, the genetic variability in the area was structured by rivers instead of by mitochondrial lineages. Relatedness and inbreeding coefficients were then calculated with a maximum-likelihood estimator. Mean relatedness was found to be very high in the area. Individuals also showed high inbreeding levels. The reliability of these estimates was assessed with bioinformatics simulations based on artificial pedigrees that included as founders actual genotypes of the studied population. The relatedness networks showed a low level of contemporary inter-river dispersal compared to intra-river dispersal, indicating poor connectivity between rivers.
Then, kinship relationships and pedigrees of Pyrenean desmans of two rivers of the northwestern part of the Iberian Peninsula (Zamora) were inferred. The ddRAD protocol was modified to allow processing each sample independently, which enabled the use of minimally invasive hair samples. Mean relatedness and inbreeding coefficients obtained from the SNPs were much lower than those from La Rioja. In addition, relatedness was higher for female dyads than for male dyads, suggesting a higher degree of female philopatry. Kinship categories were determined and their reliability was assessed using bioinformatics simulations based on artificial pedigrees. Using these kinship categories, pedigrees were reconstructed and their congruence was evaluated with the age of the individuals, the mitochondrial haplotypes, and the inbreeding coefficients. Pedigrees allowed the estimation of the average dispersal distance per generation, as well as preliminary data about the reproductive biology of the species.
Finally, the assortativity coefficient obtained from the kinships networks was used to quantify the effect of specific barriers on the dispersal of individuals in the two rivers studied of Zamora, the Tera and the Tuela. The most important barrier found with this approach was the watershed divide between both rivers, followed by a dam located in one of them. These results were highly congruent with those obtained from the population structure analysis.
The information obtained with the approaches presented in this thesis can be used to unravel fundamental aspects about the biology of endangered species, such as their dispersal patterns and their reproductive biology, as well as to quantify the effect of potential barriers on dispersal. These data may be fundamental to develop conservation plans aimed at improving the connectivity between populations.<br>El conocimiento de las relaciones genealógicas entre individuos de una población y sus patrones de dispersión son esenciales en muchos estudios sobre especies amenazadas, especialmente de aquellas con poblaciones pequeñas y fragmentadas. El objetivo principal de esta tesis doctoral es utilizar datos genómicos obtenidos con técnicas de secuenciación de última generación para inferir patrones de dispersión contemporánea de las especies a partir de redes de parentesco, construir pedigríes a partir de categorías de parentesco y cuantificar el efecto de barreras antropogénicas y geográficas en la dispersión de individuos, usando como modelo el desmán ibérico (Galemys pyrenaicus), un pequeño mamífero semi-acuático endémico de la Península Ibérica.
En primer lugar, se estudió la zona de contacto entre dos linajes de desmán ibérico en el Sistema Ibérico (La Rioja) usando SNPs obtenidos mediante bibliotecas genómicas ddRAD (DNA asociado a sitios de restricción con doble digestión). De acuerdo con el árbol genómico, el análisis de componentes principales y el análisis de estructura poblacional, la variabilidad genética en el área estudiada resultó estar estructurada por ríos en lugar de por linajes mitocondriales. A continuación, los coeficientes de parentesco y de consanguinidad fueron calculados con un estimador de máxima verosimilitud. La media del coeficiente de parentesco encontrada en el área fue muy alta. Los individuos también mostraron altos niveles de consanguinidad. La fiabilidad de estas estimaciones se comprobó mediante simulaciones bioinformáticas basadas en pedigríes artificiales que incluían como fundadores genotipos reales de la población estudiada. Las redes de parentesco construidas mostraron un bajo nivel de dispersión contemporánea entre ríos en comparación con la dispersión dentro de ríos, lo que indicaba una mala conectividad entre los ríos del Sistema Ibérico.
Después se infirieron las relaciones de parentesco y los pedigríes de desmanes ibéricos de dos ríos del noroeste de la Península Ibérica (Zamora). El protocolo de ddRAD se modificó y optimizó para poder procesar cada muestra de forma independiente, lo que permitió el uso de muestras de pelo mínimamente invasivas. Las medias de los coeficientes de parentesco y de consanguinidad obtenidos a partir de los SNPs fueron mucho más bajas que en La Rioja. Además, la media del coeficiente de parentesco fue mayor para las parejas de hembras que para las de machos, lo que sugiere un mayor grado de filopatría de las hembras. Se determinaron las categorías de parentesco existentes y se evaluó su fiabilidad con simulaciones bioinformáticas basadas en pedigríes artificiales. Usando estas categorías de parentesco, se reconstruyeron pedigríes y se evaluó su congruencia mediante la comprobación de la edad de los individuos, los haplotipos mitocondriales y los coeficientes de consanguinidad. La reconstrucción de pedigríes permitió estimar el promedio de la distancia de dispersión por generación, así como datos preliminares sobre la biología reproductiva de la especie.
Por último, se usó el coeficiente de asortatividad obtenido a partir de las redes de parentesco para cuantificar el efecto de barreras específicas en la dispersión de los individuos en los dos ríos estudiados en Zamora, el Tera y el Tuela. La barrera más importante encontrada en el área fue la divisoria de aguas entre ambos ríos, seguida por una presa situada en uno de ellos. Estos resultados fueron altamente congruentes con los obtenidos con el análisis de estructura poblacional.
La información obtenida con el enfoque metodológico presentado en esta tesis puede ser usada para desentrañar aspectos fundamentales sobre la biología de especies amenazadas, como pueden ser sus patrones de dispersión y su biología reproductiva, así como para cuantificar el efecto de barreras potenciales en la dispersión. Estos datos pueden ser fundamentales para desarrollar planes de conservación dirigidos a la mejora de la conectividad entre diferentes poblaciones.
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Costa, Débora dos Santos. "Estudo da frequência do fenótipo mutador para resistência aos antibióticos beta-lactâmicos em linhagens de E. coli patogênicas." Universidade de São Paulo, 2013. http://www.teses.usp.br/teses/disponiveis/42/42132/tde-18062013-110739/.
Full textAbstract:
Atualmente a alta incidência de isolados multirresistentes a antibióticos utilizados na clinica tem-se tornado alarmante. Estudos recentes demonstraram que um dos motivos que contribuem para o aumento da resistência a antibióticos em bactérias é a ocorrência de linhagens que apresentam o fenótipo mutador. Deficiências nos genes do complexo mut, que incluem os sistemas de reparo dependente de metilação (MMR do inglês methyl-directed mismatch repair), e o sistema de reparo oxidativo (GO) podem gerar linhagens com um fenótipo mutador, que, por sua vez, levam ao aumento das taxas mutacionais espontâneas. Isolados clínicos com fenótipo mutador foram descritos em amostras de Escherichia coli patogênica empregando-se a resistência para antibióticos como rifampicina, cloranfenicol e quinolonas mas não há registros de estudos envolvendo o possível impacto do fenótipo mutador sobre a frequência de mutações espontâneas que levem à resistência aos antibióticos beta-lactâmicos. No presente trabalho estudamos a ocorrência do fenótipo mutador frente à resistência aos antibióticos beta-lactâmicos de amplo uso clínico em linhagens de E. coli patogênicas de origem humana, incluindo 48 amostras de E. coli uropatogênica (UPEC) e 5 amostras de E. coli associadas a infecções entéricas. Foram utilizadas como controles positivos para o fenótipo mutador linhagens de E. coli K12 deficientes nos genes mutY ou mutS. Testes qualitativos revelaram a ocorrência de 6 amostras de UPEC e 2 amostras de EHEC com fenótipo mutador para cefalotina, ceftazidima e rifampicina. Entre as amostras estudadas, 3 linhagens de UPEC (amostras 29, 32 e 47) e 1 linhagem de EHEC (amostra 80) apresentaram fenótipo mutador frente à cefalotina e à ceftazidima confirmado em testes quantitativos com valores de mutação espontânea variando entre 0,68 x 10-4 e 0,8 x 10-6. Os resultados baseados em amplificação por PCR revelaram ausência de alterações estruturais em 3 genes do complexo mut (mutS, mutY e mutL) nos quatro isolados que apresentaram fenótipo mutador. O trabalho também envolveu a determinação dos níveis de resistência em clones derivados das 4 linhagens mutadoras após exposição à cefalotina ou à ceftazidima. As colônias obtidas também foram analisadas para a determinação da natureza de mutação que resultou na resistência aos antibióticos beta-lactâmicos. Os resultados obtidos apontam para aumento nos níveis de expressão de uma beta-lactamase para os derivados resistentes da amostra 32, possíveis alterações de permeabilidade do envoltório celular, e, indiretamente, modificação dos alvos celulares para esses antibióticos. Esses resultados revelam a elevada ocorrência do fenótipo mutador entre amostras de UPEC oriundas da clínica e destacam a importância do fenômeno sobre a ocorrência de resistência aos beta-lactâmicos de uso clínico.<br>Currently the high incidence of isolates with multiple resistance to antibiotics used in the clinic is alarming. Recent studies show that one of the reasons that may contribute to increased resistance in bacteria is the occurrence of strains with the mutator phenotype. Deficiencies in mut genes complex including methylation-dependent repair system (MMR from English methyl-directed mismatch repair) and oxidative repair system (GO) can generated strains with a mutator phenotype, which in turn leads to increasing spontaneous mutation rates. Clinical isolates with the mutator phenotype were reported among pathogenic Escherichia coli with antibiotics such as rifampicin, chloramphenicol and quinolones. Nonetheless, there are no studies describing the involvement of the possible impact of the mutator phenotype on the frequency of spontaneous mutations leading to resistance to beta-lactam antibiotics. In this work we studied the occurrence of the mutator phenotype leading toresistance to beta-lactam antibiotics use in clinics. For this purpose we tested a set of pathogenic E. coli strains of human origin, including 48 strains of uropathogenic E. coli (UPEC) and 5 strains of E. coli associated with enteric infections. As positive controls for the mutator phenotype we used E. coli K12 strains deficient in mutY or mutS genes. Qualitative tests revealed 6 UPEC samples and 2 EHEC strains with mutator phenotype for cephalothin, ceftazidime and rifampicin. Three UPEC strains (samples 29, 32 and 47) and one EHEC strain (sample 80) showed spontaneous mutation frequencies ranging from 0.68 x 10-4 to 0.8 x 10-6 to cephalothin and ceftazidime. The results based on PCR amplification revealed no structural changes in the mut gene complex (mutS, mutY and mutL) in the four mutator strains. The work also involved determination of the resistance level to cephalothin or ceftazidime of clones derived from the mutator strains. The colonies obtained were also analyzed to determine the nature of mutation leading to beta-lactam resistance. The results indicated increased expression levels of of a beta-lactamase in derivatives of the 32 strain, possible reduction in cell envelope permeability and, indirectly ,modification of cellular targets for these antibiotics. These results showed the high occurrence of mutator phenotype among UPEC strains derived from clinical settings and highlight the importance of the phenomenon on the occurrence of resistance to beta-lactam antibiotics in clinical use.
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Dhladhla, Busisiwe I. R. "Enumeration of insect viruses using microscopic and molecular analyses: South African isolate of cryotophlebia leucotreta granulovirus as a case study." Thesis, Nelson Mandela Metropolitan University, 2012. http://hdl.handle.net/10948/d1008395.
Full textAbstract:
Baculoviruses have been used as biocontrol agents to control insect pests in agriculture since the 1970s. Out of the fifteen virus families known to infect insects, baculoviruses offer the greatest potential as insect biopesticides, due to their high host specificity which makes them extremely safe to humans, other vertebrates, plants and non-target microorganisms. They comprise of two genera: nucleopolyhedroviruses (NPVs) and granuloviruses (GVs). The South African isolate of Cryptophlebia leucotreta granulovirus (CrleGV-SA) which is infectious for the false codling moth (FCM), Thaumatotibia leucotreta, (Meyrick) (Lepidoptera: Tortricidae), has been successfully developed into two commercial biopesticides; Cryptogran® and Cryptex®, for the control of FCM in citrus crops. The current method of enumeration used for CrleGV-SA virus particles in routine experiments during the production of the GV as biopesticides, is dark field microscopy. However, due to the small size of GVs (300-500 nm in length), the technique is not easy to perform on these viruses, and no systemic comparison has been made of potential alternative methods. Therefore, the main objective of this study was to develop a quantitative enumeration method for CrleGV-SA occlusion bodies (OBs) which is accurate, reliable, and feasible, and compare the developed methods of enumeration to the current method. Purified and semi-purified CrleGV-SA viral stocks were prepared for enumeration studies using spectrophotometry, dark field microscopy, scanning electron microscopy (SEM) and real time qPCR. Spectrophotometry was found to be an unreliable method for enumeration of GVs in the production, standardisation, and quality control of biopesticides. Dark field microscopy and SEM were found to be accurate, and statistically comparable (p = 0.064) enumeration techniques. qPCR is currently being optimised for the enumeration of GVs. This technique was demonstrated to generate accurate standard curves for absolute quantification of virus particles for pure and semi-pure virus preparations. qPCR offers the greatest potential as an accurate enumeration method because it is not affected by contamination with non-biological contaminating debris, nor by other biological material due to the specificity of PCR primers. Further work is required to fully develop qPCR as an enumeration method for GVs. However, dark field microscopy has been successfully validated as an enumeration method. SEM, which has a high resolution compared to light microscopy, has an added advantage over dark field microscopy, which is to distinguish virus particles in semi-pure viral stock preparations during counting. Therefore, SEM currently provides the most unambiguous and feasible enumeration method for GVs in both purified and semi-purified virus samples.
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Bellerose, Michelle M. "Genetic Identification of Novel Mycobacterium tuberculosis Susceptibility and Survival Mechanisms During Antibiotic Treatment." eScholarship@UMMS, 2020. https://escholarship.umassmed.edu/gsbs_diss/1081.
Full textAbstract:
Effective treatment of tuberculosis requires at least six months of combination therapy involving four antibiotics. Alterations in the physiological state of Mycobacterium tuberculosis during infection may reduce drug efficacy and prolong treatment, but these adaptations are incompletely defined. To investigate the mechanisms limiting antibiotic efficacy, I performed a comprehensive genetic study to identify M. tuberculosis genes and pathways important for bacterial survival during antibiotic treatment in vivo. First, I identified mutants in the glycerol kinase enzyme, GlpK, that promote survival under combination therapy. Similar glycerol catabolic mutants are enriched in extensively drug-resistant clinical isolates, indicating that these mutations may promote survival and the development of resistance in humans. A majority of these mutations are frameshifts within a homopolymeric region of the glpK gene, leading to the hypothesis that M. tuberculosis may reversibly produce drug-tolerant phenotypes through genetic variation introduced at homopolymer sites as a strategy for survival during antibiotic treatment. Second, I identified bacterial mutants with altered susceptibility to individual first-line anti-mycobacterial drugs. Many of these mutations did not have obvious effects in vitro, demonstrating that a wide variety of natural genetic variants can influence drug efficacy in vivo without altering standard drug-susceptibility tests. A number of these genes are enriched in drug-resistant clinical isolates, indicating that these genetic variants influence treatment outcome. Together, these data suggest new targets for improving therapy, as well as mechanisms of genetic adaptations that can reduce antibiotic efficacy and contribute to the evolution of resistance.
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Krallis, Myrsini. "Isolation and identification of Beta-Lactam Producing Microorganisms using PCR based methodologies." Thesis, Rhodes University, 1997. http://hdl.handle.net/10962/d1018237.
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The polymerase chain reaction (PCR) was investigated as a potential tool in microbial screening for 13-lactam. producing organisms. Optimization of PCR conditions and the addition of acetamide to the PCR reaction allowed for the successful amplification of the isopenicillin N synthetase (lPNS) gene in S. clavuligerus, S. tanashiensis, S. griseus, S. olivaceus, S. lipmanii, and S. chartreusis. PCR was used to produce a radiolabelled probe from S. clavuligerus that was used to detect analogous genes in bacteria and fungi. Southern blot and dot blot analysis using the lPNS probe revealed the presence of IPNS-like sequences in seventeen organisms. Fourteen of these sequences belonged to known 13-lactam. producing organisms; one unidentified soil isolate; and two non-/3-lactam. producing organisms viz. S. venezuelae ATCC 10712 and S. hygroscopicus ATCC 21703. The lPNS gene was also detected in a 13-lactam producer (S. chartreusis) that had lost its ability to produce antibiotic. It would therefore have been overlooked in a conventional antibiotic screening program. The use of PCR, coupled with Southern hybridization and dot blot analysis, increased the sensitivity and specificity of the antibiotic screening procedures and allowed for the investigation of evolutionary relationships between the eukaryotes and the prokaryotes. A preliminary investigation into the potential use of RAPD PCR and protein fmgerprinting as tools for solving discrepancies in streptomycete identification was conducted. A variety of streptomycete species that were chosen as being representative of a number of numerical taxonomic classes were amplified using various RAPD primers. Streptomycetes appear to be genetically diverse organisms as was reflected by their RAPD and protein profiles. The application of PCR in an antibiotic screening program showed great potential as a specific and sensitive tool in the detection of /3-lactam producers and in the elimination of duplicate strains.
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Dalmolin, Keina Poliana Pivarro. "Clonagem de um alelo do gene SPT15 em Saccharomyces cerevisiae para aumento da produção de etanol." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-15092011-142026/.
Full textAbstract:
Alper e colaboradores demonstraram que a linhagem BY4741 recombinante portadora de cópias adicionais de um alelo SPT15 mutagenizado em três diferentes posições (spt15-300) utiliza mais rapidamente a glicose e aumenta a produção de etanol. Neste trabalho, foi realizada a clonagem deste alelo, aqui chamado spt15*. Inicialmente o DNA genômico da linhagem S. cerevisiae S288C foi utilizado como molde para amplificação por SOEing-PCR. O alelo spt15* foi clonado no plasmídeo pGEMT-Easy e, em seguida, introduzido no plasmídeo epissomal pMA91. Após construções moleculares, foi obtido o fragmento de DNA dpPGKspt15*tPGKd, empregado na transformação genética, da linhagem S. cerevisiae YPH252, por d-integração. Os clones recombinantes YHP252/pMA91spt15* e YHP252/dpPGKspt15*tPGKd consomem mais eficientemente a glicose e aumentam a produção de etanol. O seqüenciamento do alelo SPT15 da levedura industrial PE-2 revelou 100% de identidade com o alelo das linhagens BY4741 e S288C, criando ótimas perspectivas para trabalhos futuros.<br>Alper and colleagues demonstrated that the yeast S. cerevisiae BY4741 recombinant strain carrying additional copies of a SPT15 allele mutagenized in three different positions (spt15-300) uses glucose more speedily and produces more ethanol. In this work, this allele, here called spt15*, was cloned. The genomic DNA of the S. cerevisiae S288C strain was used for amplification through SOEing-PCR. The spt15* allele was cloned in the plasmid pGEMT-Easy and introduced in the episomal plasmid pMA91. After molecular constructions the DNA dpPGKspt15*tPGKd fragment was obtained to be employed in the genetic transformation of laboratory S. cerevisiae strains using d-integration. Both recombinant clones YHP252/pMA91spt15* and YHP252/dpPGKspt15*tPGKd consume glucose more speedily and produce more ethanol. The sequencing of the SPT15 allele of the industrial yeast PE-2 revealed 100% identity with BY4741 and S288C alleles, creating huge perspectives for future works.
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Laubscher, Inge. "Characterisation of plasmid p31T1 isolated from Aeromonas." Thesis, Stellenbosch : Stellenbosch University, 2013. http://hdl.handle.net/10019.1/85725.
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Thesis (MSc)--Stellenbosch University, 2013.<br>ENGLISH ABSTRACT: Plasmids are an integral part of the horizontal gene pool and, therefore, are the main
vectors for the spread of antibiotic and heavy metal resistance genes in the
environment. Functional and taxonomic characterization of novel plasmids is, therefore,
central to our general understanding of plasmid biology and their contribution to
microbial evolution. Two 14-kb mobilizable plasmids, p31T1 and p36T2, conferring
resistance to tetracycline were isolated from the opportunistic fish pathogens
Aeromonas sobria and Aeromonas hydrophila and were found to have indistinguishable
restriction fragment length polymorphism (RFLP) patterns (Marx, MSc Thesis). DNA
sequence analysis of the two isogenic plasmids (only p36T2 was sequenced) revealed
the presence of 18 putative open reading frames (ORFs), of which the tetAR
tetracycline resistance genes, associated with a truncated Tn1721, were the only ORFs
with significant similarity to known sequences within the NCBI database. Putative
functions were assigned to 10 of the ORFs based on their distant homology with
proteins of known function. Six of the 18 ORFs, spanning 5.7-kb, were found to
comprise the minimal region required for replication (minimal replicon) by means of
deletion analysis using derivatives of p31T1. Of the six ORFs, ORF2 and ORF4 were
found to be essential for plasmid replication. Inactivation of ORF3 resulted in an
increase of plasmid copy number (PCN) from ~3 to ~7 plasmids per chromosome and a
decrease in plasmid stability from ~80 % to 16 % over approximately 127 generations (7
days). Furthermore, by means of β-galactosidase promoter fusion assays it was shown
that ORF3 autoregulated its own promoter. These results, therefore, suggested that
although ORF3 was not essential for replication, it may be involved in plasmid copy
number regulation and control. Host range analysis indicated that p31T1 was able to
replicate in two other members of the γ-proteobacteria group (Escherichia coli and
Pseudomonas putida) but was unable to do so in an α-proteobacterium strain, thus
suggesting a limited host range. Furthermore, p31T1 was mobilized only at low
frequencies (5.4 x 10-5 transconjugants per donor) by an IncP-1 conjugative system
though it is possible that the mobilization system of these plasmids is adapted to function optimally with alternate conjugative systems. Given the unique PCN, stability,
host range and mobilization characteristics determined for p31T1 and that no other
plasmid replication and mobilization systems with significant sequence similarity to
these plasmids have yet been identified, it is likely that these two plasmids are the first
representative members of a new family of plasmids found within aquacultureassociated
Aeromonas species and which are involved in the spread of tetracycline
resistance.<br>AFRIKAANSE OPSOMMING: Plasmiede vorm ‘n integrale deel van die horisontale geen poel en vorm daarom die
hoof vektore vir die verspreiding van antibiotika- en swaarmetaal-weerstandbiedende
gene in die omgewing. Funksionele en taksonomiese karakterisering van nuwe
plasmiede is belangrik in die begrip van plasmied biologie en hul bydrae tot mikrobiese
evolusie. Twee 14-kb mobiliseerbare plasmiedes, p31T1 en p36T2, met tetrasiklien
weerstandigheid was vanaf die opportunistiese vis patogene Aeromonas sobria en
Aeromonas hydrophila geïsoleer en het identiese restriksie fragment lengte
polimorfisme (RFLP) patrone. DNA volgorde analise van die twee isogeniese plasmiede
(slegs die volgorde van p36T2 was bepaal) het die teenwoordigheid van 18 moontlike
oop leesrame (OLR) getoon. Die tetAR tetrasiklien weerstandbiedende gene, wat met ‘n
verkorte Tn1721 transposon geassosieerd is, was die enigste OLR wat beduidende
volgorde ooreenkoms met bekende volgordes binne die NCBI databasis getoon het.
Moontlike funksies was toegeken aan 10 van die OLRe en was gebasseer op vêrlangse
homologie met proteïene met bekende funksies. Ses van die 18 OLRe strek oor ‘n 5.7-
kb minimale replikon fragment wat benodig word vir replisering en is deur middel van
delesie analises van p31T1 derivate gevind. Van hierdie ses OLRe, word OLR2 en
OLR4 benodig vir plasmied replisering. Inaktivering van OLR3 het ‘n toename in
plasmied kopiegetal (PKG) vanaf ~3 tot ~7 plasmiede per kromosoom en ‘n afname in
stabiliteit vanaf ~80% tot 16% oor 127 generasies (7 dae) tot gevolg gehad. Verder kon
daar deur middel van β-galaktosidase fusie analises getoon word dat OLR3 sy eie
promotor outoreguleer. Hierdie resultate stel dus voor dat alhoewel OLR3 nie benodig
was vir replikasie nie, mag dit dalk by plasmied kopiegetal regulering en beheer
betrokke wees. Bakteriële gasheer analises het getoon dat p31T1 in 2 addisionele lede
van die γ-proteobakterieë groep (Escherichia coli en Pseudomonas putida) kon
repliseer, maar nie in ‘n α-proteobacterium nie. Verder kon p31T1 teen ‘n lae frekwensie
(5.4 x 105) gemobiliseer word deur ‘n IncP-1 konjugasie sisteem, maar dit mag wees dat
die mobilisering eerder optimaal kan plaasvind met ‘n alternatiewe konjugasie sisteem.
Na aanleiding van die unieke PKG, stabiliteit, gasheer en mobilisering eienskappe wat vir p31T1 bepaal is en die feit dat geen ander replisering en mobilisering sisteme met
noemenswaardige volgorde homologie tot hierdie plasmiede gevind kon word nie, blyk
dit dat hierdie van die eerste lede van ‘n nuwe familie van plasmiede binne die
akwakultuur-geassosieerde Aeromonas spesies is, wat betrokke is by die verspreiding
van tetrasiklien weerstandbiedendheid.
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Sinotte, Christopher Matthew. "Construction, expression, and purification of soluble CD16 in bacteria." Thesis, Available online, Georgia Institute of Technology, 2006, 2006. http://etd.gatech.edu/theses/available/etd-05142006-222347/.
Full textAbstract:
Thesis (M.S.)--Bioengineering, Georgia Institute of Technology, 2007.<br>Zhu, Cheng, Committee Chair ; Selvaraj, Periasamy, Committee Member ; Orville, Allen, Committee Member ; Butera, Robert, Committee Member.
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Barcellos, Fernando Gomes. "Caracterização genética e citológica da recombinação somática em Trichoderma pseudokoningii." Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-04112002-164855/.
Full textAbstract:
Com o objetivo de se caracterizar o processo de recombinação somática em Trichoderma pseudokoningii foram feitos cruzamentos via anastomose de hifas entre duas linhagens contrastantes para quatro marcadores de auxotrofia, coloração dos conídios e marcadores de RAPD. Foram feitos quatro cruzamentos, sendo analisados um total de 1052 colônias obtidas a partir de suspensões de conídios provenientes das colônias heterocarióticas. Sessenta e oito colônias recombinantes foram analisadas quanto às marcas de auxotrofia em quatro gerações de crescimento, sendo observado que 58 mantiveram o fenótipo recombinante, enquanto que as colônias restantes reverteram para um dos parentais. A maioria das colônias recombinantes se mostrou instável. Entretanto, após 4 gerações de crescimento estas colônias se tornaram estáveis para as marcas de auxotrofia avaliadas. As colônias recombinantes instáveis apresentaram bordas de crescimento irregular, esporulação esparsa e a freqüente formação de setores. Estas colônias recombinantes foram analisadas quanto aos marcadores RAPD, tendo mostrado grande similaridade, em relação ao perfil de bandas apresentado, com a maioria dos primers analisados. Somente com um primer foi possível visualizar a presença de uma banda polimórfica entre os recombinantes e a presença de bandas nos parentais não existentes em alguns recombinantes. Cinco colônias recombinantes foram analisadas quanto ao perfil de bandas cromossomais (PFGE), tendo sido observado que 2 colônias apresentaram padrões cromossomais igual a um dos parentais e 3 colônias apresentaram padrões recombinantes. Nos estudos citológicos verificou-se a formação de conídios uninucleados na conidiogênese, e a presença de conídios verdes maduros multinucleados, devido a prováveis divisões nucleares durante o processo de maturação dos conídios. Observou-se durante a formação dos heterocários a ocorrência de anastomoses e a passagem de núcleos, tendo sido observado a presença de núcleos com várias conformações, sugerindo um movimento ativo dos mesmos. Os resultados acima sugerem a ocorrência de mecanismos de recombinação no heterocário (recombinação somática), diferentes daqueles descritos para o ciclo parassexual ou parameiose, sendo proposto a ocorrência da degradação, no heterocário, dos núcleos de um dos parentais envolvidos nos cruzamentos (parental não prevalente) e a incorporação de segmentos destes em núcleos íntegros do parental prevalente. Se estes eventos realmente estiverem ocorrendo, sugere-se que estes sejam devido a possíveis reações limitadas de incompatibilidade vegetativa, ocasionando processos de lise e morte celular em algumas regiões do micélio heterocariótico.<br>To understand the somatic recombination process in Trichoderma pseudokoningii, auxotrophic complementary mutant strains were used to produce 4 heterokaryons. These strains were contrasting for four auxotrophic markers, conidia colors and for some RAPD markers. It was analyzed a total of 1052 colonies obtained from conidial suspensions of the heterokaryotic colonies. Stability of auxotrophic markers was evaluated in 68 recombinant colonies after four growing generations. In this analysis, 58 colonies kept the recombinant phenotype, while 10 reverted to one parental strain. Most of the recombinant colonies were initially unstable, but after at least 4 growing generations these recombinants became stable for auxotrophic markers. The unstable recombinant colonies showed irregular growing borders, sparse sporulation and frequent sector formation. The recombinant colonies were analyzed by RAPD technique. These colonies showed high similarity for the most of used primers. However, one primer showed a polymorphic band and some recombinants missing bands observed in parental strains. Chromosomal band profile of 5 recombinants and two parental strains were analyzed by Pulsed Field Gel Electrophoresis technique (PFGE). Two recombinants showed parental profiles and 3 showed recombinant profiles, respectively. In cytological studies of the conidiogenesis was observed the formation of only uninucleated conidia. However, presence of multinucleated mature green conidia was evident, probably due to nuclear divisions in course of maturing process of the conidia. During the process of heterokaryotic mycelium formation was possible to observe the occurrence of anastomosis that showed nuclear transfer. The presence of nuclei in several conformations was observed at the different regions of the heterokaryon, suggesting an active movement. The results presented in this study suggest the occurrence of recombination mechanisms in the heterokayon (somatic recombination), different from those described in classic parasexual cycle or parameiosis. Thus, it was proposed that may occur during this recombinant process the degradation of nuclei from one parental (non-prevalent parental) in the heterokaryon, and that the resulting chromosomal fragments may be incorporated into whole nuclei of the another parental (prevalent parental). If this natural transformation is occurring during this recombination process could be suggested that this event is due to a limited incompatible vegetative reactions, generating cellular lyses and death in some regions of the heterokaryotic mycelium.
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Keiser, Tracy Lynn. "Biosynthesis of mannose-containing cell wall components important in Mycobacterium tuberculosis virulence." The Ohio State University, 2014. http://rave.ohiolink.edu/etdc/view?acc_num=osu1397762377.
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Ho, Brian Thomas. "Characterization of the Antibacterial Activity of the Type VI Secretion System." Thesis, Harvard University, 2013. http://dissertations.umi.com/gsas.harvard:11241.
Full textAbstract:
This dissertation summarizes advances made toward understanding of the composition, structure, mechanism, and regulation of the bacterial type VI secretion system (T6SS). The T6SS is a widely conserved bacterial nanomachine used by Gram-negative bacteria to deliver toxic effector proteins into the extracellular environment or into adjacent target cells. Systematic deletion of open reading frames present in the Vibrio cholerae T6SS gene cluster revealed the genes essential for T6SS activity and provided key insights into understanding the mechanism by which this organelle is assembled and its components are recycled. Characterization of one phage-related T6SS component yielded insight into the mechanism by which many effectors associate with the T6SS organelle and are delivered into target cells. This T6SS component serves both to sharpen the tip of the membrane-puncturing T6SS spike complex and as a vehicle for attaching a diverse set of effector proteins. Time-lapse fluorescence microscopy of GFP-labeled T6SS components revealed key insights into the behavior and regulation of the T6SS in Pseudomonas aeruginosa. The T6SS in this organism assembled in response to exogenous T6SS attack by adjacent sister cells as well as heterologous T6SS+ species V. cholerae and Acinetobacter baylyi. This retaliatory T6SS counterattack was precisely aimed and caused no collateral damage to neighboring, non-aggressive bacteria. These counterattacks are mediated by phosphorylation cascade that recognizes exogenous attacks and post-translationally activates the T6SS in P. aeruginosa. Deletion of genes in this pathway eliminated the retaliatory response while retaining T6SS functionality. This pathway also induced T6SS counterattacks in response to mating pair formation associated with type IV secretion system (T4SS)-mediated DNA conjugation as well as treatment with membrane-disrupting natural product polymyxin B, suggesting that the signal needed to induce T6SS activity was mechanical perturbation of the P. aeruginosa cell membrane. Interestingly, these T4SS-induced counterattacks were able to confer resistance to the acquisition of horizontally transferred foreign DNA by selectively killing conjugative donor cells. As such, the T6SS of P. aeruginosa may represent a type of general bacterial innate immune system capable of responding to a wide range of exogenous threats.
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Cordova, Caio Mauricio Mendes de. "Desenvolvimento de plasmídeos replicativos artificiais para transformação de Mycoplasma pulmonis, M. capricolum e M. mycoïdes subsp. mycoïdes, e dirupção do gene da hemolisina A de M. pulmonis por recombinação homóloga." Universidade de São Paulo, 2002. http://www.teses.usp.br/teses/disponiveis/9/9136/tde-20082008-112933/.
Full textAbstract:
Os micoplasmas são os menores microrganismos capazes de autoreplicação conhecidos na natureza, responsáveis por uma série de doenças no homem e nos animais, infectando ainda plantas e insetos. Constituem um grande grupo de bactérias, ordenadas em diferentes gêneros na classe Mollicutes, cuja principal característica em comum, além do genoma reduzido, é a ausência de parede celular. Mycoplasma mycoïdes subsp. mycoïdes SC, responsável pela Pleuropneumonia Contagiosa Bovina, foi o primeiro microrganismo desta classe de bactérias a ser identificado. Esta é uma doença bastante grave, com altas taxas de morbidade e mortalidade. A variedade Mycoplasma mycoïdes subsp. mycoïdes LC é responsável principalmente por casos de Pleuropneumonia Contagiosa Caprina, mastite no gado bovino, e ainda artrite em ovinos e caprinos em menor extensão. M. capricolum é um patógeno caprino, responsável principalmente por casos de artrite com grande importância econômica na medicina veterinária. M. pulmonis é um patógeno de roedores, considerado como o melhor modelo experimental para o estudo das micoplasmoses respiratórias. M. genitalium, o menor microrganismo conhecido capaz de se autoreplicar, é um patógeno humano responsável por casos de uretrite não gonocócica, cujo seqüenciamento completo do cromossomo tornou-se um marco na era da genômica. O estudo funcional do genoma destes micoplasmas, para a compreensão de sua biologia e patogenicidade, requer o desenvolvimento de ferramentas genéticas eficientes. No presente trabalho, análises in silico das seqüências na região das prováveis origens de replicação cromossômica (oriC) destes micoplasmas demonstraram a existência de possíveis DnaA boxes localizados em torno do gene dnaA. Estas regiões oriC foram caracterizadas funcionalmente após sua clonagem em vetores artificiais e a transformação dos micoplasmas com os plasmídeos recombinantes resultantes. O plasmídeo pMPO1, contendo a região oriC de M. pulmonis, sofreu integração no cromossomo do micoplasma por recombinação homóloga após poucas passagens in vitro. A redução desta oriC para o fragmento contendo somente os DnaA boxes localizados nas estremidades 5´ou 3´do gene dnaA não foi capaz de produzir plasmídeos replicativos em M. pulmonis, exceto quando estes dois fragmentos foram clonados no mesmo vetor, espaçados pelo determinante de resistência à tetraciclina tetM. Um fragmento interno do gene da hemolisina A (hlyA) de M. pulmonis foi clonado nestes plasmídeos oriC, e os vetores resultantes foram utilizados para transformar o micoplasma. A integração destes vetores por um crossing-over com o gene hlyA, causando a sua dirupção, foi documentada. Deste modo, estes plasmídeos oriC podem vir a se tornar ferramentas genéticas valiosas para o estudo do papel de genes específicos, notadamente aqueles potencialmente envolvidos na patogênese.<br>Mycoplasmas are the smallest microorganisms capable of self replication known to date, responsible for many diseases in man and animals, infecting also plants and insects. They constitute a large group of bacteria, classified in different genera in the class Mollicutes, which main common characteristic, besides the small genome, is the absence of a cell wall. Mycoplasma mycoïdes subsp. mycoïdes SC, responsible for the Bovine Contagious Pleuropneumonia, was the first microorganism of this class of bacteria to be identified. That is a quite severe disease, with high morbidity and mortality rates. Mycoplasma mycoïdes subsp. mycoïdes LC is responsible mainly for cases of Caprine Contagious Pleuropneumonia, mastitis in cattle, and also arthritis in goats and sheep in less extension. M. capricolum is a pathogen of goats, responsible mainly by cases of arthritis with large economic impact in veterinary medicine. M. pulmonis is a rodent pathogen, considered to be the best experimental model for studying respiratory mycoplasmoses. M. genitalium, the smallest microorganism capable of self replication, is an human pathogen responsible for cases of non gonococcal urethritis, which complete chromosome sequencing has become a benchmark in the era of genomics. Functional studies of these mycoplasma genomes, for comprehension of their biology and pathogenicity, requires the development of efficient genetic tools. In the present work, in silico analysis of sequences of the putative origin of chromosome replication (oriC) region of these mycoplasmas demonstrates the existence of putative DnaA boxes located around the dnaA gene. These oriC regions were functionally characterized after cloning into artificial vectors and transformation of mycoplasmas with the resulting recombinant plasmids. The plasmid pMPO1, which contains the M. pulmonis oriC region, has integrated into the mycoplasma chromosome by homologous recombination after a few in vitro passages. Reduction of this oriC to the fragment containing only the DnaA boxes located upstream or downstream the dnaA gene could not produce plasmids able to replicate in M. pulmonis, except when these two fragments were cloned in the same vector, spaced by tetracycline resistance gene tetM. An internal fragment of the M. pulmonis hemolysine A gene (hlyA) was cloned into these oriC plasmids, and the resulting vectors were used to transform the mycoplasma. Integration of these disruption vectors by one crossing-over with the hlyA gene could be documented. Therefore, these oriC plasmids may become valuable genetic tools for studying the role of specific genes of mycoplasmas, specially those potentially involved in pathogenesis.
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Silva, Carlos Henrique Domingues da. "Fungos associados a invertebrados marinhos: isolamento, seleção e avaliação da produção de enzimas celulolíticas." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-30092010-110051/.
Full textAbstract:
A micologia marinha é uma ciência relativamente recente e pouco se conhece sobre a diversidade das suas comunidades. Assim, o isolamento, triagem e preservação de fungos derivados do mar podem levar à descoberta de novas tecnologias. O objetivo deste estudo foi conhecer a diversidade de fungos filamentosos derivados marinhos e selecionar isolados capazes de produzir enzimas celulolíticas. Para tanto, foram isolados seletivamente fungos filamentosos a partir de amostras de macro-organismos marinhos coletados em 2007 e 2008. Os resultados demonstraram uma ampla diversidade de fungos potencialmente celulolíticos, pertencentes ao filo Basidiomycota e Ascomycota. Nos experimentos de produção de celulases, 17 apresentaram resultados satisfatórios de CMCase e FPase e foram selecionados para a avaliação da Celobiase. Os experimentos de cinética enzimática apresentaram os melhores resultados de produção de celulases em meio contendo farelo de trigo. O trabalho demonstra o potencial para aplicação biotecnológica dos fungos e estimula novos estudos com as celulases.<br>The Marine mycology is a relatively recent and little is known about the diversity of its communities. Thus, the isolation, separation and preservation of fungi derived from the sea can lead to the discovery of new technologies. The aim of this study was the diversity of filamentous fungi isolates derived marine and select capable of producing cellulolytic enzymes. It had been selectively isolated filamentous fungi from samples of marine macro-organisms collected in 2007 and 2008. The results showed a wide range of potential cellulolytic fungi, belonging to the phylum Basidiomycota and Ascomycota. In the experiments to produce cellulases, 17 had satisfactory results of CMCase and FPase and were selected for evaluation of cellobiase. The enzyme kinetics experiments showed better results for the production of cellulases in a medium containing wheat bran. The work demonstrates the potential for biotechnological application of fungi and stimulate further research with cellulases.
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Miranda, Ceres Maciel de. "Expressão de microplusina em Aedes aegypti: avaliação do efeito sobre Plasmodium gallinaceum." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/42/42135/tde-04082011-091317/.
Full textAbstract:
A transmissão de parasitas da malária por mosquitos vetores é dependente do desenvolvimento bem sucedido das formas infectantes de Plasmodium sp., especialmente os esporozoítas, que são as formas que infectam o hospedeiro vertebrado. A manipulação genética de mosquitos vetores tem sido uma estratégia alternativa na tentativa de controle da malária. Um componente extremamente importante desta estratégia é a escolha de uma molécula efetora capaz de reduzir a transmissão do patógeno. Microplusina é um peptídeo antimicrobiano rico em cisteína, originalmente descrito como um componente antimicrobiano da hemolinfa e dos ovos de carrapato bovino Rhipicephalus (Boophilus) microplus. Testes anteriores utilizando o modelo experimental mosquito Aedes aegypti infectado por Plasmodium gallinaceum mostraram que a microplusina é altamente tóxico para esporozoítas de Plasmodium gallinaceum em concentração relativamente baixa, sem apresentar toxicidade aos mosquitos vetores Aedes aegypti. Nosso objetivo foi analisar a expressão da microplusina e seu efeito na infecção de P. gallinaceum em mosquitos transgênicos. Obtivemos quatro linhagens através da integração de um transgene contendo a região promotora do gene da vitelogenina de Ae. aegypti, peptídeo sinal maltase-like I de Ae. aegypti e a sequência codificadora da microplusina (PMOS [3xP3-EGFP-AeVg Micro]). A atividade anti esporozoítas da microplusina expressa pelos mosquitos transgênicos mostrou diferença significante as linhagens. O desenho de novas moléculas utilizando como molde moléculas efetoras existentes e testadas, possibilitará o aperfeiçoamento da expressão de genes exógenos em mosquitos transgênicos, tornando-os refratários ao parasita.<br>Transmission of malaria parasites by mosquito vectors is dependent on the successful development of Plasmodium sp. infective forms, particularly the sporozoites, which are the forms that enter the vertebrate host. The genetic manipulation of mosquito vectors has been a strategy for malaria control. An extremely important component of this strategy is the effector molecule of choice which reduces parasite transmission. Microplusin is a cysteine-rich antimicrobial peptide originally described as an hemolymph and eggs antimicrobial component of the cattle tick Boophilus microplus. Previous tests using the experimental model Plasmodium gallinaceum infected Aedes aegypti showed that microplusin is highly toxic to P. gallinaceum sporozoites in relatively low concentration, without showing toxicity to the mosquito vector A. aegypti. Our goal was to analyze transgenic mosquitoes expressing microplusin and its effect on infection of P. gallinaceum. We obtained four lines through the integration of transgene that containing the promoter region of the A. aegypti vitelogenin gene, the maltase-like I signal peptide of A. aegypti and microplusin coding sequence (pMos[3xP3-EGFPAeVg-Micro]). The activity anti sporozoites microplusin expressed by transgenic mosquitoes showed significant differences between strains. The design of effector molecules using information from existing and tested molecules as template will enable the improvement of the expression of foreign genes in transgenic mosquitoes, making them resistant to the parasite.
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Butler, Vanessa Leanne. "The effects of genetics, age and rearing environment on AvBD gene expression and gut anti-microbial activities in three chicken lines." Thesis, University of Newcastle upon Tyne, 2010. http://hdl.handle.net/10443/2240.
Full textAbstract:
The defensins have been shown to be an important component of the innate immune system across many species from plants to man. To date a total of 14 beta defensins have been identified within the chicken genome and anti-microbial activity of an array of these peptides against pathogens has been demonstrated. The innate immune system has been shown to be important in chickens during the first week of life when exposure to pathogens in the environment occurs and the adaptive immune system is not fully developed. The research presented in this thesis attempts to investigate the effects of bird age (aged 0, 7, 14 and 35 days), genetics and rearing environment on components of the innate immune system. A farm trial was performed using three lines of Aviagen birds (lines X, Y and Z) and two distinct rearing environments (low and high hygiene farms). To determine which of the 14 avian beta defensins (AvBD) to investigate, a panel of potential single nucleotide polymorphisms (SNPs) within the AvBD locus was submitted as part of an Aviagen Ltd genomics initiative. Frequencies of the polymorphisms across the three lines of birds were determined. From these data, gene expression of AvBDs1, 4 and 10 were fully investigated using end-point PCR and then quantitative real-time PCR (qRT-PCR). A pronounced finding from the qRT-PCR was the marked intra and inter-group variation in gene expression levels, which lead to few statistical significant differences. All three genes were shown to be expressed across a panel of ten tissues analysed, but distinct patterns were also seen. Significantly AvBD1 gene expression in the duodenum indicated that of the 7 day old birds the line X birds reared in the low hygiene environment had the highest level of gene expression. In relation to AvBD4 gene expression, the highest level was observed in the spleen of the 0 day old birds, but overall environment did not appear to affect AvBD4 gene expression of the tissues examined. High levels of AvBD10 gene expression were observed in bird kidney and testicle tissue, but again environment in the case of the former tissue did not appear to statistically affect gene expression levels, the YH 7 day old bird testicle samples did have statistically significant higher expression levels compared to the other groups. The SNP analysis revealed three non-synonymous polymorphisms within the AvBD1 mature peptide locus. The three lines of birds had quite different patterns of these polymorphisms and so three different forms of the peptide along with a single form of the AvBD10 peptide were synthesised using a bacterial hyper-expression system. Peptide levels were quantified using an ELISA and subsequently tested in a bacterial time-kill assay using Salmonella enterica serovar Typhimurium phoP, Staphylococcus aureus and two strains of Enterococcus faecalis. All recombinant peptides showed anti-microbial activity against the bacteria tested. The exception was a clinical isolate of E. faecalis which showed resistance to the killing activities of recombinant AvBD10 peptide. Duodenal gut protein extracts were also tested using the same bacterial assay and marked differences in the anti-microbial activities of these samples were seen. The samples taken from the day 0 birds were found to have significant anti-microbial activity compared to those of the older birds. LC/MS identified differences in the proteomes of the respective gut extracts. These data support that bird genetics, age and the environment have an effect on AvBD gene expression and gut anti-microbial activity. These differences are not uniform for all genes and groups of birds, but clear patterns were observed.
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Ferreira, Almir José. "Diversidade e estrutura da comunidade bacteriana associada às armadilhas da planta carnívora Utricularia gibba (Lentibulariaceae) e ao ambiente aquático." Universidade de São Paulo, 2011. http://www.teses.usp.br/teses/disponiveis/87/87131/tde-10022012-162729/.
Full textAbstract:
A diversidade microbiana em ambientes aquáticos e sua associação com plantas carnívoras ainda é pouco estudada. Assim, a comunidade bacteriana da planta carnívora Utricularia gibba e do seu meio aquático foi avaliada por meio do seqüenciamento em larga escala (454 Roche) de uma biblioteca do gene 16S rRNA. Os resultados indicaram que a comunidade bacteriana na água é significativamente diferente da comunidade dos utrículos. Além disso, a comunidade bacteriana da água é composta principalmente por membros dos filos Proteobacteria, Actinobacteria, Firmicutes e Verrucomicrobia, enquanto que a comunidade presente me U. gibba é composta por membros dos filos Proteobacteria, Firmicutes, Cyanobacteria e Acidobacteria. O gênero Polynucleobacter foi dominante nos dois ambientes aquáticos, mas não foi detectado no interior dos utrículos, onde Acidobacterium e Methylococcus foram os gêneros dominantes. Assim, uma comunidade bacteriana específica no interior dos utrículos deve ter sido selecionada a partir do ambiente, podendo esta atuar na degradação das presas.<br>The microbial diversity of aquatic environments and their association to carnivorous plants is still poor studied. Thus, the bacterial community associated to traps of Utricularia gibba and its aquatic environment was evaluated by large-scale sequencing (454 Roche) of 16S rRNA library from these environments. The results indicated the bacterial community in water is significantly different from the community of utricles. In addition, the bacterial community detected in water environment is mainly composed by Proteobacteria, Actinobacteria, Firmicutes and Verrucomicrobia, while in utricules of U. gibba the community is composed by Proteobacteria, Firmicutes, Cyanobacteria and Acidobacteria. The genus Polynucleobacter was dominant in water, but was not detected in association with the plant. Inside the plant, the genus Acidobacterium and Methylococcus were dominant, but were not detected in water samples. Thus, a specific bacterial community within the utricles should have been selected from the environment, and could play a role in prey degradation.
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Chella, Krishnan Karthickeyan. "Host-Pathogen Interactions Promoting Pathogen Survival and Potentiating Disease Severity & Morbidity in Invasive Group A Streptococcal Necrotizing Soft Tissue Infections." University of Cincinnati / OhioLINK, 2015. http://rave.ohiolink.edu/etdc/view?acc_num=ucin1446546952.
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Hotaling, Scott. "GENETIC PERSPECTIVES ON BIODIVERSITY IN ROCKY MOUNTAIN ALPINE STREAMS." UKnowledge, 2017. http://uknowledge.uky.edu/biology_etds/44.
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In alpine regions worldwide, climate change is dramatically altering ecosystems, affecting biodiversity across habitats and taxonomic scales. For streams, the associated recession of mountain glaciers and snowfields, paired with altered precipitation regimes, are driving shifts in hydrology, species distributions, and basal resources – often threatening the very existence of some habitats and biota. Globally, alpine streams harbor particularly substantial species and genetic diversity due to significant habitat insularity and environmental heterogeneity: however, anthropogenic warming threatens to homogenize habitats through the reduction of the cryosphere, thereby reducing biodiversity from micro- to macroscopic organisms and genes to communities. Still, alpine stream biodiversity, particularly in North America, is poorly understood, making it difficult to predict future changes without baselines for comparison.
For my dissertation, I used genetic tools to assess biodiversity in alpine streams of the central Rocky Mountains in North America. Here, I begin by reviewing the current state of alpine stream biology from an organismal perspective. Next, I provide two perspectives on macroinvertebrate diversity. The first, a population genetic comparison of three highly similar species, is followed by a fine-scale genomic study of one species, Lednia tumana. I follow these largely macroinvertebrate-centric chapters with a modern synthesis of the microbial ecology of mountain glacier ecosystems. Finally, I conclude with a study of microbial diversity that addresses how microbial diversity is shaped by geography, habitat, and hydrological source in North America.
Collectively, this research refines existing themes in alpine stream biology by revealing unexpected differences in population genetic patterns among closely related species, the influence of recent deglaciation on population genetic structure and demographic history of a threatened stonefly, and clarification of the environmental drivers shaping microbial diversity.
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Moffo, Nathan. "Differential Analysis of Unique Genes Expressed in Stenotrophomonas maltophilia Strain OR02 in Response to Selenite." Youngstown State University / OhioLINK, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=ysu15663177454459.
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Gillan, David. "Morpho-physiology, genetics and ecological aspects of marine microbial biofilms :the study of the iron-encrusted biofilm associated with Montacuta ferruginosa (Mollusca, Bivalvia)." Doctoral thesis, Universite Libre de Bruxelles, 1999. http://hdl.handle.net/2013/ULB-DIPOT:oai:dipot.ulb.ac.be:2013/211930.
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Marcangione, Luigi. "Isolation of a Pseudomonas aeruginosa PAOI gene involved in 3-hydroxybutyrate catabolism." Thesis, National Library of Canada = Bibliothèque nationale du Canada, 1999. http://www.collectionscanada.ca/obj/s4/f2/dsk1/tape2/PQDD_0031/MQ64403.pdf.
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Hartmann, Kaitlin Ash. "Cronobacter sakazakii Genes Contributing to Persistencein Low-Moisture Dairy Matrices." BYU ScholarsArchive, 2020. https://scholarsarchive.byu.edu/etd/8466.
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Cronobacter sakazakii is a gram-negative opportunistic pathogen known to survive in dry environments and food matrices, such as infant formula. This foodborne bacterium can cause fatal human infections of the blood, central nervous system, and gastrointestinal tract; it is also problematic in wounds and urinary tract infections. Preterm infants and immunocompromised individuals are in higher risk categories related to necrotizing enterocolitis, neonatal sepsis, and meningitis due to this organism. Therefore, there is a need for increased understanding of how this bacterium is able to persist in thermally treated low-moisture products that do not support growth. The objective of this research is to identify genes and mechanisms in C. sakazakii that contribute to its resistance to desiccation and survival in low-moisture food matrices, including powdered infant formula. C. sakazakii sequence type 4 (ST4) is of particular interest as it is often the cause of neonatal infections originating from contaminated feedings of powder infant formula. The method chosen to explore these genetic patterns is massively parallel transposon insertion sequencing (Tn-seq). The E. coli strain MFDpir was used to facilitate transposon insertional mutagenesis to create a library of mutated C. sakazakii. Three different C. sakazakii ST4 isolates of different origins (clinical, environmental, and infant formula-derived) were selected for this study. Once transposon mutagenesis occurred with the aid of E. coli MFDpir, the three mutant libraries were subjected to desiccation stress in a closed system equilibrated to 11.3% relative humidity. The surviving mutant genomes were analyzed with Tn-seq. The sequencing data revealed that, while transposition events did occur successfully within the genomes of each of the selected C. sakazakii isolates, these events were not dense enough to draw biological conclusions nor statistical inferences concerning which genes contribute to this organism’s uncanny desiccation tolerance. However, we concluded that the Tn-seq method is a promising tool with this organism of interest, despite incomplete results in this first round of experimentation.
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Pallu, Ana Paula de Souza. "Potencial biotecnológico de fungos de gênero Penicillium e interação com cana-de-açúcar." Universidade de São Paulo, 2010. http://www.teses.usp.br/teses/disponiveis/11/11137/tde-17092010-152316/.
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Os fungos endofíticos têm sido reconhecidos pela sua grande importância para as plantas hospedeiras, pois podem conferir proteção contra insetos herbívoros e patógenos, promover o crescimento vegetal, além de produzir metabólitos secundários com atividades biológicas diversas, entre outros. A cana-de-açúcar é uma cultura de grande importância social e econômica no Brasil, especialmente para o estado de São Paulo. Ultimamente esta cultura vem recebendo especial atenção devido ao crescente aumento da demanda de matéria prima, principalmente em função do acréscimo no consumo de etanol como biocombustível. Fungos do gênero Penicillium habitam os tecidos e a rizosfera de cana-de-açúcar, onde podem estabelecer associações mutualísticas com a planta e conferir diversos benefícios. Dentro deste contexto, estudos que avaliem a interação de Penicillium spp. com cana-de-açúcar são bastante promissores para geração de conhecimentos que auxiliem na otimização da agricultura. Dessa forma, o presente trabalho teve como objetivos a avaliação do potencial biotecnológico dos endofíticos de raiz e da rizosfera, do gênero Penicillium, pertencentes à comunidade fúngica de cana-de-açúcar, por meio de ensaios de antagonismo, produção de enzimas, solubilização de fosfato inorgânico e produção de ácido indol acético; assim como o estudo da interação de um isolado de P. pinophilum com cana-de-açúcar a partir do desenvolvimento de um sistema de transformação genética mediada pela bactéria Agrobacterium tumefaciens. Tanto a análise da atividade antimicrobiana como a produção de metabólitos apresentaram extensa variação fisiológica entre os isolados avaliados. Um isolado da espécie P. pinophilum (linhagem 44) foi escolhido para ser usado na transformação genética por mostrar-se superior estatisticamente em relação aos demais isolados nos ensaios anteriores. Para aumento da eficiência deste sistema de transformação foram avaliados diferentes parâmetros, dentre eles: tempo de co-cultivo (24 e 48 horas), concentração do indutor acetoseringona (200 M e 400 M) e tipos de membrana (papel filtro e náilon). O sistema de agrotransformação apresentou alta eficiência (482 transformantes por 107 conídios), gerando uma elevada quantidade de transformantes resistentes à higromicina B e expressando GFP. Dentre os parâmetros avaliados, a combinação que deu origem aos melhores resultados de transformação envolveu o co-cultivo por 48 horas sobre membrana de náilon, em meio de cultura contendo 200 M de acetoseringona. A interação fungo-planta foi avaliada a partir da inoculação de P. pinophilum linhagem selvagem e transformantes, em plântulas de cana-de-açúcar, seguida da análise por microscopia óptica de epifluorescência e reisolamento. Os resultados revelaram a natureza não patogênica desse fungo, uma vez que ele foi capaz de colonizar endofiticamente cana-de-açúcar e persistir nas raízes desta planta, sem levar ao desenvolvimento de qualquer sintoma de doença. Além disso, os ensaios de agrotransformação deram origem a uma biblioteca com mil e cem transformantes insercionais, o que constitui uma ferramenta importante para o estudo molecular do metabolismo secundário desse fungo endofítico e poderá contribuir para o entendimento da interação do complexo fungo-cana-de-açúcar, possibilitando no futuro a sua aplicação no melhoramento vegetal e exploração do seu potencial biotecnológico.<br>Endophytic fungi have been recognized for its great importance for the host plants, they may provide protection against herbivores and pathogens, promote plant growth, and produce secondary metabolites with biological activity, among other benefits. Sugarcane is a socially and economically important crop in Brazil, especially for the state of São Paulo. Lately, this culture has received special attention due to the growing demand for raw materials, mainly due to the increase in consumption of ethanol as a biofuel. Fungi Penicillium inhabit the tissues and rhizosphere of sugarcane, where they can establish mutualistic associations with the plant and provide several benefits. Within this context, studies evaluating the interaction of Penicillium spp. with sugar cane are very promising to generate knowledge in order to assist in the agriculture optimization. Thus, this study aimed to evaluate the biotechnological potential of endophytic Penicillium from root and rhizosphere, belonging to the fungal community of sugarcane, through tests of antagonism, enzyme production, solubilization of inorganic phosphate and indole acetic acid production, as well as studying the interaction of an isolate of P. pinophilum with sugarcane using the development of a system for genetic transformation mediated by Agrobacterium tumefaciens. Both the analysis of antimicrobial activity and the production of metabolites showed extensive physiological variation among isolates. An isolate of the species P. pinophilum (strain 44) was chosen to be used in genetic transformation for being statistically superior than the other strains in previous trials. Different parameters were evaluated to increase the efficiency of this transformation system, among them: co-culture time (24 and 48 hours), concentration of the inducer acetosyringone (200 µM and 400 µM) and types of membrane (filter paper and nylon). Agrotransformation system showed high efficiency, generating a high amount of hygromycin B resistant transformants that expressed GFP. Among the factors evaluated, the combination that showed the best results involved the transformation with a co-cultivation for 48 hours on a nylon membrane, in culture medium containing 200 µM of acetosyringone. The plant-fungus interaction was assessed from the inoculation of wild type and transformants P. pinophilum in seedlings of sugarcane followed by analysis by epifluorescence microscopy and reisolation. Results revealed the non-pathogenic nature of this fungus, since it was capable of endophytically colonize sugarcane and persisted in the roots of this plant, without developing any symptoms of illness. In addition, agrotransformation tests gave rise to a library with a thousand and one hundred insertional transformants, which is an important tool for molecular study of secondary metabolism of endophytic fungus, and may contribute to the comprehension of the complex interaction of fungus-sugarcane, allowing its future application in plant breeding and exploitation of their biotechnological potential.
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Sánchez, Vanessa. "Characterization of Rhizobial Diversity and Relationship of Rhizobial Partner and Legume Performance in Four South Florida Pine Rockland Soils." FIU Digital Commons, 2014. http://digitalcommons.fiu.edu/etd/1124.
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Pine rocklands are endangered ecosystems unique to south Florida, the Bahamas and Cuba. As a result of their karstic calcium carbonaterich soil, these systems are limited in phosphorus and nitrogen, making symbiotic associations critical to plant growth. Four leguminous species (Cajanus cajan, Chamaecrista fasciculata, Tephrosia angustissima, and Abrus precatorious) were used to determine the relationship between rhizobial partners and plant performance, and the symbiosis related gene nifH was amplified to characterize the diversity of rhizobial symbionts. Plants were grown in soils from four different south Florida pine rocklands, and a salinity treatment was added to determine how storm surge and sea level rise could affect this symbiotic relationship. While plant performance and nodulation were highly impacted by soil type, salinity did not represent a significant effect. Phylogenetic analysis determined that all four plant species were found to associate with Bradyrhizobium spp. and no rhizobial shift between salinity treatment and soil type was found.
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Ministro, Joana Henriques. "Role of ß-lactamase operon on mecA expression in Staphylococcus aureus." Master's thesis, Faculdade de Ciências Médicas, 2011. http://hdl.handle.net/10362/6657.
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RESUMO: Os Staphylococcus aureus resistentes à meticilina (MRSA, do inglês “methicillin-resistant Staphylococcus aureus”) são um dos principais agentes responsáveis por infeções
hospitalares. Os MRSA são resistentes a praticamente todos os antibióticos β-lactâmicos devido a dois mecanismos principais: produção de β-lactamase (bla), codificada pelo gene blaZ, e produção de uma proteína de ligação à penicilina (PBP2a, do inglês “penicillin binding protein 2”), codificada pelo gene mecA. Estes dois genes são regulados por sistemas homólogos, constituídos por um sensor-transdutor (BlaR1 e MecR1) e um repressor (BlaI e MecI), de tal modo que ambos os sistemas são capazes de co-regular os genes mecA e blaZ,
embora com eficiências de indução muito diferentes. De facto, a indução mediada pelo
sistema mecI-mecR1 é tão lenta que se acredita que este sistema não está funcional na maioria das estirpes MRSA.
No entanto, dados recentes do nosso laboratório, demonstram a ausência de relação entre a presença do gene mecI e o nível de resistência à meticilina em estirpes MRSA epidémicas, e também que, o fenótipo de resistência da grande maioria das estirpes não é perturbado pela
sobre-expressão em trans do repressor mecI. Curiosamente, as duas estirpes em que a
expressão da resistência foi afetada pela sobre-expressão do mecI são negativas para o locus da β-lactamase, o que sugere que este locus pode interferir diretamente com a repressão do gene mecA mediada pelo MecI.
Nesta tese de mestrado esta hipótese foi explorada usando estratégias de biologia molecular e ensaios fenotípicos da resistência aos -lactâmicos. Os resultados obtidos demonstram que a presença do plasmídeo nativo da β-lactamase não só anula a repressão mediada pelo MecI, como também aumenta o nível de resistência das estirpes parentais. Várias hipóteses foram então formuladas para explicar estas observações. Dados preliminares, em conjunto com evidências experimentais publicadas, sugerem que o BlaI forma hetero-dímeros com o
MecI que, após a indução, são inativados eficientemente pelo BlaR1. Em conclusão, estes resultados apresentam novas perspetivas para o mecanismo de regulação do mecA e para uma nova importante função do operão da β-lactamase para o fenótipo das estirpes MRSA.-------------------ABSTRACT: Methicillin-resistant Staphylococcus aureus (MRSA) is an important nosocomial pathogen
and is also emerging in the community. MRSA is cross-resistant to virtually all β-lactam
antibiotics and has acquired two main resistance mechanisms: production of β-lactamase (bla), coded by blaZ, and production of penicillin binding protein 2a (PBP2a), coded by mecA. Both genes are regulated by homologous sensor-transducers (BlaR1 and MecR1) and repressors (BlaI and MecI), and coregulation of mecA and blaZ by both systems has been demonstrated, although with remarkable different efficiencies. In fact, induction of mecA by mecI-mecR1 is so slow that it is believed it is not functional in most MRSA strains.
However, recent data from our laboratory has unexpectedly demonstrated that not only
there is no correlation between the presence of mecI gene and the resistance level in
epidemic MRSA strains, but also that for most strains there were no significant changes on the resistance phenotype upon the mecI overexpression in trans. Interestingly, the two strains in which mecI overexpression affected the resistance expression were negative for the bla locus, suggesting that this locus may interfere directly with the MecI-mediated repression of mecA and account for those puzzling observations.
In this master thesis we have explored this hypothesis using molecular biology strategies
and phenotypic analysis of -lactam resistance. The data obtained demonstrate that the presence of a wild-type plasmid containing the bla locus not only disrupts the MecImediated repression, but also significantly enhances the expression of resistance. Several preliminary hypotheses were formulated to explain these observations and preliminary data,
together with published evidence, support the working model that BlaI forms functional
hetero-dimers with MecI, which upon induction are readily inactivated by BlaR1. These results provide new insights into the regulatory mechanism(s) of mecA and open new
perspectives for the role of β-lactamase operon in the MRSA phenotype.