Tesis doctoral de Cristina Viadas Martínez
Analysis of the brucella abortus bvrr/bvrs two-component regulatory system by dna microarray. cristina viadas martínez, faculty of sciences, university of navarra (spain), october 8th, 2010. brucella is the etiological agent of brucellosis, one of the most common bacterial zoonosis worldwide. Brucella is a facultative intracellular bacteria that must coordinate an intricate network of factors to generate a suitable adaptive response to the various signals. This attribute is often accomplished by two-component transduction systems, consisting of a sensor kinase and a response regulator. These regulatory systems control gene expression in response to environmental signals. The two-component bvrr/bvrs system is essential for b. Abortus virulence. It was shown previously that its dysfunction alters the expression of some major outer membrane proteins and the pattern of acylation and phosphorylation of the lipid a in the lipopolyssacharide. To determine the genes regulated by bvrr/bvrs, we performed a whole-genome dna microarray. First, we evaluate a dna microarray constructed using the orfeome of b. Melitensis. This orfeome is a comprehensive collection of the predicted coding sequences or open reading frames (orfs). The microarray has a total of 7,680 spots and represents over 96 % of the complete coding sequences assigned to b. Melitensis. It also included several normalization controls that represented constitutively expressed brucella genes or negative controls with arabidopsis thaliana genes. A quality control regarding the position, intensity and morphology of the spots, the background and the reproducibility of the arrays was performed by ethidium bromide staining. to test the usefulness of the microarray, a direct analysis of the global transcriptional profile of b. Abortus cells grown in laboratory conditions was made. The composition of the culture concerns significantly the set of genes with a major and minor profile of expression. In general, the genes with a major profile of expression are related to constitutive functions, whereas the genes with a minor profile of expression are involved in functions not essential or related to the virulence of the bacterium. The genes with major and minor profile of expression are not distributed of equal way between both chromosomes of brucella. The genes with a major profile of expression are located for the most part in the chromosome i, whereas the majority of the genes with a minor profile of expression are located in the chromosome ii. to determine the genes regulated by bvrr/bvrs, we performed a microarray analysis using b. Abortus rna obtained from wild type and bvrr mutant cells grown in the same conditions. The statistical analysis resulted in the identification of a total of 127 genes differentially transcribed in the bvrr mutant versus the wild type. Eighty three genes were up regulated and 44 were down regulated in the bvrr mutant. These results show that the system bvrr/bvrs is involved in the regulation of components of the cell envelope, of carbon and nitrogen metabolism, of other transcriptional regulators and genes related directly to the virulence of brucella. finally, the analysis of the gene expression of b. Abortus intracellular by rt-pcr demonstrates that several genes related to the denitrification are regulated. Brucella could be neutralize the production of toxic reactive nitrogen molecules, as no, by the host.
Datos académicos de la tesis doctoral «Análisis génico del sistema regulador de dos componentes bvrr/bvrs de brucella mediante tecnología dna microarray«
- Título de la tesis: Análisis génico del sistema regulador de dos componentes bvrr/bvrs de brucella mediante tecnología dna microarray
- Autor: Cristina Viadas Martínez
- Universidad: Navarra
- Fecha de lectura de la tesis: 08/10/2010
Dirección y tribunal
- Director de la tesis
- Ignacio Lopez Goñi
- Tribunal
- Presidente del tribunal: Juan manuel García lobo
- Juan Francisco Medina cabrera (vocal)
- clara María Marín alcala (vocal)
- Antonio gerardo Pisabarro de lucas (vocal)