Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source.
A DNA microarray was developed to analyse global gene expression of the amino acid-producing bacterium Corynebacterium glutamicum. PCR products representing 93.4% of the predicted C. glutamicum genes were prepared and spotted in quadruplicate onto 3-aminopropyltrimethoxysilane-coated glass slides. The applicability of the C. glutamicum DNA microarray was demonstrated by co-hybridisation with fluorescently labelled cDNA probes. Analysis of the technical variance revealed that C. glutamicum genes detected with different intensities resulting in ratios greater than 1.52 or smaller than -1.52 can be regarded as differentially expressed with a confidence level of greater than 95%. In a validation example, we measured changes of the mRNA levels during growth of C. glutamicum with acetate and propionate as carbon sources. Acetate-grown C. glutamicum cultures were used as reference. At the 95% confidence interval, 117 genes revealed increased transcript levels in the presence of propionate, while 43 genes showed a decreased expression compared with the acetate-grown culture. Global expression profiling confirmed the induction of the prpD2B2C2 gene cluster already known to be essential for propionate degradation via the 2-methylcitrate cycle. Besides many genes of unknown function, the paralogous prpD1B1C1 gene cluster as well as fasI-B (encoding fatty-acid synthase IB), dtsR1 and dtsR2 (components of acyl-CoA carboxylases), gluABCD (glutamate transport system), putP (proline transport system), and pyc ( pyruvate carboxylase) showed significantly increased expression levels. Differential expression of these genes was confirmed by real-time reverse transcription (RT) PCR assays.[1]References
- Development of a Corynebacterium glutamicum DNA microarray and validation by genome-wide expression profiling during growth with propionate as carbon source. Hüser, A.T., Becker, A., Brune, I., Dondrup, M., Kalinowski, J., Plassmeier, J., Pühler, A., Wiegräbe, I., Tauch, A. J. Biotechnol. (2003) [Pubmed]
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