Development and Validation ofCorynebacteriumDNA Microarrays

Abstract

We have developed DNA microarray techniques for studying Corynebacterium glutamicum. A set of 52 C. glutamicum genes encoding enzymes from primary metabolism was amplified by PCR and printed in triplicate onto glass slides. Total RNA was extracted from cells harvested during the exponential-growth and lysine production phases of a C. glutamicum fermentation. Fluorescently labeled cDNAs were prepared by reverse transcription using random hexamer primers and hybridized to the microarrays. To establish a set of benchmark metrics for this technique, we compared the variability between replicate spots on the same slide, between slides hybridized with cDNAs from the same labeling reaction, and between slides hybridized with cDNAs prepared in separate labeling reactions. We found that the results were both robust and statistically reproducible. Spot-to-spot variability was 3.8% between replicate spots on a given slide, 5.0% between spots on separate slides (though hybridized with identical, labeled cDNA), and 8.1% between spots from separate slides hybridized with samples from separate reverse transcription reactions yielding an average spot to spot variability of 7.1% across all conditions. Furthermore, when we examined the changes in gene expression that occurred between the two phases of the fermentation, we found that results for the majority of the genes agreed with observations made using other methods. These procedures will be a valuable addition to the metabolic engineering toolbox for the improvement of C. glutamicum amino acid-producing strains.Corynebacterium glutamicum is used in the commercial production of lysine and glutamic acid (reviewed in reference 9). High-level amino acid biosynthesis draws resources from many different biosynthetic pathways, and it places physiological stress upon the cell (e.g., reference 6). To understand how Corynebacterium accommodates these demands, research in recent years has sought to divine the overall regulatory processes that coordinate the cell's physiology during amino acid production.During the last 5 years, Brown and coworkers (2, 11) have described methods for studying gene regulation on a global scale using DNA microarrays. These techniques should be useful for studying how gene expression changes during amino acid biosynthesis and secretion. However, the literature offers few examples of the successful application of DNA microarray technology in gram-positive bacteria (e.g., references 3, 19, and 21). In addition, we were interested in evaluating the reproducibility of microarray technologies. To address these issues, we have adapted the methods developed by the Brown lab (herein called the Stanford protocol [http://cmgm.stanford .edu/pbrown/]). We have printed and tested pilot microarrays of Corynebacterium genes and used them to evaluate the robustness and reproducibility of the technique for studying gene expression in Corynebacterium. MATERIALS AND METHODSPreparation of DNA microarrays. Oligonucleotide primers (Life Technologies, Inc., Rockville, Md.) were de...

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