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Applied and Environmental Microbiology, May 2001, p. 2310-2318, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2310-2318.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.

Development and Validation of Corynebacterium DNA Microarrays

Andrea Loos,1,2 Christoph Glanemann,1 Laura B. Willis,1 Xian M. O'Brien,1 Philip A. Lessard,1 Robert Gerstmeir,1,dagger Stéphane Guillouet,1,Dagger and Anthony J. Sinskey1,*

Department of Biology1 and BioMicro Center,2 Massachusetts Institute of Technology, Cambridge, Massachusetts 02139

Received 27 November 2000/Accepted 16 February 2001

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.

* Corresponding author. Mailing address: Department of Biology, Massachusetts Institute of Technology 68-370, 77 Massachusetts Ave., Cambridge, MA 02139. Phone: (617) 253-6721. Fax: (617) 253-8550. E-mail: asinskey{at}mit.edu.

dagger Present address: Abteilung Mikrobiologie und Biotechnologie, Universität Ulm, Ulm, Germany.

Dagger Present address: INSA de Toulouse, Department de Genie Biochimique Alimentaire, 31400 Toulouse, France.

Applied and Environmental Microbiology, May 2001, p. 2310-2318, Vol. 67, No. 5
0099-2240/01/$04.00+0   DOI: 10.1128/AEM.67.5.2310-2318.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.


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