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Construction and Validation of the Rhodobacter sphaeroides 2.4.1 DNA Microarray: Transcriptome Flexibility at Diverse Growth Modes

Christopher T. Pappas,^1, Jakub Sram,^1, Oleg V. Moskvin,¹ Pavel S. Ivanov,^1, R. Christopher Mackenzie,² Madhusudan Choudhary,² Miriam L. Land,³ Frank W. Larimer,³ Samuel Kaplan,² and Mark Gomelsky^1*

Department of Molecular Biology, University of Wyoming, Laramie, Wyoming,¹ Department of Microbiology and Molecular Genetics, University of Texas Medical School, Houston, Texas,² Genome Analysis Group, Oak Ridge National Laboratory, Oak Ridge, Tennessee³

Received 4 December 2003/ Accepted 15 April 2004

A high-density oligonucleotide DNA microarray, a genechip, representing the 4.6-Mb genome of the facultative phototrophic proteobacterium, Rhodobacter sphaeroides 2.4.1, was custom-designed and manufactured by Affymetrix, Santa Clara, Calif. The genechip contains probe sets for 4,292 open reading frames (ORFs), 47 rRNA and tRNA genes, and 394 intergenic regions. The probe set sequences were derived from the genome annotation generated by Oak Ridge National Laboratory after extensive revision, which was based primarily upon codon usage characteristic of this GC-rich bacterium. As a result of the revision, numerous missing ORFs were uncovered, nonexistent ORFs were deleted, and misidentified start codons were corrected. To evaluate R. sphaeroides transcriptome flexibility, expression profiles for three diverse growth modes—aerobic respiration, anaerobic respiration in the dark, and anaerobic photosynthesis—were generated. Expression levels of one-fifth to one-third of the R. sphaeroides ORFs were significantly different in cells under any two growth modes. Pathways involved in energy generation and redox balance maintenance under three growth modes were reconstructed. Expression patterns of genes involved in these pathways mirrored known functional changes, suggesting that massive changes in gene expression are the major means used by R. sphaeroides in adaptation to diverse conditions. Differential expression was observed for genes encoding putative new participants in these pathways (additional photosystem genes, duplicate NADH dehydrogenase, ATP synthases), whose functionality has yet to be investigated. The DNA microarray data correlated well with data derived from quantitative reverse transcription-PCR, as well as with data from the literature, thus validating the R. sphaeroides genechip as a powerful and reliable tool for studying unprecedented metabolic versatility of this bacterium.

Present address: NMCB Graduate Program, University of Arizona, Tuscon.

Present address: City of Hope National Medical Institute, Los Angeles, Calif.

Permanent address: Department of Biophysics, Faculty of Physics, Moscow State University, Moscow, Russia.

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