Construction and Evaluation of Desulfovibrio vulgaris Whole-Genome Oligonucleotide Microarrays
Desulfovibrio vulgaris Hildenborough has been the focus of biochemical and physiological studies in the laboratory, and the metabolic versatility of this organism has been largely recognized, particularly the reduction of sulfate, fumarate, iron, uranium and chromium. In addition, a Desulfovibrio sp. has been shown to utilize uranium as the sole electron acceptor. D. vulgaris is a d-Proteobacterium with a genome size of 3.6 Mb and 3584 ORFs. The whole-genome microarrays of D. vulgaris have been constructed using 70mer oligonucleotides. All ORFs in the genome were represented with 3471 (97.1%) unique probes and 103 (2.9%) non-specific probes that may have cross-hybridization with other ORFs. In preparation for use of the experimental microarrays, artificial probes and targets were designed to assess specificity and sensitivity and identify optimal hybridization conditions for oligonucleotide microarrays. The results indicated that for 50mer and 70mer oligonucleotide arrays, hybridization at 45 C to 50 C, washing at 37 C and a wash time of 2.5 to 5 minutes obtained specific and strong hybridization signals. In order to evaluate the performance of the experimental microarrays, growth conditions were selected that were expected to give significant hybridization differences for different sets of genes. The initial evaluations were performed using D. vulgaris cells grown at logarithmic and stationary phases. Transcriptional analysis of D. vulgaris cells sampled during logarithmic phase growth indicated that 25% of annotated ORFs were up-regulated and 3% of annotated ORFs were downregulated compared to stationary phase cells. The up-regulated genes included ORFs predicted to be involved with acyl chain biosynthesis, amino acid ABC transporter, translational initiation factors, and ribosomal proteins. In the stationary phase growth cells, the two most up-regulated ORFs (70-fold) were annotated as a carboxynorspermidine decarboxylase and a 2C-methyl-D-erythritol-2,4-cyclodiphosphate (MECDP) synthase. Spermidines are polyamines that are typically abundant in rapidly dividing cells and are essential growth factors in eukaryotic organisms. Polyamines are thought to stabilize DNA by the association of the amino groups with the phosphate residues of DNA and can also enhance tRNA and ribosome stability. The MECDP synthase enzyme is essential in Escherichia coli and participates in the nonmevalonate pathway of isoprenoid biosynthesis, a critical pathway present in some bacteria and apicomplexans but distinct from that used by mammals. Several of the highly up-regulated ORFs were annotated as conserved hypothetical proteins. Interestingly, an ORF that was predicted to contain a flocculin repeat domain was almost 9-fold up-regulated in stationary phase cells compared to logarithmically growing cells. The flocculin domain is commonly observed in fungi, and is thought to play a role during flocculation (non-sexual aggregation of single-cell microorganisms). These preliminary results have identified possible responses of D. vulgaris cells to stationary phase growth and suggest that polyamine production as well as cell aggregation and/or extracellular polymer production are responses of D. vulgaris during stationary phase. The initial microarray results indicate that the recently produced oligonucleotide microarrays are functional. We are currently optimizing growth conditions in order to culture D. vulgaris cells in the presence of uranium(VI) and to monitor whole-genome expression levels.
- Research Organization:
- Oak Ridge National Lab. (ORNL), Oak Ridge, TN (United States); University of Missouri, Columbia, MO; Miami University, Oxford, OH
- Sponsoring Organization:
- USDOE Office of Science (SC)
- OSTI ID:
- 895319
- Report Number(s):
- CONF-NABIR2004-27; TRN: US200702%%754
- Resource Relation:
- Conference: Annual NABIR PI Meeting, March 15-17, 2004, Warrenton, VA
- Country of Publication:
- United States
- Language:
- English
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