Construction and validation of the Rhodobacter sphaeroides 2.4.1 DNA microarray: transcriptome flexibility at diverse growth modes

Abstract

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.

FIG. 1.

Curation of the R. sphaeroides genome annotation. A representative 9-kb DNA fragment of the R. sphaeroides genome annotated by ORNL (http://genome.ornl.gov/microbial/rsph/03nov00/obsolete) is displayed in Artemis software (34). ORFs in each of the six frames identified by the original annotation are shown as colored horizontal arrows. Codon usage graphs for three reading frames (red, green, and blue) on both DNA strands (top panel, forward strand; lower panel, reverse strand) are shown. A profile of a putative ORF that has codon usage characteristic of R. sphaeroides appears alleviated compared to profiles in alternative reading frames. A start and end of an ORF are characterized by a sharp rise and fall, respectively, of a codon usage curve. “Deleted ORFs,” “New ORFs” (shown as solid black arrows), and “Start codon change” indicate changes introduced to the original ORNL annotation. “Intergenic region” (shown as a black box) indicates a sequence between or0039 and a new ORF used for probe set construction. Black vertical lines correspond to revised borders of the ORFs. or0009 was deleted because it extensively overlapped with or0031 and had codon usage uncharacteristic of R. sphaeroides. or0013 was deleted because it extensively overlapped with or0026 and or0014, both of which had proper codon usage and showed similarity to other proteins from the databases. or0030 was deleted and replaced with a new ORF on the opposite strand showing codon usage characteristic of R. sphaeroides. A new ORF was predicted between or0014 and or0016 based upon codon usage. Start codons were corrected for the overlapping ORFs, or0026 and or0014, based on their codon usage profiles, thus eliminating the overlap between them.

FIG. 2.

Expression level changes (anoxic-light versus oxic conditions) in different categories of probe sets presented on the genechip. Bars: ▪, expression increases (>5-fold); □, expression decreases (>5-fold). Old ORFs, ORFs identified in the original ORNL annotation and redefined based on codon usage; new ORFs, newly identified ORFs; M-intergenics, intergenic regions containing putative short ORFs; G-intergenics, intergenic regions with no identifiable ORFs (see the text for details).

FIG. 3.

Intrachip reproducibility. Each bar represents a standard deviation (as a percentage) from the average signal value (from five copies of a gene) for a given gene on a given genechip. Bars 1 to 3, oxic conditions; bars 4 to 6, anoxic-light conditions; bars 7 to 9, anoxic-dark-DMSO conditions. RSP #, designation of a gene (probe set) on the R. sphaeroides genechip.

FIG. 4.

Correlation between transcript abundance determined by genechips (▪) and qPCR (□). Transcript level of each gene under oxic conditions (left bars) is assigned a value of 1. The fold change ratios for each gene (compared to oxic conditions) for anoxic-light (center bars) and anoxic-dark (right bars) conditions are shown.

FIG. 5.

Expression profiles of selected genetic loci. (A) ccoNOQP-rdxBH-rdxIS; (B) Q-pufBALMX. The average gene expression levels from three genechip replicates under oxic conditions (Aero) are shown in italics. Each of these is assigned an arbitrary value of 1 for comparison purposes. The fold changes (compared to oxic conditions) under anoxic-light (Photo) or anoxic-dark (DMSO) conditions are shown. Horizontal arrows correspond to major transcripts (, ; reference and references therein).

FIG. 6.

Pairwise comparisons of transcriptome changes under diverse growth modes. The numbers of transcripts whose abundance changed are shown. Up, upregulated genes (□, ORFs; ▨, intergenic regions); Down, downregulated genes (▪, ORFs; ▧, intergenic regions); Aero, oxic conditions; Photo, anoxic-light conditions; DMSO, anoxic-dark conditions.

FIG. 7.

R. sphaeroides electron transport chain complexes and ATP synthases involved in energy generation under oxic, anoxic-light, and anoxic-dark-DMSO conditions. Arrows indicate electron flow; dashed line arrows indicate anticipated direction of electron flow. The known sites for generation of proton motive force are shown. The expression of genes under oxic conditions is set as the background for comparisons (not colored). Blue corresponds to decreased gene expression of the corresponding proteins compared to expression under oxic conditions as follows: light blue, ≤2-fold decrease; dark blue, >2-fold decrease. Pink corresponds to increased gene expression compared to the expression under oxic conditions as follows: light pink, ≤2-fold increase; dark pink, >2-fold increase. No color corresponds to expression that is not significantly different from the expression under oxic conditions. Proteins whose genes are expressed below reliable detection are not shown. DH, dehydrogenase; Q, quinone-quinol pool; LH, light-harvesting complex; RC, reaction center complex. The reaction center and light-harvesting complexes are not shown under oxic conditions because several bch genes involved in bacteriochlorophyll biosynthesis are not expressed under these conditions and no PS is made.