Whole-genome transcriptional analysis of chemolithoautotrophic thiosulfate oxidation by Thiobacillus denitrificans under aerobic versus denitrifying conditions

Abstract

Thiobacillus denitrificans is one of the few known obligate chemolithoautotrophic bacteria capable of energetically coupling thiosulfate oxidation to denitrification as well as aerobic respiration. As very little is known about the differential expression of genes associated with key chemolithoautotrophic functions (such as sulfur compound oxidation and CO2 fixation) under aerobic versus denitrifying conditions, we conducted whole-genome, cDNA microarray studies to explore this topic systematically. The microarrays identified 277 genes (approximately 10% of the genome) as differentially expressed using RMA (robust multiarray average) statistical analysis and a twofold cutoff. Genes upregulated (ca. 6- to 150-fold) under aerobic conditions included a cluster of genes associated with iron acquisition (e.g., siderophore-related genes), a cluster of cytochrome cbb3 oxidase genes, cbbL and cbbS (encoding the large and small subunits of form I ribulose 1,5-bisphosphate carboxylase/oxygenase, or RubisCO), and multiple molecular chaperone genes. Genes upregulated (ca. 4- to 95-fold) under denitrifying conditions included nar, nir, and nor genes (associated, respectively, with nitrate reductase, nitrite reductase, and nitric oxide reductase, which catalyze successive steps of denitrification), cbbM (encoding form II RubisCO), and genes involved with sulfur compound oxidation (including two physically separated but highly similar copies of sulfide:quinone oxidoreductase and of dsrC, associated with dissimilatory sulfite reductase). Among genes associated with denitrification, relative expression levels (i.e., degree of upregulation with nitrate) tended to decrease in the order nar > nir > nor > nos. Reverse transcription-quantitative PCR analysis was used to validate these trends.

FIG. 1.

Plot of log₂ probability of differential expression versus log₂ fold differential expression for all genes identified in the T. denitrificans genome. The color coding corresponds to the major categories listed in Tables 1 and 2, namely, denitrification, sulfur compound oxidation, CO₂ fixation via RubisCO (forms I and II), iron acquisition, cytochrome cbb₃ oxidase, and chaperones and stress proteins; all genes not falling within these categories in Tables 1 and 2, as well as all genes not included in Tables 1 and 2, are colored gray.

FIG. 2.

Histogram displaying fold upregulation (denitrifying versus aerobic conditions) for genes associated with denitrification, including nar cluster genes (Tbd1399-1406), nir cluster genes (Tbd0070-0079), nor cluster genes (Tbd0555-0562), and nos cluster genes (Tbd1389-1397). Structural nir, nor, and nos genes are labeled. Absolute expression levels for these genes are shown below the histogram, with green representing the lowest levels of expression, black representing intermediate levels, and red representing the highest levels. The plot of absolute expression levels shows all nine replicates for each condition.

FIG. 3.

(A) Histogram of absolute expression levels of sqr (Tbd1407), dsrC (Tbd1408), and adjacent nar genes under aerobic and denitrifying conditions. (B) Partial sequence of the intergenic region between sqr and dsrC; the putative ribosomal-binding site (RBS) and FNR box are indicated by underlining. (C) RT-qPCR results for cells exposed to aerobic and denitrifying conditions showing the relative number of transcripts that include dsrC (primers Tbd1408F and Tbd1408R2) or dsrC plus the intergenic region between sqr and dsrC (primers Tbd1407-1408IG_F and Tbd1408R). Numbers of transcripts are normalized to the largest value (dsrC; denitrifying conditions). Error bars represent 1 standard deviation based upon triplicate analyses. (D) Electropherogram of RT-PCR products from RNA extracts for cells exposed to aerobic (lanes 2 to 5) and denitrifying (lanes 6 to 9) conditions. Lane 1, Hi-Lo DNA marker, 50 bp to 10 kbp (Bionexus Inc., Oakland, CA); lanes 2 and 6, Tbd1407F/Tbd1408R2; lanes 3 and 7, Tbd1407F/Tbd1407R; lanes 4 and 8, Tbd1408F/Tbd1408R2; lanes 5 and 9, Tbd1406F/Tbd1408R2. All bands represent cDNA amplicons of the expected length and are consistent with PCRs using genomic DNA as the template (not shown). No bands were visible for negative controls lacking reverse transcriptase (not shown).

FIG. 4.

Nucleotide sequences in the promoter regions of the hemP (A) and psuA (B) genes. The putative −35 and −10 promoter sequences as well as the putative Fur box sequences for both genes are indicated.

FIG. 5.

Correlation between aerobic fold upregulation as determined by RT-qPCR versus microarray analysis for 12 genes: narG (Tbd1403), nirS (Tbd0077), norB (Tbd0561), nosZ (Tbd1389), cbbS (Tbd2623), cbbL (Tbd2624), cbbM (Tbd2638), sqr (Tbd1407), ccoN (Tbd0643), dsrC (Tbd1408), dsrC (Tbd1365), and pvuA (Tbd0722).