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J Bacteriol. Oct 2006; 188(19): 7005–7015.
PMCID: PMC1595532

Whole-Genome Transcriptional Analysis of Chemolithoautotrophic Thiosulfate Oxidation by Thiobacillus denitrificans under Aerobic versus Denitrifying Conditions


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.

Thiobacillus denitrificans is an obligately chemolithoautotrophic bacterium characterized by its ability to conserve energy from the oxidation of inorganic sulfur compounds under either aerobic or denitrifying conditions (5). As a facultative anaerobe, T. denitrificans may benefit from modulating key components of its energy metabolism, such as sulfur compound oxidation or carbon dioxide fixation, according to whether oxygen or nitrate is the terminal electron acceptor. For example, T. denitrificans can express both form I and form II ribulose 1,5-bisphosphate carboxylase/oxygenase (RubisCO), which have different relative affinities for CO2 and the competing substrate O2 and therefore may differ in CO2 fixation efficiency under aerobic versus denitrifying conditions. Also, among its large complement of genes associated with sulfur compound oxidation, T. denitrificans shares some genes with aerobic, chemolithotrophic sulfur-oxidizing bacteria and some with anaerobic, phototrophic sulfur bacteria (5). There is very little information on how (or whether) T. denitrificans modulates the expression of these sulfur-oxidizing genes as a function of the prevailing terminal electron acceptor. The recent availability of the complete genome sequence of T. denitrificans (5) and of high-density oligonucleotide microarrays provided us an opportunity to address these and other questions by systematically investigating differential expression across the entire T. denitrificans genome under aerobic versus denitrifying conditions.


Cell growth and exposure conditions.

To represent gene expression under denitrifying conditions, T. denitrificans (ATCC strain 25259, obtained from the American Type Culture Collection) was cultivated at 30°C under strictly anaerobic conditions as described previously (4) with growth medium that contained 20 mM thiosulfate, 20 mM nitrate, and 30 mM bicarbonate (pH ~7). For exposure immediately before harvesting of RNA, 1,200 ml of cells in late exponential phase (1 × 108 to 2 × 108 cells/ml) was harvested anaerobically by centrifugation (13,400 × g, 15°C, 10 min) and resuspended in modified growth medium (phosphate concentration reduced to 1.5 mM), and three 10-ml replicates (ca. 7.3 mg protein each) in sealed vials (90% N2-10% CO2 headspace) were incubated for ca. 35 min. Cell growth, resuspension, and incubation were performed in an anaerobic glove box (4).

To represent gene expression under aerobic conditions, T. denitrificans was cultivated (two successive transfers) with growth medium that differed from the denitrifying medium in several noteworthy respects: it contained no nitrate, it was equilibrated with atmospheric oxygen (rotating in a shake flask at 200 rpm), and it contained 70 mM phosphate, 0.7 μM copper (compared to 1.2 μM in denitrifying medium), and 10 μM iron (compared to 7.5 μM). The reason for using a higher phosphate buffer concentration in the aerobic medium was that, when lower phosphate concentrations were tested, the pH of aerobic growth medium dropped from ~7 to ~5 as T. denitrificans oxidized thiosulfate. This follows from the stoichiometry of thiosulfate oxidation, which yields fivefold more protons per mole of thiosulfate under aerobic than denitrifying conditions. For exposure immediately before harvesting of RNA, 1,200 ml of cells in late exponential phase was harvested by centrifugation and resuspended in aerobic growth medium, and three 10-ml replicates (ca. 3.9 mg protein each) were incubated at 30°C in 125-ml Erlenmeyer flasks rotating at 200 rpm for 60 min. The pH of the cell suspensions remained in the circumneutral range throughout the incubation period.

Metabolic activity (thiosulfate oxidation to sulfate, nitrate consumption in anaerobic cultures) was assessed in all anaerobic and aerobic suspensions by sampling each culture twice: immediately upon resuspension and immediately before harvesting for RNA. Ion chromatography was used to determine thiosulfate, sulfate, and nitrate concentrations (4). Previous experiments indicated that metabolic rates during suspensions were sufficiently linear throughout the incubation period that initial and final concentrations could be used to calculate representative specific rates. These analyses demonstrated that specific thiosulfate oxidation rates were comparable under denitrifying and aerobic conditions (0.43 ± 0.005 and 0.56 ± 0.006 μmol thiosulfate · min−1 · mg protein−1, respectively).

RNA extraction.

Immediately after exposures, two volumes of RNAprotect (QIAGEN) were added to each culture. Samples were incubated at room temperature for 12 min, split in half, and centrifuged at 4,000 rpm for 10 min. The supernatant was decanted, and the pellet was stored at −20°C until extraction. RNA extraction was carried out with a MasterPure Complete (MPC) DNA and RNA purification kit (EpiCentre) using a modified protocol. Briefly, 300 μl of lysis solution containing 112 μg proteinase K was added to the cell pellet and the sample was incubated at 65°C for 20 to 25 min. The sample was placed on ice for 3 to 5 min, and 200 μl of MPC solution was added to precipitate protein. The supernatant was recovered after centrifugation at >10,000 × g at 4°C for 10 min. Nucleic acid was subsequently precipitated from the supernatant after addition of 500 μl 99% isopropanol and centrifugation at >10,000 × g at 4°C for 10 min. The pellet was treated with DNase I for 20 min at 37°C. To this sample was added 200 μl each of 2× T&C lysis solution and MPC solution with vortexing after each addition. The samples were placed on ice for 3 to 5 min and centrifuged at >10,000 × g at 4°C for 10 min. RNA in the supernatant was recovered by isopropanol precipitation as described above. The RNA pellet was washed twice with 75% ethanol, dried briefly, suspended in water, and stored at −80°C until cDNA synthesis. Aliquots were analyzed with a Bioanalyzer (Agilent), which indicated minimal degradation and concentrations ranging from 310 to 2,000 ng/μl. A260/A280 ratios ranged from 1.7 to 2.1.

Preparation of labeled cDNA.

cDNA production and labeling were performed by NimbleGen Systems, Inc. RNA samples were thawed on ice, and 10 μg total RNA was used to perform cDNA synthesis with SuperScript II reverse transcriptase and random hexamers. After this reaction, RNases A and H were used to digest the RNA. Single-stranded cDNA was subsequently purified by phenol extraction. Glycogen (10 μg) was added as a carrier prior to precipitation with a 1/10 volume ammonium acetate and 2.5 volumes of absolute ethanol. The resulting pellet was suspended in 30 μl water. The cDNA yield was determined by UV/visible spectrophotometry at 260 nm. The cDNA was partially digested with DNase I (0.2 U) at 37°C for 13 min or until 50- to 200-base fragments were observed with the Bioanalyzer. The fragmented cDNA was end labeled using biotin-N6-ddATP and terminal deoxynucleotidyl transferase (51 U) with incubation at 37°C for 2 h. The labeled product was concentrated to 20 μl using a Microcon YM-10 10,000 MWCO filter device (Millipore) and frozen at −20°C prior to hybridization.

Array design.

The genome sequence from T. denitrificans ATCC 25259 (5) (GenBank accession no. CP000116) was submitted to NimbleGen Systems Inc. for microarray design and manufacture using maskless, digital micromirror technology. High-density (approximately 400,000-spot) microarrays employed a randomized design and a four-in-nine pattern to enhance sensitivity. Three replicates of the genome were included per chip. An average of 10 different 60-base oligonucleotides (60-mer probes) represented each open reading frame (ORF) in the genome. 60-mer probes were selected such that each probe had at least three mismatches compared to all other 60-mers in the target genome. A total of 28,320 probes were designed for the genome, which was annotated to have 2,832 ORFs at the time of microarray design (the finished genome is annotated to have 2,827 ORFs [5]). A quality control check (hybridization) was performed for each array, which contained on-chip control oligonucleotides.

Microarray hybridization and analysis.

NimbleGen Systems, Inc. performed array hybridization using their Hybriwheel technology. The arrays were prehybridized at 45°C in a 50 mM 4-morpholineethanesulfonic acid buffer containing 500 mM NaCl, 10 mM EDTA, and 0.005% Tween 20 with herring sperm DNA (0.1 mg/ml) to prevent nonspecific binding to the array. After 15 min, 4 μg of labeled cDNA in hybridization buffer was added and arrays were incubated at 45°C for 16 to 20 h. Several wash steps (initially nonstringent and later stringent conditions) removed free probe, followed by detection of bound probe with Cy3-labeled streptavidin. To amplify the signal, biotinylated anti-streptavidin goat antibody was hybridized to the array. The arrays were analyzed using an Axon GenePix 4000B scanner with associated software (Molecular Devices Corp., Sunnyvale, CA).

Microarray data analysis.

Investigation of reproducible differences between treatments was performed using the Bioconductor R software package. Data were processed using quantile normalization (7), and background correction was performed using the RMA (robust multiarray average) method. Data were visualized with box-and-whisker plots and scatter plots (volcano plots). Intensities were adjusted to have the same interquartile range. A linear model fit was determined for each gene using the LIMMA package (Linear Models for Microarray Data; Gordon K. Smyth), and lists of genes with the most evidence of differential expression were obtained.

Reverse transcription-quantitative PCR analysis.

Confirmation of transcript levels for modulated genes was performed by reverse transcription-quantitative PCR (RT-qPCR) analysis of RNA samples representing each of the two experimental conditions. Total RNA from samples used for microarray analysis was reverse transcribed and amplified using a QuantiTect SYBR Green RT-PCR kit (QIAGEN) with gene-specific primers. Each gene-specific PCR was performed in triplicate using 25-μl reaction mixtures containing ~20 ng of template on a Prism 7000 cycler (ABI). Calibration curves were performed with genomic DNA serially diluted over a range of 4 to 5 orders of magnitude. The PCR conditions were optimized to be performed as follows for all transcripts: 50°C for 30 min; 95°C for 15 min; 94°C for 15 s, 58°C for 30 s, and 72°C for 30 s; 30 to 35 cycles. The primers are listed in Table S1 of the supplemental material.

RT-PCR analysis of sqr and dsrC transcripts.

Qualitative and quantitative RT-PCR studies were performed to investigate whether a gene associated with sulfur compound oxidation, dsrC (Tbd1408), was cotranscribed with upstream genes associated with nitrate reduction (nar genes) and sulfide:quinone oxidoreductase (sqr; Tbd1407). Forward PCR primers were designed for Tbd1406 (narI) and Tbd1407 (Tbd1406F and Tbd1407F, respectively) (see Table S1 in the supplemental material), and reverse primers were designed for Tbd1408 (Tbd1408R and Tbd1408R2) (see Table S1). Control primers for the large transcript (targeting 1406 and 1407) and the Tbd1408 transcript were also designed and tested. The PCR conditions were optimized using T. denitrificans genomic DNA. cDNA was produced from RNA samples used in microarray experiments (aerobic and denitrifying conditions) with 150 to 250 ng RNA (pretreated with DNase), 100 units Retroscript reverse transcriptase (Ambion), random decamers, and incubation at 43°C for 75 min. PCR products were visualized by gel electrophoresis on a 1% agarose 1× Tris-acetate-EDTA gel with ethidium bromide staining and UV illumination. RT-qPCR analysis was performed with a forward primer from Tbd1407, the Tbd1407-1408 intergenic region, or Tbd1408 with a reverse primer for Tbd1408 (see Table S1). The primers and template were added to SYBR Green Master Mix (Bio-Rad), and reactions were run on a Cepheid SmartCycler using the following program: 98°C for 15 s; 60°C for 60 s; 40 cycles. Controls for both RT-PCR and RT-qPCR analyses included trials without reverse transcriptase and trials without template.

Microarray data accession number.

Microarray data have been deposited in the Gene Expression Omnibus database (http://www.ncbi.nlm.nih.gov/geo) under accession number GSE5256.


Genome-wide observations.

Microarray analysis identified 277 genes in T. denitrificans as differentially expressed under aerobic versus denitrifying conditions using a twofold cutoff (P < 0.0001). The top 50 upregulated genes under denitrifying conditions are listed in Table Table1,1, and the top 50 upregulated genes under aerobic conditions are listed in Table Table2.2. A large percentage of the most upregulated genes under either denitrifying or aerobic conditions occur as gene clusters and can be classified within a small number of functional categories. To illustrate, under denitrifying conditions, upregulated genes include those associated with nitrate reductase (Tbd1401 to -1406 [Tbd1401-1406]; nar cluster), nitrite reductase (Tbd0070-0077; nir cluster), nitric oxide reductase (Tbd0554-0562; nor cluster), and sulfur compound oxidation (including Tbd1407-1408, adjacent to the nar cluster). Other gene clusters with less obvious functional associations are also included among the most upregulated genes (e.g., Tbd1499-1501 and Tbd1835-1838, which includes divergently transcribed genes) and certain functions are represented by single genes (e.g., cbbM, which encodes form II RubisCO). Under aerobic conditions, upregulated genes include a large cluster putatively encoding proteins associated with iron acquisition (Tbd0705-0725, which account for more than 40% of the top 50 upregulated genes), a cytochrome cbb3 oxidase (Tbd0638-0643), multiple chaperones (including Tbd1537-1539), and form I RubisCO (cbbS and cbbL; Tbd2623-2624). Under aerobic conditions, as under denitrifying conditions, gene clusters with less obvious functional associations are also included among the most upregulated genes (e.g., Tbd2355-2358, Tbd2592-2594, and Tbd2777-2778) (Table (Table2),2), and certain functions are represented by single genes (e.g., Tbd1365, a putative dsrC presumably associated with sulfur compound oxidation).

Top 50 ORFs upregulated under denitrifying conditions
Top 50 ORFs upregulated under aerobic conditions

The observation that a relatively small number of functional categories account for the majority of the most differentially expressed genes is apparent in Fig. Fig.1,1, which plots log2 probability of differential expression versus log2 fold differential expression for all ORFs identified in the genome. The color coding in Fig. Fig.11 corresponds to the major categories listed in Tables Tables11 and and2,2, 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 Tables11 and and2,2, and all genes not included in Tables Tables11 and and2,2, are gray in Fig. Fig.11.

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 Tables11 and and ...


Although it is not surprising that genes associated with denitrification (nar, nir, and nor genes) were among the most upregulated genes under denitrifying conditions, subtler trends in expression of these genes were more novel. Most notably, relative expression levels (i.e., degree of upregulation under denitrifying conditions) tended to decrease in the order nar > nir > nor > nos (Fig. (Fig.2).2). With the exception of a few genes (primarily associated with transcriptional regulators, such as narXL and Tbd0078-0079), fold upregulation for denitrification genes fell in the following ranges: nar, 54- to 95-fold; nir, 10- to 21-fold; nor, 4- to 10-fold; nos, 0.5- to 0.9-fold. This trend was both a function of generally decreasing absolute expression levels under denitrifying conditions (except for the structural genes nirS, norCB, and nosZ) and increased expression of nos genes (especially nosZ) under aerobic conditions (Fig. (Fig.22).

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 ...

To our knowledge, this is the most complete data set for differential aerobic/denitrifying expression across the complement of denitrification genes; previous transcriptional studies have focused primarily on structural genes or on gene clusters associated with only one of the four denitrification enzymes. In a general sense, the microarray results for T. denitrificans are consistent with the well-documented transcriptional activation of denitrification genes as a function of low O2 tension and the presence of a nitrogen oxide (NO3, NO2, NO, or N2O) (reviewed in reference 36). With respect to T. denitrificans specifically, the microarray results are generally consistent with greatly increased NAR and NIR enzyme activities (in crude extracts) that were observed to accompany the transition from aerobic to denitrifying conditions in continuous culture (18). Furthermore, the decreasing trend in upregulation shown in Fig. Fig.22 could be consistent with induction of each reductase component by its cognate substrate, as one might expect the concentration pattern of [NO3] > [NO2] > [NO] > [N2O] in a denitrifying cell (although this concept clearly oversimplifies the regulation of denitrification).

However, for nos genes in particular, the results for T. denitrificans appear to deviate from findings for other denitrifying species for which data are available, namely, Pseudomonas stutzeri, Paracoccus denitrificans, and Paracoccus pantotrophus (formerly Thiosphaera pantotropha). For example, whereas expression of nos genes (including nosD) in T. denitrificans was comparable under aerobic and denitrifying conditions, the amount of nosD transcripts in P. stutzeri (revealed by Northern blot analysis) increased steadily and dramatically during the first hour following a shift from aerobic to denitrifying conditions in continuous culture (15). In another continuous culture study of P. stutzeri (20), NosZ levels were at least 10-fold greater for cells under denitrifying conditions than for cells under fully aerobic conditions (in the presence of nitrate). A continuous culture study of Paracoccus denitrificans revealed more than a 10-fold increase in the amount of nosZ transcripts during the first hour following transition from aerobic to denitrifying conditions (3). This temporal trend was qualitatively similar to those of other denitrification genes; however, narH and nirS transcript copy numbers increased more (approximately 30- to 45-fold) (3). In continuous culture and batch culture studies of P. pantotrophus (23), NosZ expression was 2- to ca. 20-fold greater under denitrifying conditions than under aerobic conditions (in the presence or absence of nitrate) and clearly decreased as a function of increasing oxygen concentration in continuous culture. Differences in experimental approach preclude a direct comparison of the results of the present study with those just cited for P. stutzeri and P. denitrificans; such differences in experimental approach include the use of continuous cultures versus batch cultures and measurement after aerobic/anaerobic transitions versus comparisons of cultures grown exclusively under aerobic or denitrifying conditions. Acknowledging this caveat, the available data suggest inconsistent trends for differential aerobic/denitrifying nos gene expression in T. denitrificans compared to other species studied. In P. stutzeri, P. denitrificans, and P. pantotrophus, there appears to be considerable upregulation of nos genes (at least nosZ and nosD) under denitrifying conditions; this is clearly not the case for T. denitrificans (indeed, there is slight upregulation of these genes under aerobic conditions) (Fig. (Fig.22).

In the absence of additional experimental evidence, we cannot explain the anomalous lack of differential transcription of nosZ and other nos genes in T. denitrificans under aerobic versus denitrifying conditions. Nonetheless, examination of promoter regions for some key genes associated with denitrification did reveal possible clues. Specifically, these promoter regions in T. denitrificans were examined with respect to potential FNR boxes (i.e., DNA-binding motifs for FNR-like transcription factors). When compared to the canonical FNR box 5′-TTGAT-N4-ATCAA-3′ described for Escherichia coli (36), slightly degenerate sequences were found upstream of narK (aTGAcATCtt, located 778 nucleotides [nt] from the translational start site of Tbd1401), nirS (TTGAcATCAA, located 76 nt from the translational start site of Tbd0077), norC (aTGAcATCAA, located 163 nt from the translational start site of Tbd0562), and nosZ (TTGAg…gTCAg, located 1,310 nt from the translational start site of Tbd1389). Two features shared by the narK, nirS, and norC versions and not in the nosZ version are the c and A shown in boldface type above. Also, upstream of nosZ, there were five additional sequences with 5′ ends that matched either the canonical FNR box or the narK, nirS, or norC FNR boxes cited above, but these were more degenerate on the 3′ end (with only 0 to 2 bases agreeing with the canonical sequence). Degeneracy at the 3′ end of FNR boxes upstream of nosZ has been observed for some denitrifying species (8, 36) but not for others (2). Considering that FNR boxes for positive regulation of denitrification genes are typically centered at a distance of −41.5 nt from the transcription start site (36), most of the putative FNR boxes just described for T. denitrificans seem to be very far upstream. It is not clear whether any of these characteristics of putative FNR boxes in T. denitrificans could explain the lack of nosZ upregulation under denitrifying conditions.

Sulfur compound oxidation.

Although a diverse complement of more than 50 genes associated with sulfur compound oxidation has been described in T. denitrificans ATCC 25259 (5), those genes associated with activity under aerobic versus denitrifying conditions have not been elucidated to date. Many of the T. denitrificans genes associated with sulfur compound oxidation (5) were not found to be differentially expressed in this study (Tables (Tables11 and and2).2). Among the genes not appearing in Tables Tables11 and and22 were clusters of sulfur compound oxidation genes that were very highly expressed under both aerobic and denitrifying conditions. These include soxXYZA (Tbd0567-0564), dsrABEFHCMKLJOP (Tbd2485-2474), and the genes encoding ATP sulfurylase and APS reductase (Tbd0874-0872). The expression levels of these genes were typically at or above the 95th percentile expression level observed across the genome. Indeed, many of these genes are likely to be constitutively expressed in T. denitrificans as, in most cases, their expression levels were similarly high under Fe(II)-oxidizing, denitrifying conditions when no sulfur-containing electron donor was present (H. Beller et al., unpublished microarray data).

Differential expression was observed for certain genes associated with sulfur compound oxidation; in some cases, the absolute expression levels of these genes when upregulated were also in the range of the 95th percentile expression level observed across the genome. Among the most differentially regulated genes putatively associated with sulfur compound oxidation, all but one were upregulated under denitrifying rather than aerobic conditions (Tables (Tables11 and and2;2; Fig. Fig.1).1). These included two copies of sulfide:quinone oxidoreductase (sqr) that share 43% amino acid identity (Tbd1407 and Tbd2225; 55- and 6.5-fold upregulated under denitrifying conditions), a rhodanese-like domain protein (Tbd1650; 8.7-fold upregulated), and two putative copies of dsrC that share 88% amino acid identity (Tbd1408 and Tbd2327; 14- and 5.7-fold upregulated). Another putative copy of dsrC (Tbd1365) was upregulated 6.9-fold aerobically. Another rhodanese copy (Tbd2399) was less upregulated aerobically (3.8-fold) but was included in a gene cluster that exhibited some stronger aerobic upregulation (Tbd2398-2401) (Table (Table22).

Inasmuch as three dsrC copies were among the most differentially regulated genes, it is noteworthy that the T. denitrificans genome includes eight putative dsrC copies overall (5); the phylogenetic relationships and genomic organization of these homologs have been presented elsewhere (5). Only one copy, Tbd2480, is located in the large gene cluster dsrABEFHCMKLJOPNR (Tbd2485-2472) and is constitutively expressed at a high level (5). Although the exact function of DsrC is not known, it is almost certainly involved with sulfur compound oxidation; the associated dsrAB genes encode a siroheme-containing sulfite reductase that has been proposed to catalyze the oxidation of certain inorganic sulfur species (e.g., hydrogen sulfide or sulfane-sulfur derived from thiosulfate) to sulfite (27, 33).

In light of the strong upregulation of Tbd1407 (sqr) and Tdb1408 (putative dsrC) under denitrifying conditions (Table (Table1),1), the genomic location of these genes is noteworthy: they are immediately downstream of the narKK2GHJI cluster (Tbd1401 -1406) (5), which encodes a membrane-bound, dissimilatory nitrate reductase (and associated nitrate/nitrite transporters) (Fig. (Fig.3A).3A). As there is not even a single intergenic base separating Tbd1406 and Tbd1407, it follows that Tbd1407 is part of a polycistronic transcript including nar genes (probably narKK2GHJI). However, the intergenic region between Tbd1407 and Tbd1408 includes a putative ribosomal-binding site and FNR box (Fig. (Fig.3B).3B). Thus, coregulation rather than cotranscription of Tbd1407 and Tbd1408 is plausible and, indeed, is suggested by the anomalously high expression of Tbd1408 relative to Tbd1407 under aerobic conditions (Fig. (Fig.3A).3A). To further investigate whether Tbd1408 was transcribed independently of Tbd1407 and upstream nar genes, RT-qPCR studies were conducted. These studies confirmed that, for the most part, Tbd1408 was transcribed separately from Tbd1407 and upstream nar genes: under denitrifying or aerobic conditions, the copy number of transcripts of Tbd1408 (dsrC) was at least 10-fold greater than the copy number of transcripts including Tbd1407 and Tbd1408 (sqr and dsrC) (Fig. (Fig.3C).3C). Although the RT-qPCR studies were constrained by amplicon length and did not address transcripts extending upstream beyond Tbd1407, semiquantitative RT-PCR analyses (Fig. (Fig.3D)3D) suggested that, at least under denitrifying conditions, the Tbd1407-1408 transcripts actually extended at least from Tbd1406 (narI) to Tbd1408 (lane 9). Overall, the microarray and RT-qPCR results suggest that the promoter(s) controlling the expression of dsrC (Tbd1408), while clearly effecting stronger activation under denitrifying than aerobic conditions, may be further enhanced by the presence of sulfur compounds.

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) ...

Carbon dioxide fixation.

The genome of T. denitrificans encodes both form I and form II RubisCO enzymes for CO2 fixation (11, 14). The microarray results show clearly that the structural genes encoding form I and II RubisCO were differentially expressed: cbbM (Tbd2638, which encodes form II) was upregulated 6-fold under denitrifying conditions (Table (Table1),1), whereas cbbL and cbbS (Tbd2624-2623, which encode the large and small subunits of form I) were upregulated 7.4- and 6.5-fold, respectively, under aerobic conditions (Table (Table2).2). The other cbb genes included in the form I and form II RubisCO gene clusters were also differentially expressed, albeit to a lesser extent than the structural genes. Thus, cbbQ and cbbO (Tbd2637 and Tbd2636) in the form II cluster were upregulated 2.7- and 3.5-fold under denitrifying conditions, whereas their homologs in the form I cluster (Tbd2622 and Tbd2621) were upregulated 5.5- and 2.6-fold under aerobic conditions.

These results are consistent with the biochemical characterization of form I and II RubisCO in T. denitrificans with respect to their relative affinities to CO2 and O2. Molecular oxygen competes with CO2 for the active site of RubisCO and thereby decreases its efficiency for carbon fixation. The relative specificity of RubisCO enzymes for CO2 and O2 (the CO2/O2 specificity factor, or τ) was determined in T. denitrificans (14); form I was shown to have considerably higher CO2/O2 specificity (τ = 46) than form II (τ = 14). Thus, expressing form I under aerobic conditions would tend to maximize the efficiency of CO2 fixation.

We are not aware of any previous studies of differential expression of form I and II RubisCO under aerobic versus denitrifying conditions. The most relevant studies are those that investigated expression of form I and II RubisCO under a variety of chemoautotrophic, chemoheterotrophic, photoautotrophic, and photoheterotrophic conditions in Rhodobacter sphaeroides and Rhodobacter capsulatus (recently reviewed in reference 10). Differential transcription of forms I and II was observed in some of these studies. In the absence of more experimental data for T. denitrificans, these existing studies allow us only to speculate about regulatory systems that might be involved in differential transcription of form I and II RubisCO in T. denitrificans.

RegB/RegA is a global, two-component, redox-responsive regulatory system that appears to have a role in differential expression of form I and II RubisCO in Rhodobacter species (reference 10 and references therein). For example, in work with regA mutants of R. sphaeroides grown under aerobic, chemoautotrophic conditions, Gibson et al. (12) indicated that RegA (PrrA) functioned as a strong activator of form II RubisCO genes but had no effect on, or acted as a mild repressor of, the form I genes. However, rocket electroimmunoassay studies of R. sphaeroides strain HR-CAC showed that approximately 2.5-fold more form I than form II RubisCO protein was expressed under aerobic chemolithoautotrophic conditions (25). RegA also influences the differential, redox-responsive transcription of other genes, including those associated with photosynthesis, cytochrome cbb3 oxidase, and Cu-containing nitrite reductase (nirK).

Several lines of evidence suggest that the RegB/RegA system is present in T. denitrificans and may contribute to transcriptional regulation of RubisCO genes: (i) genes putatively encoding RegA and RegB have been identified in T. denitrificans (Tbd2690 and Tbd2689, respectively), (ii) possible RegA-binding sites are present upstream of cbbM and cbbL, and (iii) at least one putative RegA-binding site is present in the intergenic region upstream of the aerobically upregulated ccoN gene (Tbd0643), which encodes a subunit of cytochrome cbb3 oxidase and has been associated with RegB/RegA regulation in R. capsulatus (29, 30). BLASTP searches for RegA in the T. denitrificans genome using the RegA (PrrA) sequence from R. sphaeroides (GenBank YP_351562) revealed that Tbd2690 was the best match; the deduced amino acid sequence of Tbd2690 shares 51% identity with the RegA sequence of R. sphaeroides. Alignment of these (and other) RegA sequences showed that the T. denitrificans homolog also includes the highly conserved helix-turn-helix DNA-binding motif described for a range of RegA homologs (10). RegB is putatively encoded by Tbd2689 in T. denitrificans (26% sequence identity with the RegB sequence of R. sphaeroides; GenBank YP_351564). Alignment of the deduced amino acid sequence of Tbd2689 with known RegB sequences revealed that the T. denitrificans homolog contains a highly conserved, redox-active cysteine residue that has been shown to exert control over the activity of the sensor kinase in R. sphaeroides (31). Searches for RegA-binding sites upstream of cbbM and cbbL in T. denitrificans revealed possible degenerate sequences. Laguri et al. (22) described the following main features of RegA-binding sites derived from studies of R. sphaeroides and R. capsulatus: (i) a palindromic 5′-GCGNC…GNCGC-3′ consensus, (ii) a central AT-rich section, and (iii) a variable number of bases between the 5′ and 3′ palindromic regions (with an apparent total of 9 to 15 bases in the binding site motif). Sequences conforming to these characteristics were found upstream of cbbM (5′-GCGACAGCCGC-3′) and cbbL (5′-GCGCCTCTTGTCGC-3′). Notably, both of these putative RegA-binding sites were located at least 950 nt upstream of the translational start sites of cbbM and cbbL and occurred in a complementary cbbR coding region (i.e., in a cbbR coding region on the opposite strand from cbbM and cbbL). Since the RegA-binding consensus features were based on only two bacterial species, it is possible that other RegA-binding sites occur upstream of cbbM or cbbL but could not be detected because they diverge from Rhodobacter motifs.

Transcriptional regulation of RubisCO genes is characteristically complex and is controlled by more than just the RegB/RegA system. For example, there is undoubtedly also some positive control of form I and II RubisCO expression by the LysR-type transcriptional regulator CbbR (21). Both the form I and form II operons in T. denitrificans are adjacent to divergently transcribed cbbR genes (5), and multiple putative CbbR-binding sites were found in upstream regions of both cbbM and cbbL. To illustrate, in the intergenic region between cbbL and the upstream cbbR gene, there were putative, often overlapping CbbR-binding sites located from 4 to 17 nt and 89 to 152 nt upstream from the translational start site. In the intergenic region between cbbM and the upstream cbbR gene, there were putative CbbR-binding sites located from 4 to 24 nt and 64 to 127 nt upstream from the translational start site. The motif used to identify putative CbbR-binding sites was T-N12-A, which deviates from the T-N11-A motif characteristic of LysR-type transcriptional regulators but may be more applicable to CbbR-binding sites in autotrophic bacteria (21). There is currently no evidence suggesting that CbbR influences differential expression of RubisCO genes under aerobic versus denitrifying conditions, and it is very possible that as-yet-unidentified transcriptional regulators may influence expression of RubisCO genes (9, 12).

Cytochrome cbb3 oxidase.

The gene cluster Tbd0643-0637, which includes genes putatively encoding one of two cytochrome cbb3 oxidases in T. denitrificans (5), was upregulated under aerobic conditions (Table (Table2;2; Fig. Fig.1).1). The first four genes in this cluster (Tbd0643-0640) appear to be ccoNOQP, and the entire cluster is highly similar in terms of gene sequence and organization to a cluster in the related β-proteobacterium Azoarcus sp. strain EbN1. Throughout this cluster in T. denitrificans, genes were upregulated 3.9- to 13.5-fold relative to denitrifying conditions. The highest upregulation was for ccoN (9.6-fold) and ccoQ (13.5-fold).

The results for T. denitrificans are generally consistent with those from ccoN::lacZ transcriptional fusion studies of R. capsulatus and R. sphaeroides, which showed greater expression of ccoN under aerobic, and particularly microaerophilic, conditions compared to anaerobic conditions (24, 29, 30). Studies with regA mutants of R. capsulatus suggest that RegA activates cytochrome cbb3 oxidase expression semiaerobically or aerobically but represses expression anaerobically (29, 30). In contrast, FnrL apparently activates cytochrome cbb3 oxidase expression semiaerobically or anaerobically in these two Rhodobacter species (24, 29).

The promoter region upstream of ccoN was examined for potential RegA- and FNR-binding sites, as these transcription factors have been implicated in the regulation of cytochrome cbb3 oxidases in Rhodobacter species. We focused on the promoter region of ccoN because it is the first gene in this cluster and its upstream intergenic region is nearly 500 nt long, whereas the intergenic regions upstream of ccoO, -Q, and -P only range from 0 to 11 nt. A probable RegA-binding site (5′-GCGACACGTTGGCGC-3′) was identified upstream of ccoN; this putative binding site was located much closer to the translational start site (ca. 280 nt upstream) than those we have identified in promoter regions of cbbL and cbbM (discussed previously). The most likely FNR-binding site identified in the ccoN promoter region was TTGAT…cTCgc, which was notably degenerate at the 3′ end and was located 374 nt upstream of the translational start site.

Chaperones and stress proteins.

A number of genes associated with protein folding and turnover were upregulated under aerobic conditions (Table (Table2;2; Fig. Fig.1).1). These include the genes encoding the molecular chaperones ClpB (Tbd0815; 9.6-fold upregulated), GroEL and GroES (Tbd0091-0092; 9.1- and 5.1-fold upregulated, respectively), GrpE, DnaK, and DnaJ (Tbd1537-1539; 5.1- to 9.2-fold upregulated), and IbpA (Tbd1370; 6.3-fold upregulated). Several genes occurring in a cluster with GroEL and GroES were also aerobically upregulated, albeit to a lesser extent (Tbd0094-0096; 2.1- to 2.6-fold). Other aerobically upregulated genes encoding proteins that are putatively associated with protein folding and turnover include genes for HtpG (Tbd1078; 18-fold upregulated) and Lon protease (Tbd1252; 11-fold upregulated) (Table (Table2).2). Several of these genes have been found to be regulated in Escherichia coli by sigma 32, the heat shock/stress alternative sigma factor (Tbd0345). Sigma 32-regulated genes include clpB, grpE, dnaJ/dnaK, ibpA, htpG, and lon (35). In turn, several of the proteins encoded by these genes regulate intracellular levels of sigma 32, as do GroEL and GroES (13).

GroEL, an essential chaperone, and DnaK have been shown to play a significant role in the viability of E. coli (16). In E. coli, it has been demonstrated that about 250 proteins interact with GroEL, of which several could also utilize DnaK for proper folding (19). In the current study, both DnaK and GroEL were found to be significantly upregulated under aerobic conditions along with form I RubisCO, which has been shown to be a substrate of GroEL (17).

Iron acquisition.

A cluster of 21 genes (Tbd0705 to Tbd0725), many of which are associated with Fe3+ uptake (5), includes the 16 most aerobically upregulated genes observed in this study (Table (Table2).2). In fact, all 21 genes in the cluster are among the top 50 aerobically upregulated genes (Table (Table2).2). The level of upregulation within the cluster varies widely, ranging from 6.1-fold upregulation for Tbd0707 to 159-fold upregulation for Tbd0725. Aerobic upregulation of iron transport genes in bacteria occurs in response to limited iron availability due to the lower solubility of Fe(III) species compared to Fe(II) species (1). To illustrate for the conditions used in this study, although the amounts of iron added to the aerobic and denitrifying cultures were similar (10 and 7.5 μM, respectively), equilibrium geochemical modeling (26) indicated that the amounts of dissolved iron under these two conditions differed dramatically. Whereas all of the 7.5 μM iron would be present in solution under denitrifying conditions (~75% as FeHCO3+), less than 0.7 μM would be soluble under aerobic conditions [>93% of the Fe would be present as Fe(OH)3 precipitate].

Genes found in the cluster include those that encode proteins involved in siderophore biosynthesis and export (Tbd0716-0721), Fe3+-siderophore uptake across the outer membrane (Tbd0711-0713, Tbd0715, and Tbd0722), iron storage and mobility (Tbd0705), and heme uptake (Tbd0725). Systems involved in iron acquisition have been found to be regulated by the ferric uptake regulator protein, Fur (Tbd1123), which acts as a repressor in the presence of Fe2+ and a derepressor in the absence of Fe2+ (1). Putative DNA-binding sites allowing for Fur-dependent regulation (1, 34) were identified upstream of two genes in the cluster (Tbd0725 and Tbd0715) and overlapped with an E. coli-type sigma 70 promoter sequence for both genes (Fig. (Fig.4).4). The most highly upregulated gene, Tbd0725, encodes a putative homolog of HemP, a Fur-regulated protein associated with heme uptake in Yersinia enterocolitica (28). Although other genes associated with heme uptake are not found in this gene cluster, they are found scattered throughout the T. denitrificans genome. The second gene, Tbd0715, encodes a homolog of PsuA, a Fur-regulated Fe3+-siderophore outer membrane receptor in Vibrio parahaemolyticus found associated with siderophore biosynthesis genes similar to those occurring in this T. denitrificans cluster (32).

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.

Sequences closely matching the E. coli sigma 70 “consensus” sequences were also found in promoter regions of several other aerobically upregulated genes (Table (Table3),3), including bfd (Tbd0705), groES (Tbd0092), lon (Tbd1252), dnaK (Tbd1538), and ccoN (Tbd0643). As discussed previously, these genes encode proteins involved in a variety of functions, including iron uptake and storage, protein folding and turnover, and aerobic respiration via cytochrome cbb3 oxidase. In contrast, promoter regions for anaerobically upregulated genes, including those involved in denitrification (narK, nirS, and norC) and carbon fixation (cbbM), did not have sequences that closely matched the E. coli consensus sequence. Interestingly, the promoter region for nosZ, which was slightly upregulated aerobically despite being associated with denitrification (Fig. (Fig.2),2), also contains sequences similar to the E. coli sigma 70 consensus sequences (Table (Table33).

E. coli-like sigma 70 consensus sequences identified in promoter regions of selected genes that were upregulated aerobically in T. denitrificans

RT-qPCR validation of microarray trends.

Twelve genes were selected for analysis by RT-qPCR to confirm that differential expression indicated by the microarray data was supported by an independent method. The selected genes (listed in the legend for Fig. Fig.5)5) cover a wide range of expression and include genes that were most upregulated under aerobic conditions and under denitrifying conditions. Overall, the RT-qPCR data and microarray data were very consistent (Fig. (Fig.5);5); the data were highly correlated (r2 = 0.95) and had a slope that approached unity (1.085).

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), ...

Concluding remarks.

As one of the first whole-genome transcriptional studies of a chemolithotrophic bacterium, and one of the few studies addressing transcriptional analysis of genes associated with chemolithotrophic sulfur compound oxidation, this study provides a number of novel findings, including the following: (i) strong upregulation under denitrifying conditions of two copies of sqr (which is explained by genomic location adjacent to the nar gene cluster for only one sqr copy), (ii) a variety of expression behaviors for the eight dsrC copies (ranging from aerobic upregulation to anaerobic upregulation to constitutive expression at a high level), (iii) consistently high-level expression under aerobic and denitrifying conditions of several important gene clusters associated with sulfur compound oxidation (including soxXYZA, dsrABEFHCMKLJOP, and the genes encoding ATP sulfurylase and APS reductase), (iv) differential expression of genes putatively encoding rhodanese (an enzyme function previously lacking direct evidence for its involvement in thiosulfate oxidation), and (v) differential expression of form I and II RubisCO under aerobic versus denitrifying conditions. Whereas this study provides some insight into the unusual ability of T. denitrificans to oxidize sulfur compounds under aerobic and denitrifying conditions, additional whole-genome transcriptional studies by our group will provide information on other unusual abilities of this bacterium, namely, catalysis of anaerobic, nitrate-dependent Fe(II) and U(IV) oxidation. Combining these microarray results with the use of a newly developed genetic system in T. denitrificans (T. Letain, S. Kane, T. Legler, H. Beller, E. Salazar, and P. Agron, unpublished data) will facilitate better understanding of the biochemical and genetic basis of the oxidative metabolism of this widespread but unusual bacterium.

Supplementary Material

[Supplemental material]


We thank NimbleGen Systems, Inc. and Feliza Bourguet (Lawrence Livermore National Laboratory) for their valuable experimental contributions, Donovan Kelly (University of Warwick, United Kingdom) for insightful comments on the manuscript, and Alex Beliaev (Pacific Northwest National Laboratory) for useful discussions about RNA handling.

This work was performed under the auspices of the U.S. Department of Energy by the University of California, Lawrence Livermore National Laboratory under contract no. W-7405-Eng-48.


Supplemental material for this article may be found at http://jb.asm.org/.


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