Chlorobaculum tepidum TLS displays a complex transcriptional response to sulfide addition

Abstract

Chlorobaculum tepidum is a green sulfur bacterium (GSB) that is a model system for phototrophic sulfur oxidation. Despite over 2 decades of research, conspicuous gaps exist in our understanding of its electron donor metabolism and regulation. RNA sequencing (RNA-seq) was used to provide a global picture of the C. tepidum transcriptome during growth on thiosulfate as the sole electron donor and at time points following the addition of sulfide to such a culture. Following sulfide addition, 121 to 150 protein-coding genes displayed significant changes in expression depending upon the time point. These changes included a rapid decrease in expression of thiosulfate and elemental sulfur oxidation genes. Genes and gene loci with increased expression included CT1087, encoding a sulfide:quinone oxidoreductase required for growth in high sulfide concentrations; a polysulfide reductase-like complex operon, psrABC (CT0496 to CT0494); and, surprisingly, a large cluster of genes involved in iron acquisition. Finally, two genes that are conserved as a cassette in anaerobic bacteria and archaea, CT1276 and CT1277, displayed a strong increase in expression. The CT1277 gene product contains a DNA-binding domain, suggesting a role for it in sulfide-dependent gene expression changes.

Fig 1

Comparison of gene expression during the thiosulfate-to-sulfide transition. Differentially expressed genes (P < 0.05 using DESeq) are labeled with open squares. Expression values are log₂ transformed and quantile normalized, genes with no coverage are listed as −5, and black lines show linear regressions with the indicated R² values. (A) Library T0 versus T.5; (B) T0 versus T1; (C) T0 versus T2; (D) T.5 versus T1; (E) T.5 versus T2; (F) T1 versus T2.

Fig 2

Comparison of gene expression ratios relative to sigA measured by qRT-PCR and RNA-seq during growth on thiosulfate alone (○) and 30 min after the addition of sulfide to 1.6 mM (□). Targets for qRT-PCR were chosen to cover a wide range of expression and large differences in expression between growth conditions measured by RNA-seq. Linear regression lines: solid line, T0; dashed line, T.5.

Fig 3

Response of C. tepidum sulfur oxidation pathways 30 min after sulfide addition to a culture growing on thiosulfate. Pathways of sulfur are shown with black arrows, and electron transfer is shown with purple arrows. Multisubunit enzymes are shown as one unit. Flavocytochrome c sulfide dehydrogenase (FccAB-2) expression is from CT2080 and CT2081.

Fig 4

Log₂ transcript abundances inferred from RNA-seq over time following sulfide addition. (A) Single gene normalized absolute expression values for SQR homologs corresponding to CT0117 (▲), CT0876 (■), CT1087 (●), and nifB (△, dashed line). For psrABC (CT0496 to CT0494; ♦, dotted line), the mean expression value ± SD across all three genes is shown. (B) Concentrations of sulfide (○) and elemental sulfur (△) throughout the experiment. Concentrations of thiosulfate and sulfate remained constant following sulfide addition (data not shown).

Fig 5

Fe acquisition gene expression measured by RNA-seq and qRT-PCR. (A) RNA-seq expression values following sulfide addition relative to T0 for the Fe acquisition operon (CT1738-CT1745; ●, mean ± SD for 8 genes), the DtxR homolog corresponding to CT1737 (▲), and 59 ribosomal proteins (, dashed line; mean ± SD of 59 genes). (B) qRT-PCR expression analysis of RNA harvested from cultures grown on thiosulfate (black) or 30 min after addition of sulfide to 2 mM (white) or 6 mM (stripes) and trace metal limitation (gray). Expression is reported as log₂ of the expression ratio relative to sigA, and values are means ± SDs among all technical replicates (n ≥ 5).

Fig 6

Identification of a putative regulatory cassette. (A) Expression of proteins corresponding to CT1276 (♦) and CT1277 (■) and all 59 ribosomal proteins (mean ± SD) (, dashed line) measured by RNA-seq following the addition of sulfide. (B) qRT-PCR expression analysis of RNA harvested from cultures grown on thiosulfate (black) or 30 min after addition of sulfide to 2 mM (white) or 6 mM (stripes) and trace metal limitation (gray). Expression ratios are relative to sigA and are means ± SDs among all technical replicates (n ≥ 5).

Fig 7

Maximum likelihood phylogenies of the potential regulatory gene cassette. (A) CT1276, part of the NifX/NifB superfamily, based on 122 positions in the final data set. (B) CT1277, part of the helix-turn-helix xenobiotic response element-like superfamily, based on 129 positions in the final data set. Percent bootstrap support from 1,000 iterations is indicated if the node was observed in >50% of all trees. Members of the Chlorobi are indicated with an asterisk. Genus abbreviations: Ace., Acetohalobium; Cal., Calditerrivibrio; Cba., Chlorobaculum; Chl., Chlorobium; Chp., Chloroherpeton; Mca., Methanocella; Mtb., Methanothermobacterium; Pld., Pelodictyon; Ptc., Prosthecochloris; Ssp., Sulfurospirillum; and Syn., Syntrophobacter.