Differential RNA Sequencing Implicates Sulfide as the Master Regulator of S 0 Metabolism in Chlorobaculum tepidum and Other Green Sulfur Bacteria

The green sulfur bacteria (Chlorobiaceae ) are anaerobes that use electrons from reduced sulfur compounds (sulfide, S0 , and thiosulfate) as electron donors for photoautotrophic growth.Chlorobaculum tepidum , the model system for theChlorobiaceae , both produces and consumes extracellular S0 globules depending on the availability of sulfide in the environment. These physiological changes imply significant changes in gene regulation, which has been observed when sulfide is added toCba. tepidum growing on thiosulfate. However, the underlying mechanisms driving these gene expression changes, i.e., the specific regulators and promoter elements involved, have not yet been defined. Here, differential RNA sequencing (dRNA-seq) was used to globally identify transcript start sites (TSS) that were present during growth on sulfide, biogenic S0 , and thiosulfate as sole electron donors. TSS positions were used in combination with RNA-seq data from cultures growing on these same electron donors to identify both basal promoter elements and motifs associated with electron donor-dependent transcriptional regulation. These motifs were conserved across homologousChlorobiaceae promoters. Two lines of evidence suggest that sulfide-mediated repression is the dominant regulatory mode inCba. tepidum . First, motifs associated with genes regulated by sulfide overlap key basal promoter elements. Second, deletion of theCba. tepidum 1277 (CT1277 ) gene, encoding a putative regulatory protein, leads to constitutive overexpression of the sulfide:quinone oxidoreductase CT1087 in the absence of sulfide. The results suggest that sulfide is the master regulator of sulfur metabolism inCba. tepidum and theChlorobiaceae . Finally, the identification of basal promoter elements with differing strengths will further the development of synthetic biology inCba. tepidum and perhaps otherChlorobiaceae .IMPORTANCE Elemental sulfur is a key intermediate in biogeochemical sulfur cycling. The photoautotrophic green sulfur bacteriumChlorobaculum tepidum either produces or consumes elemental sulfur depending on the availability of sulfide in the environment. Our results reveal transcriptional dynamics ofChlorobaculum tepidum on elemental sulfur and increase our understanding of the mechanisms of transcriptional regulation governing growth on different reduced sulfur compounds. This report identifies genes and sequence motifs that likely play significant roles in the production and consumption of elemental sulfur. Beyond this focused impact, this report paves the way for the development of synthetic biology inChlorobaculum tepidum and otherChlorobiaceae by providing a comprehensive identification of promoter elements for control of gene expression, a key element of strain engineering.