Genomic reconstruction of σ54 regulons in Clostridiales

Abstract

Background: The σ 54 factor controls unique promoters and interacts with a specialized activator (enhancer binding proteins [EBP]) for transcription initiation. Although σ 54 is present in many Clostridiales species that have great importance in human health and biotechnological applications, the cellular processes controlled by σ 54 remain unknown. Results: For systematic analysis of the regulatory functions of σ 54 , we performed comparative genomic reconstruction of transcriptional regulons of σ 54 in 57 species from the Clostridiales order. The EBP-binding DNA motifs and regulated genes were identified for 263 EBPs that constitute 39 distinct groups. The reconstructed σ 54 regulons contain the genes involved in fermentation and amino acid catabolism. The predicted σ 54 binding sites in the genomes of Clostridiales spp. were verified by in vitro binding assays. To our knowledge, this is the first report about direct regulation of the Stickland reactions and butyrate and alcohols synthesis by σ 54 and the respective EBPs. Considerable variations were demonstrated in the sizes and gene contents of reconstructed σ 54 regulons between different Clostridiales species. It is proposed that σ 54 controls butyrate and alcohols synthesis in solvent-producing species, regulates autotrophic metabolism in acetogenic species, and affects the toxin production in pathogenic species. Conclusions: This study reveals previously unrecognized functions of σ 54 and provides novel insights into the regulation of fermentation and amino acid metabolism in Clostridiales species, which could have potential applications in guiding the treatment and efficient utilization of these species.

Diverse Energy-Conserving Pathways in Clostridium difficile: Growth in the Absence of Amino Acid Stickland Acceptors and the Role of the Wood-Ljungdahl Pathway

“…In the leucine-rich BHIS, it is entirely consumed and almost exclusively reduced to isocaproic acid, with less than 2% undergoing oxidation, as judged by the relative amounts of isocaproic and isovaleric acids found (see Table S2 in the supplemental material). This is consistent with the likely function of leucine as a positive activator of the reductive catabolic pathway, which is thought to be regulated by a leucine-responsive bacterial enhancer binding protein LeuR required for 54 -dependent transcription of the hadAIBC-acdB-etfBA operon (64). The reductive pathway relies on a "radicals by reduction" (86) strategy that involves ATP-dependent low-potential activation of the key enzyme 2-hydroxyisocaproyl-CoA dehydratase, whose catalytic role is to generate a remarkable ketyl radical intermediate from the substrate 2-hydroxyisocaproyl-CoA, which can then undergo dehydration and subsequent conversion to the product isocaprenoyl-CoA (87,88).…”

“…PR is a selenoenzyme complex encoded by the prd operon CD3237 to CD3247 that is upregulated in a proline-responsive manner by PrdR (49). Acting as a 54 -dependent activator protein, PrdR is a member of the bacterial enhancer binding proteins (bEBPs), many of which respond to environmental signals through ligand-binding regulatory domains (63,64). Our finding of high PR activity in the absence of proline indicates that transcription of the prd operon is unlikely to be activated solely by a PrdR-dependent mechanism and suggests the additional involvement of 70 or a different alternative sigma factor.…”

Genome-Wide Transcription Start Site Mapping and Promoter Assignments to a Sigma Factor in the Human Enteropathogen Clostridioides difficile

“…To account for uneven GC-content, for each upstream region, the set of random sequences was constructed by shuffling of its sequence 10000 times. In the case of sigma 54 (SigL), raw scores of sites that did not include known important dinucleotides GG and GC in the distal and proximal boxes, respectively (Nie et al, 2019), were artificially decreased by the maximal achievable score for this matrix for each mismatch. We considered independently two variants of PWMs for SigA, the standard two-box one and the extended one without the distal box, but with invariant TG preceding the proximal box.…”

Control of solvent production by sigma‐54 factor and the transcriptional activator AdhR in Clostridium beijerinckii

“…Electrophoretic mobility shift assays were also performed to assess the predicted SigL-binding sites upstream of the ADH-encoding genes in other clostridia. Conserved À12 and À24 elements were identified in the promoter region of 17 adhA genes encoding iron-containing/activated ADHs and of 20 adhP genes encoding zinc-dependent ADHs in our previous study (Nie et al, 2019). Among them, five DNA fragments were amplified from the promoter regions of adhA or adhP genes in Clostridium saccharobutylicum, Clostridium saccharoperbutylacetonicum, Clostridium ljungdahlii and Clostridium autoethanogenum (Fig.…”

“…Three AdhR-binding sites (i.e. UAS sites) upstream of the r 54 promoter elements was predicted for both adhA1 and adhA2 genes in our previous study (Nie, et al, 2019). To test whether these UAS sites are involved in expression of adhA1 and adhA2, mutations were introduced into individual UAS (Fig.…”