PTS regulation domain‐containing transcriptional activator CelR and sigma factor σ⁵⁴ control cellobiose utilization in Clostridium acetobutylicum

Abstract

The phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS) regulation domain (PRD)-containing enhancer binding proteins (EBPs) are an important class of σ(54) -interacting transcriptional activators. Although PRD-containing EBPs are present in many Firmicutes, most of their regulatory functions remain unclear. In this study, the transcriptional regulons of about 50 PRD-containing EBPs in diverse Firmicutes species are reconstructed by using a comparative genomic approach, which contain the genes associated with utilization of β-glucosides, fructose/levan, mannose/glucose, pentitols, and glucosamine/fructosamine. We then present experimental evidence that the cel operon involved in cellobiose utilization is directly regulated by CelR and σ(54) (SigL) in Clostridium acetobutylicum. The predicted three CelR-binding sites and σ(54) promoter elements upstream of the cel operon are verified by in vitro binding assays. We show that CelR has an ATPase activity, which is strongly stimulated by the presence of DNA containing the CelR-binding sites. Moreover, mutations in any one of the three CelR-binding sites significantly decreased the cel promoter activity probably due to the need for all three DNA sites for maximal ATPase activity of CelR. It is suggested that CelR is regulated by PTS-mediated phosphorylation at His-551 and His-829, which exerts a positive effect and an inhibitory effect, respectively, on the CelR activity.

σ54 (σL) plays a central role in carbon metabolism in the industrially relevant Clostridium beijerinckii

“…σ 54 was also shown to control sugar uptake and usage, consistently with what Nie et al predicted for the Clostridium genus (41). In particular, growth was limited on cellobiose and lactose in the AA strain, though cellobiose inhibition could only be observed on minimal medium.…”

Genomic reconstruction of σ54 regulons in Clostridiales

“…The majority (431 out of 490; 77%) of the identified EBPs in Clostridiales consist of a central AAA + domain, an N-terminal regulatory domain, and a DNA-binding domain (DBD) at the C-terminus (Additional file 1: Table S2). Forty-eight EBPs possess the PRD domain at the C-terminus [21] terminal regulatory domain, which is similar to PspF from E. coli [23].…”

“…The remaining EBPs lack orthologs in the sequenced Clostridales genomes, thus comparative genomics approach cannot be applied reliably. Among the 39 orthologous groups of EBPs, four groups are PRDcontaining EBPs, for which the UAS motifs have been identified previously [21]. We named the individual EBP groups based on the functional content analysis of the reconstructed regulons as described below.…”

“…Our previous study has identified some σ 54 -dependent genes in several Clostridium species, which are activated by the phosphoenolpyruvate-dependent phosphotransferase system regulation domain (PRD)-containing EBPs and involved in utilization of β-glucosides, fructose/ levan, pentitols, and glucosamine/fructosamine [21]. However, the regulatory functions of the majority of the EBPs in Clostridiales species remain unknown.…”

Studies of the <b><i>Listeria monocytogenes</i></b> Cellobiose Transport Components and Their Impact on Virulence Gene Repression

“…A palindromic sequence located further upstream in both operons contains the core sequence TGTGTA(X) n TACACA. It probably functions as target site (upstream activating sequence, UAS) [Martin-Verstraete et al, 1994] for the LevR-like transcription activator CelR [Nie et al, 2016]. The presumed monocistronic lmo2708 gene [Toledo-Arana et al, 2009] encodes a protein resembling EIIC Cel1 (Fig.…”