Investigation Of The σ/anti-σ Factor Of Clostridium Using The Bacillus Subtilis Heterologous Reporter System

Objective:In order to investigate the pathogenic mechanism of TcdR in Clostridium difficile and the molecular mechanism of fiber body regulation by the structure and function of regulatory factors SigIl and RsgI in Clostridium thermocellum,this study developed a Bacillus subtilis heterologous reporter system to circumvent the problems of strict growth conditions,complex genetic background,and limited genetic manipulation in C.difficile and C.thermocellum.Methods:1.Construction of a Heterologous Reporter System in Bacillus subtilis.The construction of a heterologous reporter system in Bacillus subtilis involved integrating the Clostridial σ-factor into a plasmid carrying a xylose-inducible promoter through homologous recombination.Subsequently,a promoter-driven LacZ reporter system was created,which was recognizable by the σ-factor(TcdR and SigI6).The reporter system was then integrated into the Bacillus subtilis genomic amyE locus using Spc resistance as a selection marker.For the reporter system,the E.coli β-galactosidase LacZ was used as a reporter gene,which was regulated by TcdR and SigI6-dependent promoters.The successful plasmid chromosome integration was confirmed using colony PCR to validate double exchange.Additionally,the strength of the promoter recognized by the σ-factor(TcdR/SigI6)was evaluated using xylose-induced LacZ activity of cell lysates.2.Studying the Molecular Basis of Toxin-Producing TcdR and Its Recognition Promoter in Clostridium difficile Using a Bacillus subtilis Heterologous Reporter System.This study aimed to investigate the molecular basis of toxin-producing TcdR and its recognition promoter in Clostridium difficile using the Bacillus subtilis heterologous reporter system.Plasmid vectors were constructed from E.coli containing the toxin promoters TcdA and TcdB,as well as TcdR’s own promoter,and carrying the reporter gene LacZ.These vectors were then integrated into the amyE locus of Bacillus subtilis.Point mutations were introduced into the toxin TcdA,TcdB,and the promoter-10 element of TcdR to explore the mechanism by which TcdR recognizes TcdA,TcdB,and its own promoter.For comparative analysis,we used AlphaFold2 to predict the structural model of TcdR and the reported structure of the ECF-type σ factor σH in the transcription initiation complex(TIC)of Mycobacterium tuberculosis RNA polymerase(PDB 5ZX2).Three truncated TcdC mutants containing residues 1-21,1-51,and 1-130 were constructed based on the AlphaFold2 predicted TcdC structure.Expression of the TcdC truncated gene was regulated by the xylose-inducible promoter PxylA,while TcdR was overexpressed in a plasmid vector with a LacZ expression cassette for the housekeeping promoter PserA.βGalactosidase activity assay revealed that TcdC had the ability to repress TcdR.3.Researching the Signalling Mechanism of Clostridium thermocellum SigI6RsgI6 Using the Bacillus subtilis Heterologous Reporter System.The aim of this study was to investigate the signalling mechanism of Clostridium thermocellum SigI6RsgI6 using the Bacillus subtilis heterologous reporter system.Plasmid vectors containing the xylose-inducible promoter PxylA and SigI6-RsgI6 were constructed.To examine the role of the extracellular substrate sensing region of RsgI6 further,the RsgI6-RBS-SigI6 expression cassette carrying the xylose-inducible promoter PxylA was truncated at key amino acid sites in the intracellular region,a transmembrane region,and periplasmic space region of RsgI6.We also mutated the amino acid at position 94 of the key breakpoint of SigI6-RsgI6 binding and similar amino acids.The strength of SigI6 recognition of PsigI6 was determined using β-galactosidase activity assay.To circumvent the effect of structural differences in the transmural region of C.thermocellum and B.subtilis on SigI6-RsgI6 signaling,we replaced the transmural region of C.thermocellum with that of B.subtilis.Furthermore,we deleted the first 50 amino acids at the N-terminal end of the RsgI6_Bsu transmural region and the last 50 amino acids at the C-terminal end of the transmural region.Finally,GH10(glycoside hydrolase)of C.thermocellum was replaced with CBM_CipA to complete the replacement.Results:1.Construction of Bacillus subtilis Heterologous Reporter System for TcdR and SigI6.Following PCR double exchange verification,TcdR and SigI6 were successfully integrated into the plasmid vector carrying the xylose-inducible promoter PxylA.The specific promoters PtcdA and PsigI6 were also successfully integrated into the expression cassette carrying the LacZ reporter gene.High expression levels were observed in the β-galactosidase activity assay,indicating successful construction of the Bacillus subtilis heterologous reporter system for TcdR and SigI6.2.The Recognition Mechanism of TcdR for Toxin Genes TcdA,TcdB,and Its Own Promoter.TcdR recognizes the toxin genes TcdA and TcdB,as well as its own promoter,differently with PtcdA>PtcdB>PtcdR.The promoter-10 element of TcdR plays a crucial role in regulating the activity of the various TcdR-dependent promoters.The structural model of TcdR is similar to the ECF σ-factor,and despite low sequence homology,both consist of two structural domains(σ2 and σ4)linked by a small helix(σ3.2).The σ4 structural domain of both TcdR and σH has a helixturned-helical motif that binds to the DNA in the large groove of the-35 promoter,but the surface residues of the motifs are different,allowing TcdR to recognize specific-35 elements.The σ2 structural domain of TcdR has a long helix with conserved residues for DNA deconvolution(N81,K77 and F84),which is similar to the ECF σ-factor.The specific loop of the ECF σ-factor recognizes T bases in the-10 element via W81,F72,and L78 in σH,and this area of TcdR has similar residues(Y74,F63,and 171)that may recognize T bases crucial for the second position of the TcdR-dependent promoter-10 region.Finally,the short helix(σ3.2)is a crucial component of the RNAP core enzyme active site cleft.All these features resemble the structural characteristics of the ECF σ factor,implying that TcdR is also an ECF σfactor.3.Investigating the Role of TcdC in TcdR Activity.TcdC is a transmembrane protein with an N-terminal portion,a putative transmembrane helix,a dimerization domain,and an OB fold domain.β-galactosidase activity assays revealed that neither full-length TcdC nor truncated TcdC mutants inhibited TcdR activity.This indicates that TcdC is not a direct inhibitor of TcdR and that it cannot act as an Anti-σ.4.Investigating the Inhibition of TcdR Activity and Role of SigI6 in Toxin Expression.SigI6 inhibits TcdR activity,thereby directly suppressing toxin expression.It is highly expressed in the Bacillus subtilis heterologous reporter system and selectively recognizes the SigI6 promoter.In the Bacillus subtilis heterologous system,rsgI6 can inhibit SigI6 activity,but variations in the trans-arm region between Clostridium thermocellum and Bacillus subtilis,as well as the intrinsic enzymatic protection mechanism,may impact RsgI6 signaling.Conclusions:In this study,a Bacillus subtilis heterologous system was constructed to investigate the regulatory mechanism of Clostridial σ factor.The recognition of C.difficile TcdR promoter for C.difficile toxin expression and the results revealed the molecular basis of different promoter activities.Structural analysis showed that TcdR has a structural domain organization and promoter recognition mechanism consistent with ECF σ factors and should be classified as ECF σ factors.TcdR can be used as a favorable tool to study the Clostridium thermocellum σ factor.