Inactivation of hbd Gene in Clostridia

The oligonucleotides and plasmids used in this study are listed in Table S1 and S4, respectively. The mobile group II intron system was used as described previously to inactivate the hbd.^{34 (link),48 (link)} The algorithm available on the TargeTron Design Site (http://www.clostron.com/clostron2.php) was used to identify the intron insertion site within hbd (position 414) and design primers to retarget the group II intron in hbd (c-hbd-414|415s-IBS, c-hbd-414|415s-EBS1d, and c-hbd-414|415s-EBS2). These primers were used with EBS universal primers and intron template DNA to generate a DNA fragment by overlap PCR as recommended by manufacturers. The PCR product was purified using QIAquick PCR Purification Kit (Qiagen, Courtaboeuf, France) and digested by BsrGI and HindIII restriction enzymes. Plasmid pMTL007C-E2-hbd was developed by ligating (T4 DNA ligase) (Sigma Aldrich Chimie, Saint-Quentin-Fallavier, France) the digested PCR product and pMTL007C-E2 digested with BsrGI/HindIII and purified with QIAEX II Gel Extraction kit (Qiagen). The ligation mixture was introduced into E. coli TOP10 by electroporation. After extracting the plasmid (QIAprep Spin Miniprep kit, Qiagen) and amplifying the insert by FastStart High Fidelity PCR System (Sigma Aldrich Chimie,) with primers pMTLCE2seqF and pMTLseqR, the cloned insert was verified by DNA sequencing using the same primers (Genewiz, Takeley, UK). The plasmid pMTL007C-E2-hbd was transformed into the conjugative donor E. coli HB101 (RP4) and transferred into the C. butyricum 1002 and C. neonatale 250.09 strains via conjugation. The transconjugants were selected on BHI agar supplemented with cycloserine and thiamphenicol. Inactivation of hbd was verified by screening transconjugants for erythromycin resistance and thiamphenicol sensitivity, and the resultant strains were named CbuCB1002-hbd415s::CT and Cne205.09-hbd415s::CT. Further, genomic DNA was extracted (InstaGene Matrix Kit) (Bio-Rad, Marnes-la-Coquette, France) and subjected to PCR using primers flanking hbd (hbdF and hbdR) to verify the intron insertion into the correct target gene. Moreover, the presence of the ermB was confirmed by PCR using primers RAMFCE2 and RAMRCE2, and the orientation of insertion was verified by PCR with a combination of hbdF, hbdR, and EBS Universal primers.