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"All primers used for the construction of mutants and for the verification of mutant strains are listed in Supplementary Table 2. All G. metallireducens mutants were constructed following a previously published method [21]. To construct the G. metallireducens strain deficient in the type IV pilus biogenesis ATPase (PilB, GenBank locus Gmet_1393), GS-∆pilB, three fragments were prepared: first, the primer pairs gsBupf/gsBupr and gsBdnf/gsBdnr were used to amplify the sequences 500 bp upstream and 500 bp downstream, respectively, of Gmet_1393, using G. metallireducens genomic DNA as template and the primer pair spef/sper was used to amplify the spectinomycin resistance cassette flanked by loxP sites from plasmid pRG5. These three fragments were connected and inserted into the In-Fusion Cloning site of linear plasmid pUC19 (included in the kit) using the In-Fusion HD Cloning Kit (Takara Biomedical Technology, Beijing, China), generating the plasmid pUC-GspilB. The plasmid was linearized with NcoI (New England BioLabs (NEB), Ipswich, MA, USA) and then electroporated into electrocompetent G. metallireducens GS15. The deletion of pilB was confirmed by PCR (Supplementary Fig. S1). To construct the pilB and Gmet_2896 double-deletion strain of G. metallireducens, named GS-∆pilB∆Gmet_2896, the spectinomycin resistance cassette (sp-loxP) carried by GS-∆pilB was first excised from its chromosome by expressing the Cre recombinase from the plasmid pCM158 using a published procedure [21]. The excision of the antibiotic marker was confirmed by PCR with primer pairs verGmet1393F/verGmet1393R (Supplementary Fig. 1), generating the strain GS-∆pilB2. Two fragments, the sequences 500 bp upstream and downstream of gene Gmet_2896, were amplified from the G. metallireducens genome using the primer pairs Gs2896upf/Gs2896upr and Gs2896dnf/Gs2896dnr, respectively, and then, together with the fragment of sp-loxP, they were inserted into the pUC19 In-Fusion Cloning site using the In-Fusion HD Cloning Kit, generating the plasmid pUC-GS2896. After linearizing with NcoI (NEB), the plasmid was electroporated into the electrocompetent G. metallireducens strain GS-∆pilB2. The pilA (Gmet_1399) deletion strain of G. metallireducens was constructed following the same method, using the primer pairs GspilAupf/GspilAupr and GspilAdnf/GspilAdnr to amplify sequences 500 bp upstream and downstream of the gene Gmet_1399, and then connecting them with sp-loxP and the linearized plasmid pUC19, generating plasmid pUC-GspilA. The plasmid was linearized with NcoI (NEB) and then electroporated into electrocompetent G. metallireducens.
A previously published procedure was used to construct the G. sulfurreducens mutants [22]. To construct the G. sulfurreducens strain deficient in the type IV pilus biogenesis ATPase (PilB, GenBank locus GSU1491), PCA-∆pilB, three fragments were prepared: first, the primer pairs ppilBupf/ppilBupr and ppilBdnf/ppilBdnr were used to amplify the sequences 500 bp upstream and downstream, respectively, of GSU1491 with G. sulfurreducens genomic DNA as the template, and then, the primer pair gmf/gmr was used to amplify the gentamycin-resistance cassette flanked by loxP sites (Gm-loxP) from the plasmid pCM351. These fragments were connected with the linearized plasmid pUC19 using In-Fusion HD Enzyme, generating the plasmid pUC-PCApilB. The plasmid was linearized with NcoI (NEB) and then electroporated into electrocompetent G. sulfurreducens PCA. To construct the formate dehydrogenase (fdnG gene, GSU0777), uptake hydrogenase (hybL gene, GSU0785) and PilB triple-deficient strain, as well as the citrate synthase (gltA gene, GSU1106) and PilB double-deficient strain, of G. sulfurreducens, strains PCA-∆pilB∆fdnG∆hybL and PCA-∆pilB∆gltA, respectively, the gentamycin-resistance cassette (Gm-loxP) carried in the chromosome of PCA-∆pilB was excised by expressing the Cre recombinase from plasmid pCM158 following a published procedure [21]. The removal of Gm-loxP was confirmed by PCR with primer pairs verpilBf/verpilBr (Supplementary Fig. S1), generating strain PCA-∆pilB2. The primer pairs hybLupf/hybLupr and hybLdnf/hybLdnr were used to amplify the sequences 500 bp upstream and downstream of GSU0785, and the primer pairs gltAupf/gltAupr and gltAdnf/gltAdnr were used to amplify the corresponding sequences 500 bp upstream and downstream of GSU1106, each from the G. sulfurreducens genome. These fragments, together with Gm-loxP, were connected with the linearized plasmid pUC19 by In-Fusion HD Enzyme (Takara), generating the plasmids pUC-hybL and pUC-gltA, respectively. Plasmids were linearized by NcoI (NEB) and electroporated into the electrocompetent G. sulfurreducens strain PCA-∆pilB2, generating the strains PCA-∆ pilB∆hybL and PCA-∆ pilB∆gltA, respectively. The PilA-deficient strain of G. sulfurreducens, PCA-∆pilA, was constructed in a similar manner as a previous report [23] by in-frame deleting the pilA (GSU1496) and using the primer pairs pilAupf/pilAupr, pilAdnf/InpilAr and InpilAf/pilAdnr to amply the sequences last 500 bp of pilR, promoter of GSU1496 and downstream of GSU1496, respectively. To construct strain PCA-∆pilB∆fdnG∆hybL, the sequences 500 bp upstream and downstream of GSU0777 were amplified from PCA genome with the primer pairs fdnGupf/fdnGupr and fdnGdnf/fdnGdnr, respectively, and a kanamycin resistance cassette was amplified from the plasmid pCM158 with the primer pair kanf/kanr. These three fragments were connected with pUC19, generating plasmid pUC-fdnG, and then digested with NcoI (NEB) and transformed into the electrocompetent G. sulfurreducens strain PCA-∆pilB∆hybL. All mutants were verified by PCR (Supplementary Fig. S1) and Sanger sequencing."
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