Plasmid and Strain Construction

Bacterial strains and plasmids used in this study are listed in Table 1; primer sequences are listed in Supplementary Table S1. All molecular techniques were conducted following standard protocols (Sambrook et al., 2012). S. fredii NGR234 (Stanley et al., 1988) was transformed by electroporation as described previously (Ferri et al., 2010). All constructs were verified by sequencing (Eton Bioscience, Inc., Sand Diego, United States) and all mutants were confirmed by PCR.

Strains and plasmids used in this work.

Strains with deletions in specific genes were constructed by allelic exchange and selected by sucrose sensitivity, using plasmid pK18mobSacB (Schäfer et al., 1994). The pK18mobSacB derivatives for the deletion of rpuS and crbS were constructed by overlap extension (Lee et al., 2010) of two PCR products corresponding to ~750 bp of the upstream and downstream sequence of the targeted gene, and the resulting product was re/amplified and cloned in the HindIII/EcoRI and HindIII/SmaI sites of plasmid pK18mobSacB, respectively. The pK18mobSacB derivatives for the deletion of rpuR, cbrR, NGR_RS10965, and mctP were obtained by cloning in the HindIII/EcoRI sites of plasmid pK18mobSacB a PCR product containing the gene to be deleted, together with ~750 bp of upstream and downstream sequence. Then the targeted gene was deleted by total PCR of the plasmid with phosphorylated divergent primers (Imai et al., 1991). The resulting PCR product was ligated, and the religated plasmid was recovered by transformation.

Plasmids pSRKgm-rpuR and pSRKgm-crbR were built by cloning the ORF of each gene into the NdeI/BamHI sites of plasmid pSRKgm (Khan et al., 2008). Plasmids pSRKgm-rpuS and pSRKgm-crbS were built by cloning the ORF of each gene in plasmid pSRKgm by Gibson cloning using the GeneArt Gibson Assembly HiFi Master Mix (Thermo Fisher, Waltham, United States).

The in-cis uidA transcriptional fusion of the mctP operon was constructed by cloning a PCR product of the putative promoter region in the SalI/BamHI sites of plasmid pVMG (Gao et al., 2019) producing plasmid pVMG-PrmctP. Mutagenesis of the mctP operon promoter was achieved by the quick-change strategy (Agilent, Santa Clara, United States) using plasmid pVMG-PrmctP as a template. After electroporation, plasmid cointegration and orientation were verified by PCR as described previously (Gao et al., 2019).

The in-trans uidA transcriptional fusion of the mctP operon promoter was constructed by cloning a PCR product of the putative promoter region in the BcuI/BamHI sites of plasmid pBBR53Gus (Girard et al., 2000). Transcriptional fusions used to map the NGR_RS10970 operon promoter were built by amplifying plasmid pBBR53g-PrmctP with divergent primers carrying a BcuI site or a BamHI site. The resulting PCR product was digested with the corresponding restriction enzyme, ligated, and transformed for the recovery of the reconstituted plasmid.