Construction of mutant Rhodobacter sphaeroides strains including the ΔcrtB deletion strain

A construct containing the upstream and downstream flanking regions of the gene was created to delete crtB. A 361 bp fragment upstream of crtB and the first 7 bp of the crtB gene flanked with EcoRI and XbaI restriction sites (underlined) was amplified using the primers crtBKOUF (5′-CCGGAATTCCACATCACCATCACCACGGCG-3′) and crtBKOUR (5′-GCGCTCTAGAGATCTAGGTTCTCATGAAGGTATACCG-3′). A second fragment was amplified using the primers crtBKODF (5′-GCGCTCTAGAGGCAATCATTCCGCGGCAAGC-3′) and crtBKODR (5′-CCCCGCATGCGGCTGTGGCCGAGCCCTA-3′) producing a downstream flanking fragment of 363 bp, which included 9 bp at the end of the crtB gene, flanked with XbaI and SphI restriction sites (underlined). Following digestion with the indicated enzymes, the fragments were sequentially cloned into the same restriction sites of the suicide vector pK18mobsacB. The resulting plasmid was introduced into Escherichia coli S17-1 competent cells and transferred to Rba. sphaeroides WT via conjugation, with first and second recombination events selected for as detailed in ref. ³¹. Transconjugant Rba. sphaeroides colonies were selected on M22 agar plates with 25 μg ml⁻¹ kanamycin. Single colonies were grown and scaled up to an 80 ml semi-aerobic culture. The cells were serially diluted 10⁻², 10⁻³ and 10⁻⁴ onto M22 agar containing 1 % (w/v) sucrose and incubated until single colonies appeared after 4–6 days. Single colonies were replica plated onto M22 sucrose plates, with and without 25 μg ml⁻¹ kanamycin. Colonies that grew on the antibiotic-free plate but not on the kanamycin plate were analysed by PCR to identify successful mutants.Transconjugants were confirmed by DNA sequencing; the modified DNA sequence at the location where crtB is deleted is shown in Supplementary Fig. 9.

The RC/YFP fusion strain was created using a pk18mobsacB construct designed to fuse the gene for SYFP2 (ref. ²¹) to the 3′ end of the puhA gene encoding the RC-H subunit. To create a C-terminal YFP fusion protein, firstly a PCR fragment was generated using primers puhAYFPUF (5′-CCGGAATTCTCGGCCGGCAAGAACCCGATCGG-3′) and puhAYFPUR (5′-GCTCCTCGCCCTTGCTCACCATGGCGTATTCGGCCAGCATCGCCG-3′). This fragment contained the last 354 bp of the puhA ORF (excluding the stop codon), with a 5′ EcoRI restriction site (underlined) and 3′ sequence complementary to the start of the syfp2 gene (italics). A second PCR with primers puhAYFPFor (5′-CGGCGATGCTGGCCGAATACGCCATGGTGAGCAAGGGCGAGGAGCTGTTCAC-3′) and puhAYFPRev (5′-GCGCTCTAGATCATTACTTGTACAGCTCGTCCATGCCGAGAGTGAT-3′) amplified the syfp2 ORF with 5′ sequence complementary to the end of the puhA gene (italics) and a 3′ XbaI restriction site (underlined). The two fragments were used as a template for overlap extension (OLE)-PCR with primers puhAYFPUF and puhAYFPRev, generating a fragment in which the end of the puhA gene was joined in frame with the syfp2 gene with EcoRI and XbaI sites at the 5′ and 3′ ends respectively. A third fragment in which the 354 bp immediately downstream of the puhA stop codon was flanked by XbaI and HindIII restriction sites (underlined) was amplified using the primers puhAYFPDF (5′-GCGCTCTAGATCCCCGCATGGCGCGGCCC-3′) and puhAYFPDR (5′-CCCCAAGCTTTAGGGCACCGCATAGGCCACCGC-3′). Following XbaI/HindIII digestion, the downstream fragment was cloned into the suicide vector pK18mobsacB. The resulting plasmid was opened with EcoRI and XbaI and the puhA-sfYFP2 product cut with the same enzymes was inserted. This plasmid contained a total insert of 1,428 bp. The fragments were inserted in this order as the XbaI site in the OLE-PCR product is blocked by overlapping dam methylation once inserted into a plasmid. Conjugative transfer and screening of possible mutants was performed as described for the crtB deletion.