S, we introduced the strong promoter of the puc ML390 biological activity operon [36] (which encodes the light-harvesting complexes (LHII) of R. sphaeroides 2.4.1) into the broad-host-range plasmid pBBR1MCS-2 [24]. The PstI-DraII regulatory region containing the puc promoter was then PCR-amplified from the plasmid pPS400 [37] with the primers SacIpuc and RXbaIpuc (Additional file 5: Table S2), and cloned into PCR2.1-TOPO (Invitrogen). The resulting plasmid was digested with XbaI and SacI and the 0.7 kb fragment was cloned into pBBR1MCS-2.yedY cloningStrains and plasmids used in this study are listed PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/28128382 in Additional file 4: Table S1.Untagged YedYZ: A 2246 bp DNA fragment containing yedYZ (596 bp upstream of the yedY start codon) was PCR-amplified from R. sphaeroides f. sp. denitrificans genomic DNA with the “yedYZ” and “RevyedYZ” primers (Additional file 5: Table S2) and cloned into PCR2.1-TOPO (Invitrogen). The plasmid wasSabaty et al. BMC Biochemistry 2013, 14:28 http://www.biomedcentral.com/1471-2091/14/Page 10 ofsubsequently digested with XbaI and HindIII and the fragment was cloned into pBBR1MCS-2. The resulting plasmid (pSM120) allows for the expression of YedY and YedZ under the control of their own promoter. C-ter His-tagged YedY: yedY was PCR-amplified from R. sphaeroides f. sp. denitrificans with the primers pINDyed and RpINDyed (Additional file 5: Table S2). PCR product was cloned into PCR2.1-TOPO (Invitrogen) and subsequently digested with BfuAI and HindIII. The 0.9 kb fragment was cloned into pIND4 [19] previously digested with NcoI and HindIII. The resulting plasmid (pSM88) that encodes YedY with a 6 His-tag at the C-terminus was introduced into R. sphaeroides f. sp. denitrificans by conjugation. N-ter His-tagged YedY: yedY was PCR-amplified from R. sphaeroides f. sp. denitrificans with the primers petYED and RpetYed (Additional file 5: Table S2). PCR product was cloned into PCR2.1-TOPO (Invitrogen) and digested with NdeI and HindIII. The DNA fragment was cloned into pET-TEV [23] previously digested with the same enzymes. The resulting plasmid (pSM179) encodes the mature form of YedY with a 6 His-tag and the TEV motif ENLYFQ (for cleavage with TEV protease) at the N-terminus. The DNA fragment corresponding to the yedY signal sequence was PCR-amplified with the primers YedSS and RYedSS (Additional file 5: Table S2) and cloned into PCR2.1-TOPO. The plasmid was digested with NcoI, and the fragment was cloned in the correct direction into pSM179 previously linearized with NcoI. The subsequent plasmid (pSM189) encodes a protein with a 6 His-tag and a TEV protease motif between the signal sequence and the mature protein sequence. The protein is then cleaved by a signal peptidase (AFA/ MGS) after its translocation into the periplasm. For expression in E. coli, both plasmids were introduced into E. coli BL21(DE3) by standard transformation procedure. For expression in R. sphaeroides, the two plasmids (pSM179 and pSM189) were digested with XbaI and HindIII and cloned into pMS742. The resulting respective plasmids, pSM181 and pSM196, were introduced into R. sphaeroides by triparental conjugation [38].Nucleotide sequence accession number.medium until the late exponential phase. Cells were grown with 1 mM IPTG [19] when the pIND4 derivative vector was used (C-ter tagged YedY), and harvested at the end of the exponential phase.Preparation of cell extracts”Periplasmic extracts” and “cytoplasmic extracts” were prepared using lysozyme, as previously described [39]. F.
