Supplementarydata摘要.docVIP

Supplementary data Supplementary methods Plasmids All genes were cloned into an overexpressing plasmid using a two-step strategy. bll2757 (fixK2), bll4943 (clpX), bll4944 (clpP1), bll5153 (clpA), bll5154 (clpS1) and, blr6518 (sspB() were amplified by PCR from wild-type B. japonicum genomic DNA with primers listed in Table S3. GFP proteins used in this study harbor two point mutations in the chromophore (F64L S65T). All GFP derivatives were generated by PCR using the primers listed in Table S3. PCR fragments were cloned first in the pGEM-T easy vector, digested with the respective enzymes (Table S3) and ligated into a T7-based overexpressing plasmid. clpA was cloned into pET16, clpS1, all gfp derivatives (GFP, GFPssrABj, GFPcA12, Fn9-GFP, GFP-Fc12, Fn9-GFP-Fc12) and clpP1 were cloned into pET24. As annotated in Rhizobase (http://genome.kazusa.or.jp/rhizobase/), ClpS1 from B. japonicum is 130 amino acids in length, and contrary to all other rhizobia, it possesses a non-conserved N-terminal extension of 20 amino acids. Since we failed to overexpress the full-length protein in E. coli, we cloned and overexpressed a shorter version lacking these first 20 amino acids. Curiously, ClpS1 has a second methionine in frame at position 21, which might be encoded by an alternative start codon. All experiments were done with this shorter version of the predicted ClpS1. ClpP1 was fused to a tobacco etch virus (TEV) recognition site and a His6-tag at its C-terminus. clpX and sspBα were both cloned into pProEx-HTb with an N-terminal His6-tag followed by a TEV recognition site. fixK2 was C-terminally fused to the Intein-Chitin Binding Domain tag in pTXB1. All DNA sequences were confirmed by sequencing. Protein overexpression and purification All proteins were overexpressed in BL21 (DE3) cells from bacteriophage T7 promoter-based plasmids. ClpA, ClpS1 and all GFP derivatives were purified as previously described for E. coli orthologs [1-2]. SspBα, ClpX and ClpP1 were purified over a Ni