Electronic Supplementary Material (ESI) for Metallomics. This journal is © The Royal Society of Chemistry 2019 Table S1. S. meliloti metalloproteins and transition metal transporter gene transcripts induced 4-fold in nodules vs. growth in vitro, and/or enriched (≥40%) in zones of bacteroid differentiation and nitrogen fixation. Description Metala Locus Retrived fromb Categoryc Localizatione References (gene name) Fe/Mo Nitrogenase molybdenum- Energy metabolism SMa0827 iron protein alpha chain 1, 2 C 3 (Nitrogen metabolism) (nifD) Nitrogenase molybdenum- Energy metabolism SMa0829 iron protein beta chain 1, 2 C 3 (Nitrogen metabolism) (nifK) Fe Dioxygenase reductase SMa0752 1, 4 Unclassified C 5 subunit Energy metabolism Diheme c-type cytochrome SMa0769 1, 2, 4 (Oxidative P 6 (fixP2) phosphorylation) SMa0811 Ferredoxin (fdxN) 1, 2, 4 Unclassified C 7 FeMo cofactor biosynthesis SMa0814 1, 2, 4 Energy metabolism C 8 protein (nifB) Ferredoxin-like protein SMa0816 1, 2, 4 Energy metabolism C 9 (fixX) Nitrogenase reductase Energy metabolism SMa0825 1, 4 C 10 (nifH) (Nitrogen metabolism) 1 Mn2+/Fe2+ transporter SMa1115 1, 2 Transporters IM 11 NRAMP family Iron sulfur membrane SMa1211 1, 2, 4 Unclassified IM 12 protein (fixG) Energy metabolism Di-heme cytochrome c SMa1213 1, 2, 4 (Oxidative P 6 (fixP1) phosphorylation) Iron ABC transporter ATP- SM_b20363 2 Transporters IM 13 binding protein Iron ABC transporter SM_b20364 2 Transporters IM 13 permease Iron ABC transporter SM_b20365 4 Transporters P 14 substrate-binding protein Thiamine biosynthesis Metabolism of cofactors SM_b20615 4 C 15 protein (thiC) and vitamins SM_b20819 Ferredoxin (mocE) 1, 2 Amino acid metabolism C 16 Ferredoxin reductase SM_b20820 1, 2, 4 Amino acid metabolism C 16 (mocF) Nitrite reductase Energy metabolism SM_b20984 [NAD(P)H], large subunit 1 C 17 (Nitrogen metabolism) protein (nirB) 2 Metabolism of cofactors SM_b20987 Siroheme synthase 2 C 18 and vitamins Iron ABC transporter ATP- SM_b21541 2 Transporters IM 13 binding protein Iron uptake ABC SM_b21542 2 Transporters IM 14 transporter permease Aldehyde or xanthine SM_b21558 dehydrogenase, iron-sulfur 2 Nucleotide metabolism C 19 subunit protein Ferredoxin reductase SM_b21636 electron transfer protein 2 Amino acid metabolism C 20 (paaE) 4-hydroxy-3-methylbut-2- Metabolism of SMc00016 enyl diphosphate reductase 2, 4 terpenoids and C 21 (ispH) polyketides Ubiquinol-cytochrome C Energy metabolism SMc00187 reductase iron-sulfur 2 (Oxidative C 22 subunit protein (fbcF) phosphorylation) Erythrin-vacuolar iron SMc00359 transport family protein 2 Transporters C 23 (mbfA) Iron-responsive SMc00785 transcriptional regulator 4 Transcription factors C 24 (rirA) Ferrichrome receptor SMc01611 2 Transporters OM 25 precursor protein (fhuA) 3 Succinate dehydrogenase SMc02466 2 Carbon metabolism C 26 iron-sulfur subunit (sdhB) 4-hydroxyphenylpyruvate SMc03211 1 Amino acid metabolism C 27 dioxygenase SMc03253 L-proline 3-hydroxylase 1, 4 Unclassified C 28 Iron transport regulator SMc04204 transmembrane protein 1, 2 Signaling proteins C 29 (fecR) 2 14 SMc04205 Iron/heme transport protein Transporters OM Iron ABC transporter 2 30 SMc04316 Transporters IM permease (afuB) Co ABC transporter, ATP- SM_b20058 2 Transporters IM 31 binding proteind Magnesium/cobalt 2 32 SMc00399 Transporters C transporter CorA (corA1) Vitamin B12-dependent SMc01237 ribonucleotide reductase 2 Nucleotide metabolism C 33 (nrdJ) Cobalt-precorrin-6x Metabolism of cofactors SMc03189 2 C 34 reductase (cobK) and vitamins 4 Cobalamin biosynthesis 2 Metabolism of cofactors 35 SMc03193 P protein (cobG) and vitamins Cu Energy metabolism cbb3-type cytochrome c SMa0612 2 (Oxidative IM 36 oxidase subunit I (fixN3) phosphorylation) Energy metabolism cbb3-type cytochrome c SMa0765 2 (Oxidative IM 36 oxidase subunit I (fixN2) phosphorylation) Copper translocating P- SMa1013 2 Transporters IM 37 type ATPase (actP) Cation transport P-type SMa1087 1, 2 Transporters IM 38 ATPase (copA3) Energy metabolism SMa1182 N O-reductase (nosZ) 2 P 39 2 (Nitrogen metabolism) Energy metabolism SMa1186 Copper chaperone (nosL) 2 C 40 (Nitrogen metabolism) 2 41 SMa1194 Dehydrogenase (nnrS) Defense mechanisms IM 1, 2 42 SMa1198 Copper export protein Transporters IM 1, 2, 4 38 SMa1209 FixI1 ATPase (copA2a) Transporters IM 5 Energy metabolism cbb3-type cytochrome c SMa1220 2 (Oxidative IM 36 oxidase subunit I (fixN1) phosphorylation) SMc01754 Oxidoreductase 2 Unclassified P 43 Copper-containing SMc02282 2 Unclassified P 43 oxidoreductase Copper-containing SMc02283 oxidoreductase signal 2 Unclassified C 43 peptide protein Probable outer membrane 2 44 SMc02400 Transporters OM protein (ropAe) Mn Biosynthesis of amino SMa1711 Arginase (argI2) 2 C 45 acids 3,4-dihydroxy-2-butanone- Metabolism of cofactors SMc00008 2 C 46 4-phosphate synthase (ribA) and vitamins Superoxide dismutase, Fe- SMc00043 2 Oxidoreductase C 47 Mn family (sodB) Glutathione synthetase SMc00419 2 Amino acid metabolism C 48 (gshB1) SMc00472 Hypothetical protein 2 Nucleotide metabolism C 49 6 Isocitrate dehydrogenase Carbohydrate SMc00480 2 C 50 (icd) metabolism Energy metabolism D-ribulose-5-phosphate 3- (Carbon fixation in SMc00511 2 C 51 epimerase (rpe) photosynthetic organisms) Manganese/iron transport SMc02506 system ATP-binding 2 Transporters IM 52 protein (sitD) Manganese/iron transport SMc02507 system ATP-binding 2 Transporters IM 52 protein (sitC) Manganese/iron transport SMc02508 system ATP-binding 2 Transporters IM 52 protein (sitB) Manganese/iron transport SMc02509 system ATP-binding 2 Transporters P 52 protein (sitA) Mo SMa0873 Nitrogenase molybdenum- cofactor biosynthesis 1, 2, 4 Energy metabolism C 53 protein (nifN) Aldehyde or xanthine dehydrogenase, SM_b21557 2 Nucleotide metabolism C 19 molybdopterin binding subunit protein Molybdenum cofactor Metabolism of cofactors SMc00144 biosynthesis protein A 2 C 8 and vitamins (moaA) 7 Formate dehydrogenase Carbohydrate SMc02524 4 C 54 subunit gamma (fdsG) metabolism Zn Zinc dependent SMa0041 oxidoreductase-related 2 Unclassified C 55 quinone oxidoreductase SMa1126 Protease 1, 4 Unclassified IM 55 Methionine aminopeptidase SM_b21002 2 Peptidases C 56 (map2) Conserved putative SM_b21211 membrane protein, metal- 2 Unclassified C 57 dependent hydrolase Formamidopyrimidine- DNA replication and SMc01154 2 C 58 DNA glycosylase (fpg) repair Histidinol dehydrogenase SMc02307 2 Amino acid metabolism C 59 (hisD) Zinc-type alcohol SMc02771 2 Unclassified C 55 dehydrogenase Probable high-affinity zinc uptake system SMc04243 2 Transporters IM 60 membrane ABC transporter (znuB) 8 Probable high-affinity zinc uptake system ATP- SMc04244 2 Transporters C 60 binding ABC transporter (znuC) Probable high-affinity SMc04245 zinc uptake system ABC 2 Transporters P 60 transporter (znuA) aTransported substrate, enyme co-factor or co-enzyme bLegume-Rhizobium model: M. truncatula cv. Jemalongg A17 - S. meliloti 2011 strain GMI11495 (Roux et al., 2014); M. truncatula cv. AS17 - S. meliloti Rm1021 (Barnett et al., 2004); M. truncatula J6 - S. meliloti Rm1021 (Capela et al., 2006) cLocus and descriptions in bold indicates that the transcripts of these candidates were present in differentiation and fixation nodule zones at values between 30% and 40 % of the total counts obtained from the whole nodule dBased on KEGG Orthology (https://www.genome.jp/kegg/ko.html) eLocalization in the bacterial cell: Outer membrane (OM), Inner membrane (IM), Cytoplasm (C), Periplasm (P) 9 References 1. D. Capela, C. Filipe, C. Bobik, J. Batut and C. Bruand, Sinorhizobium meliloti differentiation during symbiosis with alfalfa: a transcriptomic dissection, Mol Plant Microbe Interact, 2006, 19, 363-372. 2. B. Roux, N. Rodde, M. F. Jardinaud, T. Timmers, L. Sauviac, L. Cottret, S. Carrere, E. Sallet, E. Courcelle, S. Moreau, F. Debelle, D. Capela, F. de Carvalho-Niebel, J. Gouzy, C. Bruand and P. Gamas, An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing, Plant J, 2014, 77, 817-837. 3. R. Fani, R. Gallo and P. Lio, Molecular evolution of nitrogen fixation: the evolutionary history of the nifD, nifK, nifE, and nifN genes, J Mol Evol, 2000, 51, 1-11. 4. M. J. Barnett, C. J. Toman, R. F. Fisher and S. R. Long, A dual-genome Symbiosis Chip for coordinate study of signal exchange and development in a prokaryote-host interaction, Proc Natl Acad Sci U S A, 2004, 101, 16636-16641. 5. M. J. Wargo, B. S. Szwergold and D. A. Hogan, Identification of two gene clusters and a transcriptional regulator required for Pseudomonas aeruginosa glycine betaine catabolism, J Bacteriol, 2008, 190, 2690-2699. 6. H. Hennecke, in Biological Nitrogen Fixation for the 21st Century: Proceedings of the 11th International Congress on Nitrogen Fixation, Institut Pasteur, Paris, France, July 20–25, 1997, eds. C. Elmerich, A. Kondorosi and W. E. Newton, Springer Netherlands, Dordrecht, 1998, DOI: 10.1007/978-94-011-5159-7_271, pp. 429-434. 7. W. Klipp, H. Reilander, A. Schluter, R. Krey and A. Puhler, The Rhizobium meliloti fdxN gene encoding a ferredoxin-like protein is necessary for nitrogen fixation and is cotranscribed with nifA and nifB, Mol Gen Genet, 1989, 216, 293-302. 8. G. Schwarz, R. R. Mendel and M. W. Ribbe, Molybdenum cofactors, enzymes and pathways, Nature, 2009, 460, 839-847. 9. M. Gubler, T. Zurcher and H. Hennecke, The Bradyrhizobium japonicum fixBCX operon: identification of fixX and of a 5' mRNA region affecting the level of the fixBCX transcript,