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Jasmin Hubbard
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1 1 2 Supplemental materials 3 4 The terminal oxidase cbb 3 functions in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense 5 Running title: Oxygen respiration and magnetite biomineralization Yingjie Li a, Oliver Raschdorf a,b, Karen T. Silva a, and Dirk Schüler a# a LudwigMaximiliansUniversität München, Department Biologie I, Mikrobiologie, Großhaderner Str. 24, PlaneggMartinsried, Germany; b Max Planck Institute of Biochemistry, Department of Molecular Structural Biology, Am Klopferspitz 18, Martinsried, Germany # To whom correspondence should be addressed. Dirk Schüler, Biozentrum der LMU, Großhaderner Str.24, PlaneggMartinsried, Tel.: ; Fax: ; dirk.schueler@lrz.unimuenchen.de. 1

2 16 17 Fig. S1 Biomineralization phenotypes of Δcbb 3 and Δaa 3 Δcbb 3 mutants incubated for 24 h (A and C) and 35 h (B and C) in microaerobic nitrate medium. Scale bar, 500 nm. 18 2

3 19 Table S1 Bacterial strains and plasmids used in this work Strain or plasmid Description Reference Strains E. coli DH5α F Φ80dlacZΔM15Δ(lacZYAargF)U169 deor reca1 enda1 hsdr17 (r k, m k +) phoa supe44 λ thi1 gyra96 rela1 E. coli BW29427 dap auxotroph derivative of E. coli strain B2155 Invitrogen K. Datsenko and B. L. Wanner, unpublished MSR1 WT Wild type R3/S1, but Rif r, Sm r (1) ΔMgfnr R3/S1, ΔMgfnr (Y. Li et al., submitted) Δaa 3 R3/S1, Δaa 3 This study Δbd R3/S1, Δbd This study Δcbb 3 R3/S1, Δcbb 3 This study ΔbdΔaa 3 R3/S1, ΔbdΔaa 3 This study Δaa 3 Δcbb 3 R3/S1, Δaa 3 Δcbb 3 This study ΔbdΔcbb 3 R3/S1, ΔbdΔcbb 3 This study Plasmids pbbr1mcs2 Km r, mobilizable broadhostrange vector (2) por093 mamx CXXCH (65,104)>AXXAH, pk19mobgii derivative, Km r (3) plyj97 pbbr1mcs2 plus gusa from pk19mobgii (4) plyj115 plyj97 plus cbb 3 promoter region This study plyj128 por093 plus 2kb fused flanking section of cconoqp This study plyj129 por093 plus 2kb fused flanking section of coxbac This study plyj130 por093 plus 2kb fused flanking section of cydab This study plyj135 plyj97 plus aa 3 promoter region This study plyj137 plyj97 plus bd promoter region This study plyj138 pbbr1mcs2 plus cbb 3 with its own promoter This study plyj139 pbbr1mcs2 plus aa 3 with its own promoter This study plyj140 pbbr1mcs2 plus bd with its own promoter This study 3

4 20 Table S2 BlastP analyses of operons encoding terminal oxidases in MTB and nonmtb using MSR1 as a query Protein in MSR1 mgr_2544 mgr_2545 mgr_2546 mgr_2547 Mgr_0911 Mgr_0912 Mgr_0914 Mgr_0697 Encoded gene product (aa, kda, pi) CcoN (532, 60.6, 9.09) CcoO (243, 27.0, 6.30) CcoQ (36, 4.4, 5.09) CcoP (291, 31.2, 5.90) CoxB (269, 30.1, 6.29) CoxA (526, 57.4, 7.96) CoxC (264, 29.5, 6.97) CydB (382, 41.3, 6.06) AMB1 (evalue, Amb4363 (4e137, 86%) Amb4364 (3e09, 91%) Amb4365 (8e133, 76%) Amb2170 (1e108, 76%) Amb2169 (0.0, 89%) Amb2168 (2e115, 79%) MS1 (evalue, WP_ (0.0, 93%) WP_ (7e137, 86%) MC1 (evalue, Mmc1_2353 (0.0, 80%) Mmc1_2354 (1e94, 70%) WP_ (1e134, 77%) WP_ (3e113, 77%) WP_ (0.0, 89%) WP_ (8e104, 78%) Mmc1_2355 (4e52, 49%) gene encoding the catalytic subunit of cbb 3 oxidase is not found in M. magneticum while the ccoq gene encoding a small membrane protein of unknown function is missing in Mc. marinus and the incomplete genome assembly of M. magnetotacticum. Mc. marinus strain RS1 harbors two cydab operons as well as another operon cyoabcde encoding a quinol oxidase bo 3 oxidase (DMR_14870, _14880, _14890, _14900, and _14910). RS1 (evalue, DMR_06970 (1e63, 54%) DMR_28310 (3e23, 43%) DMR_06960 (2e61, 46%) DMR_28300 Best hit in nonmtb (evalue, (0.0, 87%) (2e124, 82%) Rhodospirillum rubrum F11 (2e11, 88%) Phaeospirillum molischianum (5e123, 73%) (6e105, 77%) (0.0, 88%) Caenispirillum salinarum (7e105, 72%) (0.0, 87%) CydA Mgr_0698 (518, 57.6, 8.66) (0.0, 92%) (4e108, 55%) AMB1: Magnetospirillum magneticum; MS1: Magnetospirillum magnetotacticum; MC1: Magnetococcus marinus; RS1: Desulfovibrio magneticus.the ccon 4

5 25 26 Table S3 Growth (OD 565 nm ), magnetic response (C mag ), and magnetosome crystal size of various terminal oxidase mutants under different conditions. Strain OD 565 nm C mag Crystal size (nm) n 0% O 2 WT 0.13 ± ± ± Δaa ± ± ± Δbd 0.13 ± ± ± ΔbdΔaa ± ± ± Δcbb ± ± ± Δaa 3 Δcbb ± ± ± ΔbdΔcbb ± ± ± % O 2, +NO 3 WT 0.22 ± ± ± Δaa ± ± ± Δbd 0.20 ± ± ± ΔbdΔaa ± ± ± % O 2, +NH 4 WT 0.11 ± ± 0.1 Δaa ± ± 0.0 Δbd 0.10 ± ± 0.1 ΔbdΔaa ± ± 0.1 Number of crystals measured for each strain (n) is presented in the last column. 28 References Schultheiss D, Kube M, Schüler D Inactivation of the flagellin gene flaa in Magnetospirillum gryphiswaldense results in nonmagnetotactic mutants lacking flagellar filaments. Appl. Environ. Microbiol. 70: Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM Four new derivatives of the broadhostrange cloning vector pbbr1mcs, carrying different antibioticresistance cassettes. Gene 166: Raschdorf O, Müller FD, Posfai M, Plitzko JM, Schüler D The magnetosome proteins MamX, MamZ and MamH are involved in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense. Mol. Microbiol. 89: Li Y, Katzmann E, Borg S, Schüler D The periplasmic nitrate reductase Nap is required for anaerobic growth and involved in redox control of magnetite biomineralization in Magnetospirillum gryphiswaldense. J. Bacteriol. 194:

Optimization of gene expression cassette for M. gryphiswaldense

Optimization of gene expression cassette for M. gryphiswaldense A) 325 bp P mamdc 270 bp 170 bp 102 bp 45 bp egfp Gradual truncation P mamdc45 egfp minimal transcriptionally active promoter B) P mamdc45