Optimization of gene expression cassette for M. gryphiswaldense
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1 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 mamdc P mamdc45 Optimization length of RBS egfp orbs egfp bp E) P tet Transposon mediated chromosomal insertion of (inducible) expression cassettes 8 magegfp tetr P Neo C) Optimization codon usage of GFP D) tetr P mamab tetr P Neo tetr P mamdc P mamdc45 8 magegfp Magnetospirillum-optimized EGFP Introduction of a tet-inducible expression system Introduction of tetr repressor under control of different promoters P tet 8 magegfp inducible / tunable promoter Suppl. Fig. S1: Optimization strategy of (inducible) expression cassette.
2 Suppl. Fig S2: Sequence alignment of non-optimzed egfp and Magnetospirillum-optimized magegfp using CLUSTALW (version 2.1, Sequence similarity is 89 %, start and stop codon are overlined.
3 A B wt EGFP MagEGFP 27 kda Suppl. Fig S3: (A) Fluorescence of Magnetospirillumoptimized (MagEGFP) versus non-optimized GFP (EGFP) expressed from the P mamdc45 promoter. Fluorescence was normalized to the cell density and described as relative fluorescence units (RFU). Error bars represent standard deviations, calculated from three independent experiments. (B) Western blot of whole M. gryphiswaldense cells expressing EGFP or MagEGFP from the control of the P mamdc45. (Mag)EGFP was detected using rabbit αgfp IgG as primary, and goat anti-rabbit IgG alkaline phosphatase antibodies as secondary antibody. PageRuler Prestained Protein Ladder from fermentas was used as a standard.
4 A B C D Suppl. Fig. S4: Fluorescence micrographs of M. gryphiswaldense C strains carrying chromosomal insertions of (A) P mamdc45 -mamcmagegfp (JH1) (B) P tet -mamc-magegfp (JH2) induced with 70 ng ml -1 Atet, (C) uninduced and (D) P mamdc45 -mamc-egfp-magegfp (JH3) expression cassettes. White bar corresponds to 2 µm. Suppl. Fig. S5: Fluorescence and DIC micrographs of isolated magnetosomes from M. gryphiswaldense MSR- 1 C (A) JH2 (induced) (B) JH1 and (C) JH3. White bar corresponds to 2 µm.
5 A B Suppl. Fig. S6: A) Transmission electron micrographs of M. gryphiswaldense SB6, expressing MagEGFP chromosomally from P mamdc45. The black scale bar represents 200 nm. B) Optical density (OD 565 ) after overnight growth of strains SB6, SB7 and SB8 in comparison to wt. Error bars represent standard deviations calculated from triplicate cultures, experiment was repeated three times, data is from one representative experiment. A B C d = 37.5 nm Volume sphere = 1/6*d*π Suppl. Fig. S7: (A) Quantitative Western blot of GFP standard curve (marked by x ) and MM samples from strains JH1 (black square), JH2 (grey square) and JH3 (light grey square, 10x diluted). (B) Corresponding GFP protein concentrations of strains JH1, JH2 and JH3. (C) Schematic drawing of approximation of magnetosome size.
6 Suppl. Tab. S1: Plasmids used in Plasmid name Description Source or reference pjet1.2/blunt Cloning vector; Amp R Fermentas pbbr-mcs2 Mobilizable broad-host-range Kovach, M. E., et al, vector; Km R pbam1 Km R, Amp R, orir6k, tnpa Martinez-Garcia, E., et al., 2011 p11aagjzc Amp R,ColE1 ori, orbs, magegfp GeneArt (Invitrogen), life technologies, Darmstadt pap150 pbbr-mcs2, with P mamdc45, egfp, A. Pollithy (unpublished) terminator-fragment; Km R pap158 pbbr-mcs2, with P tet, egfp, A. Pollithy (unpublished) terminator-fragment,p mamab -TetR; Km R pap159 pbbr-mcs2, with P tet, egfp, A. Pollithy (unpublished) terminator-fragment,p mamdc -TetR; Km R pap160 pbbr-mcs2, with P tet, egfp, A. Pollithy (unpublished) terminator-fragment,p Neo -TetR; Km R pap161 pbbr-mcs2, with P mamdc325, A. Pollithy (unpublished) egfp, terminator-fragment; Km R pap162 pbbr-mcs2, with P mamdc102, A. Pollithy (unpublished) egfp, terminator-fragment; Km R pap163 pbbr-mcs2, with P mamdc170, A. Pollithy (unpublished) egfp, terminator-fragment; Km R pap164 pbbr-mcs2, with P mamdc270, A. Pollithy (unpublished) egfp, terminator-fragment; Km R plyj97 pbbr-mcs2 with gusa Li, Y., et al., 2012 psb1 pbbr-mcs2, with P mamdc45, magegfp, terminator-fragment; Km R psb6 pbam1 with P mamdc45, magegfp, Km R, Amp R psb7 pbam1 with P tet, magegfp, P Neo - TetR, Km R, Amp R psb8 pbam1 with P tet, gusa, P Neo -TetR, Km R, Amp R pjh1 pbam1 with P mamdc45, mamcmagegfp, Km R, Amp R pjh2 pbam1 with P tet, mamc-magegfp, P Neo -TetR, Km R, Amp R pjh3 pbam1 with P mamdc45, mamcmagegfp-egfp, Km R, Amp R
7 Suppl. Tab. S2: Strains used in Strain Description Source or reference Escherichia coli DH5α F - supe44 lacu169 (Φ 80 laczdm15) hsdr17 reca1 enda1 gyra96 thi-1 rela1 WM3064 thrb1004 pro thi rpsl hsds lacz M15 RP (arabad)567 W. Metcalf, unpublished BW29427 Magnetospirillum gryphiswaldense M. gryphiswaldense MSR-1 R3/S1 dapa1341::[erm pir] DAP auxotroph derivative of E. coli strain B2155 K. Datsenko and B. L. Wanner, unpublished Rif R, Sm R spontaneous mutant, lab strain D. Schultheiss, et al., 2003 M. gryphiswaldense C mamc A. Scheffel, et al., 2007 M. gryphiswaldense (pap150) Km R, conjugated with A. Pollithy, unpublished pap150 M. gryphiswaldense (pap160) Km R, conjugated with A. Pollithy, unpublished pap160 M. gryphiswaldense (psb1) Km R, conjugated with psb1 M. gryphiswaldense MSR-1 SB6 Km R, transposon mutant with inserted magegfp from P mamdc45 M. gryphiswaldense MSR-1 SB7 Km R, transposon mutant with inserted magegfp from P tet M. gryphiswaldense MSR-1 SB8 Km R, transposon mutant with inserted gusa from M. gryphiswaldense MSR-1 JH1 Km R, transposon mutant with inserted mamcmagegfp from P mamdc45 M. gryphiswaldense MSR-1 JH2 Km R, transposon mutant with inserted mamcmagegfp from P tet M. gryphiswaldense MSR-1 JH3 Km R, transposon mutant with inserted mamcmagegfp-egfp from P mamdc45 P tet
8 Suppl. Tab. S3: Primers used in. Restriction sites indicated in bold Primer name Sequence Restriction site oegfp BamHI Rev cgaacggatcctcacttatacagctcg BamHI oegfp HindIII Fw cggctcaagcttaggagatcagcatatg HindIII pbam_pap160 Fw atcgggaccccttccggctggctggttt SanDI pbam_ Tet w/o Term Rev atcgaattcggcggatttgtcctactca EcoRI pbam_pap150 Fw atcgggacccggatcctcacttatacagct SanDI pbam_dc w/o Term Rev gcgaattcctcgagctttttcgctttac EcoRI GusA BamHI Fw gtggatcccccgggtcattgtttgcc BamHI GusA NdeI Rev ttcatatgttacgtcctgtagaaa NdeI optgfp San/Bam Fw gagggacccggatcctcacttatacagctcgtcc BamHI/SandDI optgfp linker Rev ggaggcggaggcggtggcggaggtggcggaatcgatatg ClaI gtgtcgaagggcga mamc ov linker Fw cgccaccgcctccgcctccatgggccaattcttccctca NcoI mamc NdeI Rev tacatatgagctttcaacttgcgcc NdeI optgfp2x Fw agtggatcctcacttatacagctcgtcca BamHI optgfp2x Rev ctgtgcctgcagggcgagatggtgtcgaagggcg PstI egfp overl Fw atctcgccctgcaggcacagcttgtacagctcgtccatgc PstI mamc_rbs Rev cgaagcttaggagatcagcatatgagctttcaact HindIII Suppl. Tab. S4: Insertion sites of expression cassettes in M. gryphiswaldense strains Strain Gene Putative function MSR-1 SB6 K7 MGR_1519 hypothetical protein MSR-1 SB6 K8 Inter region between MGR_ D-alanine-D-alanine ligase MGR_ acyl carrier protein MGR_1092 and MGR_2997 MSR-1 SB7 K1 MGR_1581 sugar kinase, ribokinase family MSR-1 SB7 K2 MGR_1519 hypothetical protein MSR-1 SB8 MGR_1702 transposase IS3/IS911 MSR-1 JH1 MGR_3776 insertion element ISR1 from not characterized 10 kda protein A3 MSR-1 JH2 MGR_3148 TorC, trimethylaminoxide (TMAO)-reductase I, cytochrom C subunit MSR-1 JH3 MGR_612 hypothetical protein
9 References Supplements: 1. Kovach ME, Elzer PH, Hill DS, Robertson GT, Farris MA, Roop RM, Peterson KM New Derivatives of the Broad-Host-Range Cloning Vector pbbr1mcs, Carrying Different Antibiotic-Resistance Cassettes. Gene 166: Martinez-Garcia E, Calles B, Arevalo-Rodriguez M, de Lorenzo V pbam1: an all-synthetic genetic tool for analysis and construction of complex bacterial phenotypes. Bmc Microbiol Li YJ, 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: Schultheiss D, Schüler D Development of a genetic system for Magnetospirillum gryphiswaldense. Arch Microbiol 179: Scheffel A, Gärdes A, Grünberg K, Wanner G, Schüler D The major magnetosome proteins MamGFDC are not essential for magnetite biomineralization in Magnetospirillum gryphiswaldense but regulate the size of magnetosome crystals. J Bacteriol 190:
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