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Supplemental material 1
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Table S1: Oligonucleotides used in this study. Underlined sequences indicate restriction sites. 3
Primer Nucleotide sequence (5´-3´)a Product IB009-for CGCACAAGCTGTTCACCAAGG flanking region of the recA
gene IB010-rev TCATCATCAATCTGGCGGTCA
IB011-for CGCACCGAGGCATAGAACT flanking region of the recA gene IB012-rev TCGAGGAAACGCTCAGACG
IB013-for GACGTCGGCGTCGATCACCTTCCAGAA upstream fragment of the recA gene IB014-rev AGATCTTGCCAATCCCGCTCGGTC
IB015-for ACGCGTCATCCCAAGGTACATGAT downstream fragment of the recA gene IB016-rev GAGCTCGATTCCGACGGTATCATC
IB017-for TCAATCCTCGGCGGCGTT recA gene
IB018-rev ATGTCTCAGGCTGCGTTGCG
IB056-fw ATGGAACCTGGCAGATCAGAAGT mamH-mamE
IB057-rev TCAAAGAACAATCCAGAACTCTTGG
IB058-fw ATGAGGAAGAGCGGTTGCGC mamM-mamN
IB059-rv TCATCCTGCGAGAACGGCGA
IB060-fw ATGATTGAAATTGGCGAGACCA mamO-mamA
IB061-rv CTCAATGAGACCTTCTACATCGACTG
IB062-fw ATGGCAGTAAGCGATGCGG mamQ-mamS
IB063-rv TCACTGCACGGTCATCCACA
IB064-fw ATGGGTACGCCAGGGGG mamT-mamU
IB065-rv TTATTTCGGAACCAGTATGGAAAGC
IB072-for GGTACCTCAATCCTCGGCGGCGT recA fragment containing the native promoter IB073-rev CTCGAGCTACCCACCGCCCCCGA
pJet 1.2-for CGACTCACTCACTATAGGGAGAGCGGC sequencing primers (Fermentas) pJet 1.2-rev AAGAACATCGATTTTCCAATGGCAG
EK_JKL_f CGAGACTTTTATGGCTCCG mamJ-mamL (kindly provided by E. Katzmann) EF_JKL_r ACTTCAACCTCGGCATCC
61 fw GCAACACCTTCTTCACG pCM184 loxP sites (kindly provided by René Uebe) 54 rv GAGATTTTGAGACACAACGTG
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Figure S1. Genomic neighborhood of recA in M. gryphiswaldense and scheme for 6
construction of a recA mutant. A. Amplification of the flanking regions and construction of 7
the suicide vector pCM184recAflank. B. Conjugative transfer and double crossover C. 8
Generation of a recA mutant containing a kanamycin resistance cassette. D. Removal of the 9
cassette via Cre recombinases encoded on the plasmid pCM157, and generation of an 10
unmarked deletion mutant. E. Confirmation of the recA deletion by Southern blot of KpnI-11
digested genomic WT (lane 2) and ΔrecA DNA (lane 1). The blot was hybridized with α-32P-12
dATP labeled recA probe (primers IB017, IB018; product size 1077 bp). The fragment 13
indicates presence of the recA gene, whereas no signal verifies the deletion mutant. IB013, 14
IB014, IB015, IB016: primer binding sites. IB013+IB014, IB015+IB016: flanking regions of 15
recA. Kan: kanamycin resistance cassette. loxP: loxP sites. 16
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Figure S2: A. Experimental scheme for the induced mutagenesis of IK-1 and the WT. Cells 18
were incubated for 7 days at 4 °C under microaerobic conditions. Afterwards, cultures were 19
passaged every 48 hours six times without shaking. Cells were spotted in different dilutions 20
on Agar plates. Colonies were visually screened and white colonies were picked and further 21
screened via PCR and Cmag measurements. B. Visual screening for spontaneous non-magnetic 22
mutants: (→) indicate abberant white colonies. 23
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Figure S3: PCR analysis of the spontaneous nonmagnetic mutants (mutants: 1-23, (+): WT, (-25
): negative control) via PCR. Several regions within the mamAB-operon were tested. After 26
amplification, PCR products were loaded on a 1% agarose gel. A. Results for amplification of 27
the region mamH-mamE (primers IB056, IB057); expected PCR product: 3.8 kb. B. Results 28
for amplification of the region mamJ-mamL (primers EK_mamJKL_f, EK_mamJKL_r), 29
expected product: 2.7 kb. C. Results for amplification of the region mamM-mamN (primers: 30
3
IB058, IB059); product: 2.2 kb. D. Results for amplification of the region mamO-mamA 31
(primers: IB060, IB061); product: 3.4 kb. E. Results for amplification of the region mamQ-32
mamS (primers IB062, IB063); expected product: 2.5 kb. F. Results for amplification of the 33
region mamT-mamU (primers IB064, IB065); expected product: 1.4 kb. G. Overview of the 34
mamAB operon (A-F: amplified regions) 35
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Figure S1 37
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Figure S2 39
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Figure S3 41