4
1 Supplemental material 1 2 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 GACGTC GGCGTCGATCACCTTCCAGAA upstream fragment of the recA gene IB014-rev AGATCT TGCCAATCCCGCTCGGTC IB015-for ACGCGT CATCCCAAGGTACATGAT downstream fragment of the recA gene IB016-rev GAGCTC GATTCCGACGGTATCATC 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 GGTACC TCAATCCTCGGCGGCGT recA fragment containing the native promoter IB073-rev CTCGAG CTACCCACCGCCCCCGA 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 4 5

Table S1: Oligonucleotides used in this study. Underlined … · 2011. 9. 9. · 1 1 Supplemental material 2 3 Table S1: Oligonucleotides used in this study.Underlined sequences indicate

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Page 1: Table S1: Oligonucleotides used in this study. Underlined … · 2011. 9. 9. · 1 1 Supplemental material 2 3 Table S1: Oligonucleotides used in this study.Underlined sequences indicate

 

Supplemental material 1 

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

Page 2: Table S1: Oligonucleotides used in this study. Underlined … · 2011. 9. 9. · 1 1 Supplemental material 2 3 Table S1: Oligonucleotides used in this study.Underlined sequences indicate

 

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 

 17 

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 

24 

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 

Page 3: Table S1: Oligonucleotides used in this study. Underlined … · 2011. 9. 9. · 1 1 Supplemental material 2 3 Table S1: Oligonucleotides used in this study.Underlined sequences indicate

 

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 

36 

Figure S1 37 

Page 4: Table S1: Oligonucleotides used in this study. Underlined … · 2011. 9. 9. · 1 1 Supplemental material 2 3 Table S1: Oligonucleotides used in this study.Underlined sequences indicate

 

38 

Figure S2 39 

40 

Figure S3 41