SUPPLEMENTARY MATERIAL

TABLE S1

Oligonucleotides used in this study

Oligonucleotide / Sequence (5’ → 3’) and propertiesa
Construction of pEKEx2-mtrA
OE_mtrA-fw / CCT GGG ATC CAT AAA CTA ATC CCA ATT AG (BamHI)
OE_mtrA-rv / TTA AAT GGG TAC CTT AAT CGT TGT GGC CAG (KpnI)
Construction of pEKEx2-mtrA-D53N
mtrA_Asp→Asn_1 / CCG GAT CTG ATT TTG CTC AAC TTG ATG CTT CCA GGC
mtrA_Asp→Asn_2 / GCC TGG AAG CAT CAA GTT GAG CAA AAT CAG ATC CGG
Construction of pET28b-mtrA+NHis6
mtrA-NdeI / TAT ACA TAT GTC ACA GAA AAT TCT CGT GGT TG (NdeI)
mtrA-stop-rv / TAT ACT CGA GTT AAT CGT TGT GGC CAG TTT TGT AGC C (XhoI)
Construction of pMalc-mtrBK
mtrB-pMal-BamHI-fw / TAT AGG ATC CTT GGA GGA ATA CGG CAA TCT GCA ACG (BamHI)
mtrB-pMal_BamHI-rv / TAT AGG ATC CTC ACT GCT GCT CCC CTT CCC ACG G (BamHI)
Primer extension
PE_nlpC / IRD800- GTT TGC TGA CCG AGT TAG CTG TCG GGT TCG GAG CGG
nlpC_prom_fw / ATG TCA CAT AGT GAG ATC ATT GCT G
nlpC_prom_rv / CTG CTA CAG CCT TGC GAG TTG C
PE_rpf2 / IRD800- ACG GCA CCG AGC GGG TGC TG
rpf2_prom_fw / ATG TTG CGG CGG CTT TGA ATG AG
rpf2_prom_rv / GTG ACT CCG CCG ATA AGC AAG G
PE_csbD / IRD800- GGA CTC GTT ATC AGT TGC CTC G
csbD_prom_fw / ACC GAA TTT TTC GAA CTT CAC AGG
csbD_prom_rv / GAC AGC ATC CTT GAT GTC TGC C
PE_cg1517 / IRD800- AGG GGT GAT TGC AAG TGA TAA TGC
cg1517_prom_fw / TTT TGT GGA CAT TGC ACT TTA CTG C
cg1517_prom_rv / AGC CAC TCA ACC ACC AAA CAC TC
EMSAs
nlpC_1_fw / ATG TCA CAT AGT GAG ATC ATT GCT G
nlpC_1_rv / CTG CTA CAG CCT TGC GAG TTG C
nlpC_2_rv / TTA TCG CGG ATT AAT TTG ACC GAG
nlpC_3_fw / CGG TTC GAT AAC GAC CAA TTT TTT C
nlpC_3_rv / CAG GAA CAC GTT TGG TTC CCC G
nlpC_4_fw / CGG GGA ACC AAA CGT GTT CCT G
nlpC_5_fw / GTC ATC TAG CCT TCT CAG GGT TC
nlpC_5_rv / GCT GGC GGA AAT TCG GTG TCC
nlpC_6_fw / GGA CAC CGA ATT TCC GCC AGC
nlpC_6_rv / TAG CTG TCG GGT TCG GAG CGG
nlpC_7_rv / GAA AAA ATT GGT CGT TAT CGA ACC G
nlpC_8_fw / TTG GGC TAG ACA AGT GTT GTT GC
mepA_1_fw / CAG AGA CAT CCG AAA ACC GTG C
mepA_1_rv / ACG AAA GCA GCA ATT GCT AGT CG
mepA_2_fw / CGA GCT CAT AGC CCG GGA TCG
mepA_2_rv / CAC GCC GTG CTA GGG GTG TC
mepA_3_fw / GAC ACC CCT AGC ACG GCG TG
mepA_4_fw / GTT CGG CAT GCA CAT CCG CTA C
mepA_4_rv / GCC ATT TAT TAA CCA GTC CGT TAT C
proP_1_fw / TTG GAG CAA AAT CCG ATT CTT GCC
proP_1_rv / TCA TCG ACT GTT AGT CTT GGT TCG
proP_2_rv / GAG CTG GAA GAA TGC AAA AAG ACC
proP_3_fw / GGT CTT TTT GCA TTC TTC CAG CTC
proP_4_rv / GGG AAA GTT ACT GTT TTC GGT GCC
proP_5_fw / GGC ACC GAA AAC AGT AAC TTT CCC
proP_6_rv / CAT TTC CAG ATC TGT CTG AAA AAT C
proP_7_fw / GAT TTT TCA GAC AGA TCT GGA AAT G
betP_1_fw / T TGG CAG AAT GGG CAC AAG ATG G
betP_1_rv / CTC TGG CTG AGC ATC CTC CAC
betP_2_fw / GTT GAA AGC GGT GTG CGC AGA C
betP_2_rv / GGT CTT TTA CAT AGC TGT TAC TTA AG
betP_3_fw / CTT AAG TAA CAG CTA TGT AAA AGA CC
betP_4_fw / CGA TGC GGT TAT TTC TGC CAG GG
betP_4_rv / CTG GAA AGC AAA CCG AAA AAT GGG
betP_5_fw / CGT CGA TTC GCT TAT CGA CGC C
betP_5_rv / CCC TGG CAG AAA TAA CCG CAT CG
nlpC1_M_fw / GTC ATC TAG CCT TCT CAG GGT TC
nlpC1_M_rv / AAA AAT TGG TCG TTA TCG AAC CGT TAT CGC GGA TTA ATT TGA CCG AGA CGC
nlpC1_M1_rv / AAA AAT TGG TCG TTA TCG AAC CGT TCG ATC GGA TTA ATT TGA CCG AGA CGC G
nlpC1_M2_rv / AAA AAT TGG TCG TTA TCG AAC CTG GAT CGC GGA TTA ATT TGA CCG AGA C
nlpC1_M3_rv / AAA AAT TGG TCG TTA TCG ACA AGT TAT CGC GGA TTA ATT TGA CCG AG
nlpC1_M4_rv / AAA AAT TGG TCG TTA TAT CAC CGT TAT CGC GGA TTA ATT TGA C
nlpC1_M5_rv / AAA AAT TGG TCG GGC GCG AAC CGT TAT CGC GGA TTA ATT TG
nlpC1_M6_rv / AAA AAT TGT GAT TTA TCG AAC CGT TAT CGC GGA TTA A
nlpC1_M7_rv / AAA AAG GTG TCG TTA TCG AAC CGT TAT CGC G
nlpC2_M_rv / CAG GAA CAC GTT TGG TTC CCC G
nlpC2_M_fw / AGT GTT GTT GCG GTT TCG TAA CCT TAT TGA GAC ATT GCG GGA CGG ACA CC
nlpC2_M1_fw / AGT GTG TGT GCG GTT TCG TAA CCT TAT TGA GAC
nlpC2_M2_fw / AGT GTT GTG TAT GTT TCG TAA CCT TAT TGA GAC ATT G
nlpC2_M3_fw / AGT GTT GTT GCG TGG GCG TAA CCT TAT TGA GAC ATT GCG G
nlpC2_M4_fw / AGT GTT GTT GCG GTT TAT GAA CCT TAT TGA GAC ATT GCG GGA C
nlpC2_M5_fw / AGT GTT GTT GCG GTT TCG TCC ACT TAT TGA GAC ATT GCG GGA CGG
nlpC2_M6_fw / AGT GTT GTT GCG GTT TCG TAA CAG GAT TGA GAC ATT GCG GGA CGG AC
nlpC1_M7_fw / AGT GTT GTT GCG GTT TCG TAA CCT TCG GTA GAC ATT GCG GGA CGG ACA CC
nlpC1_M8_fw / AGT GTT GTT GCG GTT TCG TAA CCT TAT TGC TCC ATT GCG GGA CGG ACA CCG AAT
mepA_M_rv / CTA GTC GAC GCT TCA TGT TGC GG
mepA_M_fw / GTG GTC ACA ACT TGA TAA CGG AC
mepA_M1_fw / CCC CTA CTT TGG TCA CAA CTT GAT AAC GGA CTG
mepA _M2_fw / GGT TTC ACA ACT TGA TAA CGG ACT GGT TAA TAA ATG G
mepA _M3_fw / GTG GGA CCA ACT TGA TAA CGG ACT GGT TAA TAA ATG G
mepA _M4_fw / GTG GTC AAC CCT TGA TAA CGG ACT GGT TAA TAA ATG G
mepA _M5_fw / GTG GTC ACA AAG TGA TAA CGG ACT GGT TAA TAA ATG GCC AAT C
mepA _M6_fw / GTG GTC ACA ACT GTA TAA CGG ACT GGT TAA TAA ATG GCC AAT C
mepA _M7_fw / GTG GTC ACA ACT TGC GAA CGG ACT GGT TAA TAA ATG GCC AAT C
mepA _M8_fw / GTG GTC ACA ACT TGA TCC CGG ACT GGT TAA TAA ATG GCC AAT C
mepA _M9_fw / GTG GTC ACA ACT TGA TAA ATG ACT GGT TAA TAA ATG GCC AAT C
mepA _M10_fw / GTG GTC ACA ACT TGA TAA CGT CCT GGT TAA TAA ATG GCC AAT C
mepA _M11_fw / GTG GTC ACA ACT TGA TAA CGG AAG GGT TAA TAA ATG GCC AAT CT
proP_M_rv / TGT TAA CTT CTT CCT GTA AGA CTC C
proP _M_fw / ATC TGG AAA TGT GAC CAA TTT GTA AC
proP _M1_fw / GAT TTT TCA GAC AGA TCT TTC CAT GTG ACC AAT TTG TAA CCC ACC C
proP _M2_fw / ATC TGG AAC GTG GAC CAA TTT GTA ACC CAC CCC C
proP _M3_fw / ATC TGG AAA TGT TCA AAA TTT GTA ACC CAC CCC CGC TC
proP _M4_fw / GAT TTT TCA GAC AGA TCT GGA AAT GTG AAA CCT TTG TAA CCC ACC CCC GCT CAC
proP _M5_fw / ATC TGG AAA TGT GAC CAA GGG GTA ACC CAC CCC CGC TCA CC
proP _M6_fw / ATC TGG AAA TGT GAC CAA TTT TGC CCC CAC CCC CGC TCA CCT GC
proP _M7_fw / ATC TGG AAA TGT GAC CAA TTT GTA AAA ACC CCC CGC TCA CCT GCA TGA G
proP _M8_fw / ATC TGG AAA TGT GAC CAA TTT GTA ACC CAA AAC CGC TCA CCT GCA TGA GTG TG
betP_M_fw / GTT GAA AGC GGT GTG CGC AGA C
betP _M_rv / CGG GAG CAT CAC AAT GAC ATA AC
betP _M1_rv / CGG GAG CAT CAC AAT GAC ATA ACG ATG CGG TTT CCC TGG CAG AAA TAA CC
betP _M2_rv / CGG GAG CAT CAC AAT GAC ATA AAT CGT AAT TTT CCC TGG CAG AAA TAA CC
betP _M3_rv / CGG GAG CAT CAC AAT GAC CGC CCG ATT AAT TTT CCC TGG CAG AAA TAA CC
betP _M4_rv / CGG GAG CAT CAC ACG TCC ATA ACG ATT AAT TTT CCC TGG CAG AAA TAA CC
betP _M5_rv / CGG GAG CAT ACA CAT GAC ATA ACG ATT AAT TTT CCC TGG CAG AAA TAA CC
betP _M6_rv / CGG GAG ACG CAC AAT GAC ATA ACG ATT AAT TTT CCC
betP _M7_rv / CGG TCT CAT CAC AAT GAC ATA ACG ATT AAT TTT CCC
nadE-nrdH_fw / CCA CGT TTC AAG GCT CGC CAG
nadE-nrdH_rv / GTC ATT TAC GCA GTT CGC CAC C
csbD_fw / ACC GAA TTT TTC GAA CTT CAC AGG
csbD_rv / TTC TTT AGC CTT ACC TGT GAG GTC
cg1517/1516_fw / TCA TCG AAG GGC TCC CAA TCT G
cg1517/1516_rv / TTC CTC ATG TCA CAT TCC AAT CGG
cg1612_fw / GGA CGA AGT TAC TTT AAG AGA AGT C
cg1612_rv / CAG TGA GCG GTA CGA GGA TCA G
mtrA_fw / CGT GGT TGC CCC TGA TGA AGA C
mtrA_rv / CGG GAT CAT CAT CAA CCA CGA G
lpqB_fw / CAT GGA AAT TAT CGT GAA GCA CCG
lpqB_rv / GTC ACA GAC AGC ACC GCG GTG
ppmA_fw / GTG GTG AGA ATT TGA TCA GCA ACC
ppmA_rv / ACT GGT TCT GTG GCG ACA GCT C
mscL_fw / CGG AGA CAG AGC AGA TAA TTA CAC
mscL_rv / AGT TCA ATG ACA TTT CCG CGA AGG
rpf2_fw / CGT GAG GTA GCT CAC AGT CAA TC
rpf2_rv / GTT GAT CCG TGA CTT CTG ATG GG
cg0858-cg1859_fw / GGG CTC GTC GAG TTT CTG TTT TC
cg0858-cg1859_rv / TGC ATC GTC CCA TGC ACC CAT G
mepB_fw / CGC AGC AAC TTT GAT TGC TGA GC
mepB_rv / GCT TCT GCA ATT TCG CCT GCT TC
cg0136/0135_fw / GGT GGA TGA ATG TGG AGC GTG C
cg0136/0135_rv / CCA ACA CAG TCC GTT CAG GCT G
cg0133/0134_fw / CGT TTG GAT AAG GTT TCG GAG CC
cg0133/0134_rv / GTC TGG CTG TTG TCT TTC TGA GG
cg0715_fw / GTT CAA CAT TGA CGT CAG AAC AGC
cg0715_rv / GGC GGG CAC GAT GCT TTG AAT G
cg0896_fw / CAT CGC TTT ATT CAG TGC TTG GG
cg0896_rv / TTG GGG AGG AGG TGT GAG ACC
cg1418_fw / ATG AAG TAG AAA AAC AAC GCA ACA C
cg1418_rv / TGT TTG GAA GAG TGC TCA CGT GC
cg1942_fw / AGA GCT CAC CGC TAG ACA AGT AG
cg1942_rv / GCT AGT TTC CTC ATG TCA CAT TCC
cg1943_fw / TTG AAG GAC TTA ACC GAA GTT AAG G
cg1943_rv / CAC CGT CCA GAG CCA ATA GAT TC
cg2052_fw / GTG GTT TAC TCC CCT GCT TTT CG
cg2052_rv / AGC AAC AAT TCC ACC GGT AAC TG
cg2096_fw / TAG CGC CGA ACA ACT CCG GGC
cg2096_rv / GGG TTC ACC ATA GGG GTA ATT CG
cg2777_fw / GCG ATG GCG GCT TTA CTG AGG
cg2777_rv / TCC AAT TAT TGT TGT CGT CGC CTG
cg3254_fw / GCC TGG TTT GTC TTG TAA AGA GTG
cg3254_rv / CCA CCA ATA ATG ATC CAT GTA ATC C
Test_BS_NK / TCA GGG TGG ATG AAT GTG GAG CGT GC
Test_BS_cg0715 / TCA GTA CAA CAA TTC GTT CAC GAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_cg0896 / TCA GAA TCA CGT ACA TAT AAG GAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_cg1943 / TCA GCA ACA GAT ATC GCG AAC GAG GTG GAT GAA TGT GGA GCG TGC
Test_BS_cg2777 / TCA GAA GGA CAT TGT TAT GAC AAG GTG GAT GAA TGT GGA GCG TGC
Test_BS_mepA / TCA GGG TCA CAA CTT GAT AAC GGA GGT GGA TGA ATG TGG AGC GTG C
Test_BS_nadE-nrdH / TCA GTG TAA CGA TTC TGT AAC GGA GGT GGA TGA ATG TGG AGC GTG C
Test_BS_csbD / TCA GGG TAA CGG ATT TAT ATC AAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_cg1665 / TCA GGG TAA CAG ATC TGT TTC TAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_cg2096 / TCA GCA TAA CAT TTT TGT TGC AAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_1517/1516 / TCA GAA TAT CAT TCC TAT AAC AAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_cg1942 / TCA GGA TAT CAT TCC TAT AAC AAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_cg2052 / TCA GTG TAA CAT TCT TAT TAC AAG GTG GAT GAA TGT GGA GCG TGC
Test_BS_betP / TCA GCA TCA CAA TGA CAT AAC GAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_cg3254 / TCA GCA TCA CCT TTT AAT AAC AAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_cg0133/0134 / TCA GCA TAA CAT TTG CAT TAC GGT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_0858-cg0859 / TCA GAA TAA CAA GTC AAT AAC AAA GGT GGA TGA ATG TGG AGC GTG C
Test_BS_nlpC1 / TCA GGA TAA CGG TTC GAT AAC GAC GGT GGA TGA ATG TGG AGC GTG C
Test_BS_nlpC2 / TCA GAA TAA GGT TAC GAA ACC GCA GGT GGA TGA ATG TGG AGC GTG C
Test_BS_proP / TCA GGG TTA CAA ATT GGT CAC ATT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_ppmA / TCA GAG TCA CAA AAT GGA CAC TCT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_mepB / TCA GGA TAA CAA GAT GGT CTC AAT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_cg1612 / TCA GGG TCA CAA AAT GAT TTC AGT GGT GGA TGA ATG TGG AGC GTG C
Test_BS_rpf2 / TCA GAG TCA CAA TAC GGT AAC AAA GGT GGA TGA ATG TGG AGC GTG C
BS_mepA / AAG GTG GTC ACA ACT TGA TAA CGG ACT GG
BS_mepA-i / CCA GTC CGT TAT CAA GTT GTG ACC ACC TT
BS_mepA_LTTT / AAG GTG GTC ACA ACT TTA TAA CGG ACT GG
BS_mepA_LTTT-i / CCA GTC CGT TAT AAA GTT GTG ACC ACC TT
BS_mepA_LGGG / AAG GTG GTC ACA ACG GGA TAA CGG ACT GG
BS_mepA_LGGG-i / CCA GTC CGT TAT CCC GTT GTG ACC ACC TT
BS_mepA_LTTTG / AAG GTG GTC ACA ACT TTG ATA ACG GAC TGG
BS_mepA_LTTTG-i / CCA GTC CGT TAT CAA AGT TGT GAC CAC CTT
BS_mepA_LTTTCG / AAG GTG GTC ACA ACT TTC GAT AAC GGA CTG G
BS_mepA_LTTTCG-i / CCA GTC CGT TAT CGA AAG TTG TGA CCA CCT T
BS_mepA_LTTTCTG / AAG GTG GTC ACA ACT TTC TGA TAA CGG ACT GG
BS_mepA_LTTTCTG-i / CCA GTC CGT TAT CAG AAA GTT GTG ACC ACC TT
BS_mepA_LTG / AAG GTG GTC ACA ACT GAT AAC GGA CTG G
BS_mepA_LTG-i / CCA GTC CGT TAT CAG TTG TGA CCA CCT T
BS_mepA_LG / AAG GTG GTC ACA ACG ATA ACG GAC TGG
BS_mepA_LG-i / CCA GTC CGT TAT CGT TGT GAC CAC CTT
BS_mepA_L / AAG GTG GTC ACA ACA TAA CGG ACT GG
BS_mepA_L-i / CCA GTC CGT TAT GTT GTG ACC ACC TT

a In some cases oligonucleotides were designed to introduce recognition sites for restriction endonucleases (recognition sites in bold letters, restriction endonucleases indicated in parentheses). (Putative) MtrA binding sites are italicised, mutations are underlined and the linker within the MtrA binding motif are underlined and bold.


FIG. S1. Coomassie-stained SDS-polyacrylamide gel showing the purified MtrA-His protein before (lane 2) and after cleavage of the N-terminal histidine tag by thrombin (lane 3). Lane 1 contains the protein standard.

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FIG. S2. Experimental analysis of the linker region within the MtrA binding motif. The 3-bp linker (shown in bold letters) in the MtrA binding site of the mepA promoter (1) was changed in sequence (2 and 3) and in length (4 - 9) and the corresponding DNA fragments were tested in EMSAs for MtrA binding (for experimental details see legend to Fig. 1).

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FIG. S3. Alignment of the promoter regions of nlpC, mepA, rpf2, betP, proP, csbD and cg1517 of C. glutamicum. The transcriptional start points identified by primer extension are indicated with asterisks, the -10 and -35-regions are highlighted by gray boxes and the MtrA-binding sites are encircled by a rectangle.


FIG. S4. Autophosphorylation of the kinase domains MalE-MtrBK, MalE-CitAC and MBP-PhoSD1-246 and phosphoryl group transfer to the response regulators MtrA or PhoR. The purified kinase domains (12 µM final concentration) MalE-MtrBK (A), MalE-CitAC (B) and MBP-PhoSD1-246 (C and D) were incubated with γ-[32P]-ATP (80 µM; 6.25 Ci/mmol) for 20 min (A-C) or for 1 min (D). Then the purified response regulators MtrA (A, B, C) or PhoR (D) were added (12 µM final concentration), resulting in a 2-fold dilution of the kinase domains and the ATP.