Cloning of Strains and Plasmids. As Described Earlier (1), RU1618 (TRC) Was Used to Express

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Cloning of strains and plasmids. As described earlier (1), RU1618 (TRC) was used to express PhoB at higher levels than RU1616 (LAC) but it displayed a high expression level of PhoB even in the absence of IPTG. To create a strain with a lower basal level and a wider range of expression levels than RU1618, RU1783 was generated by integration of the pRG378 plasmid containing lacIq to repress the basal expression of the trc promoter. pRG378 was created by cloning a SmaI / SacI digested lacIq fragment into a pAH63 derivative plasmid.

To create reporter plasmids, PCR fragments containing different PhoB-regulated promoters were amplified from E. coli chromosome, digested with PstI / XbaI and cloned into pJZG146 containing a promoter-less yfp. Primers used for amplification are: phoB, #592 (5’-GTCACTGCAGACGGTAGTATTGAGGAACGCC-3’) and #593 (5’-GCACTCTAGATCAG CCCTGTTGTAATAAATAGG-3’) ; phoE, RG246 (5’-GGTCCTGCAGCGAACGTTTTAGC AGGA CTG-3’) and RG247 (5’-GGACTCTAGAGCGAATATTCAGCGGGAGAGTCC-3’); ugpB, RG244 (5’-GGTCCTGCAGGCATGAAACGATGAGCAATC-3’) and RG245 (5’-GGACTCTAGACTCTTGTTGTACCGAATGCGC-3’); phnC, RG85 (5’-GCACTGCAGGAAG AAGGAAAACGCTGGTT-3’) and RG86 (5’-GCCTCTAGATCAAATCGTTTGCATGATGCA G-3’). phoA promoter variants were created by recombinant PCR using the following primers: RG84 (5’-GCACTGCAGGCAATGCTTCGCAATATGGC-3’), RG63 (5’-GCCTCTAGATCAGT CCGGGCTTTTGTCACA-3’), RG290 (5’-TGTCATAAATTTGTCATAAACGAGACTTATAGTCG-3’) and RG291 (5’-CGACTATAAGTCTCGTTTATGACAAATTTATGACA-3’), followed by digestion with PstI / XbaI and cloned into pRG161.

Sequences of DNA fragments for in vitro binding assays. DNA fragments used for biolayer interferometry were generated by PCR with the biotinylated primer RG-biotin-1 (5’-biotin-CTTGCTCACCATTTGTATATCTCCTTC-3’) and specific primers for individual promoters using corresponding reporter plasmids as templates. For EMSA assays, a fluorescein labeled primer, RG-FAM-1 (5’-FAM-GCTCACCATTTGTATATCTCCTTC-3’) was used to generate fluorescent DNA fragments. PCR products were purified with QIAquick columns (QIAGEN) and DNA concentrations were determined by absorbance reading at 260nm using a Nanodrop ND-1000 (NanoDrop Technologies, Inc.). Sequences are shown with PhoB-binding sites underlined and lower case letters indicating common DNA sequences (~54 bp) from the reporter vector. Transcription start sites are colored in blue and numbers in the parentheses mark the boundaries of DNA fragments relative to the transcription start unless specified otherwise.

For biolayer interferometry:

phoB: (-138 to +36)

GCGAGAGCTCGCATGGGCGCGATTATACCCAAACAGATGTGCCATTTGCTTTTTTCTGCGCCACGGAAATCAATAACCTGAAGATATGTGCGACGAGCTTTTCATAAATCTGTCATAAATCTGACGCATAATGACGTCGCATTAATGATCGCAACCTATTTATTACAACAGGGCtgatctagaaataattttgtttaactttaagaaggagatatacaaatggtgagc

phoE: (-200 to +33)

GGTCCTGCAGCGAACGTTTTAGCAGGACTGTCGTCGGTTGCCAACCATCTGCGAGCAAAGCATGGCGTTTTGTTGCGCGGGATCAGCAAGCCTAGCGGCAGTTGTTTACGCTTTTATTACAGATTTAATAAATTACCACATTTTAAGAATATTATTAATCTGTAATATATCTTTAACAATCTCAGGTTAAAAACTTTCCTGTTTTCAACGGGACTCTCCCGCTGAATATTCGCtgatctagaaataattttgtttaactttaagaaggagatatacaaatggtgagc

ugpB: (-197 to +44)

GGTCCTGCAGGCATGAAACGATGAGCAATCTGTAGAGTTTGATTCAGACCTTCTATATTTTCCCGCTTATCCGTGCCCCATCTCCCATTTTCCCTCACCCACGCCGTCACCGCCTTGTCATCTTTCTGACACCTTA CTATCTTACAAATGTAACAAAA AAGTTATTTTTCTGTAATTCGAGCATGTCATGTTACCCCGCGAGCATAAAACGCGTGAATTCGCGCATTCGGTACAACAAGAGtgatctagaaataattttgtttaactttaagaaggagatatacaaatggtgagc

phoA: (-134 to +108)

CCAGCATTCCTGACGACGATACGGAGCTGCTGCGCGATTACGTAAAGAAGTTATTGAAGCATCCTCGTCAGTAAAAAGTTAATCTTTTCAACAGCTGTCATAAAGTTGTCACGGCCGAGACTTATAGTCGCTTTGTTTTTATTTTTTAATGTATTTGTACATGGAGAAAATAAAGTGAAACAAAGCACTATTGCACTGGCACTCTTACCGTTACTGTTTACCCCTGTGACAAAAGCCCGGACtgatctagaaataattttgtttaactttaagaaggagatatacaaatggtgagc

phnC: (-297 to +12 relative to translation start shown in italics, transcription start is poorly defined)

GCACTGCAGGAAGAAGGAAAACGCTGGTTTGACAATCTTGCCGCTAACGGAAAAATCGAAATGGCCTGGCAGGAAACTTTCTGGGCGCATGGCTTTGGCAAAGTCACCGATAAATTTGGCGTACCGTGGATGATTAATGTCGTCAAACAACAACCAACGCAATAACCCGCCGGGAGGCCCGCCCTCCCGCACTGTCATCGAATTCCCGTTAACTCTTCATCTGTTAGTCACTTTTAATTAACCAAATCGTCACAATAATCCGCCACGATGGAGCCACTTTTTTAGGGAGGCTGCATCATGCAAACGATTtgatctagaaataattttgtttaactttaagaaggagatatacaaatggtgagc

For EMSA:

phoB: (-79 to +36)

GCCACGGAAATCAATAACCTGAAGATATGTGCGACGAGCTTTTCATAAATCTGTCATAAATCTGACGCATAATGACGTCGCATTAATGATCGCAACCTATTTATTACAACAGGGCtgatctagaaataattttgtttaactttaagaaggagatatacaaatggtgagc

phoA: (-79 to +34)

GAAGCATCCTCGTCAGTAAAAAGTTAATCTTTTCAACAGCTGTCATAAAGTTGTCACGGCCGAGACTTATAGTCGCTTTGTTTTTATTTTTTAATGTATTTGTACATGGAGAAtctagaaataattttgtttaactttaagaaggagatatacaaatggtgagc

1.Gao R., Stock A. M. 2013. Probing kinase and phosphatase activities of two-component systems in vivo with concentration-dependent phosphorylation profiling. Proc. Natl. Acad. Sci. USA 110:672-677.