Signal-dependent Binding of the Response Regulators PhoP and PmrA to Their Target Promoters in Vivo

Dongwoo Shin,Eduardo A Groisman

doi:10.1074/jbc.m412741200

Dongwoo Shin, Eduardo A Groisman

Open Access

https://doi.org/10.1074/jbc.m412741200

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Abstract

Low Mg2+ promotes phosphorylation of the response regulators PhoP and PmrA and transcription of their activated genes in Salmonella enterica. Using chromatin immunoprecipitation, we have now determined that the PhoP and PmrA proteins are recruited to the regulatory region of their target genes when bacteria experience low Mg2+ but not when they are grown in high Mg2+. Even when the PhoP protein was artificially produced at 4-fold higher levels than the wild-type strain, promoter occupancy required the low Mg2+ signal. Substitution of the predicted phosphorylation site Asp-52 with a valine residue abolished phosphorylation of the PhoP protein, resulting in loss of PhoP binding to target promoters and transcription of PhoP-activated genes. Our results indicate that the promoter binding ability of the PhoP and PmrA proteins occurring in low Mg2+ is correlated with phosphorylation of these proteins in vivo.

Highlights

The PhoP/PhoQ two-component system governs the adaptation to low Mg2ϩ, virulence in mammals as well as several other physiological functions in Salmonella enterica and other Gram-negative bacteria [1]
When PCR was performed on the DNA that was precipitated with the anti-HA antibody using primers specific for the mgtA and pmrD promoters, a dramatic enrichment of PhoP-associated DNA was observed for the epitope-tagged strain grown in low Mg2ϩ (Fig. 2, A and B)
No products were recovered when the PCR was performed using primers specific for the phoP coding region or the rpoD promoter, which lack sequences resembling the PhoP box. These results demonstrate that the PhoP and PmrA proteins bind to their target promoters in response to low Mg2ϩ

Summary

EXPERIMENTAL PROCEDURES

The CmR cassette from plasmid pKD3 was amplified using primers 2566 and 2567 and integrated at the 3Ј end of the phoP gene in the chromosome by selecting for CmR transformants [14]. Primer 2567 (5Ј-TACCACCGTACGCGGACAAGGATATCTTTTTGAATTGCGCTATCCGTATGATGTTCCTGATTATGCTAGCCTCTGATGTAGGCTGGAGCTGCTTCG-3Ј) was designed to encode the HA sequence immediately upstream of the stop codon of the phoP gene following the priming site 1 sequence of pKD3 [14]. We used primers 2491 (5Ј-TCGCGGGTTTGGCTACATGCTGGTTGCCACTGAGGAAAGCGACTACAAGGACGACGATGACAAGTGATGTAGGCTGGAGCTGCTTCG-3Ј) and 2482 (5Ј-TTAAACGCTGGCGAAGGGTCATCGCTCTTCGCTGAAAACGCATATGAATATCCTCCTTAG-3Ј) to amplify the CmR cassette from plasmid pKD3 and integrated the PCR product at the 3Ј end of the pmrA gene in a strategy analogous to that described above. To construct a strain harboring a chromosomal deletion of the phoP and phoQ genes (EG15599), the CmR cassette from plasmid pKD3 was amplified using primers 3726 and 4986 [14]. Plasmid pUHE-phoP-HA/phoQ (pEG13918) was constructed by cloning between the EcoRI and PstI sites of pUHE21–2lacIq [15] a PCR fragment containing the phoP-HA/phoQ coding region generated with

The abbreviations used are

RESULTS

DISCUSSION