Temperature and Mg2+ Sensing by a Novel PhoP-PhoQ Two-component System for Regulation of Virulence in Edwardsiella tarda

Smarajit Chakraborty,Mo Li,Chiradip Chatterjee,J Sivaraman,Ka Yin Leung,Yu-Keung Mok

doi:10.1074/jbc.m110.179150

Smarajit Chakraborty, Mo Li + Show 4 more

Open Access

https://doi.org/10.1074/jbc.m110.179150

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Abstract

The PhoP-PhoQ two-component system is commonly used by bacteria to sense environmental factors. Here we show that the PhoP-PhoQ system of Edwardsiella tarda detects changes in environmental temperature and Mg(2+) concentration as well as regulates the type III and VI secretion systems through direct activation of esrB. Protein secretion is activated from 23 to 35 °C or at low Mg(2+) concentrations, but it is suppressed at or below 20 °C, at or above 37 °C, or at high Mg(2+) concentrations. The effects of temperature and Mg(2+) concentration are additive. The PhoQ sensor domain has a low T(m) of 37.9 °C, and it detects temperatures through a conformational change of its secondary structure. Mutation of specific Pro or Thr residues increased the stability of the PhoQ sensor drastically, altering its temperature-sensing ability. The PhoQ sensor detects Mg(2+) concentration through the direct binding of Mg(2+) to a cluster of acidic residues (DDDSAD) and through changes that likely affect its tertiary structure. Here, we describe for the first time the use of PhoP-PhoQ as a temperature sensor for bacterial virulence control.

Highlights

Edwardsiella tarda is a Gram-negative bacterial pathogen that is associated with septicemia and fatal infections in a wide variety of animals including fish and humans [1, 2]
Identification of the PhoP-PhoQ Two-component System— Using genome walking with degenerate primers derived from the conserved nucleotide sequences of related bacterial species, the phoP and phoQ genes of E. tarda PPD130/91 (GenBankTM accession code GU324976) were identified and sequenced
Using RT-PCR experiments on RNA isolated from E. tarda PPD130/91, the phoP and phoQ genes are found to be transcribed as an operon

Summary

EXPERIMENTAL PROCEDURES

Cloning of the PhoP-PhoQ Two-component System in E. tarda PPD130/91—Bacterial genomic DNA was extracted using the Wizard genomic DNA purification kit (Promega, Madison, WI). Samples containing 20 ␮M PhoQ sensor and various amounts of urea were prepared in a buffer containing 20 mM sodium phosphate, pH 6.5, and 100 mM NaCl in the presence or absence of 10 mM Mg2ϩ. Samples containing 2 ␮M PhoQ sensor and different concentrations of urea were prepared in a buffer containing 20 mM sodium phosphate, pH 6.5, and 100 mM NaCl in the presence or absence of 10 mM Mg2ϩ. For the construction of phoPi, an internal fragment of phoP was amplified from E. tarda genomic DNA with the primer pair phoPmut (supplemental Table 1), which contains a KpnI restriction enzyme site. To obtain phoPi ϩ phoP, the complete phoP gene was prepared by PCR using the primer pair phoPfull (supplemental Table 1) from E. tarda PPD130/91 genomic DNA. The protein concentration was determined with a Bio-Rad protein assay kit using bovine serum albumin as the standard

RESULTS

Thr and Pro Residues Are Responsible for Temperature

DISCUSSION