Decrypting the H-NS-dependent regulatory cascade of acid stress resistance in Escherichia coli

Abstract

Background: H-NS regulates the acid stress resistance. The present study aimed to characterize the H-NS-dependent cascade governing the acid stress resistance pathways and to define the interplay between the different regulators.

Results: We combined mutational, phenotypic and gene expression analyses, to unravel the regulatory hierarchy in acid resistance involving H-NS, RcsB-P/GadE complex, HdfR, CadC, AdiY regulators, and DNA-binding assays to separate direct effects from indirect ones. RcsB-P/GadE regulatory complex, the general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways plays a central role in the regulatory cascade. However, H-NS also directly controls specific regulators of these pathways (e.g. cadC) and genes involved in general stress resistance (hdeAB, hdeD, dps, adiY). Finally, we found that in addition to H-NS and RcsB, a third regulator, HdfR, inversely controls glutamate-dependent acid resistance pathway and motility.

Conclusions: H-NS lies near the top of the hierarchy orchestrating acid response centred on RcsB-P/GadE regulatory complex, the general direct regulator of glutamate-, arginine- and lysine-dependent acid resistance pathways.

Figure 1

Gel mobility shift assays with GadE/RcsB_D56E complex, HdfR and AdiY. A. Gel mobility shift assays with GadE/RcsB_D56E complex and new DNA targets. Proteins were incubated with DNA targets during 30 min at 25°C in the final reaction mixture volume of 15 μl. 900 ng of each GadE and RcsB_D56E protein are used for yhiM and aslB. B. Gel mobility shift assays with HdfR or AdiY proteins. Quantities of purified HdfR or AdiY proteins are indicated above each lane (in ng). Gel mobility shift assays (A and B) were performed with 0.1 ng [γ³²P]-labelled DNA fragment and loaded on a 6% polyacrylamide native gel. An arrow points out the position of the DNA-regulatory protein complex. An asterisk marks the position of the unbound probe.

Figure 2

Competitive gel mobility shift assay with H-NS, target promoter fragments and restriction fragments derived from plasmid pBR322. The cleaved plasmid and promoter fragments were incubated with the indicated concentrations of purified H-NS protein (in μM). After protein-DNA complex formation, the fragments were resolved on a 3% (A) or 4% (B) MetaPhor agarose gel. An asterisk indicates the position of the target promoter fragments. "bla" indicates the bla promoter (positive control), the other fragments of plasmid DNA correspond to negative controls. The specific binding of H-NS is observed when bands corresponding to bla and target promoter disappear with increasing concentration of H-NS, the H-NS-DNA complex being difficult to visualize under these conditions.

Figure 3

Model of the H-NS-dependent regulatory network in flagella and acid stress control. At the top, H-NS positively controls motility and represses acid stress resistance. Genes in cross symbol are directly activated by H-NS; in rectangle: directly repressed by H-NS; in circle: indirectly repressed by H-NS. Regulatory proteins are indicated with upper case. Orange filling: flagellum synthesis process; Pink filling: glutamate-dependent acid resistance process; Blue filling: arginine-dependent acid resistance process; Red filling: lysine-dependent acid resistance process; Green filling: genes involved in three different acid resistance processes. Gene names in yellow indicate the direct targets of RcsB-P/GadE complex placed at the centre of this regulatory cascade. A positive effect on transcription is indicated by arrows and a negative regulatory effect is indicated by blunt ended lines. Direct regulation is indicated by solid lines. Indirect regulation is indicated by dashed lines. Previously published results are included in the scheme: [1-3,5-7,10,16,32-40].