Escherichia coli HdeB Is an Acid Stress Chaperone

Abstract

We cloned, expressed, and purified the hdeB gene product, which belongs to the hdeAB acid stress operon. We extracted HdeB from bacteria by the osmotic-shock procedure and purified it to homogeneity by ionexchange chromatography and hydroxyapatite chromatography. Its identity was confirmed by mass spectrometry analysis. HdeB has a molecular mass of 10 kDa in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, which matches its expected molecular mass. We purified the acid stress chaperone HdeA in parallel in order to compare the two chaperones. The hdeA and hdeB mutants both display reduced viability upon acid stress, and only the HdeA/HdeB expression plasmid can restore their viability to close to the wild-type level, suggesting that both proteins are required for optimal protection of the bacterial periplasm against acid stress. Periplasmic extracts from both mutants aggregate at acidic pH, suggesting that HdeA and HdeB are required for protein solubilization. At pH 2, the aggregation of periplasmic extracts is prevented by the addition of HdeA, as previously reported, but is only slightly reduced by HdeB. At pH 3, however, HdeB is more efficient than HdeA in preventing periplasmic-protein aggregation. The solubilization of several model substrate proteins at acidic pH supports the hypothesis that, in vitro, HdeA plays a major role in protein solubilization at pH 2 and that both proteins are involved in protein solubilization at pH 3. Like HdeA, HdeB exposes hydrophobic surfaces at acidic pH, in accordance with the appearance of its chaperone properties at acidic pH. HdeB, like HdeA, dissociates from dimers at neutral pH into monomers at acidic pHs, but its dissociation is complete at pH 3 whereas that of HdeA is complete at a more acidic pH. Thus, we can conclude that Escherichia coli possesses two acid stress chaperones that prevent periplasmic-protein aggregation at acidic pH.

Role of the Multidrug Resistance Regulator MarA in Global Regulation of the hdeAB Acid Resistance Operon in Escherichia coli

“…Both chaperones are only active below pH 3 and prevent irreversible aggregation of acid-denatured periplasmic proteins (14,21,23,28). HdeA seems to play a major role at pH 2, whereas HdeB is more active at pH 3 (23). The determined HdeA crystal structure and the model HdeB structure are similar (14,45), in agreement with their genetic and functional similarities.…”

“…Both chaperones are only active below pH 3 and prevent irreversible aggregation of acid-denatured periplasmic proteins (14,21,23,28). HdeA seems to play a major role at pH 2, whereas HdeB is more active at pH 3 (23).…”

Comparative proteomics reveal distinct chaperone–client interactions in supporting bacterial acid resistance

“…This stress-specific unfolding mechanism enables the exposure of these monomers' hydrophobic surfaces to interact with an array of client proteins in a promiscuous fashion (13-15); however, this unique disorder-triggered interaction between chaperones and clients makes direct characterization of the recognition mechanism very challenging, especially within native cellular contexts. HdeA has been recognized as the major acid chaperone, whereas HdeB has a smaller hydrophobic surface with weaker chaperoning activity in vitro (6). In particular, although HdeB has been suggested to have certain functions together with HdeA at pH 3, no distinct HdeB clients have been identified to date.…”

Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

“…Some of the best-characterized systems involve amino acid decarboxylases, which consume protons during amino acid decarboxylation, and antiporters that exchange product for substrate (29). Finally, DNA repair systems (10,81,82) and chaperones (8,31,41,46,50) appear to be important for the reparation of cellular material damaged by exposure to acid.…”