Molecular Characterization of the Acid-InducibleasrGene ofEscherichia coliand Its Role in Acid Stress Response

Enterobacteria have developed numerous constitutive and inducible strategies to sense and adapt to an external acidity. These molecular responses require dozens of specific acid shock proteins (ASPs), as shown by genomic and proteomic analysis. Most of the ASPs remain poorly characterized, and their role in the acid response and survival is unknown. We recently identified an Escherichia coli gene, asr (acid shock RNA), encoding a protein of unknown function, which is strongly induced by high environmental acidity (pH < 5.0). We show here that Asr is required for growth at moderate acidity (pH 4.5) as well as for the induction of acid tolerance at moderate acidity, as shown by its ability to survive subsequent transfer to extreme acidity (pH 2.0). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis and Western analysis of acid-shocked E. coli cells harboring a plasmid-borne asr gene demonstrated that the Asr protein is synthesized as a precursor with an apparent molecular mass of 18 kDa. Mutational studies of the asr gene also demonstrated the Asr preprotein contains 102 amino acids. This protein is subjected to an N-terminal cleavage of the signal peptide and a second processing event, yielding 15- and 8-kDa products, respectively. Only the 8-kDa polypeptide was detected in acid-shocked cells containing only the chromosomal copy of the asr gene. N-terminal sequencing and site-directed mutagenesis revealed the two processing sites in the Asr protein precursor. Deletion of amino acids encompassing the processing site required for release of the 8-kDa protein resulted in an acid-sensitive phenotype similar to that observed for the asr null mutant, suggesting that the 8-kDa product plays an important role in the adaptation to acid shock. Analysis of Asr:PhoA fusions demonstrated a periplasmic location for the Asr protein after removal of the signal peptide. Homologues of the asr gene from other Enterobacteriaceae were cloned and shown to be induced in E. coli under acid shock conditions.