The yhiM gene codes for an inner membrane protein involved in GABA export in Escherichia coli

In order to survive the exposure to acid pH, Escherichia coli activates molecular circuits leading from acid tolerance to extreme acid resistance (AR). The activation of the different circuits involves several global and specific regulators affecting the expression of membrane, periplasmic and cytosolic proteins acting at different levels to dampen the harmful consequences of the uncontrolled entry of protons intracellularly. Many genes coding for the structural components of the AR circuits (protecting from pH ≤ 2.5) and their specific transcriptional regulators cluster in a genomic region named AFI (acid fitness island) and respond in the same way to global regulators (such as RpoS and H-NS) as well as to anaerobiosis, alkaline, cold and respiratory stresses, in addition to the acid stress. Notably some genes coding for structural components of AR, though similarly regulated, are non-AFI localised. Amongst these the gadBC operon, coding for the major structural components of the glutamate-based AR system, and the ybaS gene, coding for a glutaminase required for the glutamine-based AR system. The yhiM gene, a non-AFI gene, appears to belong to this group. We mapped the transcription start of the 1.1 kb monocistronic yhiM transcript: it is an adenine residue located 22 nt upstream a GTG start codon. By real-time PCR we show that GadE and GadX equally affect the expression of yhiM under oxidative growth conditions. While YhiM is partially involved in the RpoS-dependent AR, we failed to detect a significant involvement in the glutamate- or glutamine-dependent AR at pH ≤ 2.5. However, when grown in EG at pH 5.0, the yhiM mutant displays impaired GABA export, whereas when YhiM is overexpressed, an increases of GABA export in EG medium in the pH range 2.5–5.5 is observed. Our data suggest that YhiM is a GABA transporter with a physiological role more relevant at mildly acidic pH, but not a key component of AR at pH < 2.5.