Substrate selectivity in glutamate-dependent acid resistance in enteric bacteria

Abstract

The bacterial antiporter GadC plays a central role in the glutamate (Glu)-dependent acid resistance system, which protects enteric bacteria against the extreme acidity of the human stomach. Upon acid shock, GadC imports Glu into the cytoplasm, where Glu decarboxylases consume a cytoplasmic proton, which ends up as a “virtual” proton in the decarboxylated product γ-aminobutyric acid (GABA) and is then exported via GadC. It was therefore proposed that GadC counters intracellular acidification by continually pumping out virtual protons. This scenario, however, is oversimplified. In gastric environments, GadC encounters substrates in multiple carboxyl protonation forms (outside: Glu − , Glu 0 , Glu + ; inside: GABA 0 , GABA + ). Of the six possible combinations of antiport partners, Glu + /GABA 0 results in proton influx, Glu 0 /GABA 0 and Glu + /GABA + are proton neutral, and Glu − /GABA 0 , Glu − /GABA + , or Glu 0 /GABA + lead to proton extrusion. Which of these exchanges does GadC catalyze? To attack this problem, we developed an oriented GadC liposome system holding a three-unit inward pH gradient to mimic the conditions facing bacteria in the stomach. By assessing the electrogenicity of substrate transport, we demonstrate that GadC selectively exchanges Glu − or Glu 0 with GABA + , resulting in effective proton extrusion of >0.9 H + per turnover to counter proton invasion into acid-challenged bacteria. We further show that GadC selects among protonated substrates using a charge-based mechanism, rather than directly recognizing the protonation status of the carboxyl groups. This result paves the way for future work to identify the molecular basis of GadC’s substrate selectivity.

Molecular mechanism of pH-dependent substrate transport by an arginine-agmatine antiporter

“…Considerable investigative efforts have focused on a potential role of the substrate molecules Glu and GABA, because the pK a values of their carboxylate side chains are close to the pH range in which GadC undergoes activation transition. Gratifyingly, GadC selectively transports Glu and GABA with their carboxylate side chains fully protonated (22,23). Because protonation of the carboxylate is favored by decreasing the pH value from 6.5 to 5.5 or lower, Glu and GABA clearly play a role in their pH-dependent transport.…”

Structural and Mechanistic Basis of Proton-Coupled Metal Ion Transport in the SLC11/NRAMP Family

“…This was likely caused by the high protein to lipid ratio, the choice of lipids used during reconstitution, and the use of comparably high ion concentrations required due to the slow kinetics of transport. To overcome these limitations we investigated the reconstitution of EcoDMT with different lipids at lower protein to lipid ratio 20 and we found conditions where we detected little activity in the absence of ionophores and a strong enhancement of Mn 2 þ transport, on addition of valinomycin, which dissipates the membrane potential established by the electrogenic transporter ( Supplementary Fig. 2b).…”

“…Proteoliposomes containing EcoDMT were prepared with destabilized liposomes 57 . Synthetic phospholipids dissolved in chloroform (POPE, POPG from Avanti Polar Lipids) at a w/w ratio of 3:1 were used as lipid source 20 . Lipids were dried, washed with diethylether and dried by exsiccation.…”

The effect of Mongolian medical acupuncture on cytokines and neurotransmitters in the brain tissue of insomniac rats

“…GABA stems from Glu under the action of glutamic acid decarboxylase[28][29][30][31][32][33]. It exerts its biological functions by binding with the GABA receptor.Both jointly adjust the excitability and inhibition of the nervous system for sleep regulation.The Interleukin, IL-1 is the earliest-discovered sleep-promoting cytokine.…”