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Substrate selectivity in arginine-dependent acid resistance in enteric bacteria.

ABSTRACT: To successfully colonize the human gut, enteric bacteria must activate acid resistance systems to survive the extreme acidity (pH 1.5-3.5) of the stomach. The antiporter AdiC is the master orchestrator of the arginine-dependent system. Upon acid shock, it imports extracellular arginine (Arg) into the cytoplasm, providing the substrate for arginine decarboxylases, which consume a cellular proton ending up in a C-H bond of the decarboxylated product agmatine (Agm(2+)). Agm(2+) and the "virtual" proton it carries are exported via AdiC subsequently. It is widely accepted that AdiC counters intracellular acidification by continuously pumping out virtual protons. However, in the gastric environment, Arg is present in two carboxyl-protonation forms, Arg(+) and Arg(2+). Virtual proton pumping can only be achieved by Arg(+)/Agm(2+) exchange, whereas Arg(2+)/Agm(2+) exchange would produce no net proton movement. This study experimentally asks which exchange AdiC catalyzes, an issue previously unapproachable due to the absence of a reconstituted system mimicking the situation of bacteria in the stomach. Here, using an oriented liposome system able to hold a three-unit pH gradient, we demonstrate that Arg/Agm exchange by AdiC is strongly electrogenic with positive charge moved outward, and thus that AdiC mainly mediates Arg(+)/Agm(2+) exchange to support effective virtual proton pumping. Further experiments reveal a mechanistic surprise--that AdiC selects Arg(+) against Arg(2+) on the basis of gross valence, rather than by local scrutiny of protonation states of the carboxyl group, as had been suggested by Arg-bound AdiC crystal structures.

SUBMITTER: Tsai MF

PROVIDER: S-EPMC3625328 | biostudies-other | 2013 Apr

REPOSITORIES: biostudies-other

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Substrate selectivity in arginine-dependent acid resistance in enteric bacteria.

Tsai Ming-Feng MF Miller Christopher C

Proceedings of the National Academy of Sciences of the United States of America 20130325 15

To successfully colonize the human gut, enteric bacteria must activate acid resistance systems to survive the extreme acidity (pH 1.5-3.5) of the stomach. The antiporter AdiC is the master orchestrator of the arginine-dependent system. Upon acid shock, it imports extracellular arginine (Arg) into the cytoplasm, providing the substrate for arginine decarboxylases, which consume a cellular proton ending up in a C-H bond of the decarboxylated product agmatine (Agm(2+)). Agm(2+) and the "virtual" pr ...[more]

PMID: 23530220

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Project description:Neutralophilic bacteria have developed several strategies to overcome the deleterious effects of acid stress. In particular, the amino acid-dependent systems are widespread, with their activities overlapping, covering a rather large pH range, from 6 to <2. Recent reports showed that an acid resistance (AR) system relying on the amino acid glutamine (AR2_Q), the most readily available amino acid in the free form, is operative in Escherichia coli, Lactobacillus reuteri, and some Brucella species. This system requires a glutaminase active at acidic pH and the antiporter GadC to import L-glutamine and export either glutamate (the glutamine deamination product) or GABA. The latter occurs when the deamination of glutamine to glutamate, via acid-glutaminase (YbaS/GlsA), is coupled to the decarboxylation of glutamate to GABA, via glutamate decarboxylase (GadB), a structural component of the glutamate-dependent AR (AR2) system, together with GadC. Taking into account that AR2_Q could be widespread in bacteria and that until now assays based on ammonium ion detection were typically employed, this work was undertaken with the aim to develop assays that allow a straightforward identification of the acid-glutaminase activity in permeabilized bacterial cells (qualitative assay) as well as a sensitive method (quantitative assay) to monitor in the pH range 2.5-4.0 the transport of the relevant amino acids in vivo. The qualitative assay is colorimetric, rapid and reliable and provides several additional information, such as co-occurrence of AR2 and AR2_Q in the same bacterial species and assessment of the growth conditions that support maximal expression of glutaminase at acidic pH. The quantitative assay is HPLC-based and allows to concomitantly measure the uptake of glutamine and the export of glutamate and/or GABA via GadC in vivo and depending on the external pH. Finally, an extensive bioinformatic genome analysis shows that the gene encoding the glutaminase involved in AR2_Q is often nearby or in operon arrangement with the genes coding for GadC and GadB. Overall, our results indicate that AR2_Q is likely to be of prominent importance in the AR of enteric bacteria and that it modulates the enzymatic as well as antiport activities depending on the imposed acidic stress.

Dataset Information

Substrate selectivity in arginine-dependent acid resistance in enteric bacteria.

Publications

Substrate selectivity in arginine-dependent acid resistance in enteric bacteria.

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