The Glutaminase-Dependent Acid Resistance System: Qualitative and Quantitative Assays and Analysis of Its Distribution in Enteric Bacteria

Abstract

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.

Microbial interaction between the succinate‐utilizing bacterium Phascolarctobacterium faecium and the gut commensal Bacteroides thetaiotaomicron

“…B . thetaiotaomicron possesses this system (Pennacchietti et al, 2018). The difference in pH values between the monoculture (pH 4.9) and co‐culture (pH 5.3) resulted in the downregulation of the glutamate‐dependent acid resistance system‐related genes in the co‐culture.…”

Understanding How Microorganisms Respond to Acid pH Is Central to Their Control and Successful Exploitation

“…Second, cells can deploy reactions that produce basic compounds to help neutralize the low pH. The production of ammonia from urea or amine-containing amino acids such as arginine or glutamine is commonly used to counteract acidity ( Krulwich et al, 2011 ; Pennacchietti et al, 2018 ). Third, many microbial cell types eliminate protons from the cells at the expense of ATP consumption.…”

Regulation of γ-Aminobutyrate (GABA) Utilization in Corynebacterium glutamicum by the PucR-Type Transcriptional Regulator GabR and by Alternative Nitrogen and Carbon Sources

“…An antiporter takes up L-glutamate and exports GABA. This acid stress response can also start from L-glutamine, which is first converted to L-glutamate and ammonia by a glutaminase (Pennacchietti et al, 2018). The human gut microbiome contains several GABA-producing or GABA-consuming species, and there might be a connection between GABA production in the gut and depressive disorders (Strandwitz et al, 2019).…”