Table_2_The Glutaminase-Dependent Acid Resistance System: Qualitative and Quantitative Assays and Analysis of Its Distribution in Enteric Bacteria.PDF

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.

嗜中性菌（Neutralophilic bacteria）已演化出多种策略以抵御酸胁迫（acid stress）带来的有害影响。其中，氨基酸依赖系统（amino acid-dependent systems）分布广泛，其活性存在重叠，可覆盖6至<2的宽泛pH区间。近期研究表明，一种依赖氨基酸谷氨酰胺（glutamine）的酸抗性（acid resistance, AR）系统（AR2_Q）——谷氨酰胺是游离形式下最易获取的氨基酸——在大肠杆菌（Escherichia coli）、罗伊氏乳杆菌（Lactobacillus reuteri）以及部分布鲁氏菌属（Brucella）物种中发挥作用。该系统需要在酸性pH下具有活性的谷氨酰胺酶（glutaminase），以及反向转运蛋白（antiporter）GadC来摄取L-谷氨酰胺，并输出谷氨酸（谷氨酰胺脱氨产物）或γ-氨基丁酸（γ-aminobutyric acid, GABA）。当通过酸性谷氨酰胺酶（acid-glutaminase，YbaS/GlsA）将谷氨酰胺脱氨为谷氨酸的过程，与通过谷氨酸脱羧酶（glutamate decarboxylase，GadB）——谷氨酸依赖型酸抗性（AR2）系统的结构组分，与GadC协同发挥作用——将谷氨酸脱羧为γ-氨基丁酸的过程偶联时，就会生成后者。鉴于AR2_Q可能广泛存在于细菌中，且此前通常采用基于铵离子检测的检测方案，本研究旨在开发可直接鉴定透化细菌细胞中酸性谷氨酰胺酶活性的定性检测法（qualitative assay），以及一种灵敏的定量检测法（quantitative assay），以在2.5–4.0的pH范围内体内监测相关氨基酸的转运情况。该定性检测法为比色法，具备快速、可靠的特点，还可提供多项额外信息，例如同一细菌物种中AR2与AR2_Q的共现情况，以及评估可在酸性pH下支持谷氨酰胺酶最大表达的培养条件。该定量检测法基于高效液相色谱（high performance liquid chromatography, HPLC），可在体内同步测定谷氨酰胺的摄取量，以及GadC介导的谷氨酸和/或γ-氨基丁酸的输出量，且该过程受外界pH影响。此外，大规模生物信息学基因组分析显示，编码AR2_Q相关谷氨酰胺酶的基因，通常与编码GadC和GadB的基因相邻，或与其处于同一操纵子（operon）结构中。综上，我们的研究结果表明，AR2_Q可能在肠杆菌（enteric bacteria）的酸抗性中发挥关键作用，且其可根据施加的酸胁迫调控酶活性及反向转运活性。