University of Alberta, Department of Agricultural, Food and Nutritional Science, Edmonton, Canada.
Microb Cell Fact. 2011 Aug 30;10 Suppl 1(Suppl 1):S8. doi: 10.1186/1475-2859-10-S1-S8.
Acid stress impacts the persistence of lactobacilli in industrial sourdough fermentations, and in intestinal ecosystems. However, the contribution of glutamate to acid resistance in lactobacilli has not been demonstrated experimentally, and evidence for the contribution of acid resistance to the competitiveness of lactobacilli in sourdough is lacking. It was therefore the aim of this study to investigate the ecological role of glutamate decarboxylase in L. reuteri.
A gene coding for a putative glutamate decarboxylase, gadB, was identified in the genome of L. reuteri 100-23. Different from the organization of genetic loci coding for glutamate decarboxylase in other lactic acid bacteria, gadB was located adjacent to a putative glutaminase gene, gls3. An isogenic deletion mutant, L. reuteri ∆gadB, was generated by a double crossover method. L. reuteri 100-23 but not L. reuteri ∆gadB converted glutamate to γ-aminobutyrate (GABA) in phosphate butter (pH 2.5). In sourdough, both strains converted glutamine to glutamate but only L. reuteri 100-23 accumulated GABA. Glutamate addition to phosphate buffer, pH 2.5, improved survival of L. reuteri 100-23 100-fold. However, survival of L. reuteri ∆gadB remained essentially unchanged. The disruption of gadB did not affect growth of L. reuteri in mMRS or in sourdough. However, the wild type strain L. reuteri 100-23 displaced L. reuteri ∆gadB after 5 cycles of fermentation in back-slopped sourdough fermentations.
The conversion of glutamate to GABA by L. reuteri 100-23 contributes to acid resistance and to competitiveness in industrial sourdough fermentations. The organization of the gene cluster for glutamate conversion, and the availability of amino acids in cereals imply that glutamine rather than glutamate functions as the substrate for GABA formation. The exceptional coupling of glutamine deamidation to glutamate decarboxylation in L. reuteri likely reflects adaptation to cereal substrates.
酸应激会影响乳酸菌在工业酸面团发酵和肠道生态系统中的持久性。然而，谷氨酸对乳酸菌的抗酸性的贡献尚未通过实验证明，并且缺乏抗酸性对乳酸菌在酸面团中竞争力的贡献的证据。因此，本研究旨在研究谷氨酸脱羧酶在罗伊氏乳杆菌中的生态作用。
在罗伊氏乳杆菌 100-23 的基因组中鉴定出一个编码假定谷氨酸脱羧酶的基因 gadB。与其他乳酸菌中编码谷氨酸脱羧酶的遗传基因座的组织不同，gadB 位于一个假定谷氨酰胺酶基因 gls3 的旁边。通过双交叉方法生成了罗伊氏乳杆菌 ∆gadB 的基因缺失突变体。与罗伊氏乳杆菌 ∆gadB 不同，罗伊氏乳杆菌 100-23 在磷酸盐黄油（pH 2.5）中将谷氨酸转化为 γ-氨基丁酸（GABA）。在酸面团中，两种菌株都将谷氨酰胺转化为谷氨酸，但只有罗伊氏乳杆菌 100-23 积累 GABA。向 pH 2.5 的磷酸盐缓冲液中添加谷氨酸可使罗伊氏乳杆菌 100-23 的存活率提高 100 倍。然而，罗伊氏乳杆菌 ∆gadB 的存活率基本不变。gadB 的缺失不影响罗伊氏乳杆菌在 mMRS 或酸面团中的生长。然而，在回灌酸面团发酵的 5 个循环中，野生型菌株罗伊氏乳杆菌 100-23 取代了罗伊氏乳杆菌 ∆gadB。
罗伊氏乳杆菌 100-23 将谷氨酸转化为 GABA 有助于其在工业酸面团发酵中的抗酸性和竞争力。谷氨酸转化基因簇的组织以及谷物中氨基酸的可用性意味着谷氨酰胺而不是谷氨酸是 GABA 形成的底物。罗伊氏乳杆菌中谷氨酸脱酰胺与谷氨酸脱羧酶的异常偶联可能反映了对谷物底物的适应。
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