嗜盐四联球菌arc operon多拷贝基因在精氨酸代谢中的作用

Role of multiple copies of arc operon genes in arginine metabolism of Tetragenococcus halophilus

摘要:

【背景】嗜盐四联球菌(Tetragenococcus halophilus)是一类存在于发酵食品中的耐盐乳酸菌，研究其精氨酸(arginine，Arg)代谢对解析食品发酵过程中氨基甲酸乙酯(ethyl carbamate，EC)前体积累机制和保障食品安全具有重要意义。【目的】研究酱醪来源嗜盐四联球菌精氨酸脱亚氨基(arginine deiminase，ADI)途径的基因构成，揭示这些基因对菌株精氨酸代谢和氨基甲酸乙酯前体瓜氨酸(citrulline，Cit)利用与积累的影响。【方法】采用PCR扩增与测序分析不同菌株的ADI途径基因组成，通过比较ADI途径关键基因转录水平和关键酶活性，探究环境因素对嗜盐四联球菌代谢氨基酸能力的影响及各拷贝基因参与氨基酸代谢的功能。【结果】酱醪来源嗜盐四联球菌基因组中ADI途径基因类型主要有两大类：以菌株R23为代表含有完整arc操纵子(operon)基因且具有最多基因拷贝数；以菌株C3为代表缺失arcA和arcB但含有多拷贝arcB和arcC。基因组中有arcA的菌株才具有利用精氨酸能力，并通过利用精氨酸生成瓜氨酸。体系中精氨酸含量和乙醇与脂肪酸的存在均可影响嗜盐四联球菌利用精氨酸积累中间产物瓜氨酸。当精氨酸含量大于5 g/L或体系中含有乙醇与脂肪酸时，嗜盐四联球菌会利用精氨酸积累中间产物瓜氨酸。脂肪酸和乙醇对ADI途径的3个关键酶均有显著抑制作用，可使精氨酸脱亚氨基酶(arginine deiminase，ADI)、鸟氨酸氨甲酰基转移酶(ornithine transcarbamylase，OTC)和氨甲酰磷酸激酶(carbamate kinase，CK)的活性分别降低41.0%、46.4%和60.0%。嗜盐四联球菌中arcB转录水平分别是其拷贝arcB₁和arcB₂的10.5倍和29.8倍，arcC的转录水平分别是arcC₁、arcC₂、arcC₃的17.6、20.3、23.9倍，说明arcB和arcC在瓜氨酸代谢中起主要作用。【结论】精氨酸含量和乙醇加脂肪酸是影响嗜盐四联球菌代谢精氨酸能否积累瓜氨酸的关键环境因素。嗜盐四联球菌arc operon的多拷贝基因中，arcB和arcC基因在瓜氨酸代谢中起主要作用。

关键词:嗜盐四联球菌;精氨酸代谢;瓜氨酸;氨基甲酸乙酯;精氨酸脱亚氨基途径

Abstract:

[Background] Tetragenococcus halophilus is a halophilic lactic acid bacterium detected in fermented food. Investigating the arginine (Arg) metabolism of this bacterium is of great importance for disclosing the mechanism of the accumulation of ethyl carbamate (EC) precursors during food fermentation and ensuring food safety. [Objective] To identify the key genes of arginine deiminase (ADI) pathway in the genome of T. halophilus strains isolated from soy sauce moromi mash, and to reveal the roles of these genes in arginine metabolism and utilization and accumulation of citrulline (the EC precursor). [Methods] The key genes involved in ADI pathway were identified for different strains by PCR amplification and sequencing. The transcription levels of key genes of ADI pathway and the activities of corresponding enzymes were compared to reveal the effect of environmental factors on amino acid metabolism and the role of each gene. [Results] The genes associated with the ADI pathway in the genome of T. halophilus included two categories. One contained intact arc operon genes and had the most copies, as represented by strain R23, and the other lacked arcA and arcB but had multiple copies of arcB and arcC, as represented by strain C3. Only the strains with arcA in their genomes can utilize arginine to produce citrulline. Whether the intermediate product citrulline was accumulated with the utilization of arginine by T. halophilus was affected by the content of arginine and the presence of ethanol and fatty acids in the medium. T. halophilus accumulated citrulline when arginine was more than 5 g/L or ethanol and fatty acids were present in the medium. In the presence of fatty acids and ethanol, the activities of ADI, ornithine transcarbamylase (OTC), and carbamate kinase (CK), the key enzymes of ADI pathway, were significantly reduced by 41.0%, 46.4%, and 60.0%, respectively. The transcription level of arcB in T. halophilus was 10.5 and 29.8 times as high as that of arcB ₁ and arcB ₂, respectively, and the transcription level of arcC was 17.6, 20.3, and 23.9 times as high as that of arcC ₁, arcC ₂, and arcC ₃, respectively. The results demonstrated that arcB and arcC played major roles in citrulline metabolism. [Conclusion] Arginine content and the presence of ethanol and fatty acids are key environmental factors influencing whether citrulline can be accumulated by T. halophilus when metabolizing arginine. Among the multiple copies of arc operon genes in T. halophilus, arcB and arcC play key roles in citrulline metabolism.

Key words:Tetragenococcus halophilus;arginine metabolism;citrulline;ethyl carbamate;arginine deiminase pathway