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

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.