Characterization of GshAB of Tetragenococcus halophilus: a two-domain glutathione synthetase.

The γ-glutamyl tripeptide glutathione (γ-Glu-Cys-Gly) is a low molecular thiol that acts as antioxidant in response to oxidative stress in eukaryotes and prokaryotes. γ-Glutamyl dipeptides including γ-Glu-Cys, γ-Glu-Glu, and γ-Glu-Gly also have kokumi activity. Glutathione is synthesized by first ligating Glu with Cys by γ-glutamylcysteine ligase (Gcl/GshA), and then the resulting dipeptide γ-glutamylcysteine is ligated with Gly by glutathione synthetase (Gs/GshB). GshAB/GshF enzymes that contain both Gcl and Gs domains are capable of catalyzing both reactions. The current study aimed to characterize GshAB from Tetragenococcus halophilus after heterologous expression in Escherichia coli. The optimal conditions for GshAB from T. halophilus were pH 8.0 and 25 °C. The substrate specificity of the Gcl reaction of GshAB was also determined. GshAB has a high affinity to Cys. γ-Glu-Cys was the only dipeptide generated when Glu, Cys, Gly, and other amino acids were present in the reaction system. This specificity differentiates GshAB from T. halophilus from Gcl of heterofermentative lactobacilli and GshAB of Streptococcus agalactiae, which also use amino acids other than Cys as glutamyl-acceptor. Quantification of gshAB in cDNA libraries from T. halophilus revealed that gshAB was overexpressed in response to oxidative stress but not in response to acid, osmotic, or cold stress. In conclusion, GshAB in T. halophilus served as part of the oxidative stress response but this study did not provide any evidence for a contribution to the resistance to other stressors.Key points Glutathione synthesis in Tetragenococcus halophilus is carried out by the two-domain enzyme GshAB. GshAB is inhibited by glutathione and is highly specific for Cys as acceptor. T. halophilus synthesizes glutathione in response to oxidative stress.

Characterization of DinJ-YafQ toxin-antitoxin module in Tetragenococcus halophilus: activity, interplay, and evolution.

Tetragenococcus halophilus is a moderately halophilic lactic acid bacterium widely used in high-salt food fermentation because of its coping ability under various stress conditions. Bacterial toxin-antitoxin (TA) modules are widely distributed and play important roles in stress response, but those specific for genus Tetragenococcus have never been explored. Here, a bona fide TA module named DinJ1-YafQ1tha was characterized in T. halophilus. The toxin protein YafQ1tha acts as a ribonuclease, and its overexpression severely inhibits Escherichia coli growth. These toxic effects can be eliminated by introducing DinJ1tha, indicating that YafQ1tha activity is blocked by the formed DinJ1-YafQ1tha complex. In vivo and in vitro assays showed that DinJ1tha alone or DinJ1-YafQ1tha complex can repress the transcription of dinJ1-yafQ1tha operon by binding directly to the promoter sequence. In addition, dinJ1-yafQ1tha is involved in plasmid maintenance and stress response, and its transcriptional level is regulated by various stresses. These findings reveal the possible roles of DinJ1-YafQ1tha system in the stress adaptation processes of T. halophilus during fermentation. A single antitoxin DinJ2tha without a cognate toxin protein was also found. Its sequence shows low similarity to that of DinJ1tha, indicating that this antitoxin may have evolved from a different ancestor. Moreover, DinJ2tha can cross-interact with noncognate toxin YafQ1tha and cross-regulate with dinJ1-yafQ1tha operon. In summary, DinJ-YafQtha characterization may be helpful in investigating the key roles of TA systems in T. halophilus and serves as a foundation for further research. KEY POINTS: • dinJ1-yafQ1tha is the first functional TA module characterized in T. halophilus and upregulated significantly upon osmotic and acidic stress. • DinJ2tha can exhibit physical and transcriptional interplay with DinJ1-YafQ1tha. • dinJ2tha may be acquired from bacteria in distant affiliation and inserted into the T. halophilus genome through horizontal gene transfer.

Characterization of the two nonidentical ArgR regulators of Tetragenococcus halophilus and their regulatory effects on arginine metabolism.

The halophilic lactic acid bacterium Tetragenococcus halophilus has been widely used in high-salinity fermentation processes of food. Previous studies have indicated that the catabolism of arginine may contribute to the osmotic stress adaptation of T. halophilus. Unusually, in the chromosome of T. halophilus, preceding the arginine deiminase (ADI) operon, locate two co-transcribed genes, both encoding an ArgR regulator; similar structure was rarely found and the roles of the regulators have not been demonstrated. In the current study, regulatory roles of these two nonidentical ArgR regulators on the arginine metabolism of T. halophilus were investigated. The results show that these two regulators play different roles in arginine metabolism, ArgR1 acts as a negative regulator of the ADI pathway by binding to the promoter sequences and repressing the transcription of genes, and the addition of arginine or hyper-osmotic stress conditions can abolish the ArgR1 repression, whereas ArgR2 negatively regulates the genes involved in arginine biosynthesis. Our study found that despite the commonly known roles of the ArgR regulators as the activator of arginine catabolism and the repressor of arginine biosynthesis, which are found in most studied bacteria possessed one ArgR regulator, the two nonidentical ArgR regulators of T. halophilus both act as repressors, and the repression by which is regulated when sensing changes of environments. By revealing the regulation of arginine metabolism, the current study provides molecular insights and potential tools for future applications of halophiles in biotechnology. KEY POINTS: • The expression of the ADI pathway of T. halophilus is regulated by carbon sources and osmotic stress. • The arginine metabolism process of T. halophilus is fine-tuned by the two ArgR regulators. • The ADI pathway may contribute to the osmotic stress adaptation by generating more energy and accumulating citrulline which acts as compatible solute.

Identification of a GntR family regulator BusRTha and its regulatory mechanism in the glycine betaine ABC transport system of Tetragenococcus halophilus.

Glycine betaine is one of the most effective compatible solutes of the halophilic lactic acid bacterium Tetragenococcus halophilus, the transportation of which is essential for its survival under salinity stress condition. In the current study, we attempted to define a glycine betaine ABC transporter system of T. halophilus, busATha, which plays an important role in adapting to salinity condition. The expression of busATha enhanced the growth of the recombinant strain under high salinity. BusRTha, a transcription regulator that represses the expression of busATha, was characterized, and the repression was abrogated under high salinity. The binding of the regulator was demonstrated through electrophoretic mobility shift assays, and the binding sites were characterized as 5'-AAA(T/G)TGAC(C/A)(G/A)T(C/A)C-3'. This is the first studied transcription regulator of T. halophilus, and our findings provide insights into the molecular mechanism of halophilic life and tools for further application of halophiles as chassis in industrial biotechnology.

The molecular mechanism and post-transcriptional regulation characteristic of Tetragenococcus halophilus acclimation to osmotic stress revealed by quantitative proteomics.

Tetragenococcus halophilus is a moderate halophilic bacterium which was widely used in fermentation processes, growing in a broad range of salinity conditions, and can survive a saturated 26.47% w/w NaCl concentration. However, the mechanism of this outstanding ability to acclimate to extracellular osmotic stress still remains unknown. The current study firstly conducted a quantitative proteomic analysis to identify alterations of the cellular proteome under both hypo-osmotic and hyper-osmotic stress conditions. A total of 1405 proteins were identified and differentially accumulated proteins were analyzed, further functional annotations were performed using Gene Ontology and Kyoto Encyclopedia of Genes and Genomes. The results revealed that both hypo- and hyper-osmotic stresses have prominent impacts on the synthesis of proteins involving in multiple cellular functions. Further analyses of the differentially accumulated proteins suggested that the adaptation strategies T. halophilus applies to deal with hypo- and hyper-osmotic stress conditions may be distinct. Comparison of the differentially accumulated proteins in both transcriptomic and proteomic study indicated the existence of post-transcriptional modification during salinity adaptation of T. halophilus. The current study generated a proteomic atlas of differentially accumulated proteins under both hypo- and hyper-osmotic stress conditions, provided an overview of the molecular mechanism of osmotic acclimation of T. halophilus. SIGNIFICANCE: The current study aimed to reveal how the moderately halophilic Tetragenococcus halophilus adapt to extracellular salinity stress, which is the first proteomic study analyzing the differences in proteome of Tetragenococcus halophilus between hypo- and hyper-osmotic stress to our knowledge. By analyzing the differences in the accumulating levels of the proteome via isobaric labeling-based quantitative proteomic study, we identified proteins with significantly different accumulation levels which may play important roles in the adaptation process to extracellular salinity stress. Examining the cellular functions of these proteins according to Gene Ontology and Kyoto Encyclopedia of Genes and Genomes, a draft view of how the bacterium act to acclimate to osmotic stress has been drawn. Further analysis revealing the differences between the transcriptome and proteome suggested that some proteins may undergo post-transcriptional regulation during acclimation process, which still remains unstudied and needs further investigations. The results of the current study can help researchers to gain insights and further reveal the halophilic mechanism of halophiles.