A dual role of the transcriptional regulator TstR provides insights into cyanide detoxification in Lactobacillus brevis

• Published in: Molecular microbiology 2014-05, Vol.92 (4), p.853-871
• Main Authors: Pagliai, Fernando A., Murdoch, Caitlin C., Brown, Sara M., Gonzalez, Claudio F., Lorca, Graciela L.
• Format: Article
• Language: English
• Subjects: Binding Sites; Biotransformation; Cloning, Molecular; Cyanides - metabolism; Drug Tolerance; E coli; Electrophoretic Mobility Shift Assay; Escherichia coli; Escherichia coli - drug effects; Escherichia coli - genetics; Ferric Compounds - metabolism; Gene Expression; Gene Expression Regulation, Bacterial; Genes; Lactobacillus; Lactobacillus brevis; Lactobacillus brevis - drug effects; Lactobacillus brevis - metabolism; Mutagenesis; Mutagenesis, Site-Directed; Probiotics; Promoter Regions, Genetic; Protein Binding; Repressor Proteins - genetics; Repressor Proteins - metabolism; RNA-protein interactions; Sulfites - metabolism; Thiosulfate Sulfurtransferase - genetics; Thiosulfate Sulfurtransferase - metabolism; Transcription Initiation Site; Transcription, Genetic
• ISSN: 0950-382X; 1365-2958
• DOI: 10.1111/mmi.12598

Summary In this study we uncover two genes in Lactobacillus brevis ATCC 367, tstT and tstR, encoding for a rhodanese and a transcriptional regulator involved in cyanide detoxification. TstT (LVIS_0852) belongs to a new class of thiosulphate:cyanide sulphurtransferases. We found that TstR (LVIS_0853) modulates both the expression and the activity of the downstream‐encoded tstT. The TstR binding site was identified at −1 to +33, from tstR transcriptional start site. EMSA revealed that sulphite, a product of the reaction catalysed by TstT, improved the interaction between TstR:PtstR, while Fe(III) disrupted this interaction. Site‐directed mutagenesis in TstR identified M64 as a key residue in sulphite recognition, while residues H136‐H139‐C167‐M171 formed a pocket for ferric iron co‐ordination. In addition to its role as a transcriptional repressor, TstR is also involved in regulating the thiosulphate:cyanide sulphurtransferase activity of TstT. A threefold increase in TstT activity was observed in the presence of TstR, which was enhanced by the addition of Fe(III). Overexpression of the tstRT operon was found to increase the cyanide tolerance of L. brevis and Escherichia coli. The protein–protein interaction between TstR and TstT described herein represents a novel mechanism for regulation of enzymatic activity by a transcriptional regulator.