title: A dual role of the transcriptional regulator TstR provides insights into cyanide detoxification in Lactobacillus brevis.
abstract: 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:P(tstR), 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.
authors.affiliation: Department of Microbiology and Cell Science, Genetics Institute, Institute of Food and Agricultural Sciences, University of Florida, 2033 Mowry road. PO Box 103610, Gainesville, FL, 32610-3610, USA.
authors.forename: Fernando A
authors.initials: FA
authors.lastname: Pagliai
authors.forename: Caitlin C
authors.initials: CC
authors.lastname: Murdoch
authors.forename: Sara M
authors.initials: SM
authors.lastname: Brown
authors.forename: Claudio F
authors.initials: CF
authors.lastname: Gonzalez
authors.forename: Graciela L
authors.initials: GL
authors.lastname: Lorca
mesh_terms: D001665:Binding Sites; D001711:Biotransformation; D003001:Cloning, Molecular; D003486:Cyanides / Q000378:metabolism*; D004361:Drug Tolerance; D024202:Electrophoretic Mobility Shift Assay; D004926:Escherichia coli / Q000187:drug effects / Q000235:genetics; D005290:Ferric Compounds / Q000378:metabolism; D015870:Gene Expression; D015964:Gene Expression Regulation, Bacterial; D052196:Levilactobacillus brevis / Q000187:drug effects / Q000378:metabolism*; D016297:Mutagenesis, Site-Directed; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D012097:Repressor Proteins / Q000235:genetics / Q000378:metabolism*; D013447:Sulfites / Q000378:metabolism; D013884:Thiosulfate Sulfurtransferase / Q000235:genetics / Q000378:metabolism*; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
chemical_list: D003486:Cyanides; D005290:Ferric Compounds; D012097:Repressor Proteins; D013447:Sulfites; D013884:Thiosulfate Sulfurtransferase