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

Abstract

Summary In this study we uncover two genes in Lactobacillus brevis ATCC367, tstT and tstR, encoding for a rhodanese and a transcriptional regulator involved in cyanide detoxification. TstT (LVIS_0852) belongs to a new class of thiosulfate:cyanide sulfurtransferases. 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 sulfite, a product of the reaction catalyzed 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 sulfite recognition, while residues H136-H139-C167-M171 formed a pocket for ferric iron coordination. In addition to its role as a transcriptional repressor, TstR is also involved in regulating the thiosulfate:cyanide sulfurtransferase activity of TstT. A 3-fold 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.

Identification of a Ligand Binding Pocket in LdtR from Liberibacter asiaticus

“…Here, we performed a detailed structure based site-directed mutagenesis in LdtR to show that benzbromarone may bind to residues T43, L61, F64 in Benz1 (equivalent to SAL1 pocket), while residues in the predicted SAL2 pocket were not involved in ligand binding. These results are in agreement with our previous findings in Lactobacillus brevis , where mutations in the LVIS553 predicted SAL1 pocket impaired binding to its native ligand novobiocin, and mutations in the TstR SAL1 equivalent pocket impaired binding to sulfite ( Pagliai et al, 2010 , 2014b ). Although the decreased interaction between the ligand and the mutants T43A, L61A, and F64A may suggest that these residues are involved in the interaction with benzbromarone, the possibility that the decreased interaction is due to changes in the overall structure of the dimer cannot be ruled out.…”

“…The binding pockets Benz1 and Benz2 are located in the dimerization domains of LdtR. Previous studies of MarR homologs have reported that that mutagenesis in the dimerization interface can directly affect DNA binding activity ( Saridakis et al, 2008 ; Pagliai et al, 2010 , 2014b ; Gupta and Grove, 2014 ). Based on these observations, the purified LdtR mutant variants were tested for DNA binding activity on the promoter region of the downstream gene ldtP ( P ldtP ; Pagliai et al, 2014a ).…”

“…Upon binding of a signal molecule in the dimer interface, these transcription factors lose DNA binding capabilities, allowing transcription to occur. In contrast, only few reports have described MarR family members acting as transcriptional activators ( Baumgarth et al, 2005 ; Manna and Cheung, 2006 ; Di Fiore et al, 2009 ; Ludwig et al, 2014 ; Pagliai et al, 2014b ). These include ChlR, a transcription activator that utilizes an oxygen-labile [4Fe–4S] cluster to sense oxygen in Synechococcus sp.…”

“…In contrast, several MarR repressors have been described at the molecular level, in presence and absence of inducer molecules ( Evans et al, 2001 ; Wilkinson and Grove, 2006 ; Saridakis et al, 2008 ; Kumarevel et al, 2009 ; Perera et al, 2009 ; Pagliai et al, 2014b ). The structure of MTH313 from Methanothermobacter thermautotrophicus was elucidated in presence of salicylate in the dimerization interface, in two asymmetrical pockets.…”

Zinc is an inhibitor of the LdtR transcriptional activator

“…Therefore a manual search along the model of LdtR [ 28 ] was conducted to identify potential residues capable of interacting with Zn 2+ , such as cysteine, histidine, aspartic acid and glutamic acid. A similar manual search was positively used to identify two iron binding sites in TstR, another MarR-family member from Lactobacillus brevis [ 42 ]. It was found that LdtR possesses a cysteine (C28) and a glutamic acid (E33) residue in the α1 helix, in close proximity (less than 10 Å) to the Benz1 ( Fig 5A ).…”