Abstract
Several enzymes have evolved as sensors in signal transduction pathways to control gene expression, thereby allowing bacteria to adapt efficiently to environmental changes. We recently identified the master regulator of cysteine metabolism in Bacillus subtilis, CymR, which belongs to the poorly characterized Rrf2 family of regulators. We now report that the signal transduction mechanism controlling CymR activity in response to cysteine availability involves the formation of a stable complex with CysK, a key enzyme for cysteine biosynthesis. We carried out a comprehensive quantitative characterization of this regulator-enzyme interaction by surface plasmon resonance and analytical ultracentrifugation. We also showed that O-acetylserine plays a dual role as a substrate of CysK and as an effector modulating the CymR-CysK complex formation. The ability of B. subtilis CysK to bind to CymR appears to be correlated to the loss of its capacity to form a cysteine synthase complex with CysE. We propose an original model, supported by the determination of the intracellular concentrations of the different partners, by which CysK positively regulates CymR in sensing the bacterial cysteine pool.
Highlights
We report that CysK, the O-acetylserine thiol-lyase of B. subtilis, has acquired a regulatory function and is a trigger enzyme acting as a repressor in the regulation of cysteine metabolism by an original mechanism
On either the CysK-His6 (Fig. 2A) or the maltose-binding protein (MBP)-CysK surfaces, a consistently observed a stoichiometric binding of preformed (CymR) concentration dependence of the surface plasmon resonance (SPR) signal was observed, whereas no signal could be detected on the MccA-His6 surface
Proteins belonging to the ancient, very large, and widely distributed family of pyridoxal 5Ј-phosphate (PLP)-dependent enzymes are mostly involved in various fundamental metabolic pathways
Summary
FЈ Phi80dlacZ ⌬M15 ⌬(lacZYA-argF)U169 deoR recA1 endA1 hsdR17(rK-mKϩ) phoA supE44 lambda- thi-1. The CymR regulator has recently been characterized as the central repressor of cysteine metabolism in B. subtilis [13]. CymR controls the expression of genes involved either in cysteine synthesis from sulfide (cysK), sulfonates (ssu), or methionine (mccAB) or in cystine uptake (tcyP), by directly binding a 27-bp conserved motif in their promoter regions [13, 17]. A large set of genes is controlled by both CymR and CysK in B. subtilis [13, 18]. We show in this work that CysK of B. subtilis interacts with CymR in vivo and in vitro. This enzyme acts as a sensor of cysteine availability in the signal transduction pathway modulating CymR activity
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