Structure of the Branched-chain Amino Acid and GTP-sensing Global Regulator, CodY, from Bacillus subtilis

Abstract

CodY is a branched-chain amino acid (BCAA) and GTP sensor and a global regulator of transcription in low G + C Gram-positive bacteria. It controls the expression of over 100 genes and operons, principally by repressing during growth genes whose products are required for adaptations to nutrient limitation. However, the mechanism by which BCAA binding regulates transcriptional changes is not clear. It is known that CodY consists of a GAF (cGMP-stimulated phosphodiesterases, adenylate cyclases, FhlA) domain that binds BCAAs and a winged helix-turn-helix (wHTH) domain that binds to DNA, but the way in which these domains interact and the structural basis of the BCAA dependence of this interaction are unknown. To gain new insights, we determined the crystal structure of unliganded CodY from Bacillus subtilis revealing a 10-turn α-helix linking otherwise discrete GAF and wHTH domains. The structure of CodY in complex with isoleucine revealed a reorganized GAF domain. In both complexes CodY was tetrameric. Size exclusion chromatography with multiangle laser light scattering (SEC-MALLS) experiments showed that CodY is a dimer at concentrations found in bacterial cells. Comparison of structures of dimers of unliganded CodY and CodY-Ile derived from the tetramers showed a splaying of the wHTH domains when Ile was bound; splaying is likely to account for the increased affinity of Ile-bound CodY for DNA. Electrophoretic mobility shift and SEC-MALLS analyses of CodY binding to 19–36-bp operator fragments are consistent with isoleucine-dependent binding of two CodY dimers per duplex. The implications of these observations for effector control of CodY activity are discussed.

Genome‐wide identification of Listeria monocytogenes CodY‐binding sites

“…Based on the current results, other analyses of CodY‐binding motifs (Belitsky & Sonenshein, , ; den Hengst et al, ; Guedon et al, ; Levdikov et al, ), and the overlapping motifs model created by Wray and Fisher (), we suggest that two dimers of CodY bind in the opposite orientations to two different strands of DNA containing two overlapping sequences resembling the 15‐nt consensus CodY‐binding motif, forming a 24‐nt site (Figure ). Each of these 15‐nt motifs may contain multiple mismatches and only the adenine at position 10 (and to a lesser degree at position 11) appears to be the most critical for binding; these nucleotides create the central conserved region of the 24‐nt motif (Figure b).…”

“…Poor conservation of these nucleotides and/or the absence of a second overlapping motif may explain why many genomic sequences resembling a consensus CodY‐binding motif do not serve as actual CodY‐binding sites. However, we do not know whether CodY binding always requires the presence of two (or more) 15‐nt motifs as, at least in vitro, CodY is able to bind to a short, 19‐bp long fragment of DNA that could accommodate only a single, albeit very strong, CodY‐binding motif (Levdikov et al, ).…”

The Plant Host Induces Antibiotic Production To Select the Most-Beneficial Colonizers

“…The transcription of the pksA-R operon (the operon containing pksA and pskR and the genetic material in between) initiates from a promoter located upstream of pksC and is regulated by two master regulators (52), CodY, which senses the levels of branched amino acids (53,54), and the transition phase regulator AbrB (55). In turn, AbrB is inhibited by the master regulator of sporulation and biofilm development, Spo0A (56).…”

The branched chain aminotransferase IlvE promotes growth, stress resistance and pathogenesis ofListeria monocytogenes

“…While IlvE is needed for enzymatic incorporation of Ile into BCFAs, CodY specifically senses and binds cellular Ile (33, 34). Due to their specific relationships with Ile, we then asked how Δ ilvE and Δ codY mutants would grow when exogenous Ile is lacking but when Val and Leu are present.…”