The Aes Protein Directly Controls the Activity of MalT, the Central Transcriptional Activator of the Escherichia coliMaltose Regulon

MalK, the ATP-binding Cassette Component of the Escherichia coli Maltodextrin Transporter, Inhibits the Transcriptional Activator MalT by Antagonizing Inducer Binding

“…BL21( DE3) ⌬malT220 contains a deletion of the entire malT gene (19). Plasmids pOM163 and pOM163-T38R are pET28b(ϩ) (Novagen) derivatives producing DT1 or DT1-T38R with a Strep tag at their C terminus (DT1S) (20). Plasmid pOM174 was obtained by amplifying the MalK-encoding fragment of pKN101 (obtained from K. Nikaïdo) by PCR using the oligonucleotides KU001 (5Ј-GCGG-CGCCATGGGGACCCACGATCAGGTCGA-3Ј) and KD001 (5Ј-GGCCG-CGAAGCTTCAGGCTTTGTGTGTTTTGT-3Ј), digesting it with NcoI and HindIII, and inserting it between the NcoI and HindIII sites of pET28b(ϩ) followed by the insertion of a His tag encoding linker, HT-AG1/HTAG2 (HTAG1, 5Ј-CATGCATCATCATCACCATCA-3Ј; HTAG2, 5Ј-CATGTGATGGTGATGATGATG-3Ј), in the NcoI site of the intermediate construct.…”

“…Affinity Chromatography with Immobilized DT1-Soluble extracts containing DT1S or DT1S-T38R were prepared from BL21( DE3) ⌬malT220 harboring pOM163 or pOM163-T38R, as described (20). Affinity chromatography was performed at 4°C in Micro Biospin® BioRad columns packed with 50 l of Strep-Tactin®-Sepharose® resin (IBA), as described above.…”

“…In addition to MalK, two other proteins, namely MalY, a ␤ C-S lyase, and Aes, an acyl esterase, down-regulate MalT activity (17,18). Recent studies have revealed that both MalY and Aes interact directly with MalT and compete with maltotriose for MalT binding (19,20). Available data suggest that unliganded MalT is in equilibrium between an inactive monomeric form and an active monomeric form prone to self-association.…”

“…The requirement for ATP as a positive effector of MalT might reflect a role for the ATPase activity of MalT in the competition between positive and negative effectors. Indeed, both ATP and ADP can promote MalT multimerization or inhibitory complex formation, but ATP is more effective in driving MalT self-association, whereas ADP is more effective in promoting the formation of an inhibitory complex (16,19,20).…”

“…DT1 (residues 1-241) binds and hydrolyzes ATP (21). It also binds MalY and Aes (20,22) whereas maltotriose is bound by DT3 (residues 437-806) (21,23). DT4 (residues 807-901), which belongs to the LuxR-type family of DNAbinding domains, contains the DNA-binding site and most likely also carries a surface determinant contacting the RNA polymerase (24).…”