NgcESco Acts as a Lower-Affinity Binding Protein of an ABC Transporter for the Uptake of NN′-Diacetylchitobiose in Streptomyces coelicolor A3(2)

摘要

In the model species Streptomyces coelicolor A3(2), the uptake of chitin-degradation byproducts, mainly N,N′- diacetylchitobiose ([GlcNAc]2) and N-acetylglucosamine (GlcNAc), is performed by the ATP-binding cassette (ABC) transporter DasABC-MsiK and the sugar-phosphotransferase system (PTS), respectively. Studies on the S. coelicolor chromosome have suggested the occurrence of additional uptake systems of GlcNAc-related compounds, including the SCO6005–7 cluster, which is orthologous to the ABC transporter NgcEFG of S. olivaceoviridis. However, despite conserved synteny between the clusters in S. coelicolor and S. olivaceoviridis, homology between them is low, with only 35% of residues being identical between NgcE proteins, suggesting different binding specificities. Isothermal titration calorimetry experiments revealed that recombinant NgcE^Sco interacts with GlcNAc and (GlcNAc)2, with Kd values (1.15 and 1.53 μM, respectively) that were higher than those of NgcE of S. olivaceoviridis (8.3 and 29 nM, respectively). The disruption of ngcE^Sco delayed (GlcNAc)2 consumption, but did not affect GlcNAc consumption ability. The ngcE^Sco-dasA double mutation severely decreased the ability to consume (GlcNAc)2 and abolished the induction of chitinase production in the presence of (GlcNAc)2, but did not affect the GlcNAc consumption rate. The results of these biochemical and reverse genetic analyses indicate that NgcE^Sco acts as a (GlcNAc)2- binding protein of the ABC transporter NgcEFG^Sco-MsiK. Transcriptional and biochemical analyses of gene regulation demonstrated that the ngcE^Sco gene was slightly induced by GlcNAc, (GlcNAc)2, and chitin, but repressed by DasR. Therefore, a model was proposed for the induction of the chitinolytic system and import of (GlcNAc)2, in which (GlcNAc)2 generated from chitin by chitinase produced leakily, is mainly transported via NgcEFG-MsiK and induces the expression of chitinase genes and dasABCD.