A role for EIIANtr in controlling fluxes in the central metabolism of E. coli K12

Abstract

To investigate a possible role of the nitrogen-PTS (PTSNtr) in controlling carbon metabolism, we determined the growth of Escherichia coli LJ110 and of isogenic derivatives, mutated in components of the PTSNtr, on different carbon sources. The PTSNtr is a set of proteins homologous to the PEP-dependent phosphotransferase system (C-PTS) that transfers a phosphate group from PEP over EINtr (encoded by ptsP) and NPr (encoded by ptsO) to EIIANtr (encoded by ptsN). Strains deleted in ptsN were characterized by a high acetate production coupled to slow growth on glycolytic substrates. The ΔptsP and the ΔptsO strain showed the same behavior as the parent strain. As the phosphorylation level of EIIANtr in these mutants differed significantly from that of the parent strain, phosphorylation of EIIANtr obviously is not important for its function. During growth in minimal medium with defined carbon sources, EIIANtr was always completely phosphorylated in LJ110. Significant amounts of dephosphorylated EIIANtr were only visible in strains lacking EINtr or NPr. mRNA expression studies on glucose revealed a downregulation of genes encoding TCA cycle enzymes when EIIANtr was absent. 13C-flux analyses confirmed higher fluxes towards acetate and lower fluxes in the TCA cycle in the ptsN mutants but additionally hinted to a slightly but significantly increased flux through the pyruvate dehydrogenase complex (PDH). During growth on succinate the ΔptsN strain accumulated mutations in rpoS, while no rpoS mutants were observed for the ΔptsN-O strain. This hints to an additional function of NPr during growth with succinate.