EDL933 ΔnagA/ pJFnagAED grew on GlcNAc which was expected but interestingly EDL933 ΔnagA/ pJFagaAED also grew on GlcNAc showing that agaA restored growth of a ΔnagA mutant on GlcNAc (Figure 4B). When EDL933 ΔagaA ΔnagA was complemented Necrostatin-1 with either pJFnagAED or pJFagaAED growth was restored on both GlcNAc and xAga plates (Figures 4A and 4B). The plates shown in

Figure 4 were incubated without IPTG indicating that the basal level of expression of NagA and AgaA from pJFnagAED and pJFagaAED, repectively, were sufficient for complementation for growth on GlcNAc and Aga. Growth on GlcNAc and Aga plates at IPTG concentrations of 10, 50 and 100 μM was similar to that without IPTG indicating that higher levels of expression of agaA and nagA were not detrimental to the cells (data not shown). Identical results as those shown in Figure 4 were obtained in complementation experiments with E. coli C ΔagaA, ΔnagA, and ΔagaA ΔnagA mutants with plasmids, pJFagaAC and pJFnagAC (data not shown). Figure 4 Complementation of Δ nagA and Δ agaA Δ nagA mutants of EDL933 on Aga and GlcNAc plates. Wild type EDL933 and knockout mutants derived from it

harboring the indicated plasmids GSK872 cost were streaked out on MOPS minimal agar plates with ampicillin containing Aga (A) and GlcNAc (B) and incubated at 37°C for 48 h. The description of the strains with various plasmids in the eight sectors of the plates is indicated in the diagram below (C). Thus far, several lines of evidence using knockout mutants, complementation studies with these mutants, and measuring the relative expression of relevant genes in these mutant strains and in the wild type strains indicate that NagA coded by nagA and AgaA coded by agaA can function in both the GlcNAc and Aga pathways. In this context it is pointed out that it was reported P-type ATPase in E. coli K92, growth on Aga not only

induced the Aga transport system but also induced the GlcNAc transport system [9]. From this observation it was proposed that an unidentified epimerase converts Aga-6-P to GlcNAc-6-P which then induces the GlcNAc transport system that is part of the nag regulon [9]. Our data differ in that, nagA and nagB and Mdivi1 supplier Therefore the nag regulon were induced only in ΔagaA mutants and not in wild type E. coli C and EDL933 (Table 1). Furthermore, epimerases usually carry out substrate concentration dependent reversible reactions. Therefore, the high intracellular concentration of GlcNAc-6-P that accumulate in glucose grown nagA mutant (3.2 mM) [2], which should be about the same in our glycerol grown ΔnagA mutants (discussed above), should have epimerized to Aga-6-P. Aga-6-P which is the likely inducer of the aga/gam regulon [11] would then induce the aga/gam regulon but we show that it was not induced (Table 1). Instead, nagB was highly induced and agaA and agaS were induced only 2-fold in EDL933 ΔnagA but not in E. coli C ΔnagA (Table 1).