coli C grown on GlcNAc In GlcNAc grown EDL933 ∆agaA, the express

coli C grown on GlcNAc. In GlcNAc grown EDL933 ∆agaA, the expression levels of nagA SAHA HDAC and nagB were about the same as that of EDL933 grown on GlcNAc and the expression of agaS is slightly elevated but it is only about 1% of that in Aga grown EDL933. In E. coli C ∆agaA grown on GlcNAc the expression levels of nagA and nagB were 40% of that

in E. coli C and the expression of agaS is about 3-fold higher than that grown in glycerol but it is about 5% of the level expressed in Aga grown E. coli C and E. coli C ∆agaA. What is noteworthy is that unlike in Aga grown wild type EDL933 and E. coli C where nagA and nagB were not induced, their respective ∆agaA mutants when grown on Aga induced nagA and nagB to levels that were comparable to the induced levels in GlcNAc grown in the wild type and the ∆agaA buy KU-57788 mutants of these strains. Importantly, this data shows that NagA is indeed present in Aga grown ΔagaA mutants and therefore it lends additional support to the genetic data (Figure 2) from which we concluded that ∆agaA

mutants of EDL933 and E. coli C were able to grow on Aga (Figure 2) because NagA can substitute for the absence of AgaA. This observation leads to the question how do ΔagaA mutants grown on Aga induce nagA and nagB and thereby the nag regulon. A probable explanation is that when ΔagaA mutants grow on Aga they accumulate Aga-6-P which induces the nag regulon and upon synthesis of NagA it deacetylates Aga-6-P. It has been shown that the inducer of the nag regulon is GlcNAc-6-P and not GlcN, GlcNAc, GlcN-6-P, and G-1-P [4]. There is also indirect evidence

that Aga-6-P is the inducer of the aga/gam regulon [11] but whether Aga-6-P can also induce the nag regulon has not been demonstrated. When nagA and nagB expression levels were examined in glycerol grown ΔnagA mutants it was found that expression of nagA was not detected as expected, and agaA and agaS were expressed at very low levels. However, nagB was induced 61-fold in EDL933 find more ΔnagA and 19-fold in E. coli C ΔnagA whereas, in their respective wild type parents grown on glycerol it was not induced (Table 1). These expression levels of nagB in glycerol grown EDL933 ΔnagA and E. coli C ∆agaA were about 250% and 80%, respectively, of their respective wild type strains grown in GlcNAc. This is significantly high considering that the expression of nagB remains at the uninduced levels in the wild type strains grown on glycerol. This phenomenon of nagB induction in nagA mutants of E. coli K-12 grown on glucose has been reported earlier [2, 4]. It has been explained that this happens because of the endogenous synthesis of GlcNAc-6-P, the inducer of the nag regulon, that accumulates in nagA mutants which in turn induces the nag regulon [2, 4]. It was also reported that this accumulated substance in ΔnagA mutants disappeared upon incubation of a cell extract with overexpressed GlcNAc-6-P deacetylase [4].

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