coli C ∆agaI ∆nagB would have been affected (Figure 5). In addition, as shown above, agaI cannot substitute for the absence of nagB, because pJFagaI could not complement ΔnagB and ΔagaI ΔnagB mutants of E. coli C. Together, these results show that agaI and nagB are not involved in Aga and Gam utilization. These results show that first three of the four proposals that we proposed above, do not hold true. Therefore, our fourth proposal that agaI and nagB are not essential for Aga and Gam utilization and that selleck kinase inhibitor some other gene carries out the deamination/isomerization step holds true. So it poses the question which gene
is involved in this step of the Aga/Gam pathway. The loss of agaS affects Aga and Gam utilization The agaS gene in the selleck chemical aga/gam cluster has not been assigned to any of the steps in the catabolism of Aga and Gam (Figure 1) [1, 6]. Since agaS has homology to sugar isomerases [1] it was tested if deleting agaS would affect Aga and Gam utilization. EDL933 ΔagaS and E. coli C ΔagaS, did not grow on Aga plates but their parent strains
grew (Figure 7A). On Gam plates, wild type E. coli C grew but E. coli C ΔagaS did not grow (Figure 7B). EDL933 and EDL933 ΔagaS were streaked on Gam plates but they were not expected to grow because EDL933 is Gam- (Figure 7B). The results were identical when the ΔagaS mutants Y-27632 2HCl were examined for growth on Aga and Gam plates without any added nitrogen source (data not shown). These results show that the loss of agaS affects Aga and Gam utilization and therefore AgaS plays a role in the Aga/Gam pathway. Figure 7 Growth of EDL933, E. coli C, and Δ agaS mutants on Aga and Gam. Wild type EDL933, E. coli C, and ΔagaS mutants derived from them were streaked out on MOPS minimal agar plates with Aga (A) and Gam (B) with NH4Cl as added nitrogen source. The Aga plate was incubated at 37°C for 48 h and the Gam plate was incubated at 30°C for 72 to 96 h.
The description of the strains in the four sectors of the plates is indicated in the diagram below (C). Relative expression levels of nagA, nagB, and agaA were examined by qRT-PCR in ΔagaS mutants grown on glycerol and GlcNAc. In glycerol grown ΔagaS mutants of EDL933 and E. coli C, nagA, nagB, and agaA were not induced. When grown on GlcNAc, nagA and nagB were induced about 10-fold and 23-fold, respectively, in EDL933 ΔagaS and 3-fold and 7-fold, respectively, in E. coli C ΔnagB. These expression levels of nagA and nagB in GlcNAc grown EDL933 ΔagaS are comparable to that in GlcNAc grown EDL933 ΔagaA (Table 1) but the levels of expression of these genes in GlcNAc grown E. coli C ∆agaS are lower than in GlcNAc grown E. coli C ΔagaA (Table 1). The agaA gene was not induced in GlcNAc grown ΔagaS mutants.