Publication Date:
2013-05-02
Description:
Background: The catabolic pathways of N-acetyl-D-galactosamine (Aga) and D-galactosamine (Gam) in E. coli were proposed from bioinformatic analysis of the aga/gam regulon in E. coli K-12 and later from studies using E. coli C. Of the thirteen genes in this cluster, the roles of agaA, agaI, and agaS predicted to code for Aga-6-P-deacetylase, Gam-6-P deaminase/isomerase, and ketose-aldolase isomerase, respectively, have not been experimentally tested. Here we study their roles in Aga and Gam utilization in E. coli O157:H7 and in E. coli C. Results: Knockout mutants in agaA, agaI, and agaS were constructed to test their roles in Aga and Gam utilization. Knockout mutants in the N-acetylglucosamine (GlcNAc) pathway genes nagA and nagB coding for GlcNAc-6-P deacetylase and glucosamine-6-P deaminase/isomerase, respectively, and double knockout mutants DeltaagaA DeltanagA and [increment]agaI [increment]nagB were also constructed to investigate if there is any interplay of these enzymes between the Aga/Gam and the GlcNAc pathways. It is shown that Aga utilization was unaffected in DeltaagaA mutants but DeltaagaA DeltanagA mutants were blocked in Aga and GlcNAc utilization. E. coli C DeltanagA could not grow on GlcNAc but could grow when the aga/gam regulon was constitutively expressed. Complementation of DeltaagaA DeltanagA mutants with either agaA or nagA resulted in growth on both Aga and GlcNAc. It was also found that [increment]agaI, [increment]nagB, and [increment]agaI [increment]nagB mutants were unaffected in utilization of Aga and Gam. Importantly, [increment]agaS mutants were blocked in Aga and Gam utilization. Expression analysis of relevant genes in these strains with different genetic backgrounds by real time RT-PCR supported these observations. Conclusions: Aga utilization was not affected in DeltaagaA mutants because nagA was expressed and substituted for agaA. Complementation of DeltaagaA DeltanagA mutants with either agaA or nagA also showed that both agaA and nagA can substitute for each other. The [increment]agaI, [increment]nagB, and [increment]agaI [increment]nagB mutants were not affected in Aga and Gam utilization indicating that neither agaI nor nagB is involved in the deamination and isomerization of Gam-6-P. We propose that agaS codes for Gam-6-P deaminase/isomerase in the Aga/Gam pathway.
Electronic ISSN:
1471-2180
Topics:
Biology
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