Why does Escherichia coli grow more slowly on glucosamine than on N-acetylglucosamine? Effects of enzyme levels and allosteric activation of GlcN6P deaminase (NagB) on growth rates

Abstract

Wild-type Escherichia coli grows more slowly on glucosamine (GlcN) than on N-acetylglucosamine (GlcNAc) as a sole source of carbon. Both sugars are transported by the phosphotransferase system, and their 6-phospho derivatives are produced. The subsequent catabolism of the sugars requires the allosteric enzyme glucosamine-6-phosphate (GlcN6P) deaminase, which is encoded by nagB, and degradation of GlcNAc also requires the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase. We investigated various factors which could affect growth on GlcN and GlcNAc, including the rate of GlcN uptake, the level of induction of the nag operon, and differential allosteric activation of GlcN6P deaminase. We found that for strains carrying a wild-type deaminase (nagB) gene, increasing the level of the NagB protein or the rate of GlcN uptake increased the growth rate, which showed that both enzyme induction and sugar transport were limiting. A set of point mutations in nagB that are known to affect the allosteric behavior of GlcN6P deaminase in vitro were transferred to the nagB gene on the Escherichia coli chromosome, and their effects on the growth rates were measured. Mutants in which the substrate-induced positive cooperativity of NagB was reduced or abolished grew even more slowly on GlcN than on GlcNAc or did not grow at all on GlcN. Increasing the amount of the deaminase by using a nagC or nagA mutation to derepress the nag operon improved growth. For some mutants, a nagA mutation, which caused the accumulation of the allosteric activator GlcNAc6P and permitted allosteric activation, had a stronger effect than nagC. The effects of the mutations on growth in vivo are discussed in light of their in vitro kinetics.

FIG. 1.

(A) Metabolism of GlcN and GlcNAc. Both sugars are PTS sugars, so that their transport across the inner membrane (Mb) into the cytoplasm (CP) by the manXYZ- and nagE-encoded transporters results in their phosphorylation. Subsequent metabolism is via the nagA- and nagB-encoded enzymes, GlcNAc6P deacetylase and GlcN6P deaminase, which results in fructose 6-phosphate. (B) Organization of the divergent nagE-BACD operons. The function of nagD is unknown.

FIG. 2.

Effects of mlc, nagC, and nagA mutations on growth rates on GlcN. Bacteria were grown in minimal MOPS medium with 0.2% GlcN as described in Materials and Methods. (A) Wild-type NagB⁺; (B) Phe174Ala mutant; (C) Tyr121Ser mutant. Representative growth curves for the nagB strains and their mlc, nagC, and nagA versions are shown. The Phe174Ala mutant did not grow on GlcN in the absence of a nagC or nagA mutation. The slowly growing Tyr121Ser and Tyr121Ser mlc strains were stored overnight at 4°C and then rediluted to an A₆₅₀ of 0.05 and regrown the next day. wt, wild type.

FIG. 3.

Western blot analysis of cultures of strains carrying a NagB⁺ allele (A) and Phe174Ala NagB (B). Additional mutations in nagC or nagA were introduced as indicated above the lanes, and bacteria were grown with the carbon sources indicated. Aliquots of sonicated extracts of the cultures were separated by sodium dodecyl sulfate-12.5% polyacrylamide gel electrophoresis, transferred to a Hybond C membrane (Amersham), treated with antibodies to NagA and NagB, and revealed with ¹²⁵I-labeled protein A. The positions of NagA and NagB on the gel are indicated. S, purified NagA and NagB proteins mixed with an extract of LAA95 (Δnag).