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.Escherichia coli is renowned for its ability to use a diverse array of organic compounds as sources of carbon and energy. However, different carbohydrates do not produce the same growth yield. One of the best carbon sources, after glucose, is N-acetylglucosamine (GlcNAc), an amino sugar. The other common amino sugar, glucosamine (GlcN), is also a source of carbon and nitrogen for E. coli, but use of this sugar results in lower growth rates than use of GlcNAc. Both GlcNAc and GlcN are phosphotransferase system (PTS) sugars (38) in E. coli so that their uptake occurs concomitantly with their phosphorylation, which produces intracellular N-acetylglucosamine 6-phosphate (GlcNAc6P) and glucosamine 6-phosphate (GlcN6P). GlcNAc6P is first deacetylated by the nagA gene product, GlcNAc6P deacetylase (NagA; EC 3.5.1.25); this produces GlcN6P, which is then subject to deamination and isomerization by the nagB-encoded GlcN6P deaminase (NagB; EC 3.5.99.6; formerly GlcN6P isomerase), resulting in fructose 6-phosphate, which enters the glycolytic pathway, and ammonia. Thus, the last amino sugar-specific step in the catabolism of both GlcN and GlcNAc depends on the nagB-encoded enzyme (Fig. 1A).GlcNAc6P has many functions during the metabolism of the amino sugars, both as a metabolite and as a regulator. As well ...

Allosteric Regulation of Glucosamine-6-Phosphate Deaminase (NagB) and Growth ofEscherichia colion Glucosamine

“…The nagA::tc and nagC::tc mutations were introduced using P1 lysates grown on LAA44 and LAA43, which also carry the adjacent nagB::cm. Growth rates were measured in minimal morpholineethanesulfonic acid (MOPS) medium with 10 mM GlcN or GlcNAc as described previously (1). Precultures were grown in minimal MOPS medium with glycerol and diluted 1/100 into the test medium.…”

“…Student's t test was used for pairwise comparisons of different culture conditions with the significance threshold set at 0.05%. Western blotting was carried out as described previously (1). ␤-Galactosidase activities were measured on MC4100 lysogenized with a carrying a nagB-lacZ fusion (MC-B1).…”

“…Growth on GlcN requires the manXYZ-encoded transporter, producing GlcN6P, and the nagB gene product. We found that growth on GlcN was limited by the quantity of the NagB enzyme, by the amount of the GlcN transporter ManXYZ, and thus by the amount of the substrate for the NagB deaminase, GlcN6P (1). The nagB gene is part of the divergent nagEnagBACD operon (Fig.…”

“…Using these con-structs, we have compared growth on GlcN and GlcNAc and tested the effect of mutations, which increased the concentration of either the substrate (GlcN6P) or the allosteric activator (GlcNAc6P). A mutation in the mlc gene, which controls the manXYZ-encoded transporter, increases transport of GlcN two-to threefold (1,19,22) and was found to improve the growth rate for all the strains carrying the different plasmid constructs. Mutations which eliminate the nagA-encoded GlcNAc6P deacetylase produce high levels of the allosteric activator GlcNAc6P (1,25,33) derived from the recycling of PG (15,17) but had no effect on the growth rate.…”

Comparative Genomics and Experimental Characterization of N-Acetylglucosamine Utilization Pathway of Shewanella oneidensis

“…Chitin, the second-most abundant organic polymer in nature after cellulose, is composed of GlcNAc residues that play an important role in supplying carbon and energy in a variety of organisms (8). Chitin is present in the cell walls of fungi as well as in cuticles and exoskeletons of worms, mollusks, and arthropods, and it constitutes a natural source of GlcNAc for many bacteria in respective ecosystems, such as large aquatic reservoirs.…”