Allosteric regulation of glucosamine-6-phosphate deaminase (NagB) and growth of Escherichia coli on glucosamine

Abstract

Growth on N-acetylglucosamine (GlcNAc) produces intracellular N-acetylglucosamine-6-phosphate (GlcNAc6P), which affects the regulation of the catabolism of amino sugars in Escherichia coli in two ways. First, GlcNAc6P is the inducing signal for the NagC repressor, and thus it increases the expression of the enzymes of the nagE-nagBACD operon. Second, it is the allosteric activator of glucosamine-6P (GlcN6P) deaminase, NagB, and thus increases the catalytic capacity of this key enzyme in the metabolism of amino sugars. We showed previously that both the level of expression of the nagB gene and the transport of glucosamine were limiting the growth rate on GlcN (L. I. Alvarez-Añorve et al., J. Bacteriol. 187:2974-2982, 2005). We were unable to conclude if the lack of allosteric activation of wild-type NagB was also contributing to the slower growth rate on GlcN. Using a single-copy plasmid, with a constitutive promoter, we have separated the effects of GlcNAc6P on the NagB protein level and on deaminase activity. We show that over a range of intracellular NagB concentrations it is the quantity of the substrate, GlcN6P, which is limiting growth rather than the concentration of the allosteric activator, GlcNAc6P. On the other hand, the F174A mutant of NagB, which requires higher concentrations of GlcNAc6P for activity in vitro, grew better on GlcN in the presence of GlcNAc6P. However, wild-type NagB behaves as if it is already fully allosterically activated during growth on GlcN, and we present evidence suggesting that sufficient GlcNAc6P for allosteric activation is derived from the recycling of peptidoglycan.

FIG. 1.

Structure of the divergent nagE-nagBACD operon and function of the encoding genes. GlcNAc enters the cell mostly by the nagE-encoded PTS transporter and is metabolized by the nagA- and nagB-encoded enzymes to fructose-6P. Expression of the genes nagE, nagA, and nagB is repressed by the NagC repressor. GlcN is transported by the manXYZ-encoded PTS transporter and requires just the nagB-encoded GlcN6P deaminase to be converted to Fru6P.

FIG. 2.

Western blot of cultures of strains carrying the different NagB-expressing plasmids. (A) LAA20 (ΔnagB::cm) carrying a pXE1-derived plasmid expressing NagB-L1, -L2, -L3, or -L4 or MC4100 carrying the vector plasmid pXE1 was grown in glucose, GlcN, or GlcNAc medium, as indicated, to late exponential phase (A₆₅₀ = 0.8). Bacteria were chilled, harvested by centrifugation, and lysed by sonication and boiling in sodium dodecyl sulfate sample buffer. Aliquots (0.25 A₆₅₀ units) were analyzed on 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis, transferred to Hybond-C membrane, and treated with anti-NagA and anti-NagB as described previously (1). S, standards (100 ng NagA and NagB); C, control LAA20 without any plasmid grown in glucose. Note that NagB is poorly retained on the membranes, resulting in a low signal. The band running just faster than NagB is due to contaminant antibodies reacting with a protein of molecular weight similar to that of NagB and which is equally present in all lanes. (B) NagB protein levels due to each construct and growing on the different media were quantified by using the ImageQuant program of PhosphorImager. For each blot, the levels of NagB were normalized to the level of the NagB protein from the NagB-L4 construct growing in glucose. The numbers are the relative levels ± standard deviations of three or four experiments.

FIG. 3.

Growth rates of the NagB constructs on GlcN and GlcNAc. LAA20 with pXEI-derived plasmids carrying the NagB-L1, -L2, -L3, and -L4 constructs expressing wt NagB to different levels, NagB-L4 with the F174A mutation, or MC4100 carrying pXE1 was grown on GlcN or GlcNAc. Growth on GlcN was also measured in the presence of the mutations mlc, ΔnagE-nagBACD, nagA, and nagC as indicated. The histogram gives the DT in minutes ± standard deviations and the means of four to eight cultures (note that the scales vary).