Repression and induction of the nag regulon of Escherichia coll K-12: the roles of nagC and nagA in maintenance of the uninduced state

The nag regulon located at 15.5 min on the Escherichia coli chromosome consists of two divergent operons, nagE and nagBACD, encoding genes involved in the uptake and metabolism of N-acetylglucosamine. Null mutations have been created in each of the genes by insertion of antibiotic resistance cartridges. The phenotypes of the strains carrying the insertions in nagE, B and A were consistent with the previous identification of gene products: nagE, EII(Nag), the N-acetylglucosamine specific transporter of the phosphotransferase system and nagB and nagA, the two enzymes necessary for the degradation of N-acetylglucosamine. Insertions in the nagC result in derepression of the nag genes, which is consistent with earlier observations that the nagC gene encodes the repressor of the regulon. Insertions in nagA also provoke a derepression, implying that nagA has a role in the regulation of the expression of the nag regulon as well as in the degradation of the amino-sugars. N-acetylglucosamine-6-phosphate, the intracellular product of N-acetylglucosamine transport and the substrate of the nagA gene product, is shown to be an inducer of the regulon and this suggests how nagA mutations result in derepression: the absence of N-acetylglucosamine-6-phosphate deacetylase allows N-acetylglucosamine-6-phosphate to accumulate and induce the regulon.     

Sequence of cloned enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:N-acetylglucosamine phosphotransferase system of Escherichia coli

In Escherichia coli, N-acetylglucosamine (nag) metabolism is joined to glycolysis via three specific enzymes that are the products of the nag operon. The three genes of the operon, nagA, nagB, and nagE, were found to be carried by a colicin plasmid, pLC5-21, from a genomic library of E. coli [Clarke, L., & Carbon, J. (1976) Cell (Cambridge, Mass.) 9,91-99]. The nagE gene that codes for enzyme IIN-acetylglucosamine of the phosphoenolpyruvate:sugar phosphotransferase system (PTS) was sequenced. The nagE sequence is preceded by a catabolite gene activator protein binding site and ends in a putative rho-independent termination site. The amino acid sequence determined from this DNA sequence shows 44% homology to enzymes IIglucose and IIIglucose of the PTS. Enzyme IIN-acetylglucosamine, which has 648 amino acids and a molecular weight of 68,356, contains a histidine at residue 569 which is homologous to the active site of IIIglc. Sequence homologies with enzymes IIglucose, II beta-glucoside, and IIsucrose indicate that residues His-190, His-213, and His-295 of enzyme IInag are also conserved and that His-190 is probably the second active site histidine. Other sequence homologies among these enzymes II suggest that they contain several sequence transpositions. Preliminary models of the enzymes II are proposed.     

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

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.     

Induction of the nag regulon of Escherichia coli by N-acetylglucosamine and glucosamine: role of the cyclic AMP-catabolite activator protein complex in expression of the regulon.

The divergent nag regulon located at 15.5 min on the Escherichia coli map encodes genes necessary for growth on N-acetylglucosamine and glucosamine. Full induction of the regulon requires both the presence of N-acetylglucosamine and a functional cyclic AMP (cAMP)-catabolite activator protein (CAP) complex. Glucosamine produces a lower level of induction of the regulon. A nearly symmetric consensus CAP-binding site is located in the intergenic region between nagE (encoding EIINag) and nagB (encoding glucosamine-6-phosphate deaminase). Expression of both nagE and nagB genes is stimulated by cAMP-CAP, but the effect is more pronounced for nagE. In fact, very little expression of nagE is observed in the absence of cAMP-CAP, whereas 50% maximum expression of nagB is observed with N-acetylglucosamine in the absence of cAMP-CAP. Two mRNA 5' ends separated by about 100 nucleotides were located before nagB, and both seem to be similarly subject to N-acetylglucosamine induction and cAMP-CAP stimulation. To induce the regulon, N-acetylglucosamine or glucosamine must enter the cell, but the particular transport mechanism used is not important.     

Identification, molecular cloning and sequence analysis of a gene cluster encoding the Class II fructose 1,6-bisphosphate aldolase, 3-phosphoglycerate kinase and a putative second glyceraldehyde 3-phosphate dehydrogenase of Escherichia coli

To investigate a possible chromosomal clustering of glycolytic enzyme genes, the complete nucleotide sequence of the 8029 bp insert of Escherichia coli DNA in the ColE1 plasmid pLC33-5 of the Clarke and Carbon collection (Clark and Carbon, 1976) was determined. Genes (pgk, fda) encoding the phosphoglycerate kinase and Class II fructose 1,6-bisphosphate aldolase, respectively, of E. coli were identified. The phosphoglycerate kinase was found to be highly homologous in primary structure to the same enzyme from eukaryotic organisms. A further large open reading frame, designated gapB, was also identified, which on the basis of sequence homology, appears to encode another glycolytic enzyme, glyceraldehyde 3-phosphate dehydrogenase. This putative gene differs significantly from that (designated gapA) already identified as coding for this enzyme in E. coli and which maps elsewhere on the chromosome. The products, if any, of several other open reading frames remain to be identified.     

A dominant mutation in the gene for the Nag repressor of Escherichia coli that renders the nag regulon uninducible.

The gene nagC encodes the repressor for the nag regulon. A point mutation within the gene, which confers a super-repressor phenotype and makes the repressor insensitive to the inducer, N-acetylglucosamine 6-phosphate, has been characterized. The mutation is semi-dominant since heterozygous diploids have reduced growth rates on glucosamine and N-acetylglucosamine compared to the wild-type strain.     

Alternative route for biosynthesis of amino sugars in Escherichia coli K-12 mutants by means of a catabolic isomerase.

By inserting a lambda placMu bacteriophage into gene glmS encoding glucosamine 6-phosphate synthetase (GlmS), the key enzyme of amino sugar biosynthesis, a nonreverting mutant of Escherichia coli K-12 that was strictly dependent on exogenous N-acetyl-D-glucosamine or D-glucosamine was generated. Analysis of suppressor mutations rendering the mutant independent of amino sugar supply revealed that the catabolic enzyme D-glucosamine-6-phosphate isomerase (deaminase), encoded by gene nagB of the nag operon, was able to fulfill anabolic functions in amino sugar biosynthesis. The suppressor mutants invariably expressed the isomerase constitutively as a result of mutations in nagR, the locus for the repressor of the nag regulon. Suppression was also possible by transformation of glmS mutants with high-copy-number plasmids expressing the gene nagB. Efficient suppression of the glmS lesion, however, required mutations in a second locus, termed glmX, which has been localized to 26.8 min on the standard E. coli K-12 map. Its possible function in nitrogen or cell wall metabolism is discussed.     

Identification of the gonococcal glmU gene encoding the enzyme N-acetylglucosamine 1-phosphate uridyltransferase involved in the synthesis of UDP-GlcNAc.

In searching for the gonococcal sialyltransferase gene(s), we cloned a 3.8-kb DNA fragment from gonococcus strain MS11 that hybridized with the oligonucleotide JU07, which was derived from the conserved C terminus of the sialyl motif present in mammalian sialyltransferases. Sequencing of the fragment revealed four putative open reading frames (ORFs), one of which (ORF-1) contained a partial sialyl motif including the amino acid sequence VGSKT, which is highly conserved among sialyltransferases. The gene was flanked by two inverted repeats containing the neisserial DNA uptake sequence and was preceded by a putative sigma 54 promoter. Database searches, however, revealed a high degree of homology between ORF-1 and the N-acetylglucosamine 1-phosphate uridyltransferase (GlmU) of Escherichia coli and Bacillus subtilis and not with any known sialyltransferase. This homology was further established by the successful complementation of an orf-1 mutation by the E. coli glmU gene. Enzyme assays demonstrated that ORF-1 did not possess sialyltransferase activity but mimicked GlmU function catalyzing the conversion of N-acetylglucosamine 1-phosphate into UDP-N-acetylglucosamine, which is a key metabolite in the syntheses of lipopolysaccharide, peptidoglycan, and sialic acids.     

N-acetylglucosamine 6-phosphate deacetylase (nagA) is required for N-acetyl glucosamine assimilation in Gluconacetobacter xylinus

Metabolic pathways for amino sugars (N-acetylglucosamine; GlcNAc and glucosamine; Gln) are essential and remain largely conserved in all three kingdoms of life, i.e., microbes, plants and animals. Upon uptake, in the cytoplasm these amino sugars undergo phosphorylation by phosphokinases and subsequently deacetylation by the enzyme N-acetylglucosamine 6-phosphate deacetylase (nagA) to yield glucosamine-6-phosphate and acetate, the first committed step for both GlcNAc assimilation and amino-sugar-nucleotides biosynthesis. Here we report the cloning of a DNA fragment encoding a partial nagA gene and its implications with regard to amino sugar metabolism in the cellulose producing bacterium Glucoacetobacter xylinus (formally known as Acetobacter xylinum). For this purpose, nagA was disrupted by inserting tetracycline resistant gene (nagA::tet(r); named as ΔnagA) via homologous recombination. When compared to glucose fed conditions, the UDP-GlcNAc synthesis and bacterial growth (due to lack of GlcNAc utilization) was completely inhibited in nagA mutants. Interestingly, that inhibition occured without compromising cellulose production efficiency and its molecular composition under GlcNAc fed conditions. We conclude that nagA plays an essential role for GlcNAc assimilation by G. xylinus thus is required for the growth and survival for the bacterium in presence of GlcNAc as carbon source. Additionally, G. xylinus appears to possess the same molecular machinery for UDP-GlcNAc biosynthesis from GlcNAc precursors as other related bacterial species.     

Mutations in two unlinked genes are required to produce asparagine auxotrophy in Escherichia coli.

Escherichia coli K-12 has two genes, asnA+ and asnB+, either one of which is able to satisfy the need of cells for asparagine. In order for a strain to have an auxotrophic requirement for asparagine, both genes must be mutationally inactivated. We obtained mutants with Tn5 inserted in asnB. asnB was mapped by conjugation and by three-factor P1 transductions at 15 min on the E coli K-12 linkage map, between ubiF and nagB. Specialized transducing phage lamba 781 supE was shown to carry asnB, as well as supE, ubiF, nagA, and nagB. asnA is the previously mapped ilv-linked asn locus, whiich is between uncB and rbs. E. coli C also has two asn genes, corresponding to asnA and asnB.     

Organisation of the Escherichia coli chromosome between genes glnS and glnU, V

Summary Analysis of bacteriophage DNA and of subcloned plasmid DNA has allowed the localisation of the following genes, located at 16 min on the Escherichia coli chromosome, within a restriction map of the region: glnS, nagE, nagB, nagA, asnB, metT, leuW, glnU, glnU, metT, glnV, glnV.     

Cloning and nucleotide sequence of the aroA gene of Bordetella pertussis.

The aroA locus of Bordetella pertussis, encoding 5-enolpyruvylshikimate 3-phosphate synthase, has been cloned into Escherichia coli by using a cosmid vector. The gene is expressed in E. coli and complemented an E. coli aroA mutant. The nucleotide sequence of the B. pertussis aroA gene was determined and contains an open reading frame encoding 442 amino acids, with a calculated molecular weight for 5-enolpyruvylshikimate 3-phosphate synthase of 46,688. The amino acid sequence derived from the nucleotide sequence shows homology with the published amino acid sequences of aroA gene products of other microorganisms.