Substrate binding in the allosteric site mimics homotropic cooperativity in the SIS-fold glucosamine-6-phosphate deaminases.

Glucosamine-6-phosphate (GlcN6P) deaminases from Escherichia coli (EcNagBI) and Shewanella denitrificans (SdNagBII) are special examples of what constitute nonhomologous isofunctional enzymes due to their convergence, not only in catalysis, but also in cooperativity and allosteric properties. Additionally, we found that the sigmoidal kinetics of SdNagBII cannot be explained by the existing models of homotropic activation. This study describes the regulatory mechanism of SdNagBII using enzyme kinetics, isothermal titration calorimetry (ITC), and X-ray crystallography. ITC experiments revealed two different binding sites with distinctive thermodynamic signatures: a single binding site per monomer for the allosteric activator N-acetylglucosamine 6-phosphate (GlcNAc6P) and two binding sites per monomer for the transition-state analog 2-amino-2-deoxy-D-glucitol 6-phosphate (GlcNol6P). Crystallographic data demonstrated the existence of an unusual allosteric site that can bind both GlcNAc6P and GlcNol6P, implying that the homotropic activation of this enzyme arises from the occupation of the allosteric site by the substrate. In this work we describe the presence of this novel allosteric site in the SIS-fold deaminases, which is responsible for the homotropic and heterotropic activation of SdNagBII by GlcN6P and GlcNAc6P, respectively. This study unveils an original mechanism to generate a high degree of homotropic activation in SdNagBII, mimicking the allosteric and cooperative properties of hexameric EcNagBI but with a reduced number of subunits.

Regulatory Interplay between RNase III and Antisense RNAs in E. coli: the Case of AsflhD and FlhD, Component of the Master Regulator of Motility.

In order to respond to ever-changing environmental cues, bacteria display resilient regulatory mechanisms controlling gene expression. At the post-transcriptional level, this is achieved by a combination of RNA-binding proteins, such as ribonucleases (RNases), and regulatory RNAs, including antisense RNAs (asRNAs). Bound to their complementary mRNA, asRNAs are primary targets for the double-strand-specific endoribonuclease, RNase III. Taking advantage of our own and previously published transcriptomic data sets obtained in strains inactivated for RNase III, we selected several candidate asRNAs and confirmed the existence of RNase III-sensitive asRNAs for crp, ompR, phoP, and flhD genes, all encoding global regulators of gene expression in Escherichia coli. Using FlhD, a component of the master regulator of motility (FlhD4C2), as our model, we demonstrate that the asRNA AsflhD, transcribed from the coding sequence of flhD, is involved in the fine-tuning of flhD expression and thus participates in the control of motility. IMPORTANCE The role of antisense RNAs (asRNAs) in the regulation of gene expression remains largely unexplored in bacteria. Here, we confirm that asRNAs can be part of layered regulatory networks, since some are found opposite to genes encoding global regulators. In particular, we show how an antisense RNA (AsflhD) to the flhD gene, encoding the transcription factor serving as the primary regulator of bacterial swimming motility (FlhD4C2), controls flhD expression, which in turn affects the expression of other genes of the motility cascade. The role of AsflhD highlights the importance of fine-tuning mechanisms mediated by asRNAs in the control of complex regulatory networks.

Complex synergistic amino acid-nucleotide interactions contribute to the specificity of NagC operator recognition and induction.

NagC is a transcription factor that represses genes involved in N-acetylglucosamine catabolism in Escherichia coli. Repression by NagC is relieved by interaction with GlcNAc6P, the product of transport of GlcNAc into the cell. The DNA-binding domain of NagC contains a classic helix-turn-helix (HTH) motif, but specific operator recognition requires, in addition, an adjacent linker sequence, which is thought to form an extended wing. Sequences in the linker region are required to distinguish NagC-binding sites from those of its paralogue, Mlc. In investigating the contribution of the HTH to operator recognition, we have identified mutations in the first two positions of the recognition helix of the DNA-binding motif of NagC, which change NagC from being a repressor, which binds in the absence of the inducing signal (GlcNAc6P), to one whose binding is enhanced by GlcNAc6P. In this case GlcNAc6P behaves as a co-repressor rather than an inducer for NagC. The NagC mutants exhibiting this paradoxical behaviour have basic amino acids, arginine or lysine, at two critical positions of the recognition helix. Introducing a third amino acid change converts NagC back to a protein, which represses in the absence of GlcNAc6P. The triple mutant also effectively represses a modified NagC operator that is not repressed by wild-type NagC, showing that this form of NagC is a more promiscuous DNA binder. Specific recognition of the NagC operator thus involves a modulation of basic amino acid-DNA interactions, which affects the ability to discriminate against other permissive sites.

Plasticity of Promoter-Core Sequences Allows Bacteria to Compensate for the Loss of a Key Global Regulatory Gene.

Transcription regulatory networks (TRNs) are of central importance for both short-term phenotypic adaptation in response to environmental fluctuations and long-term evolutionary adaptation, with global regulatory genes often being targets of natural selection in laboratory experiments. Here, we combined evolution experiments, whole-genome resequencing, and molecular genetics to investigate the driving forces, genetic constraints, and molecular mechanisms that dictate how bacteria can cope with a drastic perturbation of their TRNs. The crp gene, encoding a major global regulator in Escherichia coli, was deleted in four different genetic backgrounds, all derived from the Long-Term Evolution Experiment (LTEE) but with different TRN architectures. We confirmed that crp deletion had a more deleterious effect on growth rate in the LTEE-adapted genotypes; and we showed that the ptsG gene, which encodes the major glucose-PTS transporter, gained CRP (cyclic AMP receptor protein) dependence over time in the LTEE. We then further evolved the four crp-deleted genotypes in glucose minimal medium, and we found that they all quickly recovered from their growth defects by increasing glucose uptake. We showed that this recovery was specific to the selective environment and consistently relied on mutations in the cis-regulatory region of ptsG, regardless of the initial genotype. These mutations affected the interplay of transcription factors acting at the promoters, changed the intrinsic properties of the existing promoters, or produced new transcription initiation sites. Therefore, the plasticity of even a single promoter region can compensate by three different mechanisms for the loss of a key regulatory hub in the E. coli TRN.

Allosteric Activation of Escherichia coli Glucosamine-6-Phosphate Deaminase (NagB) In Vivo Justified by Intracellular Amino Sugar Metabolite Concentrations.

UNLABELLED: We have investigated the impact of growth on glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) on cellular metabolism by quantifying glycolytic metabolites in Escherichia coli Growth on GlcNAc increased intracellular pools of both GlcNAc6P and GlcN6P 10- to 20-fold compared to growth on glucose. Growth on GlcN produced a 100-fold increase in GlcN6P but only a slight increase in GlcNAc6P. Changes to the amounts of downstream glycolytic intermediates were minor compared to growth on glucose. The enzyme glucosamine-6P deaminase (NagB) is required for growth on both GlcN and GlcNAc. It is an allosteric enzyme in E. coli, displaying sigmoid kinetics with respect to its substrate, GlcN6P, and is allosterically activated by GlcNAc6P. The high concentration of GlcN6P, accompanied by the small increase in GlcNAc6P, drives E. coli NagB (NagBEc) into its high activity state, as observed during growth on GlcN (L. I. Álvarez-Añorve, I. Bustos-Jaimes, M. L. Calcagno, and J. Plumbridge, J Bacteriol 191:6401-6407, 2009, http://dx.doi.org/10.1128/JB.00633-09). The slight increase in GlcNAc6P during growth on GlcN is insufficient to displace NagC, the GlcNAc6P-responsive repressor of the nag genes, from its binding sites, so there is only a small increase in nagB expression. We replaced the gene for the allosteric NagBEc enzyme with that of the nonallosteric, B. subtilis homologue, NagBBs We detected no effects on growth rates or competitive fitness on glucose or the amino sugars, nor did we detect any effect on the concentrations of central metabolites, thus demonstrating the robustness of amino sugar metabolism and leaving open the question of the role of allostery in the regulation of NagB. IMPORTANCE: Chitin, the polymer of N-acetylglucosamine, is an abundant biomaterial, and both glucosamine and N-acetylglucosamine are valuable nutrients for bacteria. The amino sugars are components of numerous essential macromolecules, including bacterial peptidoglycan and mammalian glycosaminoglycans. Controlling the biosynthetic and degradative pathways of amino sugar metabolism is important in all organisms to avoid loss of nitrogen and energy via a futile cycle of synthesis and breakdown. The enzyme glucosamine-6P deaminase (NagB) is central to this control, and N-acetylglucosamine-6P is the key signaling molecule regulating amino sugar utilization in Escherichia coli Here, we investigate how the metabolic status of the bacteria impacts on the activity of NagBEc and the N-acetylglucosamine-6P-sensitive transcriptional repressor, NagC.

Dual inducer signal recognition by an Mlc homologue.

The Mlc transcription factor in Escherichia coli controls the expression of the phosphotransferase system genes implicated in the transport of glucose into the cell. Transport of glucose derepresses Mlc-repressed genes by provoking the sequestration of Mlc to the membrane, via an interaction with the dephosphorylated EIIB domain of the glucose transporter, PtsG. NagC, a paralogue of Mlc in E. coli, regulates the use of the amino sugar N-acetylglucosamine (GlcNAc). Both Mlc and NagC are members of the ROK (Repressors, ORFs and Kinases) family. Vibrio cholerae expresses a close orthologue of Mlc, VC2007, which represses the Mlc target, ptsG, in E. coli. However, VC2007 is not sensitive to growth on glucose but responds to growth on N-acetylglucosamine (GlcNAc). We show that growth on GlcNAc generates two different signals, which relieve VC2007 repression of ptsG in E. coli. The majority of the loss of repression is due to VC2007 interacting with dephosphorylated NagE, the GlcNAc-specific transporter. However, a minor part is due to VC2007 binding GlcNAc6P. These two inducing signals are independent and can be separated by mutations in VC2007 eliminating sensitivity to one or other signal. In addition we show that, although most induction of Mlc-repressed genes is dependent upon the interaction of Mlc with PtsG in E. coli, Mlc can also bind to NagE, but it is not sensitive to GlcNAc6P. These observations shed light on how ROK family homologues have evolved in their ability to sense glucose and GlcNAc and of the shift between recognition of different categories of inducer.

Regulation of the Utilization of Amino Sugars by Escherichia coli and Bacillus subtilis: Same Genes, Different Control.

Amino sugars are dual-purpose compounds in bacteria: they are essential components of the outer wall peptidoglycan (PG) and the outer membrane of Gram-negative bacteria and, in addition, when supplied exogenously their catabolism contributes valuable supplies of energy, carbon and nitrogen to the cell. The enzymes for both the synthesis and degradation of glucosamine (GlcN) and N-acetylglucosamine (GlcNAc) are highly conserved but during evolution have become subject to different regulatory regimes. Escherichia coli grows more rapidly using GlcNAc as a carbon source than with GlcN. On the other hand, Bacillus subtilis, but not other Bacilli tested, grows more efficiently on GlcN than GlcNAc. The more rapid growth on this sugar is associated with the presence of a second, GlcN-specific operon, which is unique to this species. A single locus is associated with the genes for catabolism of GlcNAc and GlcN in E. coli, although they enter the cell via different transporters. In E. coli the amino sugar transport and catabolic genes have also been requisitioned as part of the PG recycling process. Although PG recycling likely occurs in B. subtilis, it appears to have different characteristics.

Operator recognition by the ROK transcription factor family members, NagC and Mlc.

NagC and Mlc, paralogous members of the ROK family of proteins with almost identical helix-turn-helix DNA binding motifs, specifically regulate genes for transport and utilization of N-acetylglucosamine and glucose. We previously showed that two amino acids in a linker region outside the canonical helix-turn-helix motif are responsible for Mlc site specificity. In this work we identify four amino acids in the linker, which are required for recognition of NagC targets. These amino acids allow Mlc and NagC to distinguish between a C/G and an A/T bp at positions ±11 of the operators. One linker position, glycine in NagC and arginine in Mlc, corresponds to the major specificity determinant for the two proteins. In certain contexts it is possible to switch repression from Mlc-style to NagC-style, by interchanging this glycine and arginine. Secondary determinants are supplied by other linker positions or the helix-turn-helix motif. A wide genomic survey of unique ROK proteins shows that glycine- and arginine-rich sequences are present in the linkers of nearly all ROK family repressors. Conserved short sequence motifs, within the branches of the ROK evolutionary tree, suggest that these sequences could also be involved in operator recognition in other ROK family members.

Interplay of transcriptional and small RNA-dependent control mechanisms regulates chitosugar uptake in Escherichia coli and Salmonella.

Escherichia coli and Salmonella can use chitin-derived oligosaccharides as carbon and nitrogen sources. Chitosugars traverse the outer membrane through a dedicated chitoporin, ChiP, and are transported across the cytoplasmic membrane by the chitobiose transporter (ChbBCA). Previous work revealed that synthesis of the chitoporin, ChiP, requires transcription of the chbBCARFG operon. A sequence from the chbBC portion of the transcript was shown to act as a decoy target for a regulatory small RNA, ChiX, that normally blocks chiP expression. ChiX is destabilized and degraded upon pairing with chbBC RNA. Here, we show that the chiP gene, like the chbBCARFG operon, is also downregulated at the transcriptional level by the NagC repressor. NagC repression is critical in maintaining chiP mRNA levels low enough, relative to ChiX, to allow full silencing by this sRNA. We also show that pairing of ChiX to chbBC RNA downregulates chbC under uninduced conditions, that is, when ChiX is in excess to the decoy sequence. Hence, under these conditions, chbBC RNA is not just a decoy, but a true target of ChiX regulation. Altogether these findings underscore the importance of stoichiometry in dictating the strength of the sRNA response and in differentiating the regulator from the regulatory target.

Regulation of amino sugar utilization in Bacillus subtilis by the GntR family regulators, NagR and GamR.

In Bacillus subtilis separate sets of genes are implicated in the transport and metabolism of the amino sugars, glucosamine and N-acetylglucosamine. The genes for use of N-acetylglucosamine (nagAB and nagP) are found in most firmicutes and are controlled by a GntR family repressor NagR (YvoA). The genes for use of glucosamine (gamAP) are repressed by another GntR family repressor GamR (YbgA). The gamR-gamAP synton is only found in B. subtilis and a few very close relatives. Although NagR and GamR are close phylogenetically, there is no cross regulation between their operons. GlcN6P prevents all binding of GamR to its targets. NagR binds specifically to targets containing the previously identified dre palindrome but its binding is not inhibited by GlcN6P or GlcNAc6P. GamR-like binding sites were also found in some other Bacilli associated with genes for use of chitin, the polymer of N-acetylglucosamine, and with a gene for another GamR homologue (yurK). We show that GamR can bind to two regions in the chi operon of B. licheniformis and that GamR and YurK are capable of heterologous regulation. GamR can repress the B. licheniformis licH-yurK genes and YurK can repress B. subtilis gamA.

The use of amino sugars by Bacillus subtilis: presence of a unique operon for the catabolism of glucosamine.

B. subtilis grows more rapidly using the amino sugar glucosamine as carbon source, than with N-acetylglucosamine. Genes for the transport and metabolism of N-acetylglucosamine (nagP and nagAB) are found in all the sequenced Bacilli (except Anoxybacillus flavithermus). In B. subtilis there is an additional operon (gamAP) encoding second copies of genes for the transport and catabolism of glucosamine. We have developed a method to make multiple deletion mutations in B. subtilis employing an excisable spectinomycin resistance cassette. Using this method we have analysed the contribution of the different genes of the nag and gam operons for their role in utilization of glucosamine and N-acetylglucosamine. Faster growth on glucosamine is due to the presence of the gamAP operon, which is strongly induced by glucosamine. Although the gamA and nagB genes encode isozymes of GlcN6P deaminase, catabolism of N-acetylglucosamine relies mostly upon the gamA gene product. The genes for use of N-acetylglucosamine, nagAB and nagP, are repressed by YvoA (NagR), a GntR family regulator, whose gene is part of the nagAB yvoA(nagR) operon. The gamAP operon is repressed by YbgA, another GntR family repressor, whose gene is expressed divergently from gamAP. The nagAB yvoA synton is found throughout the Bacilli and most firmicutes. On the other hand the ybgA-gamAP synton, which includes the ybgB gene for a small protein of unknown provenance, is only found in B. subtilis (and a few very close relatives). The origin of ybgBA-gamAP grouping is unknown but synteny analysis suggests lateral transfer from an unidentified donor. The presence of gamAP has enabled B. subtilis to efficiently use glucosamine as carbon source.

DNA looping in prokaryotes: experimental and theoretical approaches.

Transcriptional regulation is at the heart of biological functions such as adaptation to a changing environment or to new carbon sources. One of the mechanisms which has been found to modulate transcription, either positively (activation) or negatively (repression), involves the formation of DNA loops. A DNA loop occurs when a protein or a complex of proteins simultaneously binds to two different sites on DNA with looping out of the intervening DNA. This simple mechanism is central to the regulation of several operons in the genome of the bacterium Escherichia coli, like the lac operon, one of the paradigms of genetic regulation. The aim of this review is to gather and discuss concepts and ideas from experimental biology and theoretical physics concerning DNA looping in genetic regulation. We first describe experimental techniques designed to show the formation of a DNA loop. We then present the benefits that can or could be derived from a mechanism involving DNA looping. Some of these are already experimentally proven, but others are theoretical predictions and merit experimental investigation. Then, we try to identify other genetic systems that could be regulated by a DNA looping mechanism in the genome of Escherichia coli. We found many operons that, according to our set of criteria, have a good chance to be regulated with a DNA loop. Finally, we discuss the proposition recently made by both biologists and physicists that this mechanism could also act at the genomic scale and play a crucial role in the spatial organization of genomes.

The linker sequence, joining the DNA-binding domain of the homologous transcription factors, Mlc and NagC, to the rest of the protein, determines the specificity of their DNA target recognition in Escherichia coli.

Protein-DNA recognition is fundamental to transcriptional regulation. Transcription factors must be capable of locating their specific sites situated throughout the genome and distinguishing them from related sites. Mlc and NagC control uptake and use of the sugars, glucose and N-acetylglucosamine. Both their helix-turn-helix motifs and their consensus binding sites on DNA are very similar. One distinguishing feature is that most NagC sites have a C/G bp at positions -11 and +11 from the centre of symmetry of the operator, while all Mlc sites have A/T. By constructing Mlc and NagC chimeras, we show that the helix-turn-helix motif per se is not responsible for specific recognition of Mlc or NagC sites, but that a linker, joining the DNA-binding domain to the rest of the protein, is the major determinant. We show that a change of just two amino acids in the NagC linker is sufficient to allow NagC to recognize an A/T bp at positions +/-11 and repress Mlc targets. Modelling of the NagC linker suggests that it forms an extended structure containing two arginines and we suggest that these arginines interact differently with the minor groove at positions +/-11 depending upon the presence of a C/G or A/T bp.

Allosteric kinetics of the isoform 1 of human glucosamine-6-phosphate deaminase.

The human genome contains two genes encoding for two isoforms of the enzyme glucosamine-6-phosphate deaminase (GNPDA, EC 3.5.99.6). Isoform 1 has been purified from several animal sources and the crystallographic structure of the human recombinant enzyme was solved at 1.75Å resolution (PDB ID: 1NE7). In spite of their great structural similarity, human and Escherichia coli GNPDAs show marked differences in their allosteric kinetics. The allosteric site ligand, N-acetylglucosamine 6-phosphate (GlcNAc6P), which is an activator of the K-type of E. coli GNPDA has an unusual mixed allosteric effect on hGNPDA1, behaving as a V activator and a K inhibitor (antiergistic or crossed mixed K(-)V(+) effect). In the absence of GlcNAc6P, the apparent k(cat) of the enzyme is so low, that GlcNAc6P behaves as an essential activator. Additionally, substrate inhibition, dependent on GlcNAc6P concentration, is observed. All these kinetic properties can be well described within the framework of the Monod allosteric model with some additional postulates. These unusual kinetic properties suggest that hGNPDA1 could be important for the maintenance of an adequate level of the pool of the UDP-GlcNAc6P, the N-acetylglucosylaminyl donor for many reactions in the cell. In this research we have also explored the possible functional significance of the C-terminal extension of hGNPDA1 enzyme, which is not present in isoform 2, by constructing and studying two mutants truncated at positions 268 and 275.

SlyA protein activates fimB gene expression and type 1 fimbriation in Escherichia coli K-12.

We have demonstrated that SlyA activates fimB expression and hence type 1 fimbriation, a virulence factor in Escherichia coli. SlyA is shown to bind to two operator sites (O(SA1) and O(SA2)), situated between 194 and 167 base pairs upstream of the fimB transcriptional start site. fimB expression is derepressed in an hns mutant and diminished by a slyA mutation in the presence of H-NS only. H-NS binds to multiple sites in the promoter region, including two sites (H-NS2 and H-NS3) that overlap O(SA1) and O(SA2), respectively. Mutations that disrupt either O(SA1) or O(SA2) eliminate or reduce the activating effect of SlyA but have different effects on the level of expression. We interpret these results as reflecting the relative competition between SlyA and H-NS binding. Moreover we show that SlyA is capable of displacing H-NS from its binding sites in vitro. We suggest SlyA binding prevents H-NS binding to H-NS2 and H-NS3 and the subsequent oligomerization of H-NS necessary for full inhibition of fimB expression. In addition, we show that SlyA activates fimB expression independently of two other known regulators of fimB expression, NanR and NagC. It is demonstrated that the rarely used UUG initiation codon limits slyA expression and that low SlyA levels limit fimB expression. Furthermore, Western blot analysis shows that cells grown in rich-defined medium contain ~1000 SlyA dimers per cell whereas those grown in minimal medium contain >20% more SlyA. This study extends our understanding of the role that SlyA plays in the host-bacterial relationship.

An antisense transcript from within the ptsG promoter region in Escherichia coli.

The ptsG gene, encoding the major glucose uptake system in Escherichia coli, is expressed from 2 promoters, a minor promoter p2 and a major downstream promoter p1. Transcription from both promoters is repressed by Mlc, and expression of p1 is activated by the cAMP/catabolite activator protein complex. Expression from p1 is also regulated post-transcriptionally in response to sugar stress via an sRNA, SgrS, which results in translational inhibition and mRNA degradation. Here, we demonstrate an additional level of complexity to the transcriptional pattern surrounding ptsG. A third promoter, p3, located between p1 and p2, was found to express a transcript antisense to ptsG. This promoter was detected by in vitro transcription and by RNA polymerase footprinting techniques and in vivo by S1 analysis and fusions with a lacZ reporter gene. Although the intrinsic strength of the p3 promoter was comparable to that of ptsG, it proved difficult to identify a full-length transcript. A faint transcript of greater than 400 nt could be detected. The transcript thus has more of the characteristics of a divergently expressed cryptic unstable transcript (CUT) than a prokaryotic sRNA.

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

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.

An alternative route for recycling of N-acetylglucosamine from peptidoglycan involves the N-acetylglucosamine phosphotransferase system in Escherichia coli.

A set of enzymes dedicated to recycling of the amino sugar components of peptidoglycan has previously been identified in Escherichia coli. The complete pathway includes the nagA-encoded enzyme, N-acetylglucosamine-6-phosphate (GlcNAc6P) deacetylase, of the catabolic pathway for use of N-acetylglucosamine (GlcNAc). Mutations in nagA result in accumulation of millimolar concentrations of GlcNAc6P, presumably by preventing peptidoglycan recycling. Mutations in the genes encoding the key enzymes upstream of nagA in the dedicated recycling pathway (ampG, nagZ, nagK, murQ, and anmK), which were expected to interrupt the recycling process, reduced but did not eliminate accumulation of GlcNAc6P. A mutation in the nagE gene of the GlcNAc phosphotransferase system (PTS) was found to reduce by 50% the amount of GlcNAc6P which accumulated in a nagA strain and, together with mutations in the dedicated recycling pathway, eliminated all the GlcNAc6P accumulation. This shows that the nagE-encoded PTS transporter makes an important contribution to the recycling of peptidoglycan. The manXYZ-encoded PTS transporter makes a minor contribution to the formation of cytoplasmic GlcNAc6P but appears to have a more important role in secretion of GlcNAc and/or GlcNAc6P from the cytoplasm.

Repression of galP, the galactose transporter in Escherichia coli, requires the specific regulator of N-acetylglucosamine metabolism.

Soupene et al. [J. Bacteriol. (2003) 185 5611-5626] made the unexpected observation that the presence of a mutation, in the gene for the N-acetylglucosamine repressor, nagC, increased the growth rate of Escherichia coli MG1655 on galactose, an unrelated sugar. We have found that NagC, binds to a single, high-affinity site overlapping the promoter of galP (galactose permease) gene and that expression of galP is repressed by a combination of NagC, GalR and GalS. In addition to the previously identified galOE operator, other gal operators further upstream are required for full repression. GalS has a specific role, as it binds with higher affinity to one of the upstream operators but its effect in vivo is only observed in the presence of GalR. Regulation of galP by three specific repressors, NagC, GalR and GalS is unusual in that it involves multiple, specific regulators from two different areas of metabolism. This novel regulation seems to be particular for E. coli and its nearest neighbour, Shigella. Other bacteria with galP orthologues, although retaining the metK-galP gene order, do not have the NagC site. Although quantitative effects were strain specific, nagC mutations increased the growth rate on galactose of all E. coli strains tested.

Switching control of expression of ptsG from the Mlc regulon to the NagC regulon.

The Mlc and NagC transcriptional repressors bind to similar 23-bp operators. The sequences are weakly palindromic, with just four positions totally conserved. There is no cross regulation observed between the repressors in vivo, but there are no obvious bases which could be responsible for operator site discrimination. To investigate the basis for operator recognition and to try to understand what differentiates NagC sites from Mlc sites, we have undertaken mutagenesis experiments to convert ptsG from a gene regulated by Mlc into a gene regulated by NagC. There are two Mlc operators upstream of ptsG, and to switch ptsG to the NagC regulon, it was necessary to change two different characteristics of both operators. Firstly, we replaced the AT base pair at position +/-11 from the center of symmetry of the operators with a GC base pair. Secondly, we changed the sequence of the CG base pairs in the central region of the operator (positions -4 to +4 around the center of symmetry). Our results show that changes at either of these locations are sufficient to lose regulation by Mlc but that both types of changes in both operators are necessary to convert ptsG to a gene regulated by NagC. In addition, these experiments confirmed that two operators are necessary for regulation by NagC. We also show that regulation of ptsG by Mlc involves some cooperative binding of Mlc to the two operators.