Co-ordination of the utilisation of amino sugars in the bacteria E. coli and B. subtilis – GRONAG

Amino sugars are fundamental building blocks in the construction of many biologically important molecules. The most abundant are glucosamine (GlcN), N-acetylglucosamine (GlcNAc) and the sialic acids, which are widely distributed in nature and are found in materials as diverse as the peptidoglycan of bacterial cell walls, the complex glycosaminoglycans of mammalian tissues and the chitin of crab shells. Amino sugars are also important energy sources for many tissues. The dual anabolic and catabolic roles of the amino sugars mean that their synthesis and degradation need to be carefully controlled to avoid them entering into a futile cycle, synthesising and degrading glucosamine-6P, and thus dissipating energy uselessly. In E. coli, the only species where this question has been addressed, control is exerted at, at least, two levels: at the level of gene expression by the transcription factor NagC, and at the level of enzyme activity by the allosteric control of the enzyme glucosamine-6P deaminase (NagB). Our proposal addresses the question of the coordination of the synthesis and degradation of amino sugars by complementary approaches in vivo and in vitro, and in two model organisms, Gram-positive and Gram-negative. Our previous results suggest that, in E. coli the allosteric regulation of GlcN6P deaminase could be important to keep the enzyme quiescent in the absence of exogenous amino sugars. GlcN is a worse carbon source in E. coli than GlcNAc and this is due to limited induction of NagC during growth on GlcN. GlcNAc6P is the key metabolite in controlling the activity of both NagB and NagC but they appear to respond to a different range of concentrations. We want to set up methods to measure the concentrations of GlcN6P and GlcNAc6P in E. coli, which integrated with the physiological and enzymatic data, will allow a description of amino sugar fluxes. Simultaneously we will continue our analysis of the NagC transcription factor using mutagenesis and molecular modelling and dynamics to investigate the GlcNAc6P binding site and compare it to the GlcNAc6P binding site on NagB. We will also continue the analysis of the specificity of the NagC binding site on DNA and the research for new members of the NagC regulon. We will extend our analysis to B. subtilis, the model Gram positive organism. There are several indications that the control of amino sugar metabolism is very different compared to that in E. coli. The number and organisation of the genes, identified by their similarity to the E. coli counterparts are different. In particular there are two genes encoding NagB orthologues but, unlike their E. coli counterpart, neither is allosteric. Moreover GlcN is a better carbon source than GlcNAc for B. subtilis. We will analyse the function and regulation of these genes in vivo and characterise the critical enzymes in vitro. An in silico genomic survey of the amino sugar utilisation genes in other bacteria will be interpreted in the context of our results on the model Gram positive and Gram negative organisms. Peptidoglycan is the major reserve of amino sugars in both Gram positive and negative species and peptidoglycan recycling affects amino sugar pool sizes in E. coli. Genes involved in the metabolism of peptidioglycan are known virulence determinants in several pathogenic bacteria and peptidoglycan degradation fragments are the signals initiating the host's innate immune response after infection by bacteria like Neisseria. We will initiate a study of peptidoglycan recycling in B. subtilis, the first in a Gram-positive organism, a group which includes a large number of serious pathogens.