Dissecting the nitrogen regulation system of streptomycetes

Streptomycetes are harmless soil bacteria that are the primary source of the vast majority of antibiotics that we use to cure infectious disease, as well as providing us with numerous other medicines that are used, for example, to treat cancer and to help organ transplant patients. Most of these compounds are called secondary metabolites, in that their synthesis is dispensable and often occurs late in the growth cycle. Amongst the bacteria, streptomycetes are also relatively unusual in that they have a complex life cycle during which they undergo a number of changes in cell shape and form leading to the production of a chain of robust spores that are dispersed and then germinate to repeat the cycle. Both the production of antibiotics and the differentiation process occur only under certain conditions, one of which is a limitation on the availability of nitrogen sources. In many bacteria the complex control processes that allow the cell to determine whether it is short of nitrogen are well understood, but up until now we have not determined exactly how this process works in streptomycetes. We plan to use a combination of our knowledge of how this process of 'nitrogen control' works in other bacteria, together with recent information on all the genes that are present in streptomycetes, to begin to dissect the sensors and switches that make up the nitrogen control system in these economically important bacteria. If we can achieve this, it will provide important information that will allow us to manipulate these bacteria to control when they begin to synthesise important compounds such as antibiotics, and the amounts produced.

This grant seeks to elucidate the mechanisms whereby streptomycetes respond to the availability of fixed nitrogen compounds in their environment and thereby control those metabolic pathways that are related to nitrogen metabolism. These processes relate both to the primary metabolism (synthesis and catabolism) of nitrogen compounds and to the control of synthesis of secondary metabolites. Whilst a number of nitrogen control proteins have been identified in streptomycetes, their interactions and the signal transduction pathways that regulate their expression or activities are unknown. Furthermore although synthesis of many antibiotics, is regulated by nitrogen sources in the growth medium, the links between nitrogen control and secondary metabolite synthesis are not understood. We will use Streptomyces venezuelae as our model system owing to its dispersed and rapid growth in liquid culture. The S. venezuelae genome sequence is complete and whole genome Affymetrix chips are available. We will use transcriptomics to determine the spectrum of nitrogen-controlled genes and proteomic analysis to examine the role of covalent modification in responding rapidly to nitrogen nutritional shifts. Previous data from studies in S. coelicolor and in other bacteria suggest that the GlnR, GlnRII, GlnK and AmtB proteins are likely to be major components of the nitrogen control system. We will combine our transcriptomic and proteomic studies with analysis of mutants of the structural genes for each of these proteins to determine the roles of each of these potentially key components. In particular we will seek to identify the major targets for regulation by GlnK and to dissect the way in which the activities of GlnR and GlnRII are controlled. Finally we will study the regulation of two well understood secondary metabolite pathways, those for chloramphenicol and actinorhodin production, to investigate the links between nitrogen control and the synthesis of these antibiotics.