Mechanism of global regulation of ATP dependent transporters by PTS-NTR

Bacteria are simple single celled organisms that lack the membrane bound structures found in higher cells of plants and animals. However, while bacteria may have a less complex cellular organisation they carry out a huge range of chemical reactions not found in plants and animals. Bacteria are responsible for the cycling of many nutrients such as N2 (N2 is also known as nitrogen gas and consists of two nitrogen atoms bound by a strong triple bond), which is a very inert atmospheric gas. N2 makes up 78% of the atmosphere but is very unreactive and cannot be used directly as a source of nitrogen, which is needed for amino acid, protein and DNA synthesis. However, a small number of bacteria can reduce (add hydrogen) to N2 and convert it into ammonia (NH3), which is readily incorporated into amino acids and then all the other building blocks of life, by a wide range of organisms including bacteria and plants. In many parts of the world the limitation to growth of plants, which in turn support animal life, is the supply of nitrogen as ammonia or related compounds. Since up to 65% of available nitrogen (eg ammonia) comes from bacteria this makes them essential for life on earth. Within the bacteria, most of the nitrogen is actually produced by one family known as the Rhizobiacea. This remarkable group of bacteria form a symbiotic association (both partners benefit) with plants of the legume family, that results in the formation of root nodules (on pea plants these are 2-3 mm bulbs that can easily be seen by pulling up a plant and inspecting its roots). The rhizobia are held inside the nodules where the plant provides them with an ideal environment (low O2 and lots of energy) in which they can reduce N2 to ammonia. The ammonia is supplied to the plant as its nitrogen source so this is why this is known as a symbiotic interaction. It means that the plant does need any nitrogen added to the the soil and enables rapid growth. The purpose of this research is to understand how the bacteria (rhizobia) regulate transport processes that are essential for acquiring amino acids.

The largest input of fixed nitrogen in the biosphere comes from biological reduction of atmospheric N2 to ammonium. Most of this comes from legume-Rhizobium symbioses, arising from infection of host plants and results in root structures called nodules within which bacteria reduce N2 and feed the resultant ammonia to the host. However, we showed that nutrient exchange between the plant cytosol and bacteroid is more complex with amino acid transport by two ATP binding cassette (ABC) amino acid uptake systems (Aap and Bra) being essential for N2 fixation. Plants take control of bacteroid differentiation and must provide branched chain amino acids to allow full development and persistence of bacteroids. We called this phenomenon symbiotic auxotrophy because bacteroids only become auxotrophic for branched chain amino acids in planta. We have now shown that these transporters are regulated by PTSNtr at the post translational level and the high affinity KDP system at the transcriptional level. We therefore proposed that PTSNtr is a ubiquitous phosphotransferase system that globally regulates ATP dependent transport systems, making it absolutely central to the regulation of most aspects of bacterial physiology and growth. Therefore, our objective is to elucidate the phosphotransfer pathway between PTS components and identify their target proteins. Absolutely critical to understanding the global role of PTSNtr is to determine how it senses adenylate charge and other metabolites and how this controls ATP dependent transport processes. This is likely to have a major impact in many bacteria because PTSNtr is widely distributed and essential for diverse processes ranging from nutrient acquisition to secretion of toxins, drugs, proteins, virulence factors and symbiotic N2-fixation.

Our aim is to understand regulation of amino acid transport, its role in bacteroid development and nitrogen fixation by legumes. Nitrogen is one of the main constraints on agricultural productivity so its use is essential for high crop yields. In a world where food security is now considered a national priority crop yield is of critical importance. However, the drive for yield alone has led to very high application of nitrogen with consequent nitrate contamination of groundwater and problems of eutrophication. The problem is so serious that reactive nitrogen in the biosphere has doubled from preindustrial levels primarily through massive inputs into agriculture. It also results in the production of N2O which is around 300 times more potent than CO2 as a greenhouse gas. These are problems of regional, national and international scope that require urgent amelioration and are at the forefront of the grand challenges for UK science. By improving our understanding of how rhizobia develop into N2 fixing bacteroids in legume nodule we acquire the understanding to improve the competitive success of desirable strains of Rhizobium. It also lays down a foundation of understanding for the transfer of bacteria to nodules in other plants such as cereals. These aims are long term but ultimately this work has relevance to farming practice as well as government policy in decisions about nitrogen utilization in agriculture. It is also relevant to UK attempts to reduce greenhouse emissions and produce a low carbon economy. Understanding the nitrogen fixation and its role in the nitrogen cycle in agricultural has wider benefits applicable to the UK public because of its importance in food security and meeting international obligations for mitigating the effects of climate change. We propose to reach a wide audience of farmers, the public, national and international policy makers and charitable institutions through active outreach (Friends of John Innes). We also have strong links with "The Nitrous Oxide Focus Group" and the newly formed "Consortium for Legumes in Agriculture, Society and Environment", which is an international consortium to promote understanding on the use of legumes. In addition we have broad links to the environmental impact of this work through the Earth and Life Systems Alliance between JIC and UEA (ELSA) and to the UK government via the "Living With Environmental Change program (LWEC)" which has its secretariat at UEA. The proposal is also broadly in the highlight area "Effects of environmental change on the soil-water interface: Implications for food production and water supply". This is because legume use has a substantial impact on soil and water quality particularly for nitrogen run off problems. Legumes use reduces the need for nitrogen fertilizer and therefore decreases the carbon footprint of agriculture associated with fertilizer production in the energy intensive Haber-Bosch process.
This proposal also deals more generally with the regulation of ATP-dependent processes in bacteria by PTSNtr. ABC transporters are the largest group of membrane transport systems in living organisms and are essential for diverse processes ranging from symbiotic N2-fixation and nutrient acquisition, to secretion of toxins, drugs, proteins and virulence factors. Understanding how these systems are regulated by PtsNtr in bacteria may provide drug targets for development of antimicrobials.