Rhizobium bacteroid development

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 develop inside legume root nodules. In this reserach we use peas as our model legume. Questions include how do the bacteria grow inside plants and what factors control this process? How do the bacteria know when to switch on N2 fixation?

Legume symbioses are initiated by flavonoids released by the host roots which induce transcription of the early bacterial nodulation (nod) genes that initiate the first steps in the infection process. Bacteria are trapped by curling plant root hairs which form infection threads bacteria enter and grow down, during formation of the nodule. The bacteria are engulfed by endocytosis resulting in them being surrounded by a plant derived symbiosome membrane. In nodules of galegoid legumes (a clade in the subfamily Papilionoideae e.g. Medicago, Pisum or Vicia) bacteria undergo dramatic increases in their size, shape and DNA content , before they activate the high level of expression of nif and fix genes involved in N2 fixation. Thus the plant takes control of the bacterial cell cycle by secretion of nodule cysteine rich peptides, resulting in bacteroids from galegoid legumes being terminally differentiated. As plants control bacterial cell division, provide LIV and the cofactor homocitrate for nitrogenase we proposed that bacteroids in galegoid nodules behave like organelles. To understand bacteroid development we used differential microarray analysis to identify those genes specifically up-regulated in bacteria in very early (7 d) rather than mature nodules (>15 d) or the rhizosphere. We have mutated the key 42 genes up-regulated in bacteria in 7 d nodules (16 prevent or reduce N2 fixation) of which 20 are transcriptional regulators. To understand bacteroid development we will use microarray analysis to define the control network between transcription factors and target genes. Chip-seq and 5' transcript mapping will then be used to determine whether transcription factors directly regulate their target genes. The spatial and temporal transcription pattern of development genes in nodules will then be mapped with mCherry fusions. This will be combined with detailed phenotypic analysis of the effects of these mutations on bacteroid development and symbiotic N2 fixation.

Our aim is to understand 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 environmenral 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.