Rhizobium
leguminosarum
The two plants on the left show the
bacteroids in bean nodules
effect of a metabolic block that
prevents amino acid uptake
by R. leguminosarum
email philip.poole@jic.ac.uk
phone 44-1603 450750
The general areas in which my group works are bacterial genetics and molecular biology of plant associated bacteria. Our emphasis is to study the physiology of bacterial growth and survival in the rhizosphere and how they establish symbiotic interactions with plants. A further focus of our work is the physiology and biochemistry of nitrogen fixation in legume nodules. Our current research areas are described below.
We have a long standing interest in how metabolism is regulated in pea bacteroids. This includes whether the TCA cycle is used exclusively to generate energy or whether other split pathways operate. Recent work has also focussed on the role of NAD+ malic enzyme and the PEP carboxykinase pathways in the generation of pyruvate in bacteroids of Rhizobium leguminosarum. Another question concerns the role of amino acid transport and storage polymer biosynthesis. In particular pea bacteroids must be supplied with branched chain amino acids by the plant because their synthesis is cutailed in bacteroids (2,3). This means that bacteroid metabolim can be considered as developmentally regulated and has led us to propose that bacteroids should be considered as plant organelles (4). The big question we are trying to understand is how is carbon flux and hence the rate of N2 fixation controlled within the context of the bacteroid differentiation. When are various metabolic pathways switched on and off during bacteroid development? When are key nutrient systems such as metal, vitamin and amino acid transport activated and how are these nutrients obtained from the plant?
1.
Mulley G, et al. (2010) Pyruvate is
synthesized by two pathways in pea bacteroids with different efficiencies for
nitrogen fixation. J. Bacteriol.
192(19):4944-4953.
2.
Lodwig EM, et
al. (2003) Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium
symbiosis. Nature 422:722-726.
3.
Prell J,
et al. (2009) Legumes regulate
Rhizobium bacteroid development and persistence by the supply of
branched-chain amino acids. Proc. Natl. Acad. Sci. USA 106:12477-12482.
4.
Oldroyd G, Murray J, Poole PS, & Downie JA (2011) The rules of engagement in the
legume-rhizobial symbiosis. Annual Review
of Genetics 45:119-144.
The
infection of legume hosts by rhizobia is typically initiated by rhizobia
attaching to root hairs. This is followed by a complex developmental pathway
that results in the formation of root nodules. The differentiated form of
rhizobia present in root nodules (bacteroids), obtain dicarboxylic acids
(succinate, fumarate and malate) as a carbon and energy source from the plant
(1-3). It has always been assumed that
these dicarboxylic acids are oxidised by the TCA-cycle to provide electrons and
ATP for N2-reduction to ammonium and the bacteroids simply secrete
the ammonium to the plant. A major
reassessment of this was caused by our demonstration that both ammonium
and alanine are secreted by bacteroids
(4),
which is supported by our recent work showing that bacteroids can completely
stop all assimilation of ammonium (5).
However, we also demonstrated that an even
more complex exchange is required with an obligate requirement for amino
acid uptake by nodule bacteria via the ABC transporters Aap and Bra (6).
Unravelling this conundrum was complicated by Aap and Bra transporting a wide
range of amino acids. However, by constraining the solute specificity of Bra we
showed that only branched chain amino acids need to be supplied to bacteroids by
the plant
(7-8). Preventing branched chain amino acid
uptake by bacteroids leads to amino acid starvation; causing a failure to fully
develop, reduced size and endoreduplication of their chromosomes. This
phenomenon was named symbiotic auxotrophy because
R. leguminosarum only becomes
auxotrophic when in symbiosis with the plant and is caused by the shut-down of
amino acid synthesis by bacteroids. It has led us to propose that bacteroids can
be considered to be organelles (9).
We demonstrated that this developmental pathway is regulated by a number of
factors including BacA (10) and as part of a large programme to
understand the development of bacteroids we dissected the transcriptional
changes that occur over time as bacteroids develop (10-11). A major breakthrough in this has
been to recognise that many of the early transcriptional changes in developing
bacteroids (~50%) also occur in free-living rhizosphere bacteria (12).
Once these shared transcriptional changes are removed the changes specific to
developing bacteroids are revealed. For the first time this has enabled us to
initiate a project to specifically examine the early development genes in
bacteroid formation. We are now investigating the regulatory network that
governs bacteroid development using transcriptional regulator mutants,
microarrays, Chip-seq, and biochemical analysis.
1.
Mulley G, et al. (2010) Pyruvate is
synthesized by two pathways in pea bacteroids with different efficiencies for
nitrogen fixation. J. Bacteriol.
192(19):4944-4953.
2.
Prell J & Poole P (2006) Metabolic changes of
rhizobia in legume nodules. Trends Microbiol. 14(4):161-168.
3.
White J, Prell J, James EK, & Poole P (2007)
Nutrient sharing between symbionts. Plant
Physiol. 144(2):604-614.
4.
Allaway D,
et al. (2000) Identification of alanine dehydrogenase and its role in mixed
secretion of ammonium and alanine by pea bacteroids.
Mol. Microbiol. 36(2):508-515.
5.
Mulley G,
et al. (2011) Mutation of GOGAT prevents pea bacteroid formation and N2
fixation by globally down-regulating transport of organic nitrogen sources.
Mol. Microbiol. 80:149-167.
6.
Lodwig EM,
et al. (2003) Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium
symbiosis. Nature 422:722-726.
7.
Prell J,
et al. (2009) Legumes regulate
Rhizobium bacteroid development and persistence by the supply of
branched-chain amino acids. Proc. Natl. Acad. Sci. USA 106:12477-12482.
8.
Prell J,
et al. (2010) Role of symbiotic auxotrophy in the
Rhizobium-legume symbioses.
PLoS ONE 5(11):e13933.
9.
Oldroyd G, Murray J, Poole PS, & Downie JA
(2011) The rules of engagement in the legume-rhizobial symbiosis.
Annual Review of Genetics 45:119-144.
10.
Karunakaran R,
et al. (2010) BacA Is Essential for Bacteroid Development in Nodules of
Galegoid, but not Phaseoloid, Legumes.
J. Bacteriol. 192(11):2920-2928.
11.
Karunakaran R,
et al. (2009) Transcriptomic analysis of
Rhizobium leguminosarum b.v.
viciae in symbiosis with host plants
Pisum sativum and
Vicia cracca. J. Bacteriol.
191(12):4002-4014.
12.
Ramachandran V, East
AK, Karunakaran R, Downie JA, & Poole PS (2011)
Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet
rhizospheres investigated by comparative transcriptomics.
Genome Biology, in press.
The common amino acid permease operon (aap) of R. leguminosarum codes for an ABC type transport complex, similar to that for multi-drug resistance protein and the cystic fibrosis chloride channel (CFTR). These proteins are crucial regulators of membrane function in both eukaryotes and prokaryotes and are of great importance to medicine. We have recently discovered a second general amino acid permease, Bra, which with the Aap controls most amino acid uptake by R. leguminosarum. The Aap and Bra have unique properties that have allowed us to begin to probe the way in which these transport complexes actually work. We are carrying out site directed mutagenesis on crucial residues of these complexes as well as examining the genetic regulation of their expression and the topology of their transmembrane segments. The ability of ABC transporters to catalyse both uptake and efflux is being examined and is particularly important as this challenges the dogma that they are unidirectional for either uptake or efflux. Our current focus is to examine how these transporters are regulated at the post-transcriptional level by Hfq and at the post translational level by the PtsNtr system (1).
1. Mulley, G., J. P. White, et al. (2011). Mutation of GOGAT prevents pea bacteroid formation and N2 fixation by globally downregulating transport of organic nitrogen sources. Molecular Microbiology 80(1): 149-167.
Colonisation by bacteria of the zone surrounding plant roots (rhizosphere) is crucial to plant productivity. In spite of its importance rhizosphere colonization is poorly understood but recent advances in genome sequencing and analysis makes it possible to address this complex topic in exciting new ways. Global food security depends on sustainably maximising crop yield whilst decreasing use of costly fertilizers which cause release of the potent greenhouse gas N2O from soils. The largest input of fixed nitrogen in the biosphere comes from the biological reduction of atmospheric N2 to ammonium, mainly through Rhizobium–legume symbioses, within which bacteria reduce N2 to ammonia for supply to the host. This frees many of the world’s major crops (e.g. soybeans, alfalfa, and peas) from nitrogenous fertilizer application and transferring nodulation to non-legume crops is a long term goal almost certain to trigger a second, environmentally sustainable, green-revolution. However, only the bacterial symbiont fixes N2 so for successful transfer we must also understand how rhizobia grow in the rhizosphere of plants and colonize their roots. To understand this we have produced a comprehensive transcription map of R. leguminosarum grown in the rhizosphere of 3 different plants (1) but the regulatory circuits controlling this transcription network is unknown. However, among the 200-genes 3x up-regulated in the rhizosphere of all 3 plant hosts there are 7 master regulators and we will determine how they control bacterial colonisation of the rhizosphere. This will involve genetic analysis of these regulators using mutational analysis, microarray analysis, Chip-seq, Network analysis, ligand screening and colonisation assays. Our aim therefore is to understand the chemical signals that govern warfare and symbiosis between plants and microbes. This is a joint project between Professor Philip Poole and Dr Tony Miller and will be based in Molecular Microbiology at the John Innes Centre.
1.
Ramachandran V, East
AK, Karunakaran R, Downie JA, & Poole PS (2011)
Adaptation of Rhizobium leguminosarum to pea, alfalfa and sugar beet
rhizospheres investigated by comparative transcriptomics.
Genome Biology, in press.
The rhizosphere microbial community is subjected to intense competition for resources, requiring micro-organisms to adapt to fluctuating conditions by changing gene expression. Thus the transcriptional activity of microbial communities reflects their global response to stress. Furthermore, the cycling of nutrients in soil and rhizosphere dependents on microbial activity, which will alter the response to global changes in levels of CO2 and fixed nitrogen. These are now severely perturbed by human activity and are profoundly influenced by rhizosphere microbes. Due to its complexity we do not understand how rhizosphere microbial communities are altered by plants and environmental change. However, the advent of high through put sequencing, such as 454Flx and Solexa, means we can now measure the population structure as well as gene expression of microbes in the rhizosphere even among organisms that cannot be cultured. In this project we will address two specific experimental aims: 1) How do plants alter the population structure and meta-transcriptome of rhizosphere micro-organisms, 2) Develop bioinformatic tools for meta-analysis of the datasets so transcript abundance in populations can be mapped to metabolic pathway databases. Alongside the meta-transcriptomic analysis we will isolate genomic DNA from rhizosphere samples for PCR amplification and sequencing of 16S, 18S genes and 16-23S spacer regions to measure the microbial population structure of pea, wheat and oat rhizospheres. Thus we will determine how different plants alter both the population structure and metabolic activity of rhizosphere organisms. This project involves work in plant growth and general microbiology, molecular biology, high through put sequencing, bioinformatic analysis of large datasets as well as analysis of community structure.
The rhizosphere is one of the most complex, yet important, of environments for both microbial and plant growth. One of the most powerful tools that could be developed to study this environment is a set of molecular biosensors to monitor environmental conditions temporally and spatially. Our genome analysis of Sinorhizobium meliloti, Mesorhizobium loti and Rhizobium leguminosarum reveals an explosive growth in the number of ABC (ATP binding cassette) uptake systems in these organisms, with around 160 systems present. The binding proteins of these transporters are highly solute specific and tightly induced in response to appropriate conditions (1). We studied the transcriptional induction of these transporters by creating a suite of plasmid and integrated fusions to nearly all ATP-binding cassette (ABC) and tripartite ATP-independent periplasmic (TRAP) transporters of Sinorhizobium meliloti. The putative promoter regions for the ABC and TRAP transporter operons were cloned upstream of reporter genes. In total, nearly 500 fusions were made and these were tested with 174 inducing conditions. Overall, specific inducers were identified for 76 transport systems, amounting to 47% of the ABC uptake systems and 53% of the TRAP transporters in S. meliloti. Of these transport systems, 64 were previously uncharacterized in rhizobia and 24 were induced by solutes not known to be transported by ABC- or TRAP-uptake systems in any organism. This provided a global expression map of S. meliloti. (transportome) (2). These biosensors are now being introduced into the plant rhizosphere, using an agar sandwich technique developed here, and expression of fusions monitored by fluorescence microscopy. Since they only fluoresce when induced by the appropriate solute they will allow us to precisely map the physical and chemical nature of the root environment. Our primary aim is to examine the factors that govern competition for growth in the plant rhizosphere and in nodulation competition in Rhizobium. More recently we have been developing FRET biosensors which enable the real time detection of ligands. These work by fusing the solute binding protein to yellow and cyan fluorescent protein and report on ligand binding by a change in the resonace transfer of energy from the blue to yellow fluorescent protein.
1. Arthur H.F. Hosie and Philip. S. Poole (2001) Bacterial ABC transporters of amino acids. Research in Microbiology 152:259-270
2. Mauchline et al (2006) Mapping the Sinorhizobium meliloti 1021 solute- binding protein-dependent transportome PNAS (2006) 103: 17933-17938
Poole, P.S. and Allaway, D. (2000)
Carbon and nitrogen metabolism in Rhizobium.
Advances in Microbial Physiology 43:117-163.
Allaway, D., Schofield, N.A. and Poole,
P.S. (2000) Optical traps: shedding light on biological processes.
Biotechnology Techniques. Biotechnology Letters
22:887-892.
Arthur H.F. Hosie and Philip. S. Poole (2001) Bacterial ABC transporters of amino acids. Research in Microbiology 152:259-270
Lodwig, E.M., Leonard, M., Marroqui, S.M., Wheeler,T.R., Findlay, K., Downie, J.A. and Poole, P.S. (2005). Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids. Molecular Plant Microbe Interaction 18: 67-74.
J Peter W Young, Lisa C Crossman, Andrew W. B. Johnston, Nicholas R Thomson, Zara F Ghazoui, Katherine H Hull, Margaret Wexler, Andrew R. J. Curson, Jonathan D Todd, Philip S Poole, Tim H Mauchline, Alison K East, Michael A Quail, Carol Churcher, Claire Arrowsmith, Inna Cherevach, Tracey Chillingworth, Kay Clarke, Ann Cronin, Paul Davis, Audrey Fraser, Zahra Hance, Heidi Hauser, Kay Jagels, Sharon Moule, Karen Mungall, Halina Norbertczak, Ester Rabbinowitsch, Mandy Sanders, Mark Simmonds, Sally Whitehead and Julian Parkhill. (2006) The genome of Rhizobium leguminosarum has recognisable core and accessory components. Genome Biology. 7, R34.
East, A.K., Mauchline, T.H. and Poole, P.S. (2008) Biosensors for ligand detection. Advances in Applied Microbiology 64,137-166.
More detail on our research work can be found on our posters page.
This page is maintained by Philip Poole. This page was last updated on 20-Oct-2011.