Positions
Energisation of Nitrogen
Fixation in the Rhizobium-
legume symbiosis
Post Doctoral Fellowship Available (April 2008)
This BBSRC funded position is available from April 2008 and has been advertised in www.jobs.ac.uk from where formal details of application can be found.
Nitrogen fixation within the Rhizobium-legume symbiosis is the main driver of the global nitrogen cycle. The process is driven by a supply of carbon from the plant. Therefore, by understanding this we can determine which factors regulate the total amount of nitrogen fixed. The regulation of nutrient exchange between a legume and Rhizobium is largely determined by two membrane systems, one is plant derived and the other is bacterial. Our approach to understanding the interactions within and between these membranes is to mutate and clone the bacterial transport systems important for nutrient exchange. The genetics governing regulation of expression of these transport systems is being investigated and their effects on nitrogen fixation assessed. We have shown that the plant must supply amino acids to the bacteroid to sustain nitrogen fixation (Lodwig et al 2002; Prell and Poole 2006). Our aim is to understand why the plant must supply amino acids to the root nodule bacteria and how this controls the development of symbiosis. In this project we aim to determine how amino acid metabolism and cycling between the plant and bacteria provides the energy for nitrogen fixation. Genetical and biochemical approaches will be used to examine the regulation of flux of amino acids through the bacteroid. The post holder will examine how the main amino acid permeases (Aap and Bra) of R. leguminosarum are regulated and how this alters nodule metabolism. For further information about this post contact Philip Poole (phone 01603 450750).Lodwig, E.M., Hosie, A.H.F., Bourdes, A., Findlay, K.,
Karunakaran, R., Downie, J.A. and Poole P.S. (2003) Amino-acid cycling drives
nitrogen fixation in the legume-Rhizobium symbiosis. Nature 422: 722-726.
(pdf)
Jurgen Prell and Philip Poole (2006) Metabolic changes in rhizobia. Trends in
Microbiology. 141: 161-168. (pdf)
The post holder will work closely with a team of Postdoctoral Research Assistants working in the laboratory of Philip Poole.
The Poole group works in the area of plant microbe interactions with particular emphasis on nitrogen fixation in the Rhizobium legume symbiosis. A wide variety of approaches are used including genetics, molecular biology, enzymology, bacterial physiology, plant physiology and micro array analysis. There is considerable expertise in these areas within the current group so training in new techniques will be provided.
Development of Molecular
Biosensors
Two Post Doctoral Fellowships Available (April 2008)
There are two BBSRC funded positions available from April 2008, which have been advertised in www.jobs.ac.uk from where formal details of application can be found.
Our analysis of the genomes of Sinorhizobium meliloti, Mesorhizobium loti and Rhizobium leguminosarum revealed an explosive growth in the number of ABC (ATP binding cassette) uptake systems, with 146 systems present. These transport systems have a solute binding protein (SBP) that tightly binds the solute to be transported, and confers specificity on uptake. In addition, the genes of ABC uptake systems are usually tightly induced by the solute that they transport. We therefore developed a high throughput screen where induction of all 146 ABC operons was coupled to GFP. This enabled the identification of the inducing solute for 76 ABC operons, including a wide range of sugars, amino acids, organic acids, nucleotides and metal ions (Mauchline et al., 2006). Furthermore, the solute binding capacity of an SBP can be predicted from the induction profile of the operon. A powerful application of this is the ability of SBPs to be used directly as FRET or electrical biosensors, allowing the production of real time bisosensors that can be used to measure solute and ion concentrations in real time (Looger et al., 2003; Looger et al., 2005). The identification of the induction and likely binding specificity of 76 SBPs represents a unique resource for the development of real time biosensors. The potential use of such biosensors offers exciting possibilities in environmental and biomedical monitoring. It has also been shown that the ligand recognition of solute binding proteins can be radically altered by rational computational design (Looger et al., 2003). Combining all of these approaches suggests an exciting future for binding protein dependent biosensors. There are now two Post Doctoral Fellowships available. The successful applicant for the first position will be responsible for expressing and purifying the SBPs already identified by us and examining their ligand binding specificity. A number of the proteins will then be targeted for structure determination by X-ray crystalography in collaboration with Kim Watson in the structural biology unit at Reading University. In addition the post holder will construct FRET biosensors by fusing the SBPs to different C-and N-terminal fluorescent proteins (e.g. CFP and YFP). The successful applicant for the second post will be responsible for constructing genomic libraries from a variety of sequenced micro-organisms in a GFP expressing vector. FAC sorting will then be used to identify novel genes that are induced by ligands of interest, including pharmacologically and biologically interesting compounds. Since the the libraries will be made from sequenced micro-organisms we will be able to obtain the full length clones for any genes and operons induced. These will then be further examined for the development of novel biosensors. For further information about these posts contact Philip Poole (phone 01603 450750).
Looger, et. al (2003). Nature 423, 185-190.
Looger, et. al. (2005). Plant Physiology 138, 555-557.
Mauchline, T. H., J. E. Fowler, A. K. East, A. L. Sartor, R. Zaheer, A. H. F.
Hosie, P. S. Poole, and T. M. Finan. (2006).
Mapping the Sinorhizobium meliloti 1021 solute binding protein-dependent
transportome. Proceedings on the National Academy of Sciences USA 103,
17933-17938. (pdf)
Transcriptomic analysis of
bacteroid formation
(Open trawl Ph.D. students)
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
Transcriptomic analysis of the transition from a
free-living cell to a root nodule bacteroid in
R. leguminosarum.
The sequencing of the genome of R.
leguminosarum offers us a unique opportunity to use microarrays to address
the problems both
of the final metabolic state of the bacteroid and the genetic and
developmental switches that are crucial to differentiation. By comparing
gene expression between free-living bacteria, rhizosphere bacteria and
bacteria/bacteroids from various stages of nodule development
we can begin to reconstruct the regulatory switches and developmental/
metabolic consequences of these switches.
In this project I propose to investigate two
aspects of bacteroid development and metabolism and a general aspect of
environmental
gene regulation, which are:
1) How does bacteroid metabolism differ from a free-living cell and how is this important to nitrogen fixation,
2) What are the genetic and development changes that occur during the transition of free-living bacteria to bacteroids.
Specifically I hypothesise that
the three dominant factors regulating bacteroid metabolism are the O2
tension, provision of
C4-dicarboxylates and cellular growth rate, therefore gene
expression profiles of rhizosphere bacteria and bacteroids will be compared
against free-living bacteria grown in chemostat culture limited by either
glucose, malate or O2 at intermediate and low dilution rates.
The expression data will then be mapped to the KEGG metabolic database using
the GeneSpring bionformatic analysis suite to allow
us to visualise the key metabolic changes that occur in bacteroid formation.
With regard to the second aim
above we will analyse the genetical and developmental changes that occur
during the transition
of free-living bacteria to bacteroids. This will be tackled by comparing the
gene expression profiles of free-living bacteria, fully
developed bacteroids and bacteroids arrested in development by well-defined
mutations. These mutants are lpcB, which codes for
CMP-Kdo:LPS Kdo transferase which is needed for formation of the LPS core
, dctA, which codes for the dicarboxylate transport
system (bacteroids are fully formed but cannot fix nitrogen), bacA, the
role of which is not understood, but is needed for development
of bacteroids and a double amino acid transport mutant (aap/bra) which
forms fully developed bacteroids capable of reducing N2 to
NH3 but which still forms ineffective nodules. By comparing gene
expression in cells blocked at various developmental stages I
believe we can determine what are the key changes in the transition from a
free-living cell to a bacteroid.
References (hyperlinked papers can be downloaded)
2.Poole, P.S. and Allaway, D. (2000) Carbon and nitrogen metabolism in Rhizobium. Advances in Microbial Physiology 43:117-163.
3. Arthur Hosie and Philip Poole (2001) Bacterial ABC transporters of amino acids. Current Microbiology 152:259-270.
For further information contact Philip Poole. This page was last updated on 11-Mar-2008.