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@bbsrc.ac.uk
phone 44-118-3788895
fax 44-118-3786671
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 theyestablish symbiotic interactions with plants. A further focus of our work is the physiology and biochemistry of nitrogen fixationin legume nodules. Currently we have five principal research areas, which are:
1) The investigation of the regulation of nutrient exchange in the Rhizobium-legume symbiosis.
2) Colonisation and competitive success in the plant rhizosphere.
3) Development of molecular biosensors.
4) The mechanism of solute movement by ABC transporters.
5) Differentiation of Rhizobium leguminosarum into root nodule bacteria.
The first area of investigation is motivated by the enormous importance of nitrogen
fixation by legumes to both
agriculture and the global nitrogen cycle. Nitrogen fixation by the legume-Rhizobium
symbiosis is driven by a supply
of carbon from the plant, so by understanding this process we can actually 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 In the second area of investigation we are studying
the genes that enable bacteria to
survive and reproduce in
the soil and particularly the plant rhizosphere. The rhizosphere is the region
immediately surrounding the plant root
that is an rich in nutrients and supports a large bacterial population.
Competition among bacteria for nutrients is fierce
and we are using signature tagged mutagenesis, microarray analysis, IVET and
high throughput sequencing to understand
the strategies that bacteria use to colonise this niche.
In our third area we are developing a powerful set of molecular biosensors to monitor
environmental
conditions both temporally and spatially. Our genome
analysis of
Sinorhizobium
meliloti,
Mesorhizobium
loti and
Rhizobium leguminosarum revealed 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. We were then able to identify the inducing solute for 90 binding proteins. This
included a wide rangeof sugars, amino acids, organic acids, nucleotides and metal ions. We are now
developing new FRET biosensors
to enable real time minotoring of ligands in the environment. In the fourth area, we are examining the mechanism of uptake of
solutes by ABC (ATP Binding Cassette)
transport
systems. The ABC superfamily is one of the largest of all transport
families with over 1100 identified members. While we concentrate on the
bacterial uptake systems these transporters are found widely in almost all
living organisms. They also have tremendous medical relevance with examples such
as the multi-drug resistance protein and the cystic fibrosis
chloride channel (CFTR). Our work is focused on the apparent ability of these
systems to
catalyze uptake and efflux in contradiction to the accepted dogma
that they are unidirectional.
We run the UK centre for transcriptional analysis of Rhizobium leguminosarum by the use of microarrays. Using glass slide arrays we are determining how the transition to the bacteroid is achieved at the genetic level. The expression of 7300 gene products is determined simultaneously. This is allowing us to address questions such as; What are the regulators that cause the switch in cell morphology? How is metabolism altered in a free-living cell to allow it to support bacteroid respiration and nitrogen fixation.
Symbiotic nitrogen
fixation between a host legume and rhizobia is fueled by the supply of a
dicarboxylic acid by the plant to the microsymbiont (bacteroid) and in return
the microsymbiont reduces N2 to ammonia. We have recently shown
that the fixed nitrogen is exported as a mixture of ammonium and alanine. While we have shown the bacterial
partner secretes alanine it has long
been speculated that nodule metabolism might be regulated by the supply of
amino
acids to the bacteroid by the plant. At one extreme a malate/aspartate
like shuttle might operate in the bacteroid, with both malate and glutamate
being provided to the bacteroid by the plant. We therefore began studying the
role of amino uptake by Rhizobium leguminosarum and identified the
first ABC transporter with broad specificity for amino acids (Aap) in bacteria. Mutation of Aap does not affect nodule development or
N2
reduction. Recently we identified in R. leguminosarum a second ABC
transporter with broad specificity for amino acids (Bra). Although Bra has
high sequence identity to branched chain amino acid uptake systems it has a
broad specificity for amino acids. Only when both the Aap and Bra are mutated
is transport of glutamate prevented in R. leguminosarum. The double
mutant is prototrophic for amino acid synthesis and shows no defects in
metabolism in the laboratory. Remarkably, the double mutant forms legume nodules that do not fix N2, since they are unable to supply fixed
nitrogen to the plant, which rapidly yellow. This is not an effect on
bacteroid development, since mature bacteroids develop and produce the enzyme
nitrogenase. Instead it indicates that carbon and nitrogen flow in mature
bacteroids are regulated by the supply of an amino acid(s) by the plant. This overturns our entire understanding of nodule metabolism and suggests a cycling
of both dicarboxylates and amino acids is occurring between the two symbionts.
In this project the molecular basis of this requirement for amino acids
by bacteroids will be examined. Initially, the in planta regulation of
these transport systems will be studied to determine exactly when and where
they are most actively expressed. Next we will examine the molecular
regulation of transcription and translation of both of these transporters, to determine what growth factors regulate expression of the transport complexes.
In the second part of the project the nature of the amino acids transported by
isolated wildtype and mutant bacteroids will be examined to define which are
crucial to
bacteroid carbon and nitrogen flux. The aap bra mutant will
be complemented with the E.coli GltS and GltP
transporters, which are specific for glutamate. These strains will then
be tested on plants for complementation of nitrogen fixation. Finally, the
effects of these transport mutations on bacteroid metabolism; in particular
dicarboxylate oxidation, N2 reduction and alanine secretion will be
examined. In particular this will require 15N2 labeling
to trace nitrogen export in wildtype and mutant bacteroids. This will enable
us to understand the metabolic flux occurring between the plant and bacteroid
and how it is regulated
by the supply of amino acid(s).
We have recently cloned and sequenced the mdh-sucABCD operon of R. leguminosarum (see diagram below). This operon codes for malate dehydrogenase (mdh), succinate dehydrogenase (sucAB) and succinyl-CoA synthetase (sucCD) and represents a critical regulatory point for flow of carbon through the TCA cycle.
A front view of the modeled structure of malate dehydrogenase from Rhizobium leguminosarum
This operon appears has a single promoter, upstream of mdh. We are interested in understanding how the transcriptional activity of this operon is regulated in response to nutritional and redox variations. We are also studying how the TCA cycle regulates amino acid transport and polyhydroxybutyrate biosynthesis. Recent work has focussed on the role of NAD malic enzyme and the PEP carboxykinase pathways in the generation of pyruvate in bacteroids of Rhizobium leguminosarum. The big question we are trying to understand is how is carbon flux and hence the rate of N2 fixation controlled by these pathways.
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 (Hosie and Poole
2001, Hosie et al.
2001). We were then able to
identify the inducing solute for 90 binding proteins. This included a wide range
of sugars, amino acids, organic acids, nucleotides and metal ions (Mauchline
2006). 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 (see below). 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.
There
are two further very important spin offs from this research. The first, is that
the
ability to screen for the induction of the gfp-fusions against a large array of
chemicals has
allowed us to identify the most likely solute transported for
most of the ABC uptake systems. This has greatly expand our understandingof the specificity of
bacterial ABC uptake
systems, A second powerful application is the recent
demonstration that bacterial ABC binding proteins can be used directly as
electrical biosensors (Benson
2001). This allows their incorporation into real time electrodes that can be
used to measure solute and ion concentrations directly. Our identification of
the induction and likely binding specificity of up to 160 binding proteins would
represent a unique resource for subsequent development of electrical biosensors.
The potential use of such electrodes offers exciting possibilities in environmental and biomedical monitoring. In another recent development it has
also been shown that the ligand recognition of solute binding proteins can be
radically altered by rational
computational design (Looger
at al 2003). Combining all of these approaches suggests an
exciting future
for binding protein dependent biosensors.
Above. Infection threads of R. leguminosarum. Two root hairs are being infected
by R. leguminosarum, note the very bright accumulation
of bacteria around the
curled root tip and in one case the accumulation of bacteria in the root cortex.
Infection threads are visible in both
infected root hairs running through the
middle of the root.
We have recently cloned and mutated the common amino acid permease operon (aap) of R. leguminosarum. This operon 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 PtsN system.
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.
Probably the most important environment for plant-microbe interactions is the rhizosphere. Whether due to pathogenesis, symbiosis or plant growth promotion the microbial population in the rhizosphere is a key determinant of plant productivity. A key stage in this is colonization of plant roots, which is a complex multifactorial process that has hitherto been difficult to analyse. Signature tag mutagenesis (STM) is a powerful technique for screening strains en masse to identify genes involved in rhizosphere colonization. In the Poole laboratory an STM library of R. leguminosarum was therefore generated which contains 20,788 uniquely tagged mutants. Over half of the signature tagged mutants were used to generate 96 separate input pools, each containing 108 tagged strains. In a preliminary experiment 54 of these 96 pools were screened on pea plants to identify strains that are lost after passage through the rhizosphere. This was done using a microarray with spotted oligos complementary to the unique signature tags to identify strains lost in the output pool. Ninety-four strains attenuated in rhizosphere colonization were identified and of these the 18 strains displaying the highest deviations between input and output pool were transduced into wild-type strain R. leguminosarum and retested in 1:1 competition experiments against the spontaneous streptomycin-resistant derivative strain Rlv3841. Our aim is to characterise these mutants to learn what factors alter competitive success.
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.
More detail on our research work can be found on our
posters
page. Posters can be viewed or downloaded
using an Adobe Acrobat viewer.
(Highlighted papers can be downloaded as pdf files)
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
Emma Lodwig1., Shalini Kumar1., David Allaway, Alex Bourdes, Jürgen Prell,
Ursula Priefer and Philip Poole
(2004) Regulation of L-alanine dehydrogenase in Rhizobium leguminosarum
bv. viciae and its role in pea nodules.
J.
Bacteriol. 186: 842-849
Role of polyhydroxybutyrate
and glycogen as carbon storage compounds in pea
and bean bacteroids.
Molecular Plant Microbe Interaction 18: 67-74.
Karunakaran,
R., Mauchline, T.H., Hosie, A.H,, Poole, P.S. (2005) A family of promoter probe
vectors incorporating autofluorescent and chromogenic reporter proteins for
studying gene expression in Gram-negative bacteria.
Microbiology 151: 3249-3256.
Kumar, S., Bourdes, A., Poole, P. (2005) De
novo alanine synthesis by bacteroids of Mesorhizobium loti is not
required for nitrogen transfer in the
determinate nodules of Lotus corniculatus. J Bacteriol. 2005 187: 5493-5495.
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.
This page is maintained by Philip Poole. This page was last updated on 30-Nov-2009.