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 they
establish symbiotic interactions with plants. A further focus of our work is the
physiology and biochemistry of nitrogen fixation
in 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 range
of 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.
Finally, in a very exciting development the genome sequence of R.
leguminosarum strain 3841, the one we use
at Reading, is currently being
sequenced by the Sanger center (http://www.sanger.ac.uk/Projects/R_leguminosarum/).
It is now more than 98.4% covered and this will soon rise to 99.9%. As a part of a
consortium of UK scientists
we are setting up a transcriptomic facility at
Reading to look at the global changes in gene expression that occur
in R.
leguminosarum as it differentiates from a free-living cell to the bacteroid
form that occurs in legume nodules.
Using glass slide arrays we will use these
gene chips to see how the transition to the bacteroid is achieved at the
genetic
level. The expression of up to 8000 gene products will be determined
simultaneously. This will allow 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. The intention is that
this facility at Reading will also be used by
scientists all over the UK to probe very specific questions about subjects
such
as iron regulation of gene expression, cell density dependent gene regulation,
regulation of stress genes and
the role of heat shock factors in symbiosis.
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).
For the last thirty years it has been a central dogma of symbiotic nitrogen
fixation that the nodule form of Rhizobium,
known as bacteroids, secrete nitrogen exclusively as ammonium to the plant
cytosol (reviewed by Poole and Allaway
2000). We have recently shown in peas that this is not strictly correct, instead
there is a mixed secretion of both
ammonium and alanine (Allaway et al 2000). This was shown by the use of 15N2
labeling studies and by the identification
of the pathway of alanine synthesis in R. leguminosarum. We showed
that alanine made by alanine
dehydrogenase (AldA) via the reductive amination of pyruvate, is secreted to the
plant cytosol. The gene coding for AldA
was identified, cloned, sequenced and mutated. It was then shown that
mutants of aldA result in a significant drop in plant
growth when inoculated onto peas. Thus alanine synthesis by AldA
contributes to the efficiency of symbiotic nitrogen fixation.
There are at least three amino acid transporters for alanine in R.
leguminosarum. We believe these transporters
may be responsible for the regulation of alanine export from cells of R. leguminosarum
in the legume nodule.
Export of alanine appears to be essential to regulate the flow of carbon in the TCA cycle
of the bacterial partner
in the legume nodule. We have mutated, cloned and sequenced these alanine transport systems from R. leguminosarum.
The
properties of these are now being investigated. The promoter regions will be mapped by primer extension and by
construction
of lacZ fusions. This will also enable us to study the regulation of expression of the gene by factors such as
combined nitrogen.
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.
We have developed procedures that enables us to screen genomic DNA libraries for
promoter fragments that
will drive expression of green fluorescent protein.
The procedure allows us to select for genes that are only expressed in the
environment (such
a
s the plant
rhizosphere) or under stress conditions and not in routine laboratory media.
This work is based
on a family of promoter probe vectors (pOT),
where expression of green
fluorescent protein (gfp) is driven by environmentally
regulated genes
(Allaway et al 2001).
Using a random library with 80,000 independent clones
we have screened for
fusions that are induced in the root environment and under
other stresses. A
summary of many of the clones identified can be found on our
environmental
database page. The fluorescence emitted by gfp is ideal for
quantification
of gene expression as well as visualization both on plates and using
a fluorescence
microscope. We can then identify bacteria that express green
fluorescent protein using an
epifluorescent microscope. In addition we
have built
an optical trapping microscope which allows us to use an infra-red laser to hold and
separate
individual bacteria that can be seen in the fluorescent microscope. This means
that bacteria that express the fluorescent
marker can be isolated and cultured. The
plasmid that these bacteria contain can then be recovered and the gene
fragment
responsible for driving expression of either the outer membrane protein or green
fluorescent protein
sub-cloned. From this the full length clone can be isolated from
a cosmid library, enabling a complete genetic
analysis including sequencing to be
performed. This approach enables us to directly select for genes that are expressed
and
are essential for survival in the environment. We have now isolated a number of
fusions expressed specifically in
the rhizosphere and are beginning the characterization
of them. These include genes required for vitamin
biosynthesis, homospermidine
synthesis and cyclic glucan synthesis. This approach has recently been extended
to examine how R. leguminosarum copes with osmotic and pH stress. We are
particularly interested in using the
gfp fusions induced under these stress conditions to isolate the characterize the regulatory networks
that sense
stress.
Above. Oxygen regulated gfp fusion grown under high
and low oxygen tension. Isolated during our studies on stress regulation.
Left is a picture of the optical trapping microscope that we have built.
The laser
beam comes in from the rear of the microscope.
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. 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.
We have recently cloned and sequenced a region upstream of the dicarboxylate
transport system of Rhizobium leguminosarum
that appears to code for a calcium export system. This project involves fully characterizing this calcium export system at
the
molecular level. Transposon and interposon mutants have been generated and their effects on calcium import and export are
being examined. This system may play a crucial role in proper assembly and stabilization of the outer membrane. We are
also
testing the hypothesis that it is the ratio between cell wall calcium and surface bound dicarboxylates that
regulate the formation
of the differentiated form of R. leguminosarum known as a bacteroid. Therefore we will also examine the
interaction between
calcium and dicarboxylates in the formation of bacteroid like cells in free-living culture.
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.
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)
Reid, C.J., Walshaw, D.L. and Poole, P.S. (1996)
Aspartate transport by the Dct system in Rhizobium
leguminosarum negatively affects nitrogen-regulated operons. Microbiology 142:2603-2612.
David L. Walshaw and Philip. S. Poole (1996) The
general L-amino acid permease of Rhizobium
leguminosarum is an efflux system that also influences efflux of solutes. Molecular Microbiology 21:1239-1252.
Allaway, D., Jeyaretnam, B., Carlson, R.W., and
Poole, P.S. (1996) Genetic and chemical characterisation
of a mutant that disrupts synthesis of the lipopolysaccharide core tetrasaccharide in
Rhizobium
leguminosarum. J. Bacteriology 178: 6403-6406.
David L. Walshaw, Shaun Lowthorpe, Alison East and
Philip. S. Poole (1997) Distribution of a sub-class
of bacterial ABC polar amino acid transporter and identification of an N-terminal region involved in
solute specificity. FEBS Letters. 414:397-401
David L. Walshaw and Philip. S. Poole (1997)
Regulation of the TCA cycle and
general amino acid
permease by overflow metabolism in Rhizobium leguminosarum.
Microbiology 143:2209-2221.
David Walshaw, Colm J Reid and Philip S. Poole.
(1997) The general amino acid permease of
Rhizobium leguminosarum strain 3841 is negatively regulated by the Ntr system. FEMS Micro Letts
152:57-64.
Gyaneshwar,
P., Parekh,L.J., Archana,G., Poole, P.S., Collins,M.D., Hutson,R.A. and
Kumar,G.N. (1999)
Involvement of phophate starvation inducible glucose dehydrogenase in soil P
solubilisation by
Enterobacter asburiae using buffered media conditions. FEMS Microbiology Letters 171:223-229.
Poole, P.S., Reid,C., East,A., Day,M., Leonard. And Allaway,
D. (1999) Regulation of the mdh-sucABCD
operon in Rhizobium leguminosarum. FEMS Microbiology Letters 176:247-255.
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.
This page is maintained by Philip Poole. This page was last updated on 22-Jan-2008.