Differentiation of Rhizobium:
Characterisation of Metal Transport Systems During Symbiosis
Graham Hood, Allan Downie and Philip Poole
[email protected]
Department of Molecular Microbiology, John Innes Centre, NRP, Norwich NR4 7UH, United Kingdom
Rhizobium leguminosarum bv. viciae 3841
The mutualism between Rhizobium leguminosarum bv. viciae 3841 (Rlv3841) and Pisum sativum
(pea) is an example of the highly complex and specialised Rhizobia-legume symbiosis. Bacterial
entry at the root hairs is later progressed through a plant-derived infection thread. Bacteria will
then become internalised by the peribacteroid membrane, and it is here where the bacteria become
fully differentiated into their endosymbiont-form, named the bacteroid.
+ 25µM MnSO4
WT
+ 25µM MnSO4.
4H2O
OD595
mntH::
pK19
WT
0.5µM MnSO4.4H2O
sitA:: pK19
MntH is a member of the Nramp of divalent cation
transporters and is selective for Mn2+. In
Bradyrhizobium japonicum, MntH has been shown
to be essential for Mn2+ transport but is not essential
for symbioisis with soybean3.
Rlv3841 has both putative SitABCD and MntH
transport systems. Both are transcriptionallyupregulated in early bacteroids1. When mutated
singularly, both strains grew on minimal media with
the standard 9µM MnSO4 (Fig 1A). The double
mutant however, only grew when MnSO4 was
increased (Fig 1B). The inability of the double-mutant
to grow on low-Mn was also observed on solid
media (Fig 1C).
1C.
Hours
1B.
+ 9µM MnSO4
Reactive oxygen species are produced by the plant in
early stages of symbiosis and Mn2+ has been shown to
be important in the resistance against this.
The sitABCD operon encodes an ABC transporter
selective for iron/manganese. When sitA was
mutated in Sinorhizobium meliloti 1021, it was shown
to be symbiotically defective on Medicago sativa2.
1B.
Hours
OD595
Manganese
2+
(Mn )
1A.
Bacteroids fix dinitrogen (N2) into ammonia (NH3), making it available to the plant. Both transcriptional
analysis and high-throughput mutagenesis strategies1, have identified metal transporters as having an
important role in successful symbiosis. Part of my research is devoted to the investigation of metal
transport systems used for manganese and magnesium uptake.
WT
50µM MnSO4.4H2O
sitA mntH
[mntH::ΩSp
sitA:: pK19]
1D.
mntH sitA
0.5µM MnSO4.4H2O
1E.
mntH sitA
50µM MnSO4.4H2O
On pea, the double mutant had a severe symbiotic
phenotype, generating small white nodules (c.f.
larger and pink nodules with WT), confirmed to be
Fix- by acetylene reduction (assay for nitrogen
fixation; Fig. 1D and 1E).
Magnesium
2+
(Mg )
Mg2+ is generally the most abundant divalent
cation in prokarotes, complexing with molecules
such as ATP, acting as cofactors, giving stability to
ribosomal subunits, maintaining pH balance and
also giving integrity to the outer membrane.
Fix+
WT
2A.
mntH::
pK19
sitA::
pK19
mntH::
ΩSp
mntH sitA
[mntH::ΩSp
sitA:: pK19]
2C.
2B.
Hours
+ 2mM MgSO4
nodules from WT inoculated pea
Fix- nodules from mntH sitA
inoculated pea
WT
The MgtE family is one example of Mg2+ transport
systems. It exists as a homodimer and possesses a
cytoplasmic domain that binds Mg2+, allowing gateregulation of the ion-conducting pore4.
When a homologue of mgtE was mutated in
Rlv3841, we observed a long lag phase for the freeliving culture when grown on the standard MgSO4
concentration for our minimal media (Fig 2A). This is
possibly a result of the mutant being less efficient at
acquiring Mg2+. The presence of other putative Mg2+
transport systems encoded by the Rlv3841 genome,
suggest that MgtE is not essential, but is important
to Mg2+ transport in free-living cells.
To our knowledge, MgtE is the first example of a
Mg2+ transporter shown to be essential for
Rhizobium-legume symbiosis; producing white
nodules on pea (Fig 2B), accompanied by severely
reduced nitrogen fixation rates (Fig 2C). TEMs of
nodule sections show mgtE::mTn5 is able to form
mature-looking, branched bacteroids.
OD595
mgtE:: mTn5
Fix- nodules from
mgtE::mTn5-inoculated pea
WT
Future Directions
The manganese and magnesium transporters mentioned, have
given us a great opportunity to elucidate metal transport during
symbiosis. With further investigation I aim to define time points
where R. leguminosarum acquires its essential metals during
symbiosis, through the utilisation of techniques such as GUSreporter fusions. Inoculating these mutated strains on other
compatible legumes (vetch and bean) will also help us address
the possibility that requirement for certain metal transporters
varies between different hosts. Transport studies will confirm the
affinity of these transporters for manganese/magnesium and
define their kinetics in different conditions.
mgtE:: mTn5
References
1
Karunakaran, R et al., 2009. J Bac. 191, 4002-12.
2 Davies,
BW and Walker, GC, 2007. J Bac. 189, 2101-09.
Hohle, TH and O’Brian, MR, 2009. Mol Micro. 72, 399409.
3
4 Hattori,
M et al., 2009. EMBO. 28, 3602-12.