Figures
Abstract
Background
Rhizobium leguminosarum bv. viciae mutants unable to transport branched-chain amino acids via the two main amino acid ABC transport complexes AapJQMP and BraDEFGC produce a nitrogen starvation phenotype when inoculated on pea (Pisum sativum) plants [1], [2]. Bacteroids in indeterminate pea nodules have reduced abundance and a lower chromosome number. They reduce transcription of pathways for branched-chain amino acid biosynthesis and become dependent on their provision by the host. This has been called “symbiotic auxotrophy”.
Methodology/Principal Findings
A region important in solute specificity was identified in AapQ and changing P144D in this region reduced branched-chain amino acid transport to a very low rate. Strains carrying P144D were still fully effective for N2 fixation on peas demonstrating that a low rate of branched amino acid transport in R. leguminosarum bv. viciae supports wild-type rates of nitrogen fixation. The importance of branched-chain amino acid transport was then examined in other legume-Rhizobium symbioses. An aap bra mutant of R. leguminosarum bv. phaseoli also showed nitrogen starvation symptoms when inoculated on French bean (Phaseolus vulgaris), a plant producing determinate nodules. The phenotype is different from that observed on pea and is accompanied by reduced nodule numbers and nitrogen fixation per nodule. However, an aap bra double mutant of Sinorhizobium meliloti 2011 showed no phenotype on alfalfa (Medicago sativa).
Conclusions/Significance
Symbiotic auxotrophy occurs in both determinate pea and indeterminate bean nodules demonstrating its importance for bacteroid formation and nodule function in legumes with different developmental programmes. However, only small quantities of branched chain amino acids are needed and symbiotic auxotrophy did not occur in the Sinorhizobium meliloti-alfalfa symbiosis under the conditions measured. The contrasting symbiotic phenotypes of aap bra mutants inoculated on different legumes probably reflects altered timing of amino acid availability, development of symbiotic auxotrophy and nodule developmental programmes.
Citation: Prell J, Bourdès A, Kumar S, Lodwig E, Hosie A, Kinghorn S, et al. (2010) Role of Symbiotic Auxotrophy in the Rhizobium-Legume Symbioses. PLoS ONE 5(11): e13933. https://doi.org/10.1371/journal.pone.0013933
Editor: Julian Rutherford, Newcastle University, United Kingdom
Received: July 20, 2010; Accepted: October 18, 2010; Published: November 11, 2010
Copyright: © 2010 Prell et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: This work was supported by the Biotechnology and Biological Sciences Research Council UK (http://www.bbsrc.ac.uk/) with grant no BB/F013159/1. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.
Competing interests: The authors have declared that no competing interests exist.
Introduction
The largest input of available nitrogen into the biosphere comes from biological reduction of atmospheric N2 to ammonium [3], with legume-Rhizobium symbioses responsible for much of this. These symbioses arise from infection of host plants, mainly of the legume family, by proteobacteria and results in root structures called nodules [4]. The symbiosis is initiated by flavonoids released from the plant roots which in return elicit the synthesis of lipochitooligosaccharide Nod factors by the rhizobial partner. Bacterial cells are trapped by curling root hairs and proliferate in an infection focus. This turns into an infection thread that grows into the root cortex where a zone of meristematic cells is newly induced. Here the bacteria are released from the infection threads into the plant cells by endocytosis and surrounded by a plant derived symbiosome membrane [5]. The whole structure develops into a nodule with plant cells filled with thousands of symbiosomes. Nodules can be classified as determinate or indeterminate, with those in the Inverted-Repeat-Lacking Clade (IRLC) of the legume subfamily Papilionoideae (such as Medicago, Pisum or Vicia) being indeterminate with a cylindrical shape and a persistent meristem at the tip of the nodule. Bacteria inside symbiosomes differentiate into N2 fixing bacteroids and this is accompanied by dramatic increases in size, shape and DNA content [6]. The differentiation is caused by a family of plant peptides that share similarities with antimicrobial peptides and probably interfere with the bacterial cell cycle [7]. Nodules of the Milletioides clade (such as Phaseolus, Vigna or Glycine), form spherical determinate nodules with a transient meristem; bacteria do not undergo endoreduplication and therefore do not enlarge substantially. These bacteroids retain a normal DNA content and are largely viable after isolation from nodules [6]. However, other combinations also exist, including enlarged bacteroids in determinate nodules and non-enlarged bacteroids in indeterminate nodules, with the evolutionary newer form of enlarged bacteroids evolving at least 5 times independently in the legume family [8].
The shared metabolism between the symbiotic partners has been proposed to be a simple nutrient exchange of dicarboxylates for ammonium in the nodule [9], [10], [11], but amino acid transport by bacteroids has also been shown to be essential in pea (Pisum sativum) nodules [1]. R. leguminosarum bv. viciae strains contain two broad specificity amino acid ABC (ATP binding cassette) transporters, AapJQMP and BraDEFGC. AapJQMP consists of a solute binding protein (SBP) AapJ, two permease units AapQM and an ABC subunit dimer formed by AapP for ATP hydrolysis. The BraDEFGC complex consists of SBP BraC, permeases BraDE and ABC subunits BraFG. The aap bra double mutants, RU1357 and RU1722, formed pea bacteroids that appeared morphologically normal in electron micrographs and fixed nitrogen per bacteroid at wild-type levels, but the plants were nitrogen starved [1]. However, aap bra mutant bacteroid protein levels per plant were reduced to ∼30% of wild-type. The reason for the nitrogen starved phenotype was later identified to be a limitation of branched-chain amino acid biosynthesis by developing bacteroids [2]. Branched-chain amino acid biosynthesis is transcriptionally reduced during bacteroid development and results in symbiotic auxotrophy where amino acids must be supplied by the plant.
Here we provide evidence that only a low rate of branched-chain amino acid transport is required to overcome symbiotic auxotrophy of R. leguminosarum bv. viciae strains in pea nodules. This shows that large quantities of amino acid are not needed and is consistent with symbiotic auxotrophy. Additionally we report the symbiotic phenotypes of an aap bra mutant in R. leguminosarum bv. phaseoli inoculated on French bean (Phaseolus vulgaris) forming determinate nodules and Sinorhizobium meliloti on alfalfa (Medicago sativa) forming indeterminate nodules.
Results
AapQ site directed mutants with altered transport rates
Previous bioinformatic analysis suggested that a conserved N-terminal domain of the polar amino acid transporting permease proteins (PAAT family of ABC transporters) such as AapQ and AapM might differentially affect the uptake of amino acids [12]. Residue 53 of this domain (residue 144 in AapQ) is strongly correlated with the charge of the transported solute. Thus transporters of basic amino acids have asp or glu at residue 53, while acid transporters have a branched-chain amino acid and broad specificity transporters (e.g. AapQ) have proline. In order to investigate this, a number of site directed mutants were isolated in AapQ and screened for transport specificity by expression from a plasmid in the aap bra null mutant RU1722 (data not shown). Of these mutants P144D proved most interesting, so a single copy of AapQ P144D was generated by recombination into the chromosome of Rlv3841. This was achieved by replacing the ΩSp cartridge in RU1722 aapJQM::ΩSp braEF::ΩTc with a full copy of the aapJQM operon containing the mutated AapQ P144D, generating RU1976. The braEF::ΩTc null mutation is present in all the control strains so transport occurs exclusively via Aap. The uptake properties of RU1976 (aapQ P144D braEF::ΩTc) were compared to those of RU1721 (braEF::ΩTc), which contains a wild-type copy of Aap and the aap bra deletion mutant RU1722 (aapJQM::ΩSp braEF::ΩTc) (Table 1). The maximum rate (Vmax) of transport of small amino acids such as alanine was less severely affected (51% of control) by the AapQ P144D mutation than transport of larger amino acids such as glutamate (25% of control). The lower levels of amino acid uptake observed in RU1976 could also arise from a reduction in solute affinity (Km). In order to investigate this, uptake rates were determined for glutamate, alanine, aspartate and leucine at varying concentrations in both RU1721 and RU1976. These were then used to calculate Km for amino acid uptake by these strains (Table 1). It is clear that the reduction in amino acid uptake in RU1976 is not due to reduced affinity of Aap for its solutes, indeed, the Km of AapQ P144D is slightly lower than wild-type. Rather, in each case, Vmax is lower in RU1976. Similar drops in transport rates were determined for all branched-chain amino acids (Table 2).
Plant phenotypes of P144D mutants on peas
In order to analyse if reduced transport by Aap triggers symbiotic auxotrophy, strain RU1976 and the control strains, RU1721 and RU1722 were inoculated onto pea plants. Dry weights of 6 week old pea plants inoculated with RU1976 and RU1721 were similar, while RU1722 dry weights were reduced as expected (Table 2). Thus a ∼60% reduction in the rate of branched-chain amino acid uptake in strain RU1976 compared to RU1721, did not significantly alter N2-fixation and presumably bacteroid formation. Consistent with the proposal of symbiotic auxotrophy only a low rate of branched-chain amino acid transport is needed for N2-fixation.
Transport phenotype of an aap bra mutant in R. leguminosarum bv. phaseoli
The transport specificities and symbiotic phenotypes of R. leguminosarum bv. viciae strain A34 and its aapJQM::ΩSp braE::TnphoA mutant RU1357, have been previously described [1], [13]. R. leguminosarum bv. phaseoli 4292 and R. leguminosarum bv. viciae A34 are isogenic strains that only carry different symbiotic (sym) plasmids. Strain 4292 nodulates Phaseolus while strain A34 nodulates Pisum, Vicia and Lens [14]. Mutants, aapJQM::ΩSp (RU1809), braE::TnphoA (RU1932) and aapJQM::ΩSp braE::TnphoA (RU1933) were isolated in R. leguminosarum bv. phaseoli 4292 as described in Materials and Methods. Transport of several amino acids by these strains confirmed that Aap and Bra have indistinguishable properties in R. leguminosarum bv viciae A34 and R. leguminosarum bv. phaseoli 4292 (Table 3; compare to [13]). Thus in strain 4292 glutamate transport is mainly facilitated via Aap while the branched-chain amino acid leucine is primarily transported via Bra. α-Aminoisobutyric acid (AIB), alanine and histidine are transported at similar rates by both transporters and GABA is exclusively transported via Bra. In the aap bra double mutant (RU1933) the transport of all solutes was reduced to background levels, except for alanine and histidine which under the assay conditions can still enter the cells via other transport systems [13]. It is important to note here, that branched-chain amino acid transport is abolished, which is essential to explain the previously reported phenotype of R. leguminosarum bv. viciae double transport mutants on pea plants [1], [2]. R. leguminosarum bv. viciae cells are dependent on the provision of branched-chain amino acids by the host plant to develop into mature bacteroids and probably also for their persistence [2].
Plant phenotypes of aap bra mutants on beans
Growth and symbiotic performance of French bean (P. vulgaris) was measured at different time points. Plants at 8 weeks showed no significant differences when inoculated with the wild-type (RU2222), the aap mutant (RU1809) and the bra mutant (RU1932) (Table 3). However, the aap bra double mutant, RU1933, produced significantly (p<0.001) reduced dry weights compared to wild-type RU2222 inoculated plants. Plants inoculated with RU1933 also showed signs of yellowing after 8 weeks although not as severe as uninoculated controls (Figure 1). Nitrogen fixation rates, measured as acetylene reduction (AR), and nodule numbers were determined after 4, 5 and 6 weeks of plant growth. Because bacteroid protein levels per pea plant were reduced in aap bra mutants of R. leguminosarum bv. viciae [1], [2] this was also measured in bean nodules after 4 weeks of growth. A strong reduction in nitrogen fixation rates (down to 36% of the wild-type control; Table 4) was accompanied by a reduced nodule number (down to 54%) after 4 weeks of plant growth (Table 4). In addition the level of acetylene reduction per nodule formed by RU1933 was significantly (p = 0.005) reduced after 4 weeks compared to levels in wild-type nodules (Table 4). Nitrogen fixation rates and nodule numbers were also markedly reduced on 5 and 6 weeks old plants inoculated with RU1933 compared to wild-type (Table 5), while acetylene reduction rates per nodule recovered. However, compared to 4 week old plants, nodule numbers on 5 and 6 week old plants were generally higher, which were also grown in bigger pots (see Materials and Methods).
Plants were inoculated with RU2222 (wild-type), RU1933 (aapJQM::ΩSp braE::TnphoA) or left uninoculated.
At 5 and 6 weeks the reduced symbiotic performance of strain RU1933 (Table 5) can be explained by the reduced number of nodules alone. However, at 4 weeks the reduction in nitrogen fixation rates is only partly explained by the reduced nodule number (Table 4). The nitrogen fixation rate per nodule and bacteroid is reduced as well.
TEM pictures of bacteroids formed by wild-type RU2222 and the aap bra mutant RU1933 showed remarkable differences (Figure 2). Bacteroids of determinate French bean nodules usually show an accumulation of granules of the carbon storage compound poly-hydroxybutyrate (PHB). This accumulation was enhanced in the aap bra mutant background RU1933 (Figure 2). TEM micrographs were prepared from all the three experiments harvested after 4, 5 and 6 weeks and always showed identical results.
A and C are pictures taken of 4 week old nodules, B and D of 6 week old nodules. Size bars are 500 µm.
Properties of a Sinorhizobium meliloti 2011 aap bra transport mutant
S. meliloti livHMGFK is homologous to R. leguminosarum braDEFGC [13], so for clarity we hereafter name S. meliloti livK as braC and livH as braD. The double mutants aapJ::ΩSp braC::mTn5 (LMB210) and aapJ::ΩSp braD::mTn5 (LMB211) were generated in the S. meliloti 2011 background as described in Materials and Methods. All amino acid transport was reduced to background levels in the aapJ braD mutant LMB211, which is very similar to R. leguminosarum strains (Table 6). Thus mutations in the SBP of the Aap and integral membrane proteins of the Bra abolish most amino acid transport in S. meliloti. However, strain LMB210 which contains a mutation in the gene coding for the SBP BraC, still transported isoleucine at a high rate. As previously described for R. leguminosarum bv. viciae 3841 an orphan SBP (BraC3) is able to interact with the Bra membrane complex and is very specific for the branched-chain amino acids and alanine [2]. A possible orthologue (SMc00078) of BraC3 is present in S. meliloti strain1021.
Plant phenotypes of S. meliloti aap bra mutants on alfalfa
Medicago sativa plants inoculated with wild-type 2011 or the aapJ braD double mutant LMB211 were analysed for nitrogen fixation (AR) and dry weights after 4 weeks of growth. Neither nitrogen fixation rates nor dry weights differed significantly (p>0.05) between S. meliloti 2011 and LMB211, although uninoculated control plants showed strong signs of nitrogen starvation and a corresponding drop in dry weights (Table 7).
TEM pictures of wild-type 2011 and the aapJ braD mutant LMB211 bacteroids showed no obvious differences in development (data not shown) and flow cytometric characterisation of bacteroids also failed to demonstrate developmental alterations of bacteroids formed by LMB211 (data not shown).
Discussion
Pea plants inoculated with Rhizobium leguminosarum bv. viciae strains mutated in the two broad specificity amino acid ABC uptake systems, Aap and Bra develop a severe nitrogen starvation phenotype [1], [2]. However, the very broad solute specificity of Aap and Bra made it difficult to determine which amino acids are responsible for this phenotype. Therefore, braC was mutated in an aap null mutant background and it was shown that strain RU1979 (ΔaapJ braC::ΩSpec) retains the transport of branched-chain amino acids. This is because of the presence of an orphan SBP (BraC3) which specifically binds branched-chain amino acids and interacts with the core Bra membrane complex. Strain RU1979, which only retains branched-chain amino acid transport, fixes nitrogen at wild-type rates on pea plants. In addition complementation analysis using heterologous ABC transport systems demonstrated that branched-chain amino acid transport alone rescues the starvation phenotype on pea plants. Microarray analysis of pea bacteroids showed that the pathways of branched-chain amino acid biosynthesis are transcriptionally down regulated leading to premature arrest of development in aap bra bacteroids [2].
Our first aim in this study was to determine how much branched-chain amino acid transport is needed for fully effective N2-fixation. It was already known that the single aap and bra mutants have no symbiotic phenotype so the wild-type rate of transport can be lowered by ∼50% with no effect [1]. However, we had previously identified a region likely to be important in solute specificity in AapQ [12] and mutation of P144D reduced branch chain amino acid transport by ∼60%. The absolute rates of branched-chain amino acid transport in RU1976 (aapQ P144D braEF::ΩTc) were 1–2 nmol min−1 mg protein−1, while the aap bra double mutant strains have rates below 1 nmol min−1 mg protein−1. This rate for RU1976 is roughly 10% of the maximum rates of branched-chain amino acid uptake measured in wild-type [2], [13]. However, RU1976 provided enough branched-chain amino acids for developing bacteroids to achieve a wild-type rate of nitrogen fixation. This supports the hypothesis that R. leguminosarum bacteroids are symbiotic auxotrophs and do not require amino acids for catabolism, which would need a much higher rate of transport.
A leuD mutant of R. leguminosarum strain 3841 required leucine at 100 µM added to AMS glucose/NH4Cl cultures to overcome auxotrophy and restore a wild-type rate of growth. By comparison as sole nitrogen source 10 mM leucine was needed to obtain the same growth rate as NH4Cl.
Published microarray data shows transcriptional down regulation of branched-chain biosynthetic genes occurs in S. meliloti in symbiosis with alfalfa (M. sativa) and Bradyrhizobium japonicum with soybean (Glycine max) [15], [16], [17]. Therefore, the requirement for branched-chain amino acid transport was investigated in other legume-rhizobia interactions. The first interaction investigated is that between R. leguminosarum bv. phaseoli and French bean. This was done for two reasons. First, beans have determinate nodules, while peas have indeterminate nodules. Second, R. leguminosarum bv. viciae A34 and R. leguminosarum bv. phaseoli 4292 differ only in their Sym plasmid so the same aap bra mutations can be directly compared in pea and bean hosts.
French bean plants inoculated with RU1933 (aap bra) became nitrogen starved, indicated by reduced dry weight, nitrogen fixation, and nitrogen fixation per nodule as well as yellowing and increased PHB accumulation in bacteroids. Unexpectedly, they produced fewer nodules on beans, in contrast to the increase in nodule formation on peas by equivalent aap bra mutants. Legumes usually increase nodule numbers when inoculated with fix reduced strains. The reduction in French bean nodules inoculated with RU1933 might result from limitation of branched-chain amino acids during infection or very early in bacteroid development leading to reduced success in nodule formation. Therefore, while peas and beans have different nodulation responses to aap bra mutants their different patterns of nodule development probably alter the timing of amino acid availability and the development of symbiotic auxotrophy.
In addition to reduced numbers of nodules elicited by RU1933 bacteroids had more PHB granules as visually scored from TEM pictures of 4, 5 and 6 week old nodules. Increased levels of carbon storage compounds in nodules, usually starch in the plant and PHB in bacteroids, are a typical feature of non-fixing or under-performing strains [1], [18], [19]. The plant provides carbon and reductant for nitrogen fixation, but the bacteroids of RU1933 may not use it efficiently. This suggests that in addition to less successful infection, nitrogen fixation by bean bacteroids is limited and leads to nitrogen starvation in the host plant. This might reflect, as for pea nodules, a general high demand for branched-chain amino acids [2], which are energetically expensive to make and are the most abundant amino acids in bacterial proteomes [20]. However, when inoculated with aap bra mutants, beans are less severely nitrogen starved than peas. For example French beans had dry weights reduced by 24% while peas were reduced by 59%, close to uninoculated controls [1].
Similar to R. leguminosarum Rlv3841 [2], [21], branched-chain amino acid biosynthesis is down regulated in S. meliloti [15], [16], [22]. Thus an aap bra mutant was isolated in S. meliloti 2011 and its rates of branched-chain amino acid transport were below 1 nmol min−1 mg protein−1. However, there were no differences in dry weight or acetylene reduction of alfalfa plants inoculated with mutant or wild-type. Inspection of TEM micrographs of bacteroids as well as FAC analysis of their size and DNA content did not reveal any differences between wild-type and mutant. Either the decrease in transcription of the branched-chain amino acid biosynthetic genes in S. meliloti 2011 bacteroids is not severe enough to produce symbiotic auxotrophy or residual transport rates by aap bra mutants are sufficient to support bacteroid maturation and persistence. The ability to reduce branched-chain amino acid transport to a very low rate in R. leguminosarum bv. viciae without inducing symbiotic auxotrophy is consistent with this. If the concentrations of branched-chain amino acids in alfalfa nodules are slightly higher than peas or the reduction in transcription of the genes for their biosynthesis is delayed or less severe, then symbiotic auxotrophy would not occur.
This study demonstrates that symbiotic auxotrophy is a phenotype right on the edge where it can be pushed in one direction or the other by small changes in conditions or gene expression. Perhaps the more important aspect of this is not whether a particular Rhizobium-plant association shows symbiotic auxotrophy for amino acids but, that bacteroids may shut down many aspects of metabolism as long as they retain the ability to transport essential nutrients from host plants. This emphasises the exquisite co-evolution of Rhizobium and legumes with the metabolism of bacteroids being organelle like.
Materials and Methods
Bacterial growth and media
The bacterial strains, plasmids and primers used in this study are detailed in Table 8. Rhizobium strains were grown at 28°C in either Tryptone Yeast extract (TY) [23] or acid minimal salts medium (AMS) [24] with 10 mM D-glucose as carbon sources and 10 mM NH4Cl as nitrogen source. S. meliloti was grown in M9 medium with 1.25 mM CaCl2, 2.5 mM MgSO4, 0.1 ug/ml Biotin, 10 ug/ml CoCl2, 10 mM D-glucose and 10 mM NH4Cl. Antibiotics were used at the following concentrations (µg ml−1): streptomycin (St), 500; neomycin (Nm), 80; tetracycline (Tc), 5; gentamicin (Gm), 20 and spectinomycin (Sp), 100.
Isolation of mutants
The Rhizobium leguminosarum bv. viciae AapQ site directed mutant was generated as follows. AapJQMP was amplified using primers p495 and p441. The 5 kb PCR product was TOPO cloned into pENTR/D-Topo (Invitrogen, USA) and the CCG triplet coding for proline at position 144 within AapQ replaced by a GAC coding for aspartic acid (P144D) using the Quick Change site-directed mutagenesis kit (Stratagene, USA). The region was transferred by a Gateway LR reaction into the pJQ200SK derivative, pGW1, producing pRU1247. This plasmid was conjugated into RU1722 (aapJQM::ΩSp braEF::ΩTc) to generate the homogenote RU1976 by sucrose selection and loss of the spectinomycin marker. The correct mutation was confirmed by PCR.
Rhizobium leguminosarum bv. phaseoli mutants were generated as follows. Plasmid pRU410 [25], which contains an omega spectinomycin (ΩSp) cassette cloned into an MfeI deletion (3.2 kb) spanning aapJQM, was transformed into Escherichia coli S17-1 and conjugated into R. leguminosarum bv. phaseoli 4292. This plasmid is derived from pJQ200SK [26] which contains the sac genes. Transconjugants were spread on AMS agar plates [24] containing sucrose (10%) as a carbon source and spectinomycin resistant, gentamicin sensitive colonies were selected. One colony (RU1809) was confirmed to have the correct aapJQM::ΩSp genotype by PCR mapping using primers p418 and p419. As expected from the phenotype of aap mutants of R. leguminosarum bv. viciae, strain RU1809 did not grow on minimal medium with L-glutamate as the sole carbon and nitrogen source. To generate bra mutants, cosmid pBIO206A [25] (pIJ1427 braE::TnphoA) was transformed into S17-1 and conjugated into strain 4292 and RU1809. Colonies were selected for tetracycline and kanamycin, resistance, before the chaser plasmid pPH1JI (gentamicin resistant) was then conjugated into them to allow the selection of recombinants with loss of the tetracycline marker and resistance to kanamycin and gentamicin. Plasmid pPH1JI was then removed by conjugating pLAFR1 into all strains, and selecting for loss of the gentamicin marker while retaining resistance to kanamycin and tetracycline. The braE::TnphoA insertion was confirmed by PCR mapping using primers pIS50R and p439. The braE::TnphoA single and aapJQM::ΩSp braE::TnphoA double mutants were designated as RU1932 and RU1933, respectively. Strain 4292 containing pLAFR1 was designated RU2222.
Sinorhizobium meliloti mutants were generated as follows. A series of mTn5STM mutants in the livHMGFK operon of S. meliloti strain 2011 [27] were obtained from Anke Becker at CeBiTec, Bielefeld, Germany. The S. meliloti livHMGFK is homologues to the R. leguminosarum braDEFGC operon [13]. A livK (braC) mutant 2011mTn5STM.3.12.F11 and a livH (braD) mutant 2011mTn5STM.4.05.D04 were chosen as a background for an aapJ insertion mutation using an ΩSp cassette [28]. Primers pr0163 and pr0164 were used to amplify a 2.6 kb region surrounding the S. meliloti strain 2011 aapJ gene. The resulting PCR product was cloned into pJET1.2 (Fermentas, Lithuania) resulting in plasmid pLMB111. Primers pr0165 and pr0166 were used in an inverse PCR to amplify the aapJ surrounding DNA from pLMB111 without the aapJ ORF. The resulting PCR product was digested with BamHI (underlined in primers) and religated forming plasmid pLMB112. An XbaI/XhoI fragment was then cloned from pLMB112 into pJQ200SK [26] to generate pLMB113. This vector was opened with BamHI and a ΩSp cassette was inserted producing pLMB118. This plasmid was used to generate aapJ::ΩSp homogenotes in the braC and braD mutant backgrounds to produce strains LMB210 and LMB211, respectively. The insertion was mapped from both sides using primers pOTforward and pr0240 or primer pr0241, respectively.
Transport assays
R. leguminosarum uptake assays were performed with 25 µM (4.625 kBq of 14C) solute [13], using cultures grown in AMS with 10 mM D-glucose and 10 mM NH4Cl to an OD600 of ∼0.4. S. meliloti was grown in modified M9 medium with 10 mM D-glucose and 10 mM NH4Cl to an OD600 of ∼0.4.
Plant assays
French bean plants (Phaseolus vulgaris cv. Tendergreen) were surface sterilised and grown for 5 to 6 weeks in a sterile mixture of fine gravel and vermiculite (1∶4) in 15 l pots with 7 seeds per pot in a controlled environment at 22°C and a 8:16 h day: night cycle as described earlier [29]. The 4 week experiments were performed in 8 l pots in a fine gravel and vermiculite mixture (1∶4) with 7 seeds per pot in a controlled environment at 22°C and a 8:16 h day: night cycle. Pots were open to drain into a sterile autoclave bag and watered with nitrogen free rooting solution [30]. Medicago sativa cv. Europe plants were grown in 1 l pots in vermiculite with 7 seeds per pot for 4 weeks watered with nitrogen free rooting solution [30].
Acetylene reduction was measured as previously described [31].
Bean shoot dry weights were determined after 8 weeks, pea shoot dry weights after 6 weeks and alfalfa shoot dry weights after 4 weeks of growth.
Bacteroid assays
After counting and removing all nodules from the plants, nodules were crushed in 40 mM HEPES buffer and bacteroids harvested by differential centrifugation [32]. Bacteroid pellets were then broken in a FastPrep FP120 ribolyser (Bio101, Thermo) and protein levels determined following a standard Bradford assay.
Flow cytometric analysis of S. meliloti bacteroids was performed as described elsewhere [2].
Acknowledgments
We like to thank Roy Bongaerts for the flow cytometer analysis and Sue Bunnewell for the TEM imaging.
Author Contributions
Conceived and designed the experiments: JP EL AH JW PP. Performed the experiments: JP AB SK EL AH SK JW. Analyzed the data: JP AB SK EL AH SK JW PP. Contributed reagents/materials/analysis tools: JP AB PP. Wrote the paper: JP PP.
References
- 1. Lodwig EM, Hosie AHF, Bourdès A, Findlay K, Allaway D, et al. (2003) Amino-acid cycling drives nitrogen fixation in the legume-Rhizobium symbiosis. Nature 422: 722–726.
- 2. Prell J, White JP, Bourdès A, Bunnewell S, Bongaerts RJ, 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.
- 3.
Newton WE (2000) Nitrogen fixation in perspective. In: Pedrosa FO, Hungria M, Yates MG, Newton WE, editors. Nitrogen fixation: From molecules to crop productivity. Dordrecht: Kluwer. pp. 3–8.
- 4. Oldroyd GED, Downie JA (2008) Coordinating nodule morphogenesis with rhizobial infection in legumes. Ann Rev Plant Biol 59: 519–546.
- 5. Gage DJ (2004) Infection and invasion of roots by symbiotic, nitrogen-fixing rhizobia during nodulation of temperate legumes. Microbiol Mol Biol Rev 68: 280–300.
- 6. Mergaert P, Uchiumi T, Alunni B, Evanno G, Cheron A, et al. (2006) Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium-legume symbiosis. Proc Natl Acad Sci USA 103: 5230–5235.
- 7. Van de Velde W, Zehirov G, Szatmari A, Debreczeny M, Ishihara H, et al. (2010) Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 327: 1122–1126.
- 8. Oono R, Schmitt I, Sprent JI, Denison RF (2010) Multiple evolutionary origins of legume traits leading to extreme rhizobial differentiation. New Phytol 187: 508–520.
- 9. Lodwig E, Poole P (2003) Metabolism of Rhizobium bacteroids. Crit Rev Plant Sci 22: 37–78.
- 10. Prell J, Poole P (2006) Metabolic changes of rhizobia in legume nodules. Trends Microbiol 14: 161–168.
- 11. White J, Prell J, James EK, Poole P (2007) Nutrient sharing between symbionts. Plant Physiol 144: 604–614.
- 12. Walshaw DL, Lowthorpe S, East A, Poole PS (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 Lett 414: 397–401.
- 13. Hosie AHF, Allaway D, Dunsby HA, Galloway CS, Poole PS (2002) Rhizobium leguminosarum has a second general amino acid permease with unusually broad substrate specificity and high similarity to branched-chain amino acid transporters (Bra/LIV) of the ABC family. J Bacteriol 184: 4071–4080.
- 14. Lamb JW, Hombrecher G, Johnston AWB (1982) Plasmid-determined nodulation and nitrogen-fixation abilities in Rhizobium phaseoli. Mol Gen Genet 186: 449–452.
- 15. Capela D, Filipe C, Bobik C, Batut J, Bruand C (2006) Sinorhizobium meliloti differentiation during symbiosis with alfalfa: a transcriptomic dissection. Mol Plant-Microbe Interact 19: 363–372.
- 16. Barnett MJ, Tolman CJ, Fisher RF, Long SR (2004) A dual-genome symbiosis chip for coordinate study of signal exchange and development in a prokaryote-host interaction. Proc Natl Acad Sci USA 101: 16636–16641.
- 17. Pessi G, Ahrens CH, Rehrauer H, Lindemann A, Hauser F, et al. (2007) Genome-wide transcript analysis of Bradyrhizobium japonicum bacteroids in soybean root nodules. Mol Plant-Microbe Interact 20: 1353–1363.
- 18. Finan TM, Wood JM, Jordan DC (1983) Symbiotic properties of C4-dicarboxylic acid transport mutants of Rhizobium leguminosarum. J Bacteriol 154: 1403–1413.
- 19. Ronson CW, Lyttleton P, Robertson JG (1981) C4-dicarboxylate transport mutants of Rhizobium trifolii form ineffective nodules on Trifolium repens. Proc Natl Acad Sci USA 78: 4284–4288.
- 20. Akashi H, Gojobori T (2002) Metabolic efficiency and amino acid composition in the proteomes of Escherichia coli and Bacillus subtilis. Proc Natl Acad Sci USA 99: 3695–3700.
- 21. Karunakaran R, Ramachandran VK, Seaman JC, East AK, Moushine B, et al. (2009) Transcriptomic analysis of Rhizobium leguminosarum b.v. viciae in symbiosis with host plants Pisum sativum and Vicia cracca. J Bacteriol 191: 4002–4014.
- 22. Becker A, Berges H, Krol E, Bruand C, Ruberg S, et al. (2004) Global changes in gene expression in Sinorhizobium meliloti 1021 under microoxic and symbiotic conditions. Mol Plant-Microbe Interact 17: 292–303.
- 23. Beringer JE (1974) R factor transfer in Rhizobium leguminosarum. J Gen Microbiol 84: 188–198.
- 24. Poole PS, Blyth A, Reid CJ, Walters K (1994) myo-Inositol catabolism and catabolite regulation in Rhizobium leguminosarum bv viciae. Microbiol 140: 2787–2795.
- 25. Hosie AHF, Allaway D, Jones MA, Walshaw DL, Johnston AWB, et al. (2001) Solute-binding protein-dependent ABC transporters are responsible for solute efflux in addition to solute uptake. Mol Microbiol 40: 1449–1459.
- 26. Quandt HJ, Puhler A, Broer I (1993) Transgenic root nodules of Vicia hirsuta - a fast and efficient system for the study of gene expression in indeterminate-type nodules. Mol Plant Microbe Interact 6: 699–706.
- 27. Pobigaylo N, Wetter D, Szymczak S, Schiller U, Kurtz S, et al. (2006) Construction of a large signature-tagged mTn5 transposon library and its application to mutagenesis of Sinorhizobium meliloti. Appl Environ Microbiol 72: 4329–4337.
- 28. Fellay R, Frey J, Krisch H (1987) Interposon mutagenesis of soil and water bacteria: a family of DNA fragments designed for in vitro insertional mutagenesis of gram-negative bacteria. Gene 52: 147–154.
- 29. Lodwig EM, Leonard M, Marroqui S, Wheeler TR, Findlay K, et al. (2005) Role of polyhydroxybutyrate and glycogen as carbon storage compounds in pea and bean bacteroids. Mol Plant-Microbe Interact 18: 67–74.
- 30. Allaway D, Lodwig E, Crompton LA, Wood M, Parsons TR, et al. (2000) Identification of alanine dehydrogenase and its role in mixed secretion of ammonium and alanine by pea bacteroids. Mol Microbiol 36: 508–515.
- 31. Trinick MJ, Dilworth MJ, Grounds M (1976) Factors affecting the reduction of acetylene by root nodules of Lupinus species. New Phytol 77: 359–370.
- 32. Prell J, Boesten B, Poole P, Priefer UB (2002) The Rhizobium leguminosarum bv. viciae VF39 gamma aminobutyrate (GABA) aminotransferase gene (gabT) is induced by GABA and highly expressed in bacteroids. Microbiol 148: 615–623.
- 33. Johnston AWB, Beringer JE (1975) Identification of the Rhizobium strains in pea root nodules using genetic markers. J Gen Microbiol 87: 343–350.
- 34. Downie JA, Hombrecher G, Ma QS, Knight CD, Wells B, et al. (1983) Cloned nodulation genes of Rhizobium leguminosarum determine host range specificity. Mol Gen Genomics 190: 359–365.
- 35. Freidman AM, Long SR, Brown SE, Buikema WJ, Ausubel FM (1982) Construction of the broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants. Gene 18: 289–296.
- 36. Quandt J, Hynes MF (1993) Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria. Gene 127: 15–21.
- 37. Hirsch PR, Beringer JE (1984) A physical map of pPH1JI and pJB4JI. Plasmid 12: 139–141.