Transcriptomic analysis of Rhizobium leguminosarum bacteroids in determinate and indeterminate nodules

Two common classes of nitrogen-fixing legume root nodules are those that have determinate or indeterminate meristems, as in Phaseolus bean and pea, respectively. In indeterminate nodules, rhizobia terminally differentiate into bacteroids with endoreduplicated genomes, whereas bacteroids from determinate nodules are less differentiated and can regrow. We used RNA sequencing to compare bacteroid gene expression in determinate and indeterminate nodules using two Rhizobium leguminosarum strains whose genomes differ due to replacement of the symbiosis (Sym) plasmid pRP2 (strain Rlp4292) with pRL1 (strain RlvA34), thereby switching symbiosis hosts from Phaseolus bean (determinate nodules) to pea (indeterminate nodules). Both bacteroid types have gene expression patterns typical of a stringent response, a stressful environment and catabolism of dicarboxylates, formate, amino acids and quaternary amines. Gene expression patterns were indicative that bean bacteroids were more limited for phosphate, sulphate and iron than pea bacteroids. Bean bacteroids had higher levels of expression of genes whose products are predicted to be associated with metabolite detoxification or export. Pea bacteroids had increased expression of genes associated with DNA replication, membrane synthesis and the TCA (tricarboxylic acid) cycle. Analysis of bacteroid-specific transporter genes was indicative of distinct differences in sugars and other compounds in the two nodule environments. Cell division genes were down-regulated in pea but not bean bacteroids, while DNA synthesis was increased in pea bacteroids. This is consistent with endoreduplication of pea bacteroids and their failure to regrow once nodules senesce.


INTRODUCTION
Rhizobia are a group of a-and b-proteobacteria forming symbiotic nitrogen-fixing nodules on legumes [1]. Legume nodulation is typically initiated by exchange of signalling compounds, with rhizobia attaching to root hairs and growing down plant-made infection threads into the root cortex [2]. Rhizobia are then endocytosed and surrounded by a plant-derived membrane (symbiosome membrane). The resulting structure, which resembles an organelle, is called a symbiosome [3], within which bacteria differentiate into bacteroids. N 2 reduced to ammonia is supplied from bacteroids to the plant in exchange for a carbon supply, mostly in the form of dicarboxylic acids, such as malate [3]. Bacteroids exist in a microoxic environment, essential for activity of the oxygen-sensitive nitrogenase [4,5].
Nodules on phaseoloid legumes (including Phaseolus vulgaris) are determinate with a transient meristem; the rhizobia do not terminally differentiate and nitrogen-fixing bacteroids can be cultured from mature nodules. In contrast, nodules formed on indeterminate nodules such as pea (Pisum sativum) maintain an active meristem and infection zone. In indeterminate nodules, growing infection threads release rhizobia into nodule cells; these bacteria endoreduplicate their genomes and terminally differentiate into pleiomorphic nitrogen-fixing bacteroids that cannot be cultured [6]. The main body of indeterminate nodules contains nitrogen-fixing symbiosomes [2,7]. In legumes such as Medicago truncatula and Pisum sativum, belonging to the inverted repeat-lacking clade (IRLC), there are up to 600 genes which encode nodule-specific cysteine-rich (NCR) peptides [8][9][10] that induce bacteroid differentiation [11,12].
Our aim was to compare gene expression in determinate bean and indeterminate pea bacteroids. We used two R. leguminosarum strains that efficiently nodulate and fix nitrogen in bean or pea nodules. They differ due to the replacement of the symbiosis (Sym) plasmid pRP2 (in strain Rlp4292) with pRL1 (in strain RlvA34) but share a core genome, facilitating direct comparison of transcriptomes in bacteroids from determinate and indeterminate nodules.

RNA isolation, sequencing, mapping and bioinformatic analyses
Bacteroids and free-living cells were ribolysed (MP Biomedicals FastPrep) with 300 mg 0.1 mm silica and 100 mg 0.1 mm glass beads. Cell debris and beads were removed by centrifugation and RNA purified using a Qiagen RNeasy mini-kit according to the manufacturer's instructions. Purified RNA was quantified using RNA-specific fluorescent

IMPACT STATEMENT
In N 2 -fixing symbioses between legumes and rhizobia, two different nodule types are those with determinate and indeterminate nodules as seen with bean and pea, respectively. Comparative transcriptomics was carried out using two rhizobial strains sharing a common core genome but each with a different symbiosis (Sym) plasmid conferring the ability to form efficient nitrogen-fixing nodules on either bean or pea. Bacteroids in both nodule types expressed genes related to nitrogen fixation, utilization of dicarboxylates, formate and amino acids, and induction of a stringent response. However, it was evident from the patterns of gene expression that the environments within the two nodule types were different, with respect to nutrient (P, S, Fe, Mo) limitation and utilization of carbon substrates. Bean bacteroids appeared to induce many genes predicted to be associated with detoxification. Pea bacteroids had increased expression of genes associated with central metabolism, the TCA (tricarboxylic acid) cycle and bacteroid differentiation. This study provides a window into the different environments experienced by nitrogen-fixing bacteroids in these two nodule types.
label derivatization (Qubit 2.0; Life Technologies) and quality checked using capillary electrophoresis (Bioanalyzer; Agilent). DNA libraries were produced from RNA samples by TGAC (The Genome Analysis Centre) (Norwich, UK) and the Illumina HiSeq 2000 sequencing platform used to produce 150 bp non-paired end reads.
To map the reads, Bowtie2 software was used with default parameters [29] and filtered with SAMtools to remove nonuniquely mapped reads [30]. A custom Perl script was used to calculate the coverage score for each base position. For absolute expression, these values were used to generate TPM (transcripts per kilobase million) scores for each gene . Genes are coloured according to differential expression in bean and pea bacteroids. Genes up-regulated >10-fold in bacteroids are red; >2-fold up-regulated, orange; between 2-fold up-regulated and 2-fold down-regulated, yellow; >2-fold down-regulated, light blue; >10-fold down-regulated, dark blue. Exploded regions show the names of genes discussed in the text. The prefix for each gene name is shown in brackets within each replicon and the numbers printed inside the outer ring indicate the gene (e.g. on pRP2: RHLp7097, nifB). Data for this figure is given in Tables S6 and S8. Green et al., Microbial Genomics 2019;5 [31]. Normalized differential expression of genes was calculated using DESeq in R [32] using raw coverage scores.
The Rlp4292 genome (NCBI taxonomy ID 936350) was downloaded from the IMG database [33], along with the Sym plasmid pRL1 from R. leguminosarum bv. viciae 248 (NCBI taxonomy ID 936136). The RlvA34 genome was electronically reconstituted from that of Rlp4292 by eliminating pRP2 and adding pRL1. Reciprocal BLAST searches against Rlv3841 and Rhizobium etli CFN42 genomes and subsequent parsing using BioPerl improved gene annotation. Homology based searches within Rlp4292 and RlvA34 used USEARCH [34]. Alignment identity (id) scores were calculated using LALIGN (www.ch.embnet.org/software/LALIGN_form. html) with default settings. A custom Perl script was used to generate heat map scores for pRP2 and pRL1 gene expression, allowing plasmid maps to be drawn with GenomeVx (http://wolfe.ucd.ie/GenomeVx/) [35].
Differential gene expression during growth of freeliving Rlp4292 and RlvA34 Gene expression differences in Rlp4292 and RlvA34 grown under identical in vitro conditions must be due to their Sym plasmids or to mutations within their genomes; about 97 % of the common genes showed little (<2-fold) or no differences in expression (Table S4). Some common genes (0.3 %) were differentially expressed (>5-fold). In RlvA34, this included the following genes normally induced under iron limitation: RHLa5120 (rpoI); the vicibactin synthesis and  Table S6 and for those of pRL1 (223 genes) in pea bacteroids in Table S8. Data for genes that are common (>80 % id) to both pRP2 and pRL1 (45 genes) are given in Table S9. uptake cluster RHLa5121-6 (vbsLDAGSO); the RHL2532-40 genes, including the hmuPSTU haemin uptake genes (RHL2537-40) [40,41]; and RHLa5475-7 encoding a FeCT (iron chelate family uptake) ABC transporter (Table S5. The RNA-Seq data revealed in RlvA34 (but not Rpl4292) a nucleotide (T) deletion in rirA, which encodes a repressor of iron-regulated genes [42]. The frameshift would change amino acid lysine 75 to leucine and lead to truncation of 80 amino acids from RirA (161 amino acids). We conclude that mutation of rirA caused up-regulation of this regulon of genes in RlvA34.
The only genes expressed more strongly (>5-fold) in Rlp4292 than in RlvA34 were RLH2777 encoding a putative oxidoreductase and RHL4467 of unknown function ( Table S5). The consistency of expression of shared genes in free-living cultures of Rlp4292 and RlvA34 justifies the validity of comparison of gene expression under symbiotic conditions, with the proviso that the RlvA34 carries a rirA mutation causing up-regulation of some iron-related genes. Differential expression of Sym plasmid genes in bean and pea bacteroids RNA sequence reads from Rlp4292 and RlvA34 bacteroids were compared with data from their respective free-living cells to generate normalized values for differential gene expression in bacteroids (Table S6). Fig. S2 shows the proportion of genes, by replicon, that are >2-fold differentially regulated. These data were heavily skewed, with most genes down-regulated, representing the 'real' nature of the gene expression in bacteroids, but not allowing accurate comparison of differential gene expression. Therefore, we applied DeSeq normalization to these data to allow calculation of differential gene expression (Table S7). Genes shown as differentially expressed in Table S7 show >5-fold normalized increases or decreases in RlvA34 or Rlp4292 bacteroids relative to the expression in free-living cultures of the same strain; we used this differential in our analyses described below (summarized in Figs 2 and 3). In this section, we will deal with differentially expressed Sym plasmid genes (Fig. 2), and in the next section we will consider the genes on the common genome (Fig. 3).
As the Sym plasmid plays a key role in symbiosis, differential gene expression on plasmids pRP2 (433 genes) and pRL1 (223 genes) is summarized as a heat-map ( Fig. 1, showing >2-and >10-fold differential expression, Tables S6 and S8). pRP2 and pRL1 encode 45 gene products showing >80 % amino acid id, and so we considered them as orthologues (summarized in Fig. 2). Table S9 gives their relative expression in bean and pea bacteroids.
(i) Expression of common Sym plasmid genes in bean and pea bacteroids

pRP2 genes up-regulated in bean bacteroids
Many up-regulated genes are predicted to be involved with detoxification or export of unwanted chemicals/toxins, presumably reflecting the environment within bean nodules (Table S6). Genes RHLp7122-6 and RHLp7083 (up-regulated 60-to 150-fold) encode five cytochrome P450 monooxygenase proteins and an oxidoreductase (Table S6). RHLp7189-97 (up-regulated 10-to 100-fold) encode genes for a putative ferredoxin and two copper-containing oxidases (Fig. 1, Table S6). Several of these predicted monooxygenases, oxidoreductases and oxidases could target specific nodule compounds made in bean but not pea nodules. In view of the observation that peas make NCR defensin-like peptides but beans and other determinate nodules do not [6], secondary antimicrobial metabolites may be of importance in bean to reduce infection by other bacteria (cheaters) that do not contribute to symbiotic nitrogen fixation.

Melanin
Production of melanin in Rlp4292 requires pRP2-encoded MelA (RHLp6882) [52], which oxidises tyrosine immediately prior to its polymerization into melanin. In bean nodules, melA (RHLp6882), which is co-regulated with fix genes [53], was up-regulated about 300-fold (Table S6). Melanin can trap free radicals that are produced during nitrogen fixation, possibly reflecting a different redox environment within bean and pea bacteroids.

Fe-S proteins
Genes encoding a Fe-S cluster assembly protein (RHLp7088) and a 4Fe-4S binding domain protein (RHLp6777) were up-regulated 40-and 100-fold, respectively, in bean nodules, and could be involved in electron transfer during nitrogen fixation or to other proteins, such as cytochrome P450s.
(iii) pRL1 genes differentially regulated in pea bacteroids In RlvA34 pea bacteroids, 15 % of pRL1 genes were up-regulated >5-fold, whereas 34 % were down-regulated >5-fold (the highest proportion of any replicon) (Table S7). In addition to the aforementioned nif (RHLv8080-8), fixABCX nifAB (RHLv8118-28) and the RHLv8134-8142 fix genes (shown in red in Fig. 1 Differential expression of the common genome in mature bacteroids Differential expression of the common genome (i.e. everything other than the Sym plasmids) is summarized in Fig. 3 and Table S7 shows the numbers of genes differentially expressed >5-fold, by replicon, for bean and pea bacteroids. On each replicon, similar numbers of genes were up-regulated >5-fold in both bean and pea bacteroids (between 7 % on plasmid B and 2 % on plasmid A), except for plasmid C with 27 genes up-regulated (5 %) in bean compared to only 10 (2 %) in pea bacteroids. For downregulated genes in the two bacteroid types, similar values were seen for the chromosome and plasmid B (1-2 %), but for plasmids A and C, a slightly higher percentage is down-regulated in bean (5-6 %) than in pea (1-3 %) (Table S7).
(i) Shared genes up-regulated in both bean and pea bacteroids Pathways important for symbioses in both determinate and indeterminate nodules can be deduced from Table S10 showing the 89 genes >5-fold up-regulated in both bean and pea bacteroids. For comparison, this table includes data with likely orthologues in Rlv3841 from pea nodules [13].
Calcium-binding proteins RHL1101 and RHLb6093 encoding EF-hand calcium (Ca)binding proteins were strongly up-regulated in bean and pea bacteroids (Table S10). Similar rhizobial proteins have two predicted EF-hand domains [50], one extending outside the cell. Ca-binding proteins have several roles including Ca homeostasis, signalling between bacteroid/plant and may be expressed as a result of nitrogen-starvation or stress (for reviews see [54,55]).

Repression of transcription
DksA proteins bind to RNA polymerase and mediate the stringent response induced on nutrient limitation. RHLb6067 (dksA1) and RHL1099 (dksA2) were up-regulated about 7-to 40-fold. Mutation of dksA in S. meliloti reduced symbiotic nitrogen fixation in alfalfa nodules [56].

Stress proteins
Nine genes encoding stress proteins were up-regulated in both bean and pea bacteroids (Table S10, Riley code 1.6.1). RHLb6081 (hspF) was about 400-and 70-fold up-regulated in bean and pea bacteroids, respectively; RHLb6065 was about 30-and 40-fold up-regulated in pea and bean, respectively; RHL1259 was 12-and 20-fold up-regulated in bean and pea bacteroids, respectively.

Transport systems
Transport system genes are often induced in response to the transported solute; thus, giving an indication of the chemical environment. Twenty genes whose products are predicted to be involved in transport across membranes were up-regulated >5-fold in both bean and pea bacteroids (Table S11, selecting Riley code 1.5.x). To ensure the most robust data, we chose to examine transporters that were not only up-regulated, but also highly expressed (>200 000 reads); 15 genes fulfilled these criteria (Table S12, Fig. 3) and are described below.

Bacteroids are fuelled by C4-dicarboxylates
The C4-dicarboxylate transporter RHL2260 (dctA) was upregulated about 14-and 5-fold in bean and pea bacteroids, respectively (Table S12) (appearing as bean-specific in Fig. 3 Fig. 4. Transporter systems whose genes are up-regulated in Rlp4292 and RlvA34 bacteroids. Common nodule-specific transporters (up-regulated >5-fold in nodules of both strains) are shown in black, those specific to bean (up-regulated >5-fold in bean bacteroids and <5-fold in pea bacteroids) are shown in purple and those specific to pea (up-regulated >5-fold in pea bacteroids and <5-fold in bean bacteroids) are shown in green. In addition to being up-regulated in bacteroids, all genes are also highly expressed (>200 000 reads). Data for this figure is given in Table S12. due to falling just below the 5-fold cut-off). It was the most highly expressed bacteroid transporter with >8Â10 6 and >3Â10 6 reads, respectively (Table S12); it is also the most highly expressed transporter in succinate-grown free-living RlvA34 and second most expressed in succinate-grown freeliving Rlp4292 (Table S4). Therefore, increased expression of dctA in bacteroids, relative to even succinate-grown cultures, illustrates the importance of dicarboxylates as a carbon source in nodules.

Amino acids
Four genes encoding components of the Bra (branched-chain amino-acids) transporter [ABC HAAT (hydrophobic amino acid transporter) family] were up-regulated >5-fold in bacteroids. Although Fig. 3 shows RHL2378 (braC3) and RHL2589 (braC) specifically up-regulated in bean bacteroids and RHL2591 (braG) and RHL2592 (braF) specifically in pea, closer inspection reveals that all genes are up-regulated by >2-fold in both bacteroid types (Table S12). Pea and bean bacteroids require the plant to supply branched-chain amino acids isoleucine and valine to allow bacteroid development [57]. The broad-specificity amino acid transporters Aap and Bra [57,58] are essential for bacteroid branched-chain amino acid uptake and normal nitrogen fixation in Rlv3841 [36,59]. The PAAT family ABC transporter Aap is unusual in that it transports a wide-range of substrates [60,61]. Aap components encoded by RHL1044-6 (aapPMQ) were up-regulated about 4-to 7-fold in both bean and pea bacteroids (Table S12). The importance of both transport systems in nodules is illustrated by the observation that although strains Rlv3841 and Rlp4292 mutated in both Aap and Bra form nodules on peas and beans, respectively, they fix nitrogen at only about 30 % of the wild-type rate [21,62,63]. These data are indicative that supply of these amino acids to pea and bean bacteroids is similar.  Table S17.

Quaternary amines and other nitrogenous compounds
The ABC QAT (quaternary amine transporter) family transports quaternary amines, such as histidine and choline. In both bean and pea bacteroids, RHL2371 and RHLa5629 [which encode solute binding proteins (SBPs) GbcX (QatX1) and QatX3, respectively] were up (about 7-to 30fold), as were the contiguous genes RHL2372-3 (qatW1V1) (3-to 9-fold) suggesting that choline and/or glycine betaine are used by both bean and pea bacteroids. Mutation of the QAT encoded by RL3533-5 (gbcXWV) inhibited uptake of choline and glycine betaine, and the residual low transport of glycine betaine was attributed to the Qat3 system (pRL120514-6) [64]. RHLa5629 shows 96 % id with pRL120516 and it may be that it forms part of a second glycine betaine transport system. RHL2564 encoding an SBP of an ABC transporter of the NitT (nitrate/nitrite/cyanate transporter) family was up-regulated in bacteroids of both Rlp4292 (about 13-fold) and RlvA34 (about 6-fold) (Fig. 3); the orthologous gene in S. meliloti (SMc01827) is induced by uracil and uridine [65].

Magnesium and other cations
Expression of mgtE (RHL0406), encoding a Mg ++ transporter was up-regulated about 18-fold in bean and about 4fold in RlvA34 pea bacteroids (the >5-fold cut-off making it appear bean-specific in Fig. 3). Cation-transporting ATPase proteins encoded by RHLb6066 and RHLb6068 were up-regulated about 20-to 30-fold and about 6-to 7-fold in bacteroids of Rlp4292 and RlvA34, respectively. MbfA (RHL3841), a putative rubrerythrin (contains ferritin fold) transmembrane protein, is related to proteins involved in iron and manganese transport (CCC1-like family), and RHL3841 was up-regulated about 23-fold in beans and about 5-fold in peas (Table S12, Fig. 3).

Tat secretion
The Tat protein exporter encoded by RHL0954 (tatA) and RHL0955 (tatB) was up-regulated about sixfold in RlvA34 pea bacteroids (Fig. 3) and about threefold in bean bacteroids (Table S12). This transporter secretes cell wall amidases needed for rhizobial-wall integrity and nodule infection; it also exports the periplasmic Rieske electron transport protein, required for bacteroid respiration [66,67].

Role of plasmid B in symbiosis
Within a cluster of just over 30 genes encoded on plasmid B (RHLb6065-6098), 24 are up-regulated >5-fold in both bean and pea bacteroids (Table S10). Although several genes in this cluster are related to stress responses: e.g. RHLb6065 and RHLb6072 encode universal stress proteins; RHLb6081 encodes a small heat shock protein, HspF (see section above); RHLb6066 and RHLb6068 encode cation transporters; and RHLb6098 encodes FeuP, part of a two-component sensor regulator (Table S10). The significance of this clustering of genes is not known.

(ii) Shared genes up-regulated only in bean bacteroids
One hundred and fifty genes were >5-fold up-regulated in Rlp4292 bean bacteroids and <2-fold up-regulated in RlvA34 from pea nodules (Table S13, Fig. 3). In this section, we deal with supply of P, S, Fe, Ca, C, N and O 2 , and then predicted stresses.

Bean bacteroids are sulphate-limited
RHLb6112 encoding the sulphur transport ABC component SulA was up-regulated about sixfold in bean and about sixfold down-regulated in pea bacteroids (Table S12, Fig. 4). The sulA (SMb21133) orthologue in S. meliloti is induced by sulphate limitation [65]. RHLc6500, a NitT family transporter that probably encodes an aliphatic sulphonate ABC transporter SBP, was up-regulated about sevenfold in bean bacteroids but down-regulated about threefold in pea (Fig. 4). This suggests that bean bacteroids are sulphate-limited, while those of pea are not.

Bean bacteroids are iron-limited
Several uptake systems associated with iron limitation are up-regulated in bean but not pea bacteroids; none of these genes (described below) is among those up-regulated in free-living RlvA34 compared with Rlv4292 (Table S5). This means that there are at least two sets of transcriptional responses to iron. The ABC transporter FeT [Fe (III) transport] family gene RHL2160 and RHL2001 (afuA3) were strongly up-regulated (about 20-and 40-fold, respectively) in bean but not pea bacteroids (Table S12, Fig. 4). RHL1379 (sufC), involved in [Fe-S] cluster assembly, was up-regulated about sixfold in bean and fourfold in pea bacteroids (Table S12). The PepT family dipeptide transporter, RHL3498 (dppA3), was up-regulated about sixfold in bean and threefold in pea bacteroids (Table S12). Mutation of the rhizobial dpp operon reduces uptake of the haem precursor d-aminolevulinic acid [69]. In Escherichia coli, the Dpp transport system transports iron via haem [70]. In addition to the Ca-binding proteins up-regulated in both bean and pea bacteroids (see above), RHLa5297 (casA) encoding the exported Ca-binding protein calsymin, was about 50-fold induced in bean but not pea bacteroids (Table S13). R. etli CasA (85 % id with RHLa5297) is important for the symbiosis with Phaseolus, because a casA mutation affected bacteroid development and decreased nitrogen fixation [55].

Diverse carbohydrates available in bean nodules
Expression of carbohydrate uptake transporters (CUTs) gives an insight into sugars available in nodules. There are two subclasses of these ABC transporters: CUT1, transporting diand oligo-saccharides; and CUT2, which generally transports monosaccharides. Eight genes encoding CUT1 components were up-regulated in bacteroids, seven of these in bean but not pea bacteroids, identifying six different CUT1 transporters (Fig. 4). The SBPs RHLb5675 and RHL3007, respectively, probably bind galactosamine and mannitol, which induce the orthologous genes in S. meliloti [65]. RHL4353, annotated as alpha-glucoside transporter (aglE), and RHL2640 and RHL2643, annotated as glycerol-3-phosphate transporter components (ATP-binding component and SBP, ugpC2 and ugpB2, respectively), were up-regulated about 80-fold in bean bacteroids (Table S12, Fig. 4).

Taurine in bean nodules
RHLc6333 (tauA), which encodes a predicted taurine uptake component, was up-regulated about sixfold in bean but down-regulated about eightfold in pea bacteroids (Fig. 4). This is indicative that there is taurine in bean but not pea nodules because RHLc6333 shows 40 % id with the TauT family SBP SMb21526 from S. meliloti, which is induced by taurine [65] and forms the basis of a taurine-inducible expression system in rhizobia [71].

Other transporters induced in bean nodules
Among the several putative POPT (polyamines, opines and phosphonate) family genes in the shared genome, only RHLa4848-9 encoding a putative POPT transporter was up-regulated 10-to 40-fold in bean but not RlvA34 pea bacteroids (Table S12, Fig. 4). Three PAAT predicted aminoacid SBP genes (RHL1551, RHLa5284 and RHLb5944) were up-regulated 5-to 10-fold in bean but not pea bacteroids (Fig. 4). A predicted QAT system (RHLc6320) was up-regulated about 160-fold in bean but not pea bacteroids. In all these cases it is likely that the unidentified solutes are present in bean but not pea nodules.

Cytochrome oxidases
Genes RHLc6648-53, encoding a transmembrane cytochrome d ubiquinol oxidase subunit I, a cytochrome bd-II oxidase subunit II, an ABC transporter system related to cytochrome bd export and a MarR family regulator of gene expression, were all more strongly up-regulated in bean bacteroids (about 12-to 90-fold, Table S4) than in pea bacteroids (about 2-to 5-fold) (Table S4). Genes encoding cytochrome c oxidase subunits RHL4623-4 (CtaC1 and CtaC2) were up-regulated about sixfold specifically in bean bacteroids (Table S13). Cytochrome cbb3 (encoded by the fixNOQP operon) is the high affinity oxidase essential for nitrogen fixation [72], but the expression of other respiratory pathways terminated by oxidases with a lower affinity for oxygen could be indicative that there may be a higher free oxygen level in (parts of) bean nodules.

Lipid X
Lipid A is the primary lipid in the outer layer of the Gramnegative bacterial membrane and acts as an anchor for lipopolysaccharide [73]. Synthesis of lipid A involves nine enzymes, one of which, LpxH, cleaves UDP-2,3-diacylglucosamine to 2,3-diacylglucosamine 1-phosphate (lipid X) and uracil-monophosphate (UMP) [74]. lpxH (RHL1469) was up-regulated about 70-fold in bean but not pea bacteroids. Genes encoding the other eight Lpx enzymes were not up-regulated in Rlp4292 bacteroids (Table S4). Lipid A oxidase RHL4482 (lpxQ) was about sevenfold up-regulated in bean but not pea bacteroids (Table S13).

Detoxification and stress
Nine genes encoding stress proteins were elevated specifically in bean bacteroids (Table S13, Riley code 1.6.x). RHL3015, encoding the osmotically-induced OsmC, and RHL1758 and RHLa5426, encoding two cold shock proteins, were up-regulated about 10-fold in bean but not pea bacteroids. Glutathione S-transferases (GSTs) are diverse enzymes involved in detoxification of oxidative stressors, antimicrobial agents and metabolic intermediates in bacteria. Three predicted GSTs, RHL0225, RHL346 and RHL3926, were up-regulated about 10-fold in bean but not pea bacteroids (Table S13). Export is another way of removing toxic compounds; RHL0518, RHL2130 and RHL3918 encode efflux systems that are up-regulated about 5-to 8-fold in bean but not pea bacteroids (Table S13, Riley code 1.5.5). These data could be indicative that rhizobia in bean nodules are more stressed by metabolites than rhizobia in pea nodules. Chaperonins are also often induced in response to stress [75] RH2128, encoding a DnaJ family protein, and RHL4498-9, predicted to encode the chaperonins Cpn60 and Cpn10, were about sevenfold up-regulated in bean but not pea bacteroids (Table S13).

Outer membrane ROPs
Bacterial outer membranes and their proteins are crucial for cell-cell and cell-environment interactions. In S. meliloti, a putative transmembrane b-barrel porin, RopA1, is essential for viability, despite the presence of ropA2, encoding a close homologue [76]. The Rlp4292 and RlvA34 share four homologues of RopA (RHL0466, RHL1573, RHL2878 and RHLb6113), each with about 50 % id with RopA1 or RopA2 from S. meliloti. Three of these genes, which we named ropA1 (RHL0466), ropA3 (RHL2878) and ropA4 (RHLb6113), were up-regulated (about 25-, 90-and 10-fold, respectively) in bean but not pea bacteroids (Table S4). Autoaggregation proteins RapA2 (RHLc6544) and RapB3 (RHL2729) were up-regulated 40-and 12-fold in bean but not pea bacteroids (Table S13). Possibly the presence of multiple bacteroids within one symbiosome as observed in bean but not pea nodules influences expression of genes that affect cell-cell interactions.
(iii) Shared genes up-regulated in pea but not bean bacteroids Table S15 shows the 116 genes that are >5-fold up-regulated in RlvA34 pea bacteroids and <2-fold up-regulated in Rlp4292 from bean nodules. These data are presented together with microarray data for those Rlv3841 genes showing >80 % amino acid id to those of Rlp4292/RlvA34 [13]. The pattern of up-regulated genes is different from that in bean, suggesting limitation of N and Mo and different gene induction associated with C metabolism and bacteroid development. Forty genes encoding components of solute uptake systems were up-regulated >5-fold in RlvA34 pea bacteroids and <5-fold in Rlp4292 bean bacteroids (Table S16, Riley code 1.5.0-1.5.4), with 26 of them highly expressed (Table S12, Fig. 4)
Phenylalanine could also be a source of N in pea bacteroids because RHL0852 (phhA), encoding a phenylalanine-4hydroxylase, was up-regulated about 6-fold in pea but not bean bacteroids (it is also up-regulated about 40-fold in the pea rhizosphere; Table S4). Phenylalanine is a precursor of lignin synthesis and may be available in pea nodules due to cell wall synthesis being maintained in mature indeterminate (pea) but not determinate (bean) nodules.

Pea bacteroids appear molybdate-limited
The gene encoding SBP ModA (RHL3521) was up-regulated about eightfold in pea but not bean bacteroids (Table S16) (this gene failed to meet the criterion for high expression and does not appear in Table S12 or on Fig. 4). Although in Rlv3841, expression of a molybdate transporter (MolT family of ABC transporters) encoded by modABC was also induced under low sulphate [78], the fact that ABC sulphur transporter SulT family gene RHLb6112 (sulA) was downregulated (about 10-fold) in pea bacteroids of RlvA34 and Rlv3841 (pRL110374, 97 % id) (Table S12) suggests that although RlvA34 bacteroids are molybdate-limited, they have sufficient sulphate within pea nodules.
Bacteroid development NCR peptides secreted by the plant [11] affect bacteroid development in peas but not beans, and the resulting differences in bacteroid differentiation are likely to cause differences in bean and pea bacteroid gene expression. Several lipid biosynthesis and metabolism genes were slightly upregulated (about 2-to 3-fold) in pea, but not bean bacteroids. These include genes encoding the enzymes of the lipid A biosynthetic pathway: lpxB (RHL1074), lpxD (RHL1070) and lpxK (RHL4516), lipid metabolic enzyme (RHL2015), an ABC family lipid exporter (RHL3351-2). An acyl carrier protein (RHLc6550) was up-regulated about 10-fold in pea bacteroids and about 4-fold in bean bacteroids (Tables S4  and S13). The level of induction of most of these genes was modest and not particularly different from those seen in bean bacteroids. This may reflect the fact that changes attributable to NCR peptides have already occurred by the time nodules have matured, a conclusion reached with RNA from Rlv3841 bacteroids of 7, 15, 21 and 28 days post-inoculation analysed using microarrays [13].

Detoxification and stress
A predicted salicylate hydrolase gene (RHLa4811) was about 12-fold up-regulated in RlvA34 pea bacteroids. A putative arsenate reductase gene (RHL4205) was up-regulated about sevenfold in RlvA34 (Table S15). The heat shock protein RHL2870 (IbpA) may be more important in pea bacteroids as ibpA expression was about 15-fold up-regulated in RlvA34 pea bacteroids, but only about 2-fold up-regulated in bean bacteroids (Table S4). Numerous export systems that are up-regulated in pea but not bean bacteroids may remove unwanted and/or toxic compounds; these include two pea-specific GSTs (RHL1557 and RHL3865, sharing 32 % id), which were up-regulated about 35-and 10-fold in pea RlvA34 bacteroids (Table S15). Amongst those genes with the most highly elevated expression in pea but not bean bacteroids were the RND family efflux systems encoded by RHL2076, RHL2565-6 RHL2619-20 (mexF1), RHL2967 and RHL3012 (about 5-to 8-fold up-regulated, Table S12, Fig. 3), a HlyD family efflux pump, RHL2518 (about 10-fold), contiguous with an ABC export system RHL2519-21 (about 4-to 6-fold), a TetR family transcriptional regulator (RHL2517, about 8-fold), another ABC export cluster RHL2324 (about 8-fold) and MFS (major facilitator subfamily) protein RHL2967 (about 5-fold). [For a full list of the 12 genes encoding proteins involved in export (Riley code 1.5.5) that are >5-fold elevated in pea bacteroids and <2-fold elevated in bean bacteroids, see Table S15.] Table S12 and Fig. 4 show up-regulated transporters that are also highly expressed.

Central carbon metabolism
In pea bacteroids, strongly up-regulated genes included those encoding TCA (tricarboxylic acid) cycle enzymes RHL1075 (GltA, citrate synthase), RHL3358 (AcnA, aconitase), RHL3260 and RHL3258 (SucAB, oxoglutarate dehydrogenase), RHL3261 (SucD, succinyl-CoA synthetase), RHL3268 (SdhA) and RHL3267 (SdhB) (both parts of the succinate dehydrogenase complex) and RHL3264 (Mdh, malate dehydrogenase) (Table S17, Fig. 5) were also seen in bacteroids of Rlv3841 [13]. In Rlp4292, these genes were up-regulated at most about twofold, except for citrate synthase (GltA) encoded by RHL1075, which was up-regulated about fourfold (Table S17), while RHL1300 (gltA2) and RHL1301 (citZ) were unchanged or twofold down-regulated, respectively. From these data, we can predict that bean bacteroids maintain a level of TCA cycle enzymes similar to that of their free-living cells, including glutamine/glutamate synthesis, whereas highly differentiated pea bacteroids have more perturbed central metabolism. It should be stressed that an increase in expression of genes of the TCA cycle does not give information on the flux through the cycle.
The glyoxylate cycle is unlikely to be elevated in pea or bean bacteroids as the genes encoding enzymes RHL4371 (AceA) and RL3631 (GlcB) (Fig. 5) were not differentially expressed in either Rlp4292 or RlvA34 bacteroids (Table S17). Aconitase and 2-ketoglutarate dehydrogenase enzymes are not required for nitrogen fixation in bacteroids of B. japonicum [79,80].
(iv) Shared genes down-regulated in both bean and pea bacteroids Table S18 shows 17 genes that are down-regulated >5-fold in both bean and pea bacteroids (summarized in Fig. 3) and these include the ribonucleoside reductase genes RHL3048-50 (nrdEIH) (down-regulated about 5-to-10-fold) that produce dNTPs required for DNA synthesis [82]. The six genes, RLHa5121-6 (vbsLDAGSOP), encoding the ironscavenging siderophore vicibactin and proteins for its biosynthesis and export, were among the most down-regulated (about 5-to 10-fold) in both bean and pea bacteroids (Table S18). While these genes were down-regulated by approximately the same amount in both bean and pea, the fact that the RlvA34 strain had about fivefold more expression in free-living cells than in those of Rlp4292 (Table S5) means that the absolute expression in RlvA34 bacteroids in pea is about five times higher than in Rlp4292 bean bacteroids. The down-regulation of these genes in bacteroids is indicative of a regulatory mechanism epistatic to RirA, mutation of which caused their increased expression in freeliving RlvA34. Other genes thought be affected by the rirA mutation in RlvA34 (Table S5) behaved differently; RHL2531-40 encoding haemin iron transport proteins were slightly down-regulated in bean bacteroids (about 2-fold) but not differentially regulated in RlvA34 pea bacteroids (compared to the elevated level in free-living bacteria); RHLa5475-7, encoding a FeCT ABC transporter, was down-regulated in both bean and pea bacteroids by about 3-fold (meaning that the absolute level in RlvA34 pea bacteroids was about 2-to 5-fold higher that in Rlp4292 bean bacteroids) (Table S5). However, it is important to realise that RirA may act differently in bacteroids than in free-living bacteria and this should, therefore, be taken into account.
Three genes strongly down-regulated in both pea and bean bacteroids were the quorum-sensing regulators encodes cinI and cinS, and the adjacent gene (RHL2191) encoding a putative 3-hydroxybutyryl-CoA dehydrogenase (25 % id to HbdA). RHL2814, encoding the chemotaxis transcriptional regulator CheY (98 % to RL4036), was down-regulated about fivefold, befitting an environment where chemotaxis is not possible. The polysaccharide lyase, encoded by RHL1848 (plyA2), is down-regulated about 5-to 15-fold in bean and pea bacteroids (Table S18); this lyase cleaves acidic extracellular polysaccharide [83] minimising the high viscosity caused by extracellular polysaccharide (an issue that would be unimportant in bacteroids).
Genes encoding FMN reductases, RHLc6323 and RHLc6678, were down-regulated about 5-to 8-fold in RlvA34 pea, but not Rlv4292 bean bacteroids (Table S20). The RHLb5787-9 genes, encoding a trifolitoxin-related protein and two hypothetical proteins, were down-regulated about fivefold in RlvA34 pea but slightly up-regulated in bean bacteroids.
Genes encoding components of several solute uptake systems were down-regulated in pea but not bean bacteroids; RHL1566 (NitT), RHL3478 and RHLc6677 (PAAT), RHLb6112 (SulT), RHLc6283 and RHLc6625 (CUT2), and RHLc6332-3 (unclass) (about fivefold). These genes were more highly expressed due to solutes present in the liquid media and the observation that they were not differentially regulated in bean, correlates with the wide-range of solutes present in bean nodules apparent from induction of the large number and variety of bean-specific transporters (Fig. 4).

Conclusions
(1) Against a general pattern of down-regulation of gene expression in bacteroids compared with free-living rhizobia, both bean and pea bacteroids showed increased expression of genes associated with nitrogen fixation and utilization of dicarboxylates, formate, amino acids and quaternary amines. The decreased expression of many genes may be associated with the increased expression of stringent response genes. (2) Gene expression patterns suggest that bean bacteroids were more limited for P, S and Fe than pea bacteroids, and the expression of cytochrome oxidase genes suggests that bean bacteroids are exposed to higher levels of oxygen than pea bacteroids. (3) Bacteroids in bean nodules expressed many genes whose products are predicted to be related to metabolite detoxification and export. (4) Bean bacteroids express high levels of phasin genes that are associated with storage of the large amounts of PHB that accumulate in bean nodules. (5) Pea nodules have strongly upregulated genes associated with central metabolism and this is indicative of some fundamental differences in the use of the TCA cycle. (6) Surprisingly, pea bacteroids showed evidence for expression of nitrate and nitrite reduction enzymes. (7) Expression patterns of uptake transporters are indicative that bacteroids in pea and bean nodules are exposed to different sets of substrates specific to each nodule type.