Members of the β-proteobacterial have been discovered in 2001 to be able to enter a nitrogen-fixing symbiosis with legumes [
4]. In contrast to the α-rhizobial symbiosis with host legumes, where the different steps leading to the establishment of a successful symbiosis are well studied, very little is known about the molecular mechanisms that are relevant for these steps in β-rhizobial symbioses. Here, we used an RNA-sequencing approach to provide a comprehensive view on the gene expression profile of
P. phymatum grown in either normal or nitrogen-limited free-living conditions, and as bacteroids in symbiosis with
P. vulgaris. To the best of our knowledge, this is the first transcriptome study from root nodules formed by a β-rhizobial strain. By testing the gene expression profile in nitrogen-limited conditions, we aimed to partially mimic the conditions that rhizobia encounter in soils lacking nitrogen and in our laboratory settings before colonizing the roots of legumes. Apart from genes involved in nitrogen metabolism, a gene cluster potentially involved in EPS biosynthesis (Bhy_1056-Bphy_1077) showed increased expression when nitrogen became limited. Exopolysaccharides were shown in several rhizobia to be required for root hairs attachment [
74] and infection [
75], the first two steps of the cascade. The gene that showed the most significant and the second highest upregulation during nitrogen starvation encodes for a potential methyl-accepting chemotaxis sensory transducer (Bphy_2338). The presence of another gene involved in motility among the top regulated genes (the flagellar gene
fliL) suggests that the cells react to a nitrogen-limited environment by changing their movement behavior, which is a crucial trait for the successful colonization and infection of host legume roots [
76]. The future construction and characterization of mutant strains will shed light on the importance of EPS and flagella in a nitrogen-starved environment. The expression of
nod genes, encoding the
P. phymatum Nod factor required for the recognition of the symbiotic partner, changed only slightly in response to nitrogen starvation (
Figure S1). The expression of genes in the
nif cluster did not significantly change when the cells were grown under nitrogen limitation, suggesting that—similar to the situation in α-rhizobia—the presence of a reduced amount of nitrogen is not sufficient to activate the expression of the
nif cluster. In contrast, a low-oxygen environment highly induced the expression of the genes coding for the nitrogenase (
Table 2). Among the 38 genes commonly upregulated, we found
ntrC, which codes for a transcriptional regulators known to be important for nitrogen control (Ntr) in other organisms [
64]. While the Ntr system is usually switched off during nitrogen fixation in symbiotic α-rhizobia [
36], in free-living diazotrophs such as
Azospirillum brasilense the two-component regulatory system NtrB/NtrC has been shown to be involved in the regulation of nitrogenase activity [
77]. Two genes coding for a urea ABC transporter (Bphy_2251-52) were also among the genes commonly induced under nitrogen-starving and symbiotic conditions. This may suggest that this organic compound serves an important role in free-living and symbiotic metabolism.
As expected, the expression of the
nif gene cluster (including genes from Bphy_7728 to Bphy_7755,
Figure 3A), was found significantly upregulated inside nodules induced by
P. phymatum. Unlike most α-rhizobia [
78],
P. phymatum contains a
nifV homolog in the genome (upstream of
nifB), which is also highly induced during symbiosis. This gene encodes a homocitrate synthase that synthesizes homocitrate—a component of the Fe–Mo cofactor of the nitrogenase—which has been shown to be important in diazotrophs to reduce N
2 in free-living conditions [
79,
80,
81]. The presence of
nifV in
P. phymatum may explain the ability of this bacterium to fix nitrogen in free-living conditions [
4]. Among the upregulated genes in symbiosis, we found a potential four-component oxidase cluster also annotated as a cytochrome o ubiquinol oxidase complex. This cluster (Bphy_3646-49,
cyoABCD) was also significantly upregulated in a preliminary transcriptome analysis performed on free-living cells grown in microaerobic conditions compared to cells growing aerobically, suggesting that this heme–copper respiratory oxidase could be used by
P. phymatum to respire inside root nodules. Indeed, the classical
cbb3-oxidase crucial for symbiosis in α-rhizobia was detected neither in the
P. phymatum genome nor in other symbiotic
Paraburkholderia species [
44]. The construction of a
cyoB insertion mutant and a
cyoAB deletion mutant provided proof that this cluster is indeed important for an efficient symbiotic interaction. Bean plants inoculated with these mutants showed a significantly reduced nitrogenase activity and a lower N content compared to plants infected with the wild type. In previous studies [
82,
83], three genes were shown to be important during symbiosis with
M. pudica: Bphy_0456, involved in the biosynthesis of branched-chain amino acids, Bphy_0685, coding for a fructose 1,6-bisphosphatase, and Bphy_0266 (
gpmA), coding for a phosphoglycerate mutase. The expression of these three genes was not regulated in bean root nodules compared to free-living conditions, suggesting that
P. phymatum may upregulate a different set of genes in its natural host plant
Mimosa. Interestingly, and in contrast to the situation in α-rhizobia [
84,
85], a gene coding for an isocitrate lyase (Bphy_1368) was found inside the top 500 regulated genes in
P. phymatum bacteroids, suggesting that the glyoxylate shunt pathway is active during β-rhizobial symbiosis. In previous studies on
Bradyrhizobium sp. ORS278, the
ccbL1 gene, which codes for a ribulose 1,5 bis-phosphate carboxylase oxygenase (RuBisCO) needed for carbon fixation, was proven to have a critical role in symbiotic nitrogen fixation [
86]. We noticed here that the
P. phymatum gene coding for ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO; Bphy_6497, 77% amino acid identity to
ccbL1 or BRADO1659 of
Bradyrhizobium sp. ORS278) was also upregulated inside root nodules, suggesting a possible role of this enzyme also in β-rhizobial symbiosis. The mutation of two
P. phymatum regulatory genes known to play a key role for α-rhizobial symbiosis [
38]—the alternative sigma factor RpoN and its activator NifA—showed that both regulators are also important for the regulation of nitrogenase activity (
Figure 5C). Nodules infected by a
nifA mutant strain were impaired in nitrogen fixation, even 28 dpi [
87]. An increased number of nodules in grape-like structures were produced in plants infected with the
P. phymatum nifA mutant strain. A similar phenotype was observed in the nodules of another legume, soybean, that were induced by a
Bradyrhizobium diazoefficiens nifA mutant [
88]. Using a metabolomics approach on
Bradyrhizobium diazoefficiens nodules, we previously speculated that such a phenotype could be due to a defense reaction of the legume evoked by the
nifA mutant, involving an increased production of phytoalexins [
89]. Since NifA is an activator protein of the alternative sigma factor σ
54 (RpoN), we constructed an
rpoN mutant, which indeed did not show any nitrogenase activity. In addition, the
rpoN gene was found highly expressed in all conditions tested, i.e., nitrogen-replete and -limited conditions and during symbiosis, suggesting that RpoN may play an important role not only in symbiosis but also in free-living conditions. In fact, the utilization of nitrogen sources as well as the EPS production were affected in this strain. In a closely related
Burkholderia strain belonging to the pathogenic clade, RpoN was shown to play a role in free-living conditions and also in vivo, where a mutant showed reduced virulence in the
Caenorhabditis elegans infection model [
59].
In summary, this first analysis of bacterial gene expression in symbiotic bean root nodules induced by a β-rhizobial strain revealed new insights into this recently discovered symbiosis. It provides a rich basis for a further dissection of the molecular mechanisms underlying this symbiotic association and for the elucidation of the mechanistic differences between β-rhizobial and the much better characterized α-rhizobial symbioses.