Metabolomic Profiling of Bradyrhizobium diazoefficiens-Induced Root Nodules Reveals Both Host Plant-Specific and Developmental Signatures.

Bradyrhizobium diazoefficiens is a nitrogen-fixing endosymbiont, which can grow inside root-nodule cells of the agriculturally important soybean and other host plants. Our previous studies described B. diazoefficiens host-specific global expression changes occurring during legume infection at the transcript and protein level. In order to further characterize nodule metabolism, we here determine by flow injection-time-of-flight mass spectrometry analysis the metabolome of (i) nodules and roots from four different B. diazoefficiens host plants; (ii) soybean nodules harvested at different time points during nodule development; and (iii) soybean nodules infected by two strains mutated in key genes for nitrogen fixation, respectively. Ribose (soybean), tartaric acid (mungbean), hydroxybutanoyloxybutanoate (siratro) and catechol (cowpea) were among the metabolites found to be specifically elevated in one of the respective host plants. While the level of C4-dicarboxylic acids decreased during soybean nodule development, we observed an accumulation of trehalose-phosphate at 21 days post infection (dpi). Moreover, nodules from non-nitrogen-fixing bacteroids (nifA and nifH mutants) showed specific metabolic alterations; these were also supported by independent transcriptomics data. The alterations included signs of nitrogen limitation in both mutants, and an increased level of a phytoalexin in nodules induced by the nifA mutant, suggesting that the tissue of these nodules exhibits defense and stress reactions.

A link between arabinose utilization and oxalotrophy in Bradyrhizobium japonicum.

Rhizobia have a versatile catabolism that allows them to compete successfully with other microorganisms for nutrients in the soil and in the rhizosphere of their respective host plants. In this study, Bradyrhizobium japonicum USDA 110 was found to be able to utilize oxalate as the sole carbon source. A proteome analysis of cells grown in minimal medium containing arabinose suggested that oxalate oxidation extends the arabinose degradation branch via glycolaldehyde. A mutant of the key pathway genes oxc (for oxalyl-coenzyme A decarboxylase) and frc (for formyl-coenzyme A transferase) was constructed and shown to be (i) impaired in growth on arabinose and (ii) unable to grow on oxalate. Oxalate was detected in roots and, at elevated levels, in root nodules of four different B. japonicum host plants. Mixed-inoculation experiments with wild-type and oxc-frc mutant cells revealed that oxalotrophy might be a beneficial trait of B. japonicum at some stage during legume root nodule colonization.

Copper starvation-inducible protein for cytochrome oxidase biogenesis in Bradyrhizobium japonicum.

Microarray analysis of Bradyrhizobium japonicum grown under copper limitation uncovered five genes named pcuABCDE, which are co-transcribed and co-regulated as an operon. The predicted gene products are periplasmic proteins (PcuA, PcuC, and PcuD), a TonB-dependent outer membrane receptor (PcuB), and a cytoplasmic membrane-integral protein (PcuE). Homologs of PcuC and PcuE had been discovered in other bacteria, namely PCu(A)C and YcnJ, where they play a role in cytochrome oxidase biogenesis and copper transport, respectively. Deletion of the pcuABCDE operon led to a pleiotropic phenotype, including defects in the aa(3)-type cytochrome oxidase, symbiotic nitrogen fixation, and anoxic nitrate respiration. Complementation analyses revealed that, under our assay conditions, the tested functions depended only on the pcuC gene and not on pcuA, pcuB, pcuD, or pcuE. The B. japonicum genome harbors a second pcuC-like gene (blr7088), which, however, did not functionally replace the mutated pcuC. The PcuC protein was overexpressed in Escherichia coli, purified to homogeneity, and shown to bind Cu(I) with high affinity in a 1:1 stoichiometry. The replacement of His(79), Met(90), His(113), and Met(115) by alanine perturbed copper binding. This corroborates the previously purported role of this protein as a periplasmic copper chaperone for the formation of the Cu(A) center on the aa(3)-type cytochrome oxidase. In addition, we provide evidence that PcuC and the copper chaperone ScoI are important for the symbiotically essential, Cu(A)-free cbb(3)-type cytochrome oxidase specifically in endosymbiotic bacteroids of soybean root nodules, which could explain the symbiosis-defective phenotype of the pcuC and scoI mutants.

Early expression of the type III secretion system of Parachlamydia acanthamoebae during a replicative cycle within its natural host cell Acanthamoeba castellanii.

The type three secretion system (T3SS) operons of Chlamydiales bacteria are distributed in different clusters along their chromosomes and are conserved at both the level of sequence and genetic organization. A complete characterization of the temporal expression of multiple T3SS components at the transcriptional and protein levels has been performed in Parachlamydia acanthamoebae, replicating in its natural host cell Acanthamoeba castellanii. The T3SS components were classified in four different temporal clusters depending on their pattern of expression during the early, mid- and late phases of the infectious cycle. The putative T3SS transcription units predicted in Parachlamydia are similar to those described in Chlamydia trachomatis, suggesting that T3SS units of transcriptional expression are highly conserved among Chlamydiales bacteria. The maximal expression and activation of the T3SS of Parachlamydia occurred during the early to mid-phase of the infectious cycle corresponding to a critical phase during which the intracellular bacterium has (1) to evade and/or block the lytic pathway of the amoeba, (2) to differentiate from elementary bodies (EBs) to reticulate bodies (RBs), and (3) to modulate the maturation of its vacuole to create a replicative niche able to sustain efficient bacterial growth.