Isovaleryl-homoserine lactone, an unusual branched-chain quorum-sensing signal from the soybean symbiont Bradyrhizobium japonicum.

Many species of Proteobacteria communicate by using LuxI-LuxR-type quorum-sensing systems that produce and detect acyl-homoserine lactone (acyl-HSL) signals. Most of the known signals are straight-chain fatty acyl-HSLs, and evidence indicates that LuxI homologs prefer fatty acid-acyl carrier protein (ACP) over fatty acyl-CoA as the acyl substrate for signal synthesis. Two related LuxI homologs, RpaI and BtaI from Rhodopseudomonas palustris and photosynthetic stem-nodulating bradyrhizobia, direct production of the aryl-HSLs p-coumaroyl-HSL and cinnamoyl-HSL, respectively. Here we report that BjaI from the soybean symbiont Bradyrhizobium japonicum USDA110 is closely related to RpaI and BtaI and catalyzes the synthesis of isovaleryl-HSL (IV-HSL), a branched-chain fatty acyl-HSL. We show that IV-HSL induces expression of bjaI, and in this way IV-HSL functions like many other acyl-HSL quorum-sensing signals. Purified histidine-tagged BjaI was an IV-HSL synthase, which was active with isovaleryl-CoA but not detectably so with isovaleryl-ACP. This suggests that the RpaI-BtaI-BjaI subfamily of acyl-HSL synthases may use CoA- rather than ACP-linked substrates for acyl-HSL synthesis. The bjaI-linked bjaR(1) gene is involved in the response to IV-HSL, and BjaR(1) is sensitive to IV-HSL at concentrations as low as 10 pM. Low but sufficient levels of IV-HSL (about 5 nM) accumulate in B. japonicum culture fluid. The low levels of IV-HSL synthesis have likely contributed to the fact that the quorum-sensing signal from this bacterium has not been described elsewhere.

Host-specific symbiotic requirement of BdeAB, a RegR-controlled RND-type efflux system in Bradyrhizobium japonicum.

Multidrug efflux systems not only cause resistance against antibiotics and toxic compounds but also mediate successful host colonization by certain plant-associated bacteria. The genome of the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum encodes 24 members of the family of resistance/nodulation/cell division (RND) multidrug efflux systems, of which BdeAB is genetically controlled by the RegSR two-component regulatory system. Phylogenetic analysis of the membrane components of these 24 RND-type transporters revealed that BdeB is more closely related to functionally characterized orthologs in other bacteria, including those associated with plants, than to any of the other 23 paralogs in B. japonicum. A mutant with a deletion of the bdeAB genes was more susceptible to inhibition by the aminoglycosides kanamycin and gentamicin than the wild type, and had a strongly decreased symbiotic nitrogen-fixation activity on soybean, but not on the alternative host plants mungbean and cowpea, and only very marginally on siratro. The host-specific role of a multidrug efflux pump is a novel feature in the rhizobia-legume symbioses. Consistent with the RegSR dependency of bdeAB, a B. japonicum regR mutant was found to have a greater sensitivity against the two tested antibiotics and a symbiotic defect that is most pronounced for soybean.

Global consequences of phosphatidylcholine reduction in Bradyrhizobium japonicum.

Phosphatidylcholine (PC) is the major phospholipid in eukaryotic membranes. In contrast, it is found in only a limited number of bacteria including members of the Rhizobiales. Here, PC is required for pathogenic and symbiotic plant-microbe interactions, as shown for Agrobacterium tumefaciens and Bradyrhizobium japonicum, respectively. Two different phospholipid N-methyltransferases, PmtA and PmtX1, convert phosphatidylethanolamine (PE) to PC by three consecutive methylation reactions in B. japonicum. PmtA mainly catalyzes the first methylation reaction converting PE to monomethyl PE, which then serves as substrate for PmtX1 performing the last two methylation reactions. Disruption of the pmtA gene results in a significantly reduced PC content causing a defect in symbiosis with the soybean host. A genome-wide survey for differentially expressed genes in the pmtA mutant with a custom-made Affymetrix gene chip revealed that PC reduction affects transcription of a strictly confined set of genes. Among the 11 up regulated genes were pmtX3 and pmtX4, which code for isoenzymes of PmtA. The expression of two typical two-component systems, a MarR-like regulator and two proteins of a RND-type (resistance nodulation cell division) efflux system were differentially expressed in the pmtA mutant. Our data suggests that a decrease in the PC content of B. japonicum membranes induces a rather specific transcriptional response involving three different transcriptional regulators all involved in the regulatory fine-tuning of a RND-type transport system.

The genistein stimulon of Bradyrhizobium japonicum.

An initializing step in the rhizobia-legume symbiosis is the secretion of flavonoids by plants that leads to the expression of nodulation genes in rhizobia. Here we report the genome-wide transcriptional response of Bradyrhizobium japonicum to genistein, an isoflavone secreted by soybean. About 100 genes were induced in the wild type. This included all nod box-associated genes, the flagellar cluster and several genes that are likely to be involved in transport processes. To elucidate the role of known regulators, we analysed mutant strains. This revealed that the two-component response regulator NodW is essential for induction of almost all genistein-inducible genes, with the exception of 8 genes. The phenotype of the nodW mutant could be partially suppressed by overexpression of NwsB, which is also a two-component response regulator. These data indicate that genistein has a much broader function than mere induction of nod genes.

Genome-wide transcript analysis of Bradyrhizobium japonicum bacteroids in soybean root nodules.

The transcriptome of endosymbiotic Bradyrhizobium japonicum bacteroids was assessed, using RNA extracted from determinate soybean root nodules. Results were compared with the transcript profiles of B. japonicum cells grown in either aerobic or microaerobic culture. Microoxia is a known trigger for the induction of symbiotically relevant genes. In fact, one third of the genes induced in bacteroids at day 21 after inoculation are congruent with those up-regulated in culture by a decreased oxygen concentration. The other induced genes, however, may be regulated by cues other than oxygen limitation. Both groups of genes provide a rich source for the possible discovery of novel functions related to symbiosis. Samples taken at different timepoints in nodule development have led to the distinction of genes expressed early and late in bacteroids. The experimental approach applied here is also useful for B. japonicum mutant analyses. As an example, we compared the transcriptome of wild-type bacteroids with that of bacteroids formed by a mutant defective in the RNA polymerase transcription factor sigma54. This led to a collection of hitherto unrecognized B. japonicum genes potentially transcribed in planta in a sigma54-dependent manner.

New target genes controlled by the Bradyrhizobium japonicum two-component regulatory system RegSR.

RegSR-like proteins, members of the family of two-component regulatory systems, are present in a large number of proteobacteria in which they globally control gene expression mostly in a redox-responsive manner. The controlled target genes feature an enormous functional diversity. In Bradyrhizobium japonicum, the facultative root nodule symbiont of soybean, RegSR activate the transcription of the nitrogen fixation regulatory gene nifA, thus forming a RegSR-NifA cascade which is part of a complex regulatory network for gene regulation in response to changing oxygen concentrations. Whole-genome transcription profiling was performed here in order to assess the full regulatory scope of RegSR. The comparative analysis of wild-type and delta regR cells grown under oxic and microoxic conditions revealed that expression of almost 250 genes is dependent on RegR, a result that underscores the important contribution of RegR to oxygen- or redox-regulated gene expression in B. japonicum. Furthermore, transcription profiling of delta regR bacteroids compared with wild-type bacteroids revealed expression changes for about 1,200 genes in young and mature bacteroids. Incidentally, many of these were found to be induced in symbiosis when wild-type bacteroids were compared with free-living, culture-grown wild-type cells, and they appeared to encode diverse functions possibly related to symbiosis and nitrogen fixation. We demonstrated direct RegR-mediated control at promoter regions of several selected target genes by means of DNA binding experiments and in vitro transcription assays, which revealed six novel direct RegR target promoters.

Dissection of the Bradyrhizobium japonicum NifA+sigma54 regulon, and identification of a ferredoxin gene (fdxN) for symbiotic nitrogen fixation.

Hierarchically organized regulatory proteins form a complex network for expression control of symbiotic and accessory genes in the nitrogen-fixing soybean symbiont Bradyrhizobium japonicum. A genome-wide survey of regulatory interactions was made possible with the design of a custom-made gene chip. Here, we report the first use of the microarray in a comprehensive and complete characterization of the B. japonicum NifA+sigma(54) regulon which forms an important node in the entire network. Comparative transcript profiles of anaerobically grown wild-type, nifA, and rpoN (1/2) mutant cells were complemented with a position-specific frequency matrix-based search for NifA- and sigma(54)-binding sites plus a simple operon definition. One of the newly identified NifA+sigma(54)-dependent genes, fdxN, encodes a ferredoxin required for efficient symbiotic nitrogen fixation, which makes it a candidate for being a direct electron donor to nitrogenase. The fdxN gene has an unconventional, albeit functional sigma(54 )promoter with the dinucleotide GA instead of the consensus GC motif at position -12. A GC-containing mutant promoter and the atypical GA-containing promoter of the wild type were disparately activated. Expression analyses were also carried out with two other NifA+sigma(54) targets (ectC; ahpC). Incidentally, the tiling-like design of the microarray has helped to arrive at completely revised annotations of the ectC- and ahpC-upstream DNA regions, which are now compatible with promoter locations. Taken together, the approaches used here led to a substantial expansion of the NifA+sigma(54 )regulon size, culminating in a total of 65 genes for nitrogen fixation and diverse other processes.

Bradyrhizobium japonicum senses iron through the status of haem to regulate iron homeostasis and metabolism.

The Irr protein from the bacterium Bradyrhizobium japonicum is expressed under iron limitation to mediate iron control of haem biosynthesis. The regulatory input to Irr is the status of haem and its precursors iron and protoporphyrin at the site of haem synthesis. Here, we show that Irr controls the expression of iron transport genes and many other iron-regulated genes not directly involved in haem synthesis. Irr is both a positive and negative effector of gene expression, and in at least some cases the control is direct. Loss of normal iron responsiveness of those genes in an irr mutant, as well as a lower total cellular iron content, suggests that Irr is required for the correct perception of the cellular iron status. Degradation of Irr in iron replete cells requires haem. Accordingly, control of Irr-regulated genes by iron was aberrant in a haem-defective strain, and iron replete mutant cells behave as if they are iron-limited. In addition, the haem mutant had an abnormally high cellular iron content. The findings indicate that B. japonicum senses iron via the status of haem biosynthesis in an Irr-dependent manner to regulate iron homeostasis and metabolism.

The Iron control element, acting in positive and negative control of iron-regulated Bradyrhizobium japonicum genes, is a target for the Irr protein.

Bradyrhizobium japonicum, the nitrogen-fixing soybean symbiont, possesses a heme uptake system encoded by the gene cluster hmuVUT-hmuR-exbBD-tonB. Transcription of the divergently oriented hmuT and hmuR genes was previously found to be induced by iron limitation and to depend on a 21-bp promoter-upstream iron control element (ICE). Here, we show by deletion analysis that the full-length ICE is needed for this type of positive control. Additional genes associated with ICE-like motifs were identified in the B. japonicum genome, of which bll6680 and blr7895 code for bacterioferritin and rubrerythrin homologs, respectively. Transcription start site mapping revealed that their ICEs directly overlap with either the -10 promoter region or the transcription initiation site, suggesting an involvement of the ICE in negative control of both genes. Consistent with this inference was the observed down-regulation of both genes under iron limitation, which in the case of bll6680 was shown to require an intact ICE motif. Using a yeast one-hybrid system, we demonstrated in vivo interaction of the iron response regulator (Irr) with all three ICEs. Moreover, specific in vitro binding of purified Irr protein to the ICE motifs of bll6680 and blr7895 was shown in electrophoretic mobility shift experiments. A genome-wide survey for iron-regulated genes with a custom-made Affymetrix gene chip revealed 17 genes to be induced and 68 to be repressed under iron-replete conditions. Remarkably, ICE-like motifs are associated with a large subset of those B. japonicum genes. We propose the ICE as an important cis-acting element in B. japonicum which represents the DNA-binding site for the Irr protein and, depending on its location within promoter regions, is involved in positive or negative control of the associated iron-regulated genes.