No abstract

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.

Symbiotic N 2 fixation in Bradyrhizobium japonicum is controlled by a complex transcription factor network. Part of it is a hierarchically arranged cascade in which the two-component regulatory system FixLJ, in response to a moderate decrease in oxygen concentration, activates the fixK 2 gene. The FixK 2 protein then activates not only a number of genes essential for microoxic respiration in symbiosis (fixNOQP and fixGHIS) but also further regulatory genes (rpoN 1 , nnrR, and fixK 1 ). The results of transcriptome analyses described here have led to a comprehensive and expanded definition of the FixJ, FixK 2 , and FixK 1 regulons, which, respectively, consist of 26, 204, and 29 genes specifically regulated in microoxically grown cells. Most of these genes are subject to positive control. Particular attention was addressed to the FixK 2 -dependent genes, which included a bioinformatics search for putative FixK 2 binding sites on DNA (FixK 2 boxes). Using an in vitro transcription assay with RNA polymerase holoenzyme and purified FixK 2 as the activator, we validated as direct targets eight new genes. Interestingly, the adjacent but divergently oriented fixK 1 and cycS genes shared the same FixK 2 box for the activation of transcription in both directions. This recognition site may also be a direct target for the FixK 1 protein, because activation of the cycS promoter required an intact fixK 1 gene and either microoxic or anoxic, denitrifying conditions. We present evidence that cycS codes for a c-type cytochrome which is important, but not essential, for nitrate respiration. Two other, unexpected results emerged from this study: (i) specifically FixK 1 seemed to exert a negative control on genes that are normally activated by the N 2 fixation-specific transcription factor NifA, and (ii) a larger number of genes are expressed in a FixK 2 -dependent manner in endosymbiotic bacteroids than in culture-grown cells, pointing to a possible symbiosisspecific control.

SummaryThe 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.

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.