Comprehensive Assessment of the Regulons Controlled by the FixLJ-FixK₂-FixK₁Cascade inBradyrhizobium japonicum

Abstract

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.

Posttranslational control of transcription factor FixK 2 , a key regulator for the Bradyrhizobium japonicum –soybean symbiosis

“…S1, lanes 1-3). This result was unexpected, because previous studies showed a clear induction of fixK 2 gene expression in response to decreased oxygen concentrations, although a basal level of fixK 2 expression had been noticed even in aerobiosis (16,17,20 H2O2 sensitivity of the B. japonicum WT and the C183A-FixK2-expressing mutant strain 8854. Soft agar (0.9%) plates with PSY medium were inoculated with the indicated B. japonicum strains.…”

“…Signal intensity detection, normalization and statistical analysis were done as already established (17,45). Only those probe sets that were called ''present'' or ''marginal'' in Ն67% of the replicates of each experiment were considered for further analysis.…”

“…Recently, the FixK 2 regulon was unraveled by using a transcriptomics approach (17). Also, DNA binding site predictions together with a FixK 2 -dependent in vitro transcription assay has identified 11 direct target genes or operons for FixK 2 .…”

Analysis of Two Polyhydroxyalkanoate Synthases in Bradyrhizobium japonicum USDA 110

“…Therefore, if the early increase in phaC2 transcript levels observed in this study results in a later increase in PhaC2 protein levels, this paralog would soon occupy the limited PhaC sites on the granule surface, thus disturbing the access of PhaC1, which might lead to a diminished biosynthetic activity, or it may preclude the access of a PHA depolymerase enzyme. The interplay between PhaC1 and PhaC2 might be relevant in nodules, where phaC2 expression is stimulated 6-fold by FixK2 (a transcription factor enhancer that is stimulated by low O 2 levels) (10). Surprisingly, phaC3, which was poorly expressed in the wild type, was upregulated at logarithmic growth phase in the ⌬phaC2 mutant.…”

Versatile Vectors for Efficient Mutagenesis of Bradyrhizobium diazoefficiens and Other Alphaproteobacteria

“…Escherichia coli strains grew in LB medium (18) at 37°C with the following antibiotics, added at the indicated final concentrations: ampicillin at 200 g/ml in solid medium and 100 g/ml in liquid medium, gentamicin at 10 g/ml, hygromycin B at 200 g/ml, kanamycin at 30 g/ml, streptomycin at 50 g/ml, and tetracycline at 10 g/ml. We cultivated B. diazoefficiens strains at 30°C in PSY medium (19) supplemented with 0.1% L-(ϩ)-arabinose and the following antibiotics at the indicated concentrations: chloramphenicol at 20 g/ml (for counterselection of E. coli), hygromycin B at 200 g/ml, kanamycin at 100 g/ml, spectinomycin at 100 g/ml, and tetracycline at 50 g/ml (solid medium) and 25 g/ml (liquid medium). Rhodopseudomonas palustris strains grew at 30°C in modified YPMS medium (20) (which has the same trace elements that exist in PSY medium) containing gentamicin where appropriate (800 g/ml for initial selection and 400 g/ml for routine cultures).…”