Comprehensive assessment of the regulons controlled by the FixLJ-FixK2-FixK1 cascade in Bradyrhizobium 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 symbiosis-specific control.

FIG. 1.

Schematic representation of microoxically induced B. japonicum genes that are regulated by FixJ, FixK₂, or FixK₁. The numbers of genes controlled by these regulators are circled by ovals. The fixK₁ gene and its product are highlighted with a black arrow and white letters in a solid rectangle, respectively. Details are explained in the text. Note that although the model suggests a direct hierarchical organization, the existence of additional control levels in between FixLJ and FixK₂ and in between FixK₂ and FixK₁ cannot be excluded. Therefore, the regulation of target genes by FixJ, FixK₂, or FixK₁ may be direct or indirect. +, positive regulation; −, negative regulation.

FIG. 2.

Schematic representation of soybean bacteroid-induced genes that are controlled by FixJ or FixK₂. For details, see the text and the Fig. 1 legend. A set of 166 FixK₂-activated genes is not at the same time dependent on FixJ (indicated in white letters on a black background). An unknown regulatory signal might be sensed at the level of FixK₂ (directly or indirectly). dpi, days postinoculation; +, positively regulated; −, negatively regulated.

FIG. 3.

Strategies for the identification of direct FixK₂ targets. (A) Sequence logo for the FixK₂ binding site created with “WebLogo” (16). The consensus motif is based on the sequences listed in Table S1 in the supplemental material (see also Materials and Methods). (B) Venn diagram representing FixK₂-dependent mono-, di-, or polycistronic transcription units which contain putative FixK₂ boxes in their upstream promoter regions. The left circle contains 114 transcription units induced in free-living, microoxic culture, whereas the right circle contains 99 transcription units induced in bacteroids. For further details, see the text and Table 2.

FIG. 4.

In vitro transcription activation mediated by purified FixK₂. Supercoiled template plasmids comprising the promoter regions of target genes (shown at top) and a strong transcriptional terminator were used for multiple-round in vitro transcription assays with FixK₂ protein and RNA polymerase from B. japonicum cells. FixK₂ dimer concentrations were as follows: no protein (lane 1), 1.25 μM (lane 2), and 2.5 μM (lane 3). Transcripts synthesized in vitro in the presence of [α-³²P]UTP were separated on a 6% denaturing polyacrylamide gel and visualized by phosphorimager analysis of the dried gel. RNA size markers (M1 and M2) were generated as described earlier (49). The positions of the FixK₂-dependent transcripts are marked by arrows. Also shown is a FixK₂-independent reference transcript that is encoded on the vector portion of the template plasmids. nt, nucleotides.

FIG. 5.

FixK₂-dependent transcription from the divergently oriented fixK₁ and cycS promoters. Shown are a simplified map of the B. japonicum fixK₁ and cycS genes (A), a schematic of the relevant template (B), and the results of its use for in vitro transcription (C). The single FixK₂ binding site is symbolized by a dark box. The transcription start sites of fixK₁ and cycS are marked as “+1.” The stem-loops symbolize the transcription terminators. Transcripts from the template plasmid pRJ8871 were generated by multiple-round in vitro transcription using B. japonicum RNA polymerase and purified FixK₂ (no protein, lane 1; 1.25 μM, lane 2; and 2.5 μM, lane 3). The positions and sizes of the fixK₁ transcript, the cycS transcript, and the vector-encoded reference transcript are indicated. nt, nucleotides.

FIG. 6.

Mapping of the transcription start site of cycS. Total RNA was isolated from microoxic (0.5% O₂) or anoxic (nitrate respiring) cells of the wild-type (wt) and the ΔfixK₂ strain and used for primer extension experiments with two cycS-specific primers (results are shown for only one of the primers). The sequencing ladder on the left was generated with plasmid pRJ8886 and the same primer used for transcript mapping. The relevant nucleotide sequence of the cycS promoter is shown at the bottom. The putative −10 element is overscored, the FixK₂ box is highlighted by white letters on a black background, the transcription start site is marked by “+1,” and the start codon of the cycS gene is underlined.

FIG. 7.

Heme staining of soluble proteins expressed in nitrate-respiring cells. Wild-type B. japonicum (wt) and ΔcycS strains were grown in anoxic conditions (see Materials and Methods). Proteins were separated by SDS-polyacrylamide gel electrophoresis, transferred to nitrocellulose, and stained for having covalently bound heme. Cytochromes CycC (15 kDa) and CycA (12 kDa), which were identified previously, are specified on the left. Each lane was loaded with a sample of cell extract containing 80 μg soluble proteins.