Disparate Oxygen Responsiveness of the Regulatory Cascades

Using lacZ reporter fusions, we have compared the redox responsiveness of a FixLJ-dependent gene (fixKi) with that of a NifA-dependent gene (nifH\ see also M.A. Sciotti et al. this volume). As depicted in Figure 2, FixLJ-mediated activation was observed at relatively high oxygen concentrations (<5%) whereas significant activation by NifA is observed only at oxygen concentrations of <0.5%. Induction factors were larger for the NifA- than for the FixLJ-controlled fusion because the latter was expressed at a significant level even under normal aerobic conditions. Interestingly, fixKx-lacZ expression was rather low at <0.5% oxygen, which could be a consequence of the negative autoregulation of fixKj (Nellen-Anthamatten et al. 1998). It is likely that these findings have physiological implications. During the early stages of symbiosis, i.e. infection and root nodule formation, the products of the FixLJ-FixK2 regulon enable the invading bacteria to adapt their respiratory metabolism to the decreasing oxygen supply. Synthesis of the oxygen-labile nitrogen fixation apparatus under the control of the RegSR-NifA cascade is retarded until nodule formation and leghemoglobin synthesis have progressed to the point where free oxygen levels are sufficiently low to allow proper functioning of the nitrogenase complex.

4. RegSR: A Global Regulatory System

RegS and RegR exhibit the typical structural features of two-component regulatory proteins (Bauer et al. 1998). Biochemical studies with purified proteins demonstrated that a soluble variant of RegS autophosphorylates and can donate the phosphoryl group to RegR (Emmerich et al. 1999). In addition, RegS is able to dephosphorylate RegR-phosphate. Binding of RegR to the faR-nifA upstream element is strongly stimulated by phosphorylation. A minimal RegR-binding site was defined by performing DNA binding studies with mutated derivatives of the flxR-nifA upstream activator sequence and an in vitro binding-site selection assay (SELEX; Emmerich et al. 2000a). The 'RegR box' comprises 11 critical nucleotides within a 15-bp imperfect inverted repeat.

RegSR-homologous systems are present in Rhodobacter capsulatus (RegBA), R. sphaeroides (PrrBA), Synechocystis sp. strain PCC 6803 (RppBA) and Sinorhizobium meliloti (ActSR) where they are involved in the control of such diverse processes as photosynthesis, aerobic respiration, fixation of CO2 and N2, H2 oxidation, regulation of Ci metabolism and acid tolerance (for references, see Swem et al. 2001; Emmerich et al. 2000b; Fenner et al. 2000). The functional similarity of RegR and RegA was substantiated by heterologous complementation studies. Specifically, regA was able to restore symbiotic nitrogen fixation of a B. japonicum regR mutant, and RegR activated in R. capsulatus the expression of the photosynthesis operon puc, normally a target for RegA (Emmerich et al. 2000b). These results are in good agreement with the striking similarity of the RegR box with a consensus DNA-binding site for RegA that was derived from footprinting studies with a number of RegA target promoters (Swem et al. 2001). Thus, the B. japonicum RegSR systems belong to a growing class of global regulatory systems that control diverse processes involved in redox metabolism.

5. Which Other Functions Has the RegSR System in B. japonicum"!

On the basis of its diverse regulatory functions the RegBA regulatory system of R. capsulatus is believed to play a key role in the maintenance of a balanced cellular redox state (see above; Swem et al. 2001). For example, the reductive pentose phosphate pathway (Calvin-Benson-Bassham pathway) which can serve as an electron sink during photoheterotrophic growth of R. capsulatus belongs to the RegBA regulon (Vichivanives et al. 2000; Tichi, Tabita 2000). With this in mind, we have investigated whether the ebb operon of B. japonicum encoding the enzymes of the Calvin-Benson-Bassham pathway belongs to the RegSR regulon. Unlike the results reported for

Figure 2. Disparate redox responsiveness of the B. japonicum regulatory cascades. Cultures of strains harboring the indicated translational lacZ reporter fiision-chromosomally integrated were grown at 30°C in Erlenmeyer flasks (21% oxygen) or in serum bottles that were flushed twice daily with oxygen-nitrogen gas mixtures containing the specified oxygen concentration. p-Galactosidase activity was assayed after 48 h of growth. Values are indicated as % of the maximum that each reporter strain reached.

R. capsulatus, we found no effect of a regR null mutation on the expression of the B. japonicum cbbFPTALSXE operon when assayed in a strain with an intact ebb operon (H.M. Fischer et al., unpublished). Interestingly, we observed a 5-10-fold increase of ebb expression in a strain carrying a chromosomal cbbP-lacZ fusion which reduces transcription of the distal genes, and this increase was dependent on RegR. We conclude that ebb expression is negatively autoregulated by a mechanism that involves RegR. It is likely that RegR exerts its effect through CbbR, the LysR-type activator required for ebb expression.

B. japonicum regR null mutants are symbiotically defect (Bauer et al. 1998). We were interested to find out whether this phenotype is simply a consequence of the altered fixR-nifA expression level or whether regR has additional target genes required for symbiotic nitrogen fixation. To this end, we constructed a strain lacking regR and expressing nifA from the promoter of a kanamycin resistance cassette (aphIT) inserted upstream of nifA in the fixR gene (fixR, whose function is not known, was shown previously to be dispensable for symbiotic nitrogen fixation; Fischer et al. 1986). To monitor NifA activity, a nifH-lacZ fusion was integrated into the same background. The symbiotic properties of these strains were determined in a plant infection test and nifH-lacZ expression levels were assayed in microaerobically grown cultures (Table 1).

Table 1. Symbiotic properties and microaerobic NifA activity (monitored as nifH-lacZ expression) of a B. japonicum regR mutant harboring a nifA expression cassette (aphll::nifA).


Relevant genotype

Fix phenotype nifH-lacZ expression



+ 100%


nifH-lacZ, aphII::nifA

+ 184 %


nifH-lacZ, AregR

3 %


nifH-lacZ, AregR, aphII::nifA

<1 %

Forced expression of nifA was not sufficient to correct the symbiotic defect of the regR deletion mutant, and, most notably, no NifA activity could be detected in this strain. This result suggests that RegR is not only required for proper expression levels of nifA but also for NifA activity. We speculate that regR mutants have altered cellular redox conditions which may interfere with NifA activity. The molecular basis for the link between RegR and the formation of active NifA is currently not known. Also, it remains open whether RegR controls additional functions which are essential for symbiosis but independent of NifA.

6. References

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