Institute of Microbiology, Eidgenössische Technische Hochschule, Schmelzbergstrasse 7, CH-8092 Zürich, Switzerland
The expression of nitrogen fixation genes in Bradyrhizobium japonicum depends on two parallel regulatory cascades (see also article by H.M. Fischer et al., this volume). The first one involves the products of the fixL, fixJ and flxKj genes. The oxygen-responsive two-component regulatory system FixLJ induces the expression offixK2, which is also negatively regulated by its own product. The second cascade consists to the regS and regR genes and the fixR-nifA operon. This operon harbors two overlapping promoters PI and P2. The RegSR two-component system activates the transcription of the fixR-nifA operon from P2, while transcription from PI is activated by Nif A under microaerobic conditions.
We have investigated the modulation by oxygen of the induction by FixJ offixK.2 expression, the induction by NifA of a specific target gene, nifli, and the induction by RegSR of fixR-nifA. To this aim, we translationally fused the specific target genes with a lacZ reporter gene and chromosomally integrated the fusions. The fixR-nifA promoter fused with the reporter gene harbors a TG>CT mutation in the -24 region of the a54-/NifA-dependent PI promoter. This fusion can therefore not be activated by NifA and depends exclusively on RegSR for its induction. The fusion-carrying strains were grown under different atmospheric oxygen concentrations, namely 0.1, 0.5, 2, 5, and 21%. ß-Galactosidase activity was assayed after 24, 48 and 72 hours.
Expression of nifH-lacZ is strongly induced at < 0.5 % oxygen and almost undetectable at > 5% oxygen. This further documents the redox sensitivity of B. japonicum NifA. In contrast, expression of fixKi shows significant basal activity under aerobic conditions and a three-fold increase in the range of > 2% to < 5% oxygen. Expression of P2-fixR-lacZ was also found to be regulated by the oxygen conditions. RegSR-dependent expression shows basal level of activity under aerobic conditions and a four-fold increase in the range of > 0.5% to < 2% oxygen. In a regR' background, the P2 activity is almost completely abolished. In the regS~ background, similar results were obtained, except at < 0.5% oxygen, where low but significant induction was maintained.
We conclude that the expression of the fixR-nifA operon under the control of the P2 promoter is strictly RegR-dependent. RegS is required for maximal expression from P2. Activation by RegSR is modulated by oxygen. Since disruption of the regS gene does not completely abolish the microaerobic induction and taking into account the lack of an obvious structural basis for oxygen sensing in RegS, we propose that the RegSR system senses the cellular oxygen status indirectly via an alternative mechanism.
Disparate oxygen responsiveness of the FixLJ-, NifA-, and RegSR-regulated genes may result in ordered temporal activation of the symbiotic genes during invasion of the host plant by the bacteria and formation of a functional nodule.
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