Regulatory Circuits Controlling Both Nitrogenases In Rhodobacter Capsulatus

S. Groß1, T. Drepper1, B. Masepohl1, T. Rehmann1, A.F. Yakunin2, P.C. Hallenbeck2,

W. Klipp1

1 Ruhr-Universität Bochum, LS Biologie der Mikroorganismen, D-44780 Bochum, Germany

Université de Montréal, Dépt. de Microbiologie et Immunologie, Montréal, Canada

The phototrophic purple bacterium R. capsulatus is able to reduce atmospheric dinitrogen to ammonia either via a molybdenum («¿/-encoded) or an alternative heterometal-free (a«/-encoded) nitrogenase. Expression and activity of both nitrogenase systems is controlled by ammonium on at least three different levels. At the first level, transcription of the nifAl, nifA2 and anfA genes -coding for the transcriptional activators of the other nif and anf genes - is controlled by the Ntr system in dependence on ammonium availability. As in other bacteria the R. capsulatus Ntr system consists of the two-component regulatory system NtrB/NtrC, two Pn-like proteins (GlnB and GlnK) and the Pn-modifying enzyme GlnD. In addition, two ammonium transporters (AmtB and AmtY) might play a role in ammonium-dependent signal transduction. In a glnK mutant (constitutively expressing amtB) NtrC-dependent gene expression is derepressed in the presence of ammonium, whereas in an amtY mutant NtrC-dependent gene activation is abolished in the absence of ammonium. Both the glnK-amtB operon and amtY are part of the Ntr regulon. In contrast to most NtrC-dependent genes, which are constitutively expressed in an R. capsulatus glnB mutant, glnK expression is still repressed by ammonium. However, in a glnB/glnK double mutant ammonium suppression of glnK expression is relieved.

Besides NtrB/NtrC the R. capsulatus ntr gene region codes for a second two-component system (NtrY/NtrX). The sensor kinase NtrY contains two membrane spanning elements and includes a PAS domain characteristic for redox sensing proteins. Although NtrB effectively phosphorylates and thereby activates NtrC, an ntrB mutant still exhibits nitrogenase activity albeit at a reduced level compared to the wild type. In contrast, an ntrB/ntrY double mutant no longer synthesizes nitrogenase suggesting that NtrB and NtrY can (partially) substitute for each other. A glnK-ntrB double mutant constitutively expresses both NifA and NifH, and exhibits nitrogenase activity even in the presence of ammonium. Therefore the double mutant mimics a glnB-glnK double mutant. Complementation of the glnK-ntrB double mutant with either glnK or glnB abolishes NifH synthesis due to GlnK-/GlnB-dependent ammonium regulation at the level of NifA activity.

At the second level of control, the activity of the transcriptional activators NifAl, NifA2 and AnfA is inhibited in an NtrC-independent manner. This post-translational ammonium control of NifA activity is partially released in the absence of GlnK, and completely abolished in a glnB-glnK double mutant, whereas AnfA activity is still repressed by ammonium in the glnB-glnK mutant background.

At the third level of regulation, both nitrogenase reductases (NifH and AnfH) are controlled by DraT/DraG-mediated reversible ADP-ribosylation. In the presence of ammonium, oxygen or in the dark "switch-off' occurs. Both GlnB and GlnK as well as AmtB are involved in ammonium control of the DraT/DraG system. While GlnB might affect the ADP-ribosylation reaction in response to increasing ammonium concentrations, GlnK seems to be involved in the demodification of nitrogenase under N2-fixing conditions when glnK expression is highly induced. An amtB mutant is unable to carry out the short-term ADP-ribosylation.


F. D'Aoust1, H. Ip2, A.A. Begum1, T.C. Charles2, D.L. Smith3, B.T. Driscoll1

3Dept Plant Sci., McGill U, Ste-Anne-de-Bellevue, Quebec, Canada

2Dept of Biol., U Waterloo, Waterloo, Ontario, Canada

The establishment of symbiosis between soybeans [Glycine max (L.) Merr.] and Bradyrhizobium japonicum has been shown to be elongated by suboptimal soil temperatures, and this has a negative impact on yield. The delays appear to be partially due to reduced plant-microbe signaling. B. japonicum mutants with increased sensitivity to genistein were isolated by UV mutagenesis and transformed with plasmid pZB32 (carrying a nodYr.lacZ gene fusion). The mutants were found to have higher nodY expression than the wild type in the presence of genistein. The increased sensitivity of all mutants to genistein was more apparent under suboptimal inducer concentration (0.1|xM) and/or temperature (15°C). The kinetics of nodY gene induction were determined for five strains (Bj30050, 53, 56, 57, 58) under different temperature and inducer conditions. These five strains were also found to produce more lipochito-oligosaccharide than the wild type, at both 25°C and 15°C. Three of the ten mutant strains (including Bj30056 and 57) were unable to fix nitrogen with soybeans grown at optimal temperatures. We are continuing the investigation of nod gene expression in these mutants, including the effects of cell-density, by both (3-galactosidase and semi-quantitative mRNA analyses.


W.G. Reeve1, R.P. Tiwari1, J. Castelli1, M.J. Dilworth1, A.R. Glenn2, J.G. Howieson1

'Centre for Rhizobium Studies, School Biological Sciences and Biotechnology, Murdoch

University, Murdoch, WA 6150, Australia 20ffice of the Pro Vice-Chancellor (Research), University of Tasmania, Hobart, Australia

1. Introduction

One important environmental stress encountered by root nodule bacteria is that of unfavorably low pH. The isolation of acid-tolerant Sinorhizobium meliloti strains from Sardinia and Greece (Howieson, Ewing 1986) made possible the establishment of medic pastures on over 400,000 ha of acid soils in Western Australia. An understanding of the genetics of pH response is being sought to identify the mechanisms required for successful persistence of these acid-tolerant strains of S. meliloti. One approach has been to create gusA fusions (Reeve et al. 1998, 1999) to identify genes that are low-pH activated.

2. Results and Discussion

Two such genes are phrR (pH regulated regulator) and IpiA (low pH inducible gene). The expression of phrR responds to copper, zinc, H2O2 and ethanol besides low pH. The IpiA gene encodes a transmembrane protein similar to hypothetical membrane proteins from S. meliloti, Synechocystis sp. and Escherichia coli. The fsrR (fused sensor-regulator) gene upstream to IpiA encodes a protein similar to Methanobacterium thermoautotrophicum sensory transduction regulatory proteins.

The IpiA mutant is no more sensitive to stress than the wild-type. Transcriptional activation of IpiA is specific to low pH; expression increasing at least 20-fold between pH 7.0 and 5.7. High concentrations of calcium reduced expression significantly at low pH. We have previously shown that high concentrations of calcium promote cell growth and survival at low pH (Reeve et al. 1993); the influence of calcium on the expression of a gene at low pH is an effect observed in S. meliloti at the genetic level for the first time.

Mobilization of a plasmid-borne IpiA-gusA fusion into different genetic backgrounds revealed that the low pH-specific activation was not regulated by ActR (a regulator essential for low pH tolerance of Sinorhizobium). The low-pH responsive promoter has been pinpointed within a 372 bp region upstream to the IpiA start codon. The IpiA gene is not required for symbiotic effectiveness although it is expressed in the nodule.

3. References

Howieson JG, Ewing MA (1986) Austral. J. Agric. Res. 37, 55-64 Reeve WG et al. (1993) Soil Biol. Biochem. 25, 581-586 Reeve WG et al. (1998) Microbiol. 144, 3335-3342 Reeve WG et al. (1999) Microbiol. 145,1507-1516

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