Proteinprotein Interactions Of Regulatory And Other Gene Products Involved In Nitrogen Fixation In Rhodobacter Capsulatus Identification Of New anf GENES

B. Masepohl, P. Dreiskemper, T. Drepper, S. Groß, N. Isakovic, A. Pawlowski, K. Raabe,

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

The photosynthetic purple bacterium Rhodobacter capsulatus can fix atmospheric dinitrogen either via a molybdenum («//-encoded) or an alternative heterometal-free (¿/«/-encoded) nitrogenase. Synthesis and activity of both nitrogenases is tightly 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 «//and anf genes - is controlled by the Ntr system consisting of the two-component regulatory system NtrB/NtrC and the signal transduction protein GlnB (PII). In addition, expression of glnK-amtB and amtY- coding for a PII-paralog and two putative (methyl-ammonium transporters - is also under control of the Ntr system. At the second level of regulation, activity of 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 GlnB and GlnK as well as AmtB are involved in ammonium control of the DraT/DraG system, which mediates reversible ADP-ribosylation of both nitrogenase reductases in response to changes in ammonium availability. Remarkably, in a glnB-/glnK double mutant ammonium control of the molybdenum (but not of the alternative) nitrogenase is completely relieved, leading to synthesis of active nitrogenase in the presence of high concentrations of ammonium.

To identify proteins interacting with the above-mentioned regulatory proteins, yeast two-hybrid (Y2H) studies were carried out. For this purpose, an R. capsulatus DNA library was constructed using a low-copy GAL4-based Y2H system. This library covered the R. capsulatus genome several-fold with in-frame fusions to the activating domain of the prey plasmid every 12 bp along the coding strand. To test the library, GlnB was used as a bait. As expected, the by far strongest interaction was found for NtrB confirming the current regulatory model. Weaker interactions were found for NifA2, DraT, and the ATP-dependent helicase PcrA. Using GlnK as a bait, interaction with the Ras-like protein Era could be demonstrated, whereas no interaction was found with NtrB. However, the roles of the interactions between GlnB/PcrA and GlnK/Era, respectively, remain to be elucidated. In addition, analysis of defined protein pairs demonstrated interaction of GlnB with NifAl, GlnK, and GlnB itself. Furthermore, protein interactions were found for NifAl-NifAl, NifAl-NifA2, NifAl-GlnK, NifA2-NifA2, and NifA2-GlnK. These results suggest that (i) NifAl and NifA2 may form both homodimers as well as heterodimers, and (ii) post-translational regulation of NifA activity in response to ammonium is mediated via direct interaction of the transcriptional activator with the PH-like signal transduction proteins.

Using Anfl (encoded by a gene located downstream and co-transcribed with anfHDGK) as a bait, two new putative cytoplasmatic Anf proteins were identified, called Orß49 and 0rfl065 (TetR). The role of Orf349 and 0rfl065 as Anf proteins was corroborated by analyses of in vivo nitrogenase activity of corresponding mutant strains. Both orß49 and orfl065 mutants did not reduce acetylene via the alternative nitrogenase, whereas the activity of the molybdenum nitrogenase was not affected. Surprisingly, the orf349 mutant was still able to grow diazotrophically with the Anf system albeit at a reduced level compared to the parental strain.

GLYOXYLATE CYCLE IN R. LEGUMINOSARUM: CHARACTERIZATION OF A MUTANT CARRYING A Tn5B22 INSERTION DISRUPTING THE MALATE SYNTHASE-ENCODING GENE

A. Garcia-de los Santos1, A. Morales1,1. Hernandez-Lucas1, C.Y. Yost2, S. Brom1, M.F. Hynes2

'CIFN/UNAM, Apdo. Postal 565-A, Cuernavaca, Mor. México

2Dept of Biological Sciences, University of Calgary, Calgary, AB, Canada

1. Introduction

We have been interested in isolating rhizobial genes which are turned on by environmental signals. Sugars commonly found as components of plant cell walls may be among these molecular signals. Oresnik et al. (1998) reported the isolation of a plant-inducible rhamnose locus involved in competition for nodulation. These data encouraged us to search for other genes whose expression is induced by sugars that form part of plant polysaccharides.

2. Procedures

We screened a collection of 3000 R. leguminosarum mutants carrying random insertions of the lacZ transposon Tn5B22 for induction by arabinose.

3. Results and Discussion

We identified a chromosomal locus inducible by arabinose, a component of plant polysaccharides. BLASTX analysis of the ORF disrupted by Tn5B22 showed high similarity to different bacterial malate synthase G (MSG) enzymes. MSG participates in the metabolism of glyoxylate, by catalyzing the irreversible aldol condensation of glyoxylate and acetyl CoA to form malate. The similarity with MSG sequences is reinforced by the presence of 21 residues which are strictly conserved in the 23 MSG sequences previously published (Howard et al. 2000). Furthermore, a lacZ fusion in the putative malate synthase encoding gene was also induced by glycolate, a compound which activates the transcription of MSG in E. coli (Molina et al. 1994). Pea plants inoculated with the MSG mutant were shorter than those inoculated with the wt strain and their leaves presented a marked chlorosis. The nodules induced by the mutant were white, indicating that this mutation has a negative effect on nitrogen fixation.

4. References

Howard BR et al. (2000) Biochem. 39, 3156-3168 Molina I et al. (1994) Eur. J. Biochem. 224, 541-548 Oresnik IJ et al. (1998) MPMI11, 1175-1185

5. Acknowledgements

We thank CONACYT and DGAPA for supporting the postdoctoral stay of Alejandro García de los Santos in the laboratory of Michael Hynes at the University of Calgary.

IDENTIFICATION OF GENES REGULATED BY THE ALTERNATIVE SIGMA FACTOR a54 IN RHIZOBIUMLEGUMINOSARUM BV. VICIAE VF39SM

S.R.D. Clark, M.L. Tabche, B.T. Steven, A. Cieslak, M.F. Hynes Dept of Biological Sciences, University of Calgary, Calgary, AB, Canada

1. Introduction a54 is an 'alternative' sigma factor encoded by rpoN\ recognized promoters display a highly conserved consensus -12/-24 sequence, 5'- CTGGCAC-N5-TTGCA -3' (Beynon et al. 1983). (independent systems in Rhizobium include dicarboxylic acid transport and nitrogen regulation, and the fixGHIS,fixNOQP, and fnrN genes of Rhizobium leguminosarum bv. viciae VF39SM are dependent on a54 for full induction under microaerobic conditions (Clark et al. 2001). a54 can bind DNA in the absence of core polymerase, allowing the sigma factor to be involved in negative as well as positive regulation, depending on the spacing of promoter structures (Merrick 1993).

2. Procedure

The strategy used to identify putative targets for cy54 is similar to that used by Bittinger et al. (2000). A VF39SM rpoNv.QSp mutant, RM046, was mutagenized with the transposon, Tn5-B30 (Simon et al. 1989). This element carries a promoterless neomycin gene, generating a pool of random reporter fusions. Each mutant was replica plated, and one copy received pC028 by conjugation. This plasmid carries the wild type rpoN gene, complementing the rpoN::QSp allele. Both the complemented and uncomplemented forms of each mutant were screened on TY containing increasing concentrations of neomycin; differences in resistance levels were predictive for the nature of each insert under the conditions tested (i.e. Nmr only in the presence of wt rpoN suggests (independence). Inserts cloned from total genomic DNA were identified by homology.

3. Results and Discussion

Of approximately 2400 mutants screened so far, 54 display putative, positive regulation and 21 display putative, negative regulation. Sequences obtained show homology to transposases including ISRm3 and ISRle39 (Rochepeau et al. 1997); leucine-response protein transcriptional regulators; and nuclease inhibitors. One of the transposases, as well as the LRP-type regulator, carry the insert in the opposite orientation to the host genes. Upstream flanking sequences will be obtained using a primer to the Nmr gene.

4. References

Beynon J, Cannon M, Buchanan-Wollaston V, Cannon F (1983) Cell 34, 665-671

Bittinger MA, Handelsman J (2000) J. Bacteriol. 182,1706-1713

Clark SRD, Oresnik IJ, Hynes MF (2001) Mol. Gen. Genet. 264, 623-633

Merrick MJ (1993) Mol. Microbiol. 10, 903-909

Simon R, Quandt J, Klipp W (1989) Gene 1989 80, 161-169

Rochepeau P, Selinger LB, Hynes MF (1997) Mol. Gen. Genet. 256, 387-396

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