Table Of ContentGenetics and Regulation of Nitrogen Fixation in Free-Living Bacteria
Nitrogen Fixation: Origins, Applications, and Research Progress
VOLUME 2
Genetics and Regulation
of Nitrogen Fixation
in Free-Living Bacteria
Edited by
Werner Klipp
Lehrstuhl für Biologie der Mikroorganismen,
Fakultät für Biologie, Ruhr-Universität Bochum, Germany
Bernd Masepohl
Lehrstuhl für Biologie der Mikroorganismen,
Fakultät für Biologie, Ruhr-Universität Bochum, Germany
John R. Gallon
Biochemistry Research Group,
School of Biological Sciences, University of Wales, Swansea, U.K.
and
William E. Newton
Department of Biochemistry,
Virginia Polytechnic Institute & State University, Blacksburg, U.S.A.
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v
TABLE OF CONTENTS
Series Preface . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ix
Preface. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xiii
List of Contributors. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . xvii
Dedication – Werner Klipp and John Gallon. . . . . . . . . . . . . . . . . . . . . . . . . . . xix
Chapter 1. Historical Perspective – Development of nif Genetics and
Regulation in Klebsiella pneumoniae R. Dixon . . . . . . . . . . . . . . . . . . . . . . . . . . 1
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1
2. The Early Years . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2
3. Defining the K. pneumoniae nif Genes . . . . . . . . . . . . . . . . . . . . . . . 4
4. The Recombinant DNA Era . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 5
5. nif Gene Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 6
6. Coda . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 16
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 17
Chapter 2. Genetics of Nitrogen Fixation and Related Aspects of
Metabolism in Species of Azotobacter: History and Current Status
C. Kennedy and P. Bishop . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27
1. Research on the Genus Azotobacter (1901-2003) . . . . . . . . . . . . . . 27
2. Application of the Tools of Genetics and Molecular Biology
in Species of Azotobacter . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29
3. The nif Genes encoding the Enzymes for Structure, Function, and
Biosynthesis of Mo-containing Nitrogenase . . . . . . . . . . . . . 33
4. Regulation of Expression of nif and Associated Genes by
Ammonium and O2 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35
5. Ancillary Properties of Azotobacter Species that Aid the
Efficiency of Nitrogen Fixation . . . . . . . . . . . . . . . . . . . . . . . 36
6. Discovery of Molybdenum-independent Nitrogenase
Systems in A. vinelandii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 38
7. Molybdenum-independent Nitrogenase systems in
other Azotobacter Species . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 45
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46
Chapter 3. Nitrogen Fixation in the Clostridia J.-S. Chen . . . . . . . . . . . . . . . . . 53
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 53
2. The Nitrogen-fixing Clostridia . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 54
3. Distinctive Features of the nif Genes of the Clostridia . . . . . . . . . . . 55
vi
4. Genes for Ammonia Assimilation . . . . . . . . . . . . . . . . . . . . . . . . . . 58
5. Regulation of Nitrogen Fixation and Ammonia Assimilation . . . . . 59
6. Concluding Remarks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 62
Chapter 4. Regulation of Nitrogen Fixation in Methanogenic Archaea
J. A. Leigh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
2. History and Background . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65
3. Transcriptional Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 66
4. Regulation of Nitrogenase Activity . . . . . . . . . . . . . . . . . . . . . . . . . 69
5. Summary . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 71
Chapter 5. Nitrogen Fixation in Heterocyst-Forming Cyanobacteria
T. Thiel . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73
2. Structure of Heterocysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74
3. Nitrogenase Genes . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78
4. Heterocyst Metabolism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 81
5. Genes Important for Heterocyst Formation . . . . . . . . . . . . . . . . . . . 88
6. Heterocyst Pattern Formation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95
7. Regulation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101
Chapter 6. N2 Fixation by Non-Heterocystous Cyanobacteria J. R. Gallon . . . 111
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 111
2. Non-heterocystous Cyanobacteria . . . . . . . . . . . . . . . . . . . . . . . . . . 113
3. Patterns of N2 Fixation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 126
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 133
Chapter 7. Nitrogen Fixation in the Photosynthetic Purple Bacterium
Rhodobacter capsulatus B. Masepohl, T. Drepper and W. Klipp . . . . . . . . . . . 141
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 141
2. Organization of Nitrogen-fixing Genes . . . . . . . . . . . . . . . . . . . . . . 142
3. The Nitrogen-fixation Regulon of R. capsulatus . . . . . . . . . . . . . . . 155
4. Ammonium Control of Synthesis and Activity of
both Nitrogenases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 158
5. Environmental Factors Controlling Nitrogen Fixation . . . . . . . . . . 161
6. Linkage of Nitrogen Fixation, Photosynthesis, and
Carbon Dioxide Assimilation . . . . . . . . . . . . . . . . . . . . . . . . 162
7. Nitrogen Fixation in other Photosynthetic Purple Bacteria . . . . . . . 163
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 165
vii
Chapter 8. Post-translational Regulation of Nitrogenase in
Photosynthetic Bacteria S. Nordlund and P. W. Ludden . . . . . . . . . . . . . . . . . . 175
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 175
2. Discovery of Nitrogen Fixation by Photosynthetic Bacteria . . . . . . 176
3. In vitro Studies of Nitrogenase in Photosynthetic Bacteria . . . . . . . 177
4. The Protein Era . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 178
5. Evidence for the Drat/Drag System in other Organisms . . . . . . . . . 182
6. Other ADP-Ribosylations . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 183
7. Genetics of the Drag/Drat System . . . . . . . . . . . . . . . . . . . . . . . . . . 185
8. Signal Transduction to Drat and Drat . . . . . . . . . . . . . . . . . . . . . . . 185
9. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 189
Acknowledgement . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 190
Chapter 9. Regulation of Nitrogen Fixation in Free-Living Diazotrophs
M. J. Merrick . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 197
2. General Nitrogen Control Systems . . . . . . . . . . . . . . . . . . . . . . . . . 198
3. nif-specific Nitrogen Control . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 204
4. Nitrogen Control of Nitrogenase Activity . . . . . . . . . . . . . . . . . . . . 211
5. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 214
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 215
Chapter 10. Molybdenum Uptake and Homeostasis R. N. Pau . . . . . . . . . . . . 225
1. Molybdenum Outside Cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 225
2. Transport . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230
3. Cytoplasmic Molybdate-binding Proteins . . . . . . . . . . . . . . . . . . . . 236
4. The ATP-binding Protein of the Molybdate Transporter . . . . . . . . 242
5. The Regulatory Protein, ModE . . . . . . . . . . . . . . . . . . . . . . . . . . . . 243
6. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 249
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 250
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 251
Chapter 11. Electron Transport to Nitrogenase: Diverse Routes for a
Common Destination K. Saeki . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
1. Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 257
2. Direct Electron Donors to the Fe Protein of Nitrogenase . . . . . . . . 259
3. Reduction of Electron-donor Proteins for the Fe Protein . . . . . . . . 269
4. Conclusions . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
Acknowledgements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 280
References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 281
Chapter 12. Prospects J. R. Gallon and B. Masepohl . . . . . . . . . . . . . . . . . . . . 291
Subject Index. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 297
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ix
SERIES PREFACE
Nitrogen Fixation: Origins, Applications, and Research Progress
Nitrogen fixation, along with photosynthesis as the energy supplier, is the basis of
all life on Earth (and maybe elsewhere too!). Nitrogen fixation provides the basic
component, fixed nitrogen as ammonia, of two major groups of macromolecules,
namely nucleic acids and proteins. Fixed nitrogen is required for the N-containing
heterocycles (or bases) that constitute the essential coding entities of
deoxyribonucleic acids (DNA) and ribonucleic acids (RNA), which are responsible
for the high-fidelity storage and transfer of genetic information, respectively. It is
also required for the amino-acid residues of the proteins, which are encoded by the
DNA and that actually do the work in living cells. At the turn of the millennium, it
seemed to me that now was as good a time as any (and maybe better than most) to
look back, particularly over the last 100 years or so, and ponder just what had been
achieved. What is the state of our knowledge of nitrogen fixation, both biological
and abiological? How has this knowledge been used and what are its impacts on
humanity?
In an attempt to answer these questions and to capture the essence of our
current knowledge, I devised a seven-volume series, which was designed to cover
all aspects of nitrogen-fixation research. I then approached my long-time contact at
Kluwer Academic Publishers, Ad Plaizier, with the idea. I had worked with Ad for
many years on the publication of the Proceedings of most of the International
Congresses on Nitrogen Fixation. My personal belief is that congresses, symposia,
and workshops must not be closed shops and that those of us unable to attend
should have access to the material presented. My solution is to capture the material
in print in the form of proceedings. So it was quite natural for me to turn to the
printed word for this detailed review of nitrogen fixation. Ad’s immediate
affirmation of the project encouraged me to share my initial design with many of
my current co-editors and, with their assistance, to develop the detailed contents of
each of the seven volumes and to enlist prospective authors for each chapter.
There are many ways in which the subject matter could be divided. Our
decision was to break it down as follows: nitrogenases, commercial processes, and
relevant chemical models; genetics and regulation; genomes and genomics;
associative, endophytic, and cyanobacterial systems; actinorhizal associations;
leguminous symbioses; and agriculture, forestry, ecology, and the environment. I
feel very fortunate to have been able to recruit some outstanding researchers as co-
editors for this project. My co-editors were Mike Dilworth, Claudine Elmerich,
John Gallon, Euan James, Werner Klipp, Bernd Masepohl, Rafael Palacios,
Katharina Pawlowski, Ray Richards, Barry Smith, Janet Sprent, and Dietrich
Werner. They worked very hard and ably and were most willing to keep the
volumes moving along reasonably close to our initial timetable. All have been a
pleasure to work with and I thank them all for their support and unflagging interest.
