Progress 07/01/03 to 06/30/07
Outputs
OUTPUTS: Cytochromes are necessary for ATP synthesis that supports nitrogen fixation. Cytochromes contain heme as a prosthetic group, which is an iron-containing tetrapyrrole. B. japonicum can acquire the heme precursor ALA from the plant host, and iron must be obtained from the environment. We identified a B. japonicum mutant defective in ALA transport that is likely mutated in a gene encoding the transporter subunit OppB. This finding is important because there are many oligopeptide permease homologs in the B. japonicum genome, and therefore it would have been difficult to find it based on homology searches. In a screen for ALA transport mutants, we unexpectedly picked up a fur mutant, which regulates iron metabolism in bacteria. This is a gene that we had previously found in a different context. We found that the Fur protein from B. japonicum (BjFur) recognizes a DNA sequence that differs from the so-called Fur box consensus, and is therefore novel in its DNA recognition. We found
that conserved amino acid residues proposed to be involved in metal-binding are not required for metal-dependent DNA binding or transcriptional repression in vitro. The data suggest that Fur is functionally more diverse in bacteria than has been assumed from studying model systems. We are interested in finding the link between Fur function and ALA transport, and in better understanding protein function. We found that that an operon encoding the cytochrome bc1 complex is regulated by iron in B. japonicum. Furthermore, this iron-dependent control is mediated by heme by a post-translational mechanism. Our model argues that low iron conditions result in less heme available for cytochrome formation. Thus, cytochrome polypeptide is not stable in the absence of heme adn will degrade. Since cytochromes b and c1 are only stable as a complex, heme deficiency that destabilizes one protein may cause break down of the entire complex. We know that cytochrome c1 is destabilized in the absence of
heme, and we are investigating the stability of b-type cytochromes.
PARTICIPANTS: Dr. Tao Gao was a Ph.D. student in my laboratory working on the project. He was trained in molecular biology. He is currently a postdoctoral fellow at the University of Pennsylvania. Dr. Yali Friedman was a Ph.D student in my laboratory working on the project. He was trained in molecular biology. He is currently Chief Knowledge Officer at New Economy Strategies, Washington, DC. Dr. Elena Fabiano is a collaborator at the Instituto Investigaciones Biochimica Clemente Estable in Montevideo Uruguay. This collaboration led to a Fogarty Award from the NIH.
Impacts
We expect to acquire a better understanding of how B. japonicum acquires necessary components from the environment for heme biosynthesis, and how these processes are regulated. Understanding the mechanisms and control of biosynthetic and metabolic processes should allow us to devise ways to improve nitrogen fixation. It should also allow us to understand fundamental cellular processes in the rhizobia and related bacteria.
Publications
- Friedman, Y.E. and M.R. O'Brian. 2003. A novel DNA-binding site for the ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum. J. Biol. Chem. 278: 38395-38401.
- Friedman, Y.E. and M.R. O'Brian. 2004. The ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum is an iron-responsive transcriptional repressor in vitro. J. Biol. Chem. 279: 32100-32105.
- Platero, R., L. Peixoto, M.R. O'Brian and E. Fabiano. 2004. Fur is involved in manganese-dependent regulation of mntA (sitA) expression in Sinorhizobium meliloti. Appl. Environ. Microbiol. 70: 4349-4355.
- Gao, T. and M.R. O'Brian. 2005. Iron-dependent cytochrome c1 expression is mediated by the status of heme in Bradyrhizobium japonicum. J. Bacteriol. 187: 5084-5089.
- Yang, J., Y. Friedman and M.R. O'Brian. 2005. Regulation of heme and iron metabolism in Bradyrhizobium japonicum. In Biological Nitrogen Fixation, Sustainable Agriculture and the Environment (Y.-P. Yang, M. Lin, Z.-X. Tian, C. Elmerich, W.E. Newton, eds), Springer, Dordrecht, pp. 303-306.
|
Progress 10/01/05 to 09/30/06
Outputs
Cytochromes are necessary for ATP synthesis that supports nitrogen fixation. Cytochromes contain heme as a prosthetic group, which is an iron-containing tetrapyrrole. B. japonicum can acquire the heme precursor ALA from the plant host, and iron must be obtained from the environment. We found that that an operon encoding the cytochrome bc1 complex is regulated by iron in B. japonicum. Furthermore, this iron-dependent control is mediated by heme by a post-translational mechanism. Our model argues that low iron conditions result in less heme available for cytochrome formation. Thus, cytochrome polypeptide is not stable in the absence of heme adn will degrade. Since cytochromes b and c1 are only stable as a complex, heme deficiency that destabilizes one protein may cause break down of the entire complex. We know that cytochrome c1 is destabilized in the absence of heme, and we are investigating the stability of b-type cytochromes.
Impacts
We expect to acquire a better understanding of how B. japonicum acquires necessary components from the environment for heme biosynthesis, and how these processes are regulated. Understanding the mechanisms and control of biosynthetic and metabolic processes should allow us to devise ways to improve nitrogen fixation. It should also allow us to understand fundamental cellular processes in the rhizobia and related bacteria.
Publications
- Friedman, Y.E. and M.R. OBrian. 2003. A novel DNA-binding site for the ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum. J. Biol. Chem. 278: 38395-38401.
- Friedman, Y.E. and M.R. OBrian. 2004. The ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum is an iron-responsive transcriptional repressor in vitro. J. Biol. Chem. 279: 32100-32105.
- Gao, T. and M.R. OBrian. 2005. Iron-dependent cytochrome c1 expression is mediated by the status of heme in Bradyrhizobium japonicum. J. Bacteriol. 187: 5084-5089.
|
Progress 10/01/04 to 09/30/05
Outputs
Cytochromes are necessary for ATP synthesis that supports nitrogen fixation. Cytochromes contain heme as a prosthetic group, which is an iron-containing tetrapyrrole. B. japonicum can acquire the heme precursor ALA from the plant host, and iron must be obtained from the environment. In a screen for ALA transport mutants, we unexpectededly picked up a fur mutant, which regulates iron metabolism in bacteria. We found that the Fur protein from B. japonicum (BjFur) recognizes a DNA sequeence that differs from the so-called Fur box consensus, and is therefore novel in its DNA recognition. We found that conserved amino acid residues proposed to be involved in metal-binding are not required for metal-dependent DNA binding or transcriptional repression in vitro. The data suggest that Fur is functionally more diverse in bacteria than has been assumed from studying model systems. We are interested in finding the link between Fur function and ALA transport, and in better
understanding protein function.
Impacts
We expect to acquire a better understanding of how B. japonicum acquires necessary components from the environment for heme biosynthesis. Understanding the mechanisms and control of biosynthetic and metabolic processes should allow us to devise ways to improve nitrogen fixation. It should also allow us to understand fundamental cellular processes in the rhizobia and related bacteria.
Publications
- Friedman, Y.E. and M.R. OBrian. 2003. A novel DNA-binding site for the ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum. J. Biol. Chem. 278: 38395-38401.
- Friedman, Y.E. and M.R. OBrian. 2004. The ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum is an iron-responsive transcriptional repressor in vitro. J. Biol. Chem. 279: 32100-32105.
- Platero, R., L. Peixoto, M.R. OBrian and E. Fabiano. 2004. Fur is involved in manganese-dependent regulation of mntA (sitA) expression in Sinorhizobium meliloti. Appl. Environ. Microbiol. 70: 4349-4355.
- Gao, T. and M.R. OBrian. 2005. Iron-dependent cytochrome c1 expression is mediated by the status of heme in Bradyrhizobium japonicum. J. Bacteriol. 187: 5084-5089.
|
Progress 10/01/03 to 09/30/04
Outputs
Cytochromes are necessary to produce ATP that supports symbiotic nitrogen fixation. Cytochromes contain heme as the prosthetic group, which is an iron-containing tetrapyrrole. B. japonicum can acquire the heme precursor ALA from the plant host, and it must acquire iron exogenously. The progress for ALA and iron transport is as follows: 1. We identified a B. japonicum mutant defective in ALA transport that is likely mutated in a gene encoding the transporter subunit OppB. This finding is important because there are many oligopeptide permease homologs in the B. japonicum genome, and therefore it would have been difficult to find it based on homology searches. We are in the process of characterizing the gene and mutant further. 2. Iron transport is regulated by the Fur protein in many bacteria. We are interested in understanding how iron is transported and regulated. We found that the Fur protein from B. japonicum recognizes a DNA sequence that differs from the so-called
"Fur box" consensus sequence. This is important because it will assist us in finding genes, including transporters, that are under the control of Fur.
Impacts
We expect to acquire a better understanding of how B. japonicum acquires necessary components from the environment for heme biosynthesis. Understanding the mechanisms and control of biosynthetic and metabolic processes should allow us to devise ways to improve nitrogen fixation. It should also allow us to understand fundamental cellula processes in the rhizobia and related bacteria.
Publications
- Friedman, Y.E. and M.R. O'Brian. 2003. A novel DNA-binding site for the ferric uptake regulator (Fur) protein from Bradyrhizobium japonicum. J. Biol. Chem. 278, 38395-38401.
|
|