Source: DARTMOUTH COLLEGE submitted to NRP
IRON METABOLISM IN THE BRADYRHIZOBIUM JAPONICUM/SOYBEAN SYMBIOSIS
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
COMPLETE
Funding Source
NRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
0183337
Grant No.
99-35305-8644
Cumulative Award Amt.
(N/A)
Proposal No.
1999-03686
Multistate No.
(N/A)
Project Start Date
Nov 15, 1999
Project End Date
Aug 31, 2004
Grant Year
2000
Program Code
[(N/A)]- (N/A)
Recipient Organization
DARTMOUTH COLLEGE
8000 CUMMINGS HALL
HANOVER N H,NH 03755
Performing Department
BIOLOGICAL SCIENCES
Non Technical Summary
The increasing world demand for protein and the increasing expense of producing nitrogen fertilizer has generated a widespread interest in biological nitrogen fixation. Although the nitrogen-fixing Bradyrhizobium/legume symbioses supply much of the fixed nitrogen for agriculture in the temperate regions of the world, many aspects of the symbioses are still poorly understood. The research proposed here is aimed at obtaining a better understanding of the role of iron in the symbioses, given the prominent role of iron-containing proteins such as nitrogenase and leghemoglobin in the nitrogen fixation process. Such an understanding should elucidate how rhizobia interact with their plant host to acquire enough iron to establish an effective symbiosis. Studies will be carried out on iron uptake in the Bradyrhizobium japonicum/soybeans symbiosis. The experiments are all directed at answering a central question: How do bacteroids acquire iron in planta. Specifically, we want to know 1) are bacteria using ferric or ferrous forms of iron when they are in the nodule. 2) does the iron regulatory protein Fur control genes important for the symbiosis and 3) are there nodule-specific ferric chelate reductases and iron transporters that deliver iron to the bacteria when they are living in the nodule. Our lab has recently cloned the genes encoding one of the iron-regulated outer membrane proteins of B. japonicum, the Fur protein from B. japonicum, as well as the Fe(III) chelate reductase and a presumptive Fe(II) transporter from Arabidopsis, so we are well positioned to now examine iron
Animal Health Component
(N/A)
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2031820104050%
2034010104050%
Knowledge Area
203 - Plant Biological Efficiency and Abiotic Stresses Affecting Plants;

Subject Of Investigation
1820 - Soybean; 4010 - Bacteria;

Field Of Science
1040 - Molecular biology;
Goals / Objectives
The experiments are all directed at answering a central question: How do bacteroids acquire iron in planta. Specifically, we want to know 1) are bacteria using ferric or ferrous forms of iron when they are in the nodule, 2) does the iron regulatory protein Fur control genes important for the symbiosis and 3) are there nodule-specific ferric chelate reductases and iron transporters that deliver iron to the bacteria when they are living in the nodule.
Project Methods
Our lab has recently cloned the genes encoding one of the iron-regulated outer membrane proteins of B. japonicum (FegA), the Fur protein from B. japonicum, as well as the Fe(III) chelate reductase (FRO2) and a presumptive Fe(II) transporter (IRT1) from Arabidopsis. The symbiotic phenotype of fegA and fur mutants will be determined in order to address which forms of iron are available in planta. We will also clone the genes encoding the two other iron-regulated outer membrane proteins from B. japonicum and generate mutants that no longer express each of these proteins. We will identify and clone soybean homologs of the Arabidopsis FRO2 and IRT1 genes. The role of these genes in supplying the iron required to support nitrogen fixation will be examined.

Progress 11/15/99 to 08/31/04

Outputs
Rhizobia live in the soil or enter into a nitrogen-fixing symbiosis with a suitable host plant. Each environment presents different challenges with respect to iron acquisition. The soybean symbiont, Bradyrhizobium japonicum strain 61A152 can utilize a variety of siderophores [Fe(III) specific ligands]. Purification of iron-regulated outer membrane proteins had previously allowed the cloning of a gene, fegA, from B. japonicum 61A152 whose predicted protein shares significant amino acid similarity with known TonB-dependent siderophore receptors. We have shown that fegA is in an operon with a gene fegB that is predicted to encode an inner membrane protein. Characterization of fegAB and fegB mutants shows that both fegA and fegB are required for utilization of the siderophore ferrichrome (Benson et al., 2005). Whereas the fegB mutant forms a normal symbiosis, the fegAB mutant has a dramatic phenotype in planta. Six weeks after inoculation with a fegAB strain, soybean nodules do not contain leghemoglobin and do not fix nitrogen. Infected cells contain few symbiosomes and are filled with vesicles. As ferrichrome is a fungal siderophore not likely to be available in nodules, the symbiotic defect suggests that the fegAB operon is serving a different function in planta, possibly one involved in signaling between the two partners. Certain TonB-dependent outer membrane proteins contain an additional domain at their N terminus that is involved in signal transduction. This domain interacts with an inner membrane protein that, in turn, interacts with an ECF sigma factor. FegA is predicted to have such an N terminal extension. In many bacteria, the Ferric Uptake Regulator (Fur) protein plays a central role in the regulation of iron uptake genes. We identified a fur mutant that fails to repress iron-regulated outer membrane proteins in the presence of iron (Benson et al., 2004). Unexpectedly, a wild type copy of the fur gene cannot complement the fur mutant. Expression of the fur mutant allele in wild type cells leads to a fur phenotype. Unlike a B. japonicum fur null mutant, the strain carrying the dominant negative fur mutation is unable to form functional, nitrogen-fixing nodules on soybean, suggesting a role for a Fur-regulated protein(s) in the symbiosis. We had previously identified the ZIP gene family of cation transporters. This family includes IRT1, the major transporter responsible for uptake of Fe(II) from the soil. We have identified a perbacteroid specific zinc transporter that belongs to the ZIP family, GmZIP1 (Moreau et al., 2002). Expression of GmZIP1 at the mRNA level can only be detected in nodules and antibodies raised against GmZIP1 specifically localize this protein to the peribacteroid membrane. Furthermore, antibodies to GmZIP1 inhibit zinc uptake by symbiosomes indicating that at least some of the zinc uptake observed for isolated symbiosomes can be attributed to GmZIP1. GmZIP1 is able to complement a zinc uptake mutant of yeast and not an iron uptake mutant. GmZIP1 can also transport cadmium.

Impacts
Nitrogen fixation is crucial for nutrient cycling and crop health. Our work is focused on the Bradyrhizobium japonicum/soybean symbiosis because soybean is the most important legume crop grown in the US. One of the main goals of our research is to understand how the bacterial symbionts interact with the host plant to regulate metabolic processes essential for the nitrogen-fixing symbiosis. Our studies help to elucidate how bradyrhizobia adapt to life as intracellular bacteria.

Publications

  • Moreau, S., R.W. Thomson, B.N. Kaiser, B. Trevaskis, M.L. Guerinot, M.K. Udvardi, A. Puppo, and D.A. Day. 2002. GmZIP1 encodes a symbiosis specifiic zinc transporter in soybean. J. Biol. Chem. 277: 4738-4746.
  • Benson, H., K. LeVier and M.L. Guerinot. 2004. Characterization of a dominant negative fur mutation in Bradyrhizobium japonicum. J. Bacteriol. 186: 1409-14.
  • Benson, H.P., E. Boncompagni and M.L. Guerinot. 2005. An iron uptake operon required for proper nodule development in the Bradyrhizobium japonicum/soybean symbiosis. Mol. Plant Microbe Interact. In Press.