Progress 08/15/03 to 08/14/08
Outputs
OUTPUTS: Genes for truncated hemoglobins are scattered among various bacteria, a few protozoans, and most if not all plants. All three of the recently sequenced genomes of Frankia include genes for two different types of truncated hemoglobin, HbO and HbN. The functions of truncated hemoglobins are not well known but appear to be rather variable. During the grant period we improved methods for assay of hemoglobin and the hemoglobin response in Frankia to environmental conditions. We developed an improved assay for determining hemoglobin and total protein concentration in the same extract and found that for nitrogen-fixing cultures the BCA protein assay, but not the Bradford assay, gave accurate results. Determination of the effects of oxygen concentration in Frankia is difficult because it is a filamentous bacterium and tends to form clumps when agitated by stirring or shaking. We were able to minimize this problem by using bubbling with fine capillary tubes inserted to the bottoms of long culture tubes. Determination of the effects of NO exposure was also difficult, because NO is an extremely reactive and unstable compound, and also because excessive amounts are toxic to Frankia. We solved this problem with the use of the proper concentration of DETA NONOate, which releases NO over a period of several days, thus allowing sufficient time for hemoglobin concentrations to change in response to NO. In order to study the differences in function of HbO and HbN it was necessary to separate the two proteins. After investigating a number of approaches, we achieved good separation using a high resolution ion exchange column, run in an ice bath. Using these techniques we found very large differences between nitrogen fixing and nitrogen supplied cultures of Frankia in the amounts of HbN and HbO. Nitrogen fixing cultures maintained a constant ratio of HbO to HbN throughout the life of the culture, with HbO constituting about 80-85% of the total hemoglobin. In contrast, in nitrogen supplied cultures the proportion of HbN increased as the cultures aged, and was about 70% of the total hemoglobin at the end of the observation period. The fraction of HbO in nitrogen supplied cultures increased by a factor of four when cultures bubbled with 1% oxygen were compared to those bubbled with 20% oxygen. The fraction of HbN in nitrogen fixing cultures increased by a factor of 2.5 when they were exposed to NO. To gain insight into the reason why the fraction of HbN increased in nitrogen supplied cultures with age, we assayed cultures for nitrite concentration as a function of age. We found no detectable nitrite in nitrogen fixing cultures at any age, while nitrite concentration in nitrogen supplied cultures increased as they aged. A search of the Frankia genomes revealed the presence the presence of a nitrite reductase. Good evidence exists that a similar enzyme in E. coli produces significant amounts of NO when it reduces nitrite. Thus it is possible that in nitrogen supplied cultures HbN is formed in response to NO originating from the nitrite present in these cultures. PARTICIPANTS: John Tjepkema, principal investigator Christa Schwintzer, principal investigator Pramod Dawadi, graduate student Vanessa Coats, undergraduate student David LaMarche, undergraduate student Jessica Sleeth, undergraduate student TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Not relevant to this project.
Impacts
These results are consistent with a function of HbO in adaptation to reduced oxygen concentration, and a function of HbN in NO oxidation. These two hemoglobins may be essential for the formation of nitrogen-fixing symbioses between Frankia and its various host plants. This project may lead to the creation of new nitrogen-fixing plants. Such plants will reduce the cost of agricultural inputs. They will also reduce the water pollution that occurs when nitrogen fertilizers are used. The discovery of nitrite production by Frankia, combined with the presence of an enzyme that may reduce nitrite to NO implies that HbN may be important for the disposal of NO generated by the internal metabolism of Frankia. Heretofore HbN has been thought to be important only for the oxidation of NO formed by plants during symbiosis with Frankia. These findings may also apply to the function of HbN in Mycobacterium, which is the genus responsible for tuberculosis.
Publications
- Coats, V., Schwintzer, C.R., and Tjepkema, J.D. 2008. Truncated hemoglobins in Frankia CcI3: effects of nitrogen source, oxygen concentration, and nitric oxide. Can. J. Microbiol.(pending)
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Progress 10/01/06 to 09/30/07
Outputs
OUTPUTS: Genes for truncated hemoglobins are scattered among various bacteria, a few protozoans, and most if not all plants. All three of the recently sequenced genomes of Frankia include genes for two different types of truncated hemoglobin, HbO and HbN. The functions of truncated hemoglobins are not well known but appear to be rather variable. The HbO type has been implicated in oxygen transport, NO oxidation, and photosynthesis, while the only function proposed thus far for the HbN type is in NO oxidation. The objective of our current research is to examine the functions of HbO and HbN by measuring their concentrations in cultures of Frankia grown under various conditions. Thus if HbN concentrations rise when cultures are exposed to NO, this would be consistent with a function in NO oxidation. Likewise if HbO concentrations rise when cultures are grown at limiting concentrations of oxygen, it would suggest a function in oxygen transport to respiratory enzymes. In cooperation with
another project, a method for hemoglobin extraction and separation by an ion exchange column has been developed. The crude hemoglobin extract is separated by ion exchange into two major and two minor fractions. Using this column we have compared Frankia cultures grown in the presence and absence of NO. We find that the relative proportion of hemoglobin in the two major fractions changes substantially. The sequence of HbO and HbN elution from the column can be predicted from the isoelectric points calculated from the amino acid sequences. If the two major peaks correspond to HbO and HbN, our results suggest a higher concentration of HbN in the cultures grown with NO, and a higher concentration of HbO in the controls. We have also compared Frankia cultures grown at 20% oxygen to those grown at 1% oxygen. Those grown at 1% oxygen had a much higher concentration of HbO.
PARTICIPANTS: John D. Tjepkema, PI Christa R. Schwintzer, coPI Pramod Dawadi, graduate student research assistant Vanessa Coats, undergraduate research assistant
TARGET AUDIENCES: Agricultural researchers working on the creation of new nitrogen-fixing symbioses. Medical researchers seeking to understand the diverse functions of hemoglobins.
Impacts
These results are consistent with a function of HbO in adaptation to reduced oxygen concentration, and a function of HbN in NO oxidation. This project may lead to the creation of new nitrogen-fixing plants. Such plants will reduce the cost of agricultural inputs. They will also reduce the water pollution that occurs when nitrogen fertilizers are used.
Publications
- Pawlowski K, Jacobsen KR, Alloisio N, Ford Denison R, Klein M, Tjepkema JD, Winzer T, Sirrenberg A, Guan C, Berry AM. 2007. Truncated Hemoglobins in Actinorhizal Nodules of Datisca glomerata. Plant biol (Stuttg) 9: 776-785
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Progress 10/01/05 to 09/30/06
Outputs
Genes for truncated hemoglobins are scattered among various bacteria, a few protozoans, and most if not all plants. All three of the recently sequenced genomes of Frankia include genes for two different types of truncated hemoglobin, HbO and HbN. The functions of truncated hemoglobins are not well known but appear to be rather variable. The HbO type has been implicated in oxygen transport, NO oxidation, and photosynthesis, while the only function proposed thus far for the HbN type is in NO oxidation. The objective of our current research is to examine the functions of HbO and HbN by measuring their concentrations in cultures of Frankia grown under various conditions. Thus if HbN concentrations rise when cultures are exposed to NO, this would be consistent with a function in NO oxidation. Likewise if HbO concentrations rise when cultures are grown at limiting concentrations of oxygen, it would suggest a function in oxygen transport to respiratory enzymes. In
cooperation with another project, a method for hemoglobin extraction and separation by an ion exchange column has been developed. The crude hemoglobin extract is separated by ion exchange into two major and two minor fractions. Using this column we have compared Frankia cultures grown under nitrogen fixing conditions with those grown with ammonium as a nitrogen source. We find that the relative proportion of hemoglobin in the two major fractions changes substantially. The sequence of HbO and HbN elution from the column can be predicted from the isoelectric points calculated from the amino acid sequences. If the two major peaks correspond to HbO and HbN, our results suggest a relatively higher concentration of HbO in the nitrogen-fixing cultures, and a relatively high concentration of HbN in the ammonium-fed cultures.
Impacts
This project may lead to the creation of new nitrogen-fixing plants. Such plants will reduce the cost of agricultural inputs. They will also reduce the water pollution that occurs when nitrogen fertilizers are used.
Publications
- Silvester, W.B., Berg, R.H., Schwintzer, C. R., and Tjepkema, J. D. Responses to oxygen and the role of hemoglobin. In Nitrogen-Fixing Actinorhizal Symbioses. K. Pawlowski and W. E. Newton, eds. Springer. 2006.
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Progress 10/01/04 to 09/30/05
Outputs
Work has continued on two objectives, one that focuses on the expression of the trHbO gene in Frankia and the other on the oxygenation status of the hemoglobin produced by it. The work on trHbO expression is at an early stage. The second objective is to determine the oxygenation status of Frankia hemoglobin in vivo. Most of the hemoglobin in alder nodules appears to be associated with Frankia. We have now developed a system that detects the shift from oxy- to deoxyhemoglobin in intact nodule tissue. This is being used to determine the oxygenation status of the hemoglobin under nitrogen-fixing conditions. If partial oxygenation is found, this will support a function in oxygen transport to respiratory enzymes, as has been found for the trHbO in Mycobacterium. If much or all of the hemoglobin remains in the deoxy form, this would suggest that most of it is involved in another function such as NO oxidation. We are also continuing to develop a system for measuring the
oxygenation status of hemoglobin in Frankia grown in culture. In culture there is no possibility of plant-produced hemoglobin, so this will serve to confirm our results for nodule tissue. We plan to measure respiration, nitrogen fixation, and hemoglobin oxygenation simultaneously, and predict that hemoglobin will be partially oxygenated at the oxygen concentrations that give maximal rates of nitrogen fixation. This system will also allow us to measure any changes that take place when NO-generating compounds are introduced.
Impacts
This project may lead to the creation of new nitrogen-fixing plants. Such plants will reduce the cost of agricultural inputs. They will also reduce the water pollution that occurs when nitrogen fertilizers are used.
Publications
- Schwintzer, C.R. and Tjepkema, J.D. 2005. Effect of oxygen concentration on growth and hemoglobin production in Frankia. Symbiosis 39:77-82.
- Niemann, J.M., Tjepkema, J.D. and Tisa, L.S. 2005. Identification of the truncated hemoglobin gene in Frankia. Symbiosis 39:91-95.
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Progress 10/01/03 to 09/30/04
Outputs
Current research is focused on determination of the ligand binding and oxidation state of Frankia hemoglobin in vivo. This is being pursued by spectroscopy of alder nodules and cultures of Frankia. A spectrophotometer has been obtained that focuses its light output into an area of just over 1 mm diameter, thus allowing intense illumination of nodules which are only about 2 mm in diameter. The scattered light transmitted through a nodule is received by a detector placed immediately behind the nodule. Cultures of Frankia are observed in tubes with reflective walls, with glass windows at the ends, with the detector being placed immediately behind one of the windows. Because Frankia are now known to possess genes for three different kinds of hemoglobin, it seems likely that the type of hemoglobin produced and its function will depend on the physiological status of the organism. In other work we have found that the concentration of hemoglobin produced by Frankia depends on
oxygen concentration and culture age. This suggests variations in the function of hemoglobin in Frankia, with various possible mixtures of the three kinds of hemoglobins. Our previous work suggested a predominance of the oxy and deoxy forms of ferric hemoglobin in unstressed nodules of alder. However it now known that some hemoglobins function in the oxidation of nitric oxide. The product of this reaction is ferric hemoglobin. Thus we are looking for the characteristic absorption spectrum of ferric hemoglobin as well as those of the oxygenated and deoxygenated ferrous forms. These studies will be correlated with extractions of hemoglobin to determine which of the three types of hemoglobin are present.
Impacts
This project may lead to the creation of new nitrogen-fixing plants. Such plants will reduce the cost of agricultural inputs. They will also reduce the water pollution that occurs when nitrogen fertilizers are used.
Publications
- No publications reported this period
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Progress 08/15/03 to 12/15/03
Outputs
Progress for the first 4 months of the grant: response of hemoglobin concentration to environmental variables and culture age. A. Hemoglobin assay. We have developed an improved assay by determining both hemoglobin and total protein concentration in the same extract. In a comparison of the Bradford and BCA protein assays, we found that both gave similar results for cultures grown with ammonium. However the Bradford assay underestimated the protein in nitrogen-fixing cultures. Known addition of a protein standard to extracts from nitrogen-fixing cultures resulted in a less than the expected protein concentration using the Bradford assay, but gave the expected value using the BCA assay. B. Effects of culture age and presence/absence of ammonium. Preliminary results indicate that the ratio of hemoglobin to total protein remains relatively constant as a function of culture age and the presence or absence of ammonium. These results suggest that the hemoglobin may play a
central role in Frankia metabolism, perhaps being needed under all growth conditions. C. Effects of oxygen concentration. Investigation of this question requires good control of the oxygen concentration. Because Frankia is a filamentous bacterium, it tends to form clumps when agitated by stirring or shaking. Stirring has the additional disadvantage of requiring a stirring device for each replicate. As an alternative we are developing a system that keeps the Frankia suspended, with minimal clumping, using bubbling with the desired gas mixture in large tubes.
Impacts
This project may lead to the creation of new nitrogen-fixing plants. Such plants will reduce the cost of agricultural inputs. They will also reduce the water pollution that occurs when nitrogen fertilizers are used.
Publications
- No publications reported this period
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