Progress 10/01/03 to 09/30/07
Outputs
OUTPUTS: Biological nitrogen fixation is a natural process carried out by the bacterium Azotobacter vinelandii that involves the reduction of molecular nitrogen to ammonia while producing hydrogen gas as a byproduct. A. vinelandii can synthesize three distinct nitrogenases, that can be divided into two categories; molybdenum (Mo)-independent and Mo-dependent. The Mo-independent nitrogenases, (nitrogenase 2 and nitrogenase 3) are less efficient in the production of ammonia and more efficient in producing hydrogen gas than the Mo-dependent nitrogenase (nitrogenase 1). For example, approximately 50 percent of the electron flux through nitrogenase 3 is channeled into proton (H+) reduction to generate molecular hydrogen (H2) whereas in nitrogenase 1 only 25 percent of the electron flux is channeled into H+ reduction. Our work has focused on hydrogen production via nitrogenase 3 in the previously characterized tungsten-tolerant A. vinelandii mutant CA6. Unpublished results indicate that
CA6 has mutations in an operon (mohCAB) that is believed to encode a low affinity molybdenum uptake system. Inactivation of each of the moh genes using interposon plus deletion mutations indicates that inactivation of mohC is responsible for hydrogen evolution. Preliminary evidence based on RT-PCR experiments suggests that expression of hupSL (genes encoding uptake hydrogenase subunits) requires MohC. A salient phenotypic feature of strains with mutations in the mohCAB operon is their ability to grow under nitrogen-fixing conditions in the presence of relatively high concentrations of tungstate (>1 mM). To see if tungsten could be used as a selective agent for the isolation of hydrogen-evolving spontaneous mutants from genetically uncharacterized environmental isolates (known to have genes encoding nitrogenase 3), tungsten-tolerant mutants were isolated from isolates Br5, Br6, Br7, and Mu7 (bacterial strains that are closely related to the pseudomonads). Hydrogen production by these
strains was similar to that of A. vinelandii strains CA6 and CA125. Further tests with strain CA6 demonstrated that it is capable of utilizing carbon sources for H2 production which include sugars and alcohols, making it suitable for growth on relatively cheap carbon sources such as molasses. Results of growth and hydrogen production by CA6 in a bench-top bioreactor suggest that hydrogen production experiments can be scaled up to the pilot plant level.
PARTICIPANTS: Other participants who worked on this project are: Telisa M. Loveless,Soybean and Nitrogen Fixation Unit, USDA-ARS, Raleigh, NC. Dr. Jonathan Olson, Dept. of Microbiology, North Carolina State University. Dr. Jose M. Bruno-Barcena, Bio-Manufacturing Training and Education Center and Dept. of Microbiology, North Carolina State University.
Impacts
A provisional patent has been filed by USDA-ARS (application number 60/959,940) on the production of hydrogen using Azotobacter vinelandii strain CA6 as a prototype strain and the ability to use spontaneous mutants of natural isolates (known to have Mo-independent nitrogenases) that are selected for ability to grow in the presence of high concentrations of tungstate under nitrogen-fixing conditions. We envision the use of heterotrophic nitrogen-fixing bacteria(such as Azotobacter vinelandii and other related naturally occuring bacteria) for the bioproduction of hydrogen as a partial solution for the impending global energy shortage. The advantage that a heterotrophic bacterium has over a photosynthetic organism for hydrogen production is that the heterotrophic bacterium can be cultured under conventional culture conditions using carbon substrates produced by plants (which are ideally suited for the capture of solar energy). On the other hand, the culture of a
photosynthetic microrganism requires a large surface area in order to capture sufficient solar energy for hydrogen production. This complicates both the design of the fermentation process and collection of hydrogen from a process engineering point of view.
Publications
- No publications reported this period
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Progress 10/01/04 to 09/30/05
Outputs
Azotobacter vinelandii contains three genetically distinct nitrogenases that are expressed in response to the presence or absence of molybdenum (Mo) or vanadium (V) in N-free growth medium. Mo represses expression of the two Mo-independent nitrogenases. A. vinelandii strain CA6, however, carries multiple mutations which permit diazotrophic growth in the presence of normally inhibitory concentrations of tungstate (W). This is because Mo-independent nitrogenase 3 is constitutively expressed in this strain in the presence of Mo or W. We have isolated and subcloned an l.7-kbp SalI-SphI fragment from CA6 that confers the W-tolerance phenotype. The corresponding fragment from wild-type strain CA, a 2.l-kbp SalI-SphI fragment was also subcloned. The nucleotide sequence of both fragments was determined and a sequence comparison showed a number of mutational alterations in the l.7-kbp SalI-SphI fragment from CA6. Thus the observed W-tolerance phenotype of CA6 could result from
any of these mutations. Three genes designated here as mohC, mohA, and mohB were found to correspond to the mutated region in CA6. Their deduced amino acid sequence shows extensive homology with peripheral proteins of the cytoplasmic membrane which are components of bacterial ABC transport systems. The MohC product shares 57 percent identity with FhuC, an ATP-binding component found in Yersina pestis. The MohA product shares 39 percent identity with the periplasmic component, FhuD of Rhizobium leguminosarum, and the MohB product shares 48 percent identity with the membrane component, FhuB of Yersinia pestis. Mutants with kan interposon insertions in genes mohC, mohA and mohB were designated strains CA125, CA130, and CA129 respectively. All three strains exhibit a W-tolerance phenotype similar to that observed for CA6 and molybdenum accumulation experiments indicate that Mo-uptake is impaired in CA125 and CA129, but not in CA130. Beta-Galactosidase expression from a mohC-lacZ
transcriptional fusion increased approximately two-fold in the presence of 100 mM molybdate after a 20-hr period of incubation. Diazotrophic growth of CA125 in the presence of 1 mM sodium tungstate was found to be dependent on nitrogenase 3. Competition experiments indicate that strain CA has an advantage over the tungstate-tolerance strain under diazotrophic conditions in the presence or absence of Mo. In conclusion, the results of this study suggest that mohC, mohA, and mohB are involved in Mo regulation of nitrogenase(s).
Impacts
The mhoCAB system not only appears encode a second molybdenum transport system in A. vinelandii but these genes are definitely involved in the regulation of nitrogenases by molybdenum in this nitrogen-fixing bacterium. This information is crucial to the understanding of how nitrogen fixation is influenced by environmental conditions in this organism and may prove useful in utilizing the these nitrogenases for agronomic purposes.
Publications
- No publications reported this period
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Progress 10/01/03 to 09/30/04
Outputs
Azotobacter vinelandii contains three genetically distinct nitrogenases which are expressed in response to the presence or absence of molybdenum (Mo) or vanadium (V) in N-free growth medium. Expression of the alternative nitrogenases is repressed by Mo. However, A. vinelandii strain CA6 carries a mutation which permits diazotrophic growth in the presence of normally inhibitory concentrations of tungstate (W). This is because Mo-independent nitrogenase 3 is constitutively expressed in this strain. We have isolated and subcloned a l.7-kbp SalI-SphI fragment from CA6 that confers the W-tolerance phenotype. The corresponding fragment from wild-type strain CA, a 2.l-kbp SalI-SphI fragment was also subcloned. The nucleotide sequence of both fragments was determined and a sequence comparison showed a number of mutations in the l.7-kbp SalI-SphI fragment from CA6. Thus the observed W-tolerance phenotype of CA6 could result from any of these mutations. Three genes designated
here as mohC, mohA, and mohB were found to correspond in CA to the mutated region in CA6. Their deduced amino acid sequence shows extensive homology with peripheral proteins of the cytoplasmic membrane which are components of bacterial transport systems. The MohC product shares 57 percent identity with FhuC of Yersina pestis. The MohA product shares 39 percent identity with FhuD of Rhizobium leguminosarum and the MohB product shares 48 percent identity with FhuB of Yersinia pestis. Mutants with kan interposon insertions in genes mohC, mohA and mohB were designated strains CA125, CA130, and CA129 respectively. All three strains exhibit a W-tolerance phenotype similar to that observed for CA6 and molybdenum accumulation studies of these strains indicate that Mo-uptake is impaired. Beta-Galactosidase expression from a mohC-lacZ transcriptional fusion shows that this gene is constitutively expressed in the presence or absence of ammonium with varying concentrations of molybdate, sulfate
and iron. Diazotrophic growth of CA125 in the presence of 1 millimolar sodium tungstate was found to be dependent on nitrogenase 3. Competition experiments indicate that strain CA has an advantage over the tungstate-tolerance strain under diazotrophic conditions in the presence or absence of Mo. In conclusion, the results of this study suggest that mohC, mohA and mohB are involved in Mo accumulation and that mohC is involved in the regulation of nitrogenases.
Impacts
To acquire molybdenum, A.vinelandii has a high affinity molybdenum transport system, encoded by genes modGEABC. Previous studies indicate that in A. vinelandii, a biphasic response of both nitrate reductase activity and the uptake of radioactive molybdenum to increasing molybdenum concentrations could be interpreted as the result of at least two uptake processes operating at different Mo concentrations. In this paper, we describe genes (mohCAB) for a second molybdenum transport system that could function as a low-affinity system. These genes resemble a typical ABC transport system. Studies of strains containing mutational disruption of these genes indicate that molybdenum accumulation is impaired and that the mutants are able to grow under nitrogen-fixing conditions in the presence of tungsten, an inhibitor of the molybdenum-dependent nitrogenase (nitrogenase 1). In addition, protein gel analysis indicates that in the mohC mutant, a molybdenum-independent nitrogenase
(nitrogenase 3) is used to carry out nitrogen fixation while growing in media containing tungsten. Under these growth conditions, the wild type strain is unable to fix nitrogen because nitrogenase 3 is not expressed and the molybdenum-containing nitrogenase 1 is expressed but is inactive in the presence of tungsten. This indicates that the mohCAB genes are involved with the regulation of nitrogenase genes in Azotobacter vinelandii as well as molybdenum accumulation. The results of this study lead to a better understanding of the different ways that molybdenum can be acquired by cells.
Publications
- No publications reported this period
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