Progress 04/01/02 to 03/30/07
Outputs
We have published: 1) two studies linking nitrification activities to microbial community structures in N-impacted environments, 2) a study comparing global gene regulation in a mutant strain of an ammonia-oxidizing bacterium, and 3) a study revealing the diversity of nitrite reductase gene sequences among nitrifying bacteria. 3 graduate students, 3 postdoctoral researchers, and 4 undergraduates have been supported under this project. Using molecular and traditional tools, we published a study on N-impacted forest soils showing that autotrophic ammonia-oxidizers were not responsible for the bulk of nitrifying activity in that system. Conversely, our publication on turf-covered aridisols showed that nitrification activity was dominated by autotrophic ammonia oxidizers. The strength of activity and abundance was correlated to salinity of soil or irrigation water and duration of turf management. We published a study using a genomic microarray for N. europaea to compare
global gene expression between wild-type and cells mutated in the nitrite reductase gene, nirK. This gene catalyzes the reduction of nitrite to nitric oxide under aerobic conditions as a detoxification mechanism. Genes up-regulated in NirK-deficient cells included those in the nirK operon, a cytochrome c oxidase that may catalyze nitric oxide reduction, and several genes involved in iron acquisition and metabolism. These results showed a connection between iron homeostasis and nitrosative stress response that is triggered in the absence of NirK activity. Using molecular tools, we published a study showing the unexpected diversity of nirK gene sequences among the nitrifying bacteria. This study showed that nitrous oxide-producing pathways among the nitrifiers likely evolved from multiple gene transfer and evolutionary events. Also, many investigators use the nirK gene as a proxy for denitrifying activity in the environment. Our study showed unequivocally that use of a single PCR primer
set covers only a small portion of the total diversity of this gene. In a related study (in review), we discovered an intimate connection between NirK activity and central metabolism of an ammonia-oxidizing bacterium. NirK stimulates production of nitrite from ammonia and prevents the formation of toxic N-oxide intermediates from chemical oxidation of hydroxylamine. This is the likely mechanism for protection of ammonia-oxidizers from toxicity experienced during their chemolithotrophic metabolism. We continue to have a leading role in annotation of complete genome sequences of nitrifying and methanotrophic bacteria. Genomic data continues to be crucial to design and data interpretation of on-going projects.
Impacts
Comparative physiology studies of nitrifiers and methanotrophs continue to open new and interesting avenues of inquiry. We have developed and applied cutting-edge tools to answer how and why ammonia-oxidizers and methanotrophs produce nitrous oxide, an extremely powerful greenhouse gas, and what environmental factors control their activities and population compositions. Closing these gaps in knowledge will allow us to predict, and perhaps control, the release of nitrous oxide and nitrate pollutants from agricultural lands and other N-impacted ecosystems.
Publications
- Arp, D.J. and L.Y. Stein. 2003. Metabolism of inorganic N compounds by ammonia-oxidizing bacteria. Crit. Rev. Biochem. Molec. Biol. 38(6):471-495.
- Jordan, F.L., J.J.L. Cantera, M.E. Fenn, L.Y. Stein. 2005. Autotrophic ammonia oxidizers contribute minimally to nitrification in a nitrogen-saturated forest soil. Appl. Environ. Microbiol. 71(1):197-206.
- Cantera, J.J.L., F.L. Jordan, L.Y. Stein. 2006. Effects of irrigation sources on ammonia-oxidizing bacterial communities in a managed turf-covered aridisol. Biol. Fertil. Soils. 43:247-255.
- Cho, C. M.-H, T. Yan, X. Liu, L. Wu, J. Zhou, L.Y. Stein. 2006. Transcriptome of Nitrosomonas europaea with a disrupted nitrite reductase (nirK) gene. Appl. Environ. Microbiol. 72:4450-4454.
- Cantera, J.J.L. and L.Y. Stein. 2007. Molecular diversity of nitrite reductase genes (nirK) in nitrifying bacteria. Environ. Microbiol. 9(3):765-776.
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Progress 01/01/05 to 12/31/05
Outputs
Progress: We have completed: 1) our second study linking nitrification activities to microbial community structures in N-impacted environments, and 2) a study comparing global gene regulation in a mutant strain of an ammonia-oxidizing bacterium. Current projects include: 1) investigations of the ammonia-oxidizing pathway by 4 methanotrophic strains, 2) studies of nitrous oxide-producing pathways in ammonia- and nitrite-oxidizing bacteria, and 3) studies of functional gene diversity in nitrifiers. 2 graduate students, 3 postdoctoral researchers, and 2 undergraduates have been supported under this project. Using molecular and traditional tools, our study on turf-covered aridisols showed that nitrification activity in these systems was dominated by autotrophic ammonia oxidizers. The strength of activity and abundance of the ammonia-oxidizing population was correlated to salinity of soil or irrigation water and duration of turf management. We published a study using a
genomic microarray for Nitrosomonas europaea to compare global gene expression between wild-type and cells mutated in the nitrite reductase gene, nirK. This gene catalyzes the reduction of nitrite to nitric oxide under aerobic conditions as a detoxification mechanism. Genes up-regulated in NirK-deficient cells included those in the nirK operon, a cytochrome c oxidase that may catalyze nitric oxide reduction, and several genes involved in iron acquisition and metabolism. These results showed a connection between iron homeostasis and nitrosative stress response that is triggered in the absence of NirK activity. Current projects include construction of a hydroxylamine oxidoreductase mutant in a methanotrophic bacterium to determine the role of this enzyme in the ammonia-oxidizing pathway. Secondly, we are investigating the function of nirK genes in nitrite-oxidizing bacteria that are evolutionarily related to those in Nitrosomonas europaea. Third, we are close to submitting for
publication a phylogenetic study of nirK genes in multiple nitrifying isolates which showed radiation of nirK genes into 4 distinct taxonomic clades. We continue to have a leading role in annotation of complete genome sequences of nitrifying bacteria. Genomic data continues to be crucial to design and data interpretation of on-going projects.
Impacts
Comparative physiology studies of nitrifiers and methanotrophs continue to open new and interesting avenues of inquiry. We have developed and applied cutting-edge tools to answer how and why ammonia-oxidizers and methanotrophs produce nitrous oxide, an extremely powerful greenhouse gas, and what environmental factors control their populations. Closing these gaps in knowledge will allow us to predict, and perhaps control, the release of nitrous oxide and nitrate pollutants from agricultural lands and other managed ecosystems.
Publications
- Arp, D.J., L.Y. Stein. 2003. Metabolism of inorganic N compounds by ammonia-oxidizing bacteria. Crit. Rev. Biochem. Molec. Biol. 38:471-495.
- Cantera, J.J.L., F.L. Jordan, L.Y. Stein. 2006. Effects of irrigation sources on ammonia-oxidizing bacterial communities in managed turf-covered aridisols. Biol. Fertil. Soils. In review.
- Cho, C. M.-H, T. Yan, X. Liu, L. Wu, J. Zhou, L.Y. Stein. 2006. Transcriptome of Nitrosomonas europaea with a disrupted nitrite reductase (nirK) gene. Appl. Environ. Microbiol. In press.
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Progress 01/01/04 to 12/31/04
Outputs
We have completed: 1) two studies linking nitrification activities to microbial community structures in N-impacted environments, and 2) a study comparing global gene regulation in an ammonia-oxidizing bacterium. Current projects include: 1) investigations of ammonia-oxidizing activity by 5 methanotrophic strains and 2) two studies of nitrous oxide-producing pathways in ammonia oxidizing bacteria. 1 graduate student, 3 postdoctoral researchers, and an undergraduate have been supported under this project. Using molecular and traditional tools, we published a study on N-impacted forest soils showing that autotrophic ammonia-oxidizers were not responsible for the bulk of nitrifying activity in that system. Conversely, in turf-covered aridisols we found that nitrification activity was dominated by autotrophic ammonia oxidizers. The strength of activity and abundance was correlated to salinity of irrigation water and duration of turf management. Results will be submitted
for publication soon. We successfully used a genomic microarray for N. europaea to compare global gene expression between wild-type and cells mutated in the nitrite reductase gene, aniA. This gene catalyzes the reduction of nitrite to nitric oxide under aerobic conditions to produce energy. Genes up-regulated in aniA mutated cells included those in the aniA operon, genes involved in reducing copper toxicity, sigma factors involved in iron acquisition, and a biotin biosynthesis. Genes down-regulated in aniA mutated cells included an assimilatory NADPH-nitrite reductase. These results show the connection between metal homeostasis and ammonia-oxidation. Our results will be submitted for publication soon. Current projects include constructing additional mutants in one of the three gene copies of hydroxylamine oxidoreductase in Nitrosomonas europaea to verify its role in producing nitrous oxide under microaerobic conditions. We are also generating an aniA mutant in Nitrosospira multiformis
to see whether this gene has similar function in the two ammonia-oxidizing strains. We continue to investigate ammonia- and hydroxylamine-oxidizing activities in methanotrophic bacteria, organisms closely related to ammonia-oxidizers. We are currently investigating the separate roles of particulate and soluble methane monooxygenase enzymes in mediating ammonia oxidation.
Impacts
Comparative physiology studies of nitrifiers and methanotrophs continue to open new and interesting avenues of inquiry. We have developed and applied cutting-edge tools to answer how and why ammonia-oxidizers and methanotrophs produce nitrous oxide, an extremely powerful greenhouse gas, and what environmental factors control their populations. Closing these gaps in knowledge will allow us to predict, and perhaps control, the release of nitrous oxide and nitrate pollutants from agricultural lands and other managed ecosystems.
Publications
- Jordan, F.L., Cantera, J.J.L., M.E. Fenn, L.Y. Stein. 2004. Autotrophic ammonia oxidizers contribute minimally to nitrification in a nitrogen-saturated forest soil. Appl. Environ. Microbiol. 71:197-206.
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Progress 01/01/03 to 12/31/03
Outputs
We have generated tools and techniques to: 1) explore molecular regulation of greenhouse gas production by cultures of ammonia- and methane-oxidizing bacteria, 2) unleash the power of genomic microarray technology to investigate global gene expression in Nitrosomonas europaea, and 3) connect population structure to function in N-impacted environments. 2 graduate students, 3 postdoctoral researchers, and an undergraduate have been supported under this project. Using mutated strains of Nitrosomonas europaea, we found that one of three identical gene copies of hydroxylamine oxidoreductase (hao), the enzyme catalyzing the second step in ammonia oxidation to nitrite, is involved in producing nitrous oxide under microaerobic conditions. We were surprised by this because all of the HAO enzymes produced from identical genes should catalyze identical reactions. Thus, either the position of this gene within the genome affects expression of near-by genes, or an altered pattern
of expression results in nitrous oxide production. We are currently generating more mutated strains of N. europaea to resolve this mystery. We are using the genomic microarray for N. europaea to compare global gene expression between wild-type and cells mutated in their putative nitrite reductase gene, nirK. This gene was thought to catalyze the reduction of nitrite to nitric oxide in the pathway for nitrous oxide production. However, physiological studies with the nirK mutant showed that this NirK is involved in the aerobic pathway of hydroxylamine oxidation. Experiments with the genomic micorarry will show what genes are expressed in this strain in response to increased nitrite concentrations and will illuminate the actual process requiring nirK activity. Because N. europaea is a model organism, we can not make assumptions about behavior of ammonia-oxidizing bacteria in the environment. We have conducted comparative physiology studies between N. europaea and a common terrestrial
species, Nitrosospira multiformis. We discovered that this strain also harbors three highly similar copies of the hao gene and it responds similarly to N. europaea to environmental stimuli. We investigated ammonia- and hydroxylamine-oxidizing activities in methanotrophic bacteria, organisms closely related to ammonia-oxidizers. We found that inorganic nitrogen metabolism is not evolutionarily conserved among closely related strains of methanotrophs and that aerobic production of nitrous oxide is tightly connected to their metabolism of hydroxylamine. Last, we developed molecular and traditional tools to connect the structure of ammonia-oxidizing communities to their function in soils. We completed a study in an N-impacted forest and found that autotrophic ammonia-oxidizers were not responsible for the bulk of nitrifying activity in that system. We are now applying our tools to turf-covered aridisols; i.e. golf courses. These systems are heavily influenced year-round by nitrogen
fertilizers and irrigation. We found that nitrification activity is correlated to the duration of turf management and are now determining the identity and abundance of ammonia- and nitrite-oxidizing bacteria.
Impacts
: Just like the past 50 years of research on a single strain of Escherichia coli, our physiology studies of Nitrosomonas europaea continue to open new and interesting avenues of inquiry. We have developed a new generation of tools and techniques to answer how and why N. europaea, Nitrosospira multiformis, other ammonia-oxidizers, and methanotrophic bacteria produce nitrous oxide, an extremely powerful greenhouse gas. Closing this gap in knowledge will allow us to predict, and perhaps control, the release of nitrous oxide and nitrate pollutants from agricultural lands and other managed ecosystems.
Publications
- Arp, D.J. and L.Y. Stein (2003): Metabolism of inorganic N compounds by ammonia-oxidizing bacteria. Crit. Rev. Biochem. Molec. Biol. 38:471-495.
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Progress 01/01/02 to 12/31/02
Outputs
The pathway for nitrous oxide production by ammonia oxidizing bacteria (AOB) was investigated using the model organism, Nitrosomonas europaea. Utilizing a whole cell physiology approach, I found that the presumed nitrite reductase enzyme, NirK, is not involved in nitrous oxide production by N. europaea. This result was surprising since no other genes with similarity to nitrite reductase have been identified in the completed genome sequence of this organism. Also, NirK is one of two primary classes of nitrite reductase found in traditional denitrifying bacteria. According to my experiments, NirK appears to catalyze the reverse reaction of a nitrite reductase, the oxidation of nitric oxide to nitrite, which assists in the efficient oxidation of hydroxylamine, the second step in ammonia oxidation. These results suggest that NirK is not a valid genetic marker for measuring denitrification activity in natural systems since the expression of this gene will be a product of
both nitrifying and denitrifying processes. The results from this study have been submitted to the Journal of Bacteriology for publication. The metabolic activity of Nitrosospira multiformis, a representative of the most common terrestrial genus of ammonia oxidizing bacteria, was compared to the activity of N. europaea. N. multiformis had a slowed rate of ammonia oxidation but a similar rate of hydroxylamine oxidation compared to N. europaea. This difference was especially acute with changes in pH and growth phase of the bacteria. We found that N. multiformis maintains 2 copies of the hao gene, which encodes hydroxylamine oxidoreductase. In contrast, previous studies show that N. europaea has 3 copies of hao. We are currently sequencing the hao genes of N. multiformis to compare similarities between the two copies and to the hao genes of N. europaea. Because hao is a specialized gene unique to ammonia oxidizers, we are investigating its use as a molecular probe for ecological studies
of nitrifiers. This data will be presented at the 2003 American Society for Microbiology General Meeting. We have applied quantitative polymerase chain reaction (qPCR) to enumerate populations of AOB along a nitrogen gradient in forest soils of the San Bernardino Mountains, Southern California. We used a previously published primer and probe set to amplify the 16S rRNA gene from AOB. Using different concentrations of genomic DNA from N. europaea and N. multiformis, we generated highly repeatable standard curves. We enumerated the relative abundance of AOB in the total DNA pool extracted from the forest soils. The activity, diversity, and abundance of AOB species varied along the nitrogen gradient, as expected. This data is in preparation for submission to Applied and Environmental Microbiology and will be presented at the 2003 ASM General Meeting. Current work includes the maintenance of 4 methanotrophic bacterial (MOB) strains. Ongoing studies involve determining the effects of
nitrogen on the ability of MOB to consume methane and produce nitrous oxide. Primers and probes for qPCR to enumerate 16S rRNA genes of MOB have been designed and are being optimized.
Impacts
The nitrogen cycle has been wildly altered from anthropogenic inputs, especially from agriculture and industry. Our goal is to determine how soil bacteria involved in nitrogen transformations are impacted by human activities, specifically in their production and consumption of greenhouse gases. Our research indicates that traditional views of denitrification and denitrifying enzymes do not hold true for ammonia oxidizing bacteria (AOB). Also, the unique genetic content of individual ammonia oxidizing species translates into altered rates of activity. Thus, the creation of unifying assumptions regarding ammonia oxidizers and their activities in the environment is becoming more confounding. However, we are narrowing the possibilities for how AOB, and likely methane oxidizing bacteria (MOB), generate greenhouse gases in nitrogen impacted soils. Along this line, we are rapidly developing a suite of techniques to enumerate and measure specific populations and activities of
AOB and MOB in soils. Our approach combines detailed analyses of pathways and genetic components of AOB and MOB. Our results have already changed our initial assumptions regarding how these organisms actually function in nature.
Publications
- No publications reported this period
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