COLLABORATIVE RESEARCH: HOW ARCHAEAL DIVERSITY, ABUNDANCE AND FUNCTION ARE REGULATED IN AGROECOSYSTEMS?

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: NRI COMPETITIVE GRANT
• Reporting Frequency: Annual
• Accession No.: 0215197
• Grant No.: 2008-35319-04720
• Proposal No.: 2008-04411
• Project Start Date: Sep 1, 2008
• Project End Date: Aug 31, 2013
• Grant Year: 2008
• Program Code: [51.8A]- Microbial Biology (A): Microbial Observatories

Recipient Organization
OREGON STATE UNIVERSITY
(N/A)
CORVALLIS,OR 97331

Performing Department
BOTANY AND PLANT PATHOLOGY

Non Technical Summary
Archaea are both ubiquitous and abundant in soil. Soil archaea were first discovered in the mid-1990?s. Although there are numerically fewer bacteria than archaea in soil, there is an increasing amount of evidence suggesting that the relative abundance of archaea may be far higher in cultivated soils than in uncultivated soils. The recognition that some Archaea are capable of ammonia oxidation sparked a revolution in our understanding of the distribution of archaeal ammonia oxidizers and shattered an over century-old dogma that ammonia oxidation belonged solely in the bacterial kingdom. However, a major gap in our understanding of archaeal ammonia oxidation is that we do not know the relative rates of ammonia oxidation in situ by ammonia oxidizing archaea relative to the rates by the ammonia oxidizing bacteria. Population studies could be presenting a greatly skewed picture of the relative contributions of each type if one or the other has a much higher activity per unit. This proposal contributes to improve our understanding of soil ammonia oxidizing archaea and the factors regulating their abundance, diversity, and functional capabilities in agricultural soils.

Animal Health Component

20%

Research Effort Categories

Basic

60%

Applied

20%

Developmental

20%

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
101	0110	1100	33%
101	4010	1100	34%
101	4099	1100	33%

Knowledge Area
101 - Appraisal of Soil Resources;

Subject Of Investigation
4099 - Microorganisms, general/other; 0110 - Soil; 4010 - Bacteria;

Field Of Science
1100 - Bacteriology;

Keywords

soil archaea

archaea abundance

ammonia oxidation

ammonia monooxygenase

Goals / Objectives
The research focus is to determine the ammonia oxidizing activity of soil Archaea compared to the ammonia oxidizing bacteria. The archaea currently remain a highly mysterious group of soil organisms that may be particularly important in agroecosystems. Archaea have been found in nearly every type of soil that has been examined, regardless of soil organic matter content, soil depth, or oxygen availability. The main goal of this proposal is a) to measure the relative contributions of ammonia oxidizing Archaea and ammonia oxidizing bacteria to ammonia oxidation in soils and b) to test our hypothesis that ammonia oxidation activity in cultivated soils is primarily due to the activity of ammonia oxidizing Archaea. Characterization of the differential ammonia oxidizing activities through inactivators, inhibitors, and substrates will provide a basis for discriminatory assays and provide information about the diversity of ammonia oxidation by ammonia oxidizing archaea and ammonia oxidizing bacteria. The project will produce cultures with ammonia oxidizing activity that are enriched in archaea and devoid of bacteria. The project will find properties to discriminate between archaeal and bacterial ammonia oxidizers.

Project Methods
For enrichment/Isolation: We will produce cultures with ammonia-oxidizing activities that are enriched in archaea and devoid of bacteria. We will characterize these cultures once they are established. Growth yields (mg biomass produced/mmol ammonia consumed) and growth rates will be determined. Rates of ammonia oxidation and nitrite or nitrate production will be measured. Highly enriched cultures or pure cultures will be subjected to shotgun sequencing (e.g. high throughput Illumina/Solexa sequencing at OSU) and the DNA sequences will be analyzed using BLAST in an effort to find additional clues to the physiology and growth requirements of these bacteria. For differential activities determination: Once we have active ammonia-oxidizing archaeal cultures devoid of ammonia-oxidizing bacteria, they will be used to search for properties that discriminate between archaeal and bacterial ammonia oxidizers. We will search for compounds that inactivate one ammonia oxidizer type, but not the other. We will search for inhibitors that unlike inactivators, are reversible and must remain with the sample during the assay. Inhibitors provide another potential mechanism to discriminate ammonia oxidizing bacteria and ammonia oxidizing Archaea AMO (ammonia monooxygenase) activities. We will search for substrates that are discriminatory. Discriminatory substrates would have the advantage that each activity could be measured independently. We will research discriminatory stable isotope effects between archaeal and bacterial ammonia oxidation. Potential differences would provide a mechanism to discriminate the relative contributions of each process in the field.

Progress 09/01/08 to 08/31/13

Outputs
Target Audience: Scientific community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Three undergraduate students and one postdoctoral trainee. How have the results been disseminated to communities of interest? The results from this proposal were presented in the form of posters and invited talks to local, national, international scientific meetings. The results were disseminated through peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? Aliphatic n-alkynes discriminated soil nitrification activities of AOA and AOB. Ammonia-oxidation by N. maritimus was not inhibited by octyne, but ammonia-oxidation of the AOB Nitrosomonas europaea and Nitrosospira multiformis was quickly and irreversibly inactivated. The nitrification carried out by soil-borne AOA was also insensitive to octyne. Cycloheximide (CHX) also differentiated the relative contributions of AOA and AOB to ammonia oxidation. CHX prevented in N. maritimus de novo polypeptide synthesis and recovery from acetylene (C2H2) inhibition. CHX, but not kanamycin and gentamycin, inhibited the recovery of ammonia-oxidizing activity in N.maritimus. CHX prevented the incorporation of 14CO2-labeling into cellular proteins (i.e. inhibited protein synthesis) in N. maritimus. The combination of C2H2 inactivation followed by recovery of ammonia oxidation, either in the presence of bacteria-selective protein synthesis inhibitors, or CHX, might be used to estimate the relative contributions of AOB and AOA to nitrification in natural environments. NH2OH oxidation and subsequent nitric oxide production by N. maritimus were demonstrated. The enzyme complex for the oxidation of NH2OH is not known in AOA. We showed in N.maritimus that NH2OH was both produced and consumed during the oxidation of ammonia to NO2-; that consumption was coupled to energy conversion; and that NH2OH was the most probable product of the archaeal AMO homolog. Initial oxidation of NH3 by bacteria and archaea appears mechanistically similar. They diverge, however, biochemically at the point of oxidation of NH2OH, the archaea possibly catalyzing NH2OH oxidation using a novel enzyme complex that is different to that in AOB.

Publications

• Type: Conference Papers and Presentations Status: Accepted Year Published: 2009 Citation: Michelle Land, Dr. Neeraja Vajrala, Luis Sayavedra-soto, Daniel Arp. Can you differentiate the Ammonia Oxidizers in Dirt? Poster-talk at the Subsurface Biosphere Initiative Workshop at OSU Sep 2009.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2009 Citation: Neeraja Vajrala, Anne E. Taylor, Dan Arp, Sandra Dooley, David D. Myrold and Peter J. Bottomley. Identifying the relative contributions of ammonia-oxidizing archaea and bacteria to nitrification: Pure culture and Environmental approaches. Talk at the Subsurface Biosphere Initiative Workshop at OSU Sep 2009.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2010 Citation: Spencer Whitter, Neeraja Vajrala, Luis Sayavedra-Soto, Daniel J. Arp. Can Ammonia Oxidizing Archaea (AOA) utilize Hydroxylamine as substrate? Poster-talk at the Subsurface Biosphere Initiative Workshop at OSU. Sep 2010.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Neeraja Vajrala, Luis A. Sayavedra-Soto, David A Stahl, Peter J Bottomley, Daniel J Arp Discriminatory inhibition of protein synthesis in ammonia-oxidizing bacteria and archaea At the Annual Meeting of the Center for Genome Research and Bioinformatics, OSU. Poster abstract 2012.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Neeraja Vajrala, Willm Martens-Habbena, Luis A. Sayavedra-Soto, Andrew Schauer, Peter J. Bottomley, David A. Stahl, and Daniel J. Arp. Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea. Microbiology Colloquium at OSU. Jan 2013.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Anne E. Taylor, Neeraja Vajrala, Andrew T. Giguere, David D. Myrold, Luis Sayavedra-Soto, and Peter J. Bottomley. Discriminating between the contributions of ammonia oxidizing archaea and bacteria to soil nitrification with linear C2  C9 alkynes. Microbiology Colloquium at OSU. Jan 2013.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Neeraja Vajrala, Luis A. Sayavedra-Soto, David A Stahl, Daniel J Arp, Peter J Bottomley. Hydroxylamine oxidation and subsequent nitric oxide production by the archaeal ammonia oxidizer Nitrosopumilus maritimus. Talk and poster presentation at the International Conference on Nitrification ICON3 in Tokyo Japan. Sep 2013.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2010 Citation: N. Vajrala, L. A. Sayavedra-Soto, W. Martens-Habbena, D. A. Stahl, P. J. Bottomley, D. J. Arp. Discrimination of ammonia oxidizing activity by bacteria and archaea: A pure culture approach. American Society for Microbiology General Meeting. May 2010
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: Anne E. Taylor, Neeraja Vajrala, Andrew T. Giguere, Luis Sayavedra-Soto, David D. Myrold, and Peter J. Bottomley. Discriminating between the contributions of ammonia oxidizing archaea and bacteria to soil nitrification. At the International Meeting ISME Aug 2012.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2011 Citation: Neeraja Vajrala, Willm Martens-Habbena, Luis A. Sayavedra-Soto,David A Stahl, and Daniel J Arp. Nitrosopumilus maritimus oxidizes hydroxylamine to nitrite. Oregon State University. Abstract-poster to the International Conference in Nitrification ICoN2 at the Netherlands. July 2011.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Use of aliphatic n-alkynes to discriminate soil nitrification activities of ammonia-oxidizing thaumarchaea and bacteria. Anne E. Taylor, Neeraja Vajrala, Andrew T. Giguere, Alix I. Gitelman, Daniel J. Arp, David D. Myrold, Luis Sayavedra-Soto, and Peter J. Bottomley. Talk and poster presentation at the International Conference on Nitrification ICON3 in Tokyo Japan 2013.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Peter J. Bottomley, Anne E. Taylor, Neeraja Vajrala, Andrew T. Giguere, Daniel Arp, David D. Myrold, and Luis Sayavedra-Soto. Discriminating between archaeal and bacterial contributions to soil nitrification. Invited Talk at Technicon University, Haifa, Israel. April, 2013.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2013 Citation: Anne E. Taylor, Neeraja Vajrala, Andrew T. Giguere, Alix I. Gitelman, Daniel J. Arp, David D. Myrold, Luis Sayavedra-Soto, and Peter J. Bottomley. Use of aliphatic n-alkynes to discriminate soil nitrification activities of ammonia-oxidizing thaumarchaea and bacteria. Talk and poster presentation at the International Conference on Nitrification ICON3 in Tokyo Japan 2013.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Vajrala N, Martens-Habbena W, Sayavedra-Soto LA, Schauer A, Bottomley PJ, Stahl DA, Arp DJ. Hydroxylamine as an intermediate in ammonia oxidation by globally abundant marine archaea. Proc. Natl. Acad. Sci. U S A. 2013 Jan 15;110(3):1006-11.
• Type: Conference Papers and Presentations Status: Published Year Published: 2013 Citation: Use of aliphatic n-alkynes to discriminate soil nitrification activities of ammonia oxidizing. Taylor AE, Vajrala N, Giguere AT, Gitelman AI, Arp DJ, Myrold DD, Sayavedra-Soto L, Bottomley PJ. Use of aliphatic n-alkynes to discriminate soil nitrification activities of ammonia-oxidizing thaumarchaea and bacteria. Applied Environmental Microbiology. 2013.79(21):6544-51.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Zhalnina K, de Quadros PD, Gano KA, Davis-Richardson A, Fagen JR, Brown CT, Giongo A, Drew JC, Sayavedra-Soto LA, Arp DJ, Camargo FA, Daroub SH, Clark IM, McGrath SP, Hirsch PR, Triplett EW. Ca. Nitrososphaera and Bradyrhizobium are inversely correlated and related to agricultural practices in long-term field experiments. Frontiers in Microbiology. 2013 May 1; 4:104.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2013 Citation: Neeraja Vajrala, Peter J. Bottomley, David A. Stahl, Daniel J. Arp, Luis A. Sayavedra-Soto. Cycloheximide prevents the de novo polypeptide synthesis required to recover from acetylene inhibition in Nitrosopumilus maritimus. FEMS Microbial Ecology Dec 2013. In Press.

Progress 09/01/11 to 08/31/12

Outputs
Target Audience: Scientific community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One undergraduate student and one postdoctoral trainee. How have the results been disseminated to communities of interest? The results from this proposal were presented in the form of posters and invited talks to local, national, international scientific meetings. The results were disseminated through peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Toward the elucidation of the metabolism of ammonia oxidation in AOA: Hydroxylamine oxidation and subsequent nitric oxide production by the archaeal ammonia oxidizer Nitrosopumilus maritimus. Although we were able to demonstrate that the stoichiometry of NO2- accumulation and O2 uptake during NH2OH oxidation by N. maritimus are coupled initially with longer incubation times, the rate of NH2OH oxidation in N.maritimus decreases gradually and the stoichiometry between NO2- accumulation and NH2OH consumption is lost. NH2OH rapidly disappears from the N. maritimus media, but the NO2- accumulation, O2 uptake do not follow suit. Interestingly, the NH2OH oxidation and NO2- accumulation resume when the N. maritimus cells are moved to fresh media containing NH2OH. Likely a by-product of NH2OH oxidation inhibits the further oxidation of NH2OH. To determine whether both NO and N2O are by-products of NH2OH oxidation and whether they could inhibit further oxidation of NH2OH, we monitored generation of 15NO and 15N2O during incubation of N. maritimus cells with 15N-labeled NH2OH. Our results indicated that NO is a direct by-product of NH2OH oxidation and is subsequently converted to N2O. 2. Toward the discrimination between the ammonia-oxidizing activities of AOA and AOB: Cycloheximide prevents the de novo polypeptide synthesis required to recover from acetylene inhibition in Nitrosopumilus maritimus. We developed a potential method to differentiate the relative contributions of ammonia-oxidizing thaumarchaea (AOA) and ammonia-oxidizing bacteria (AOB) to ammonia (NH3) oxidation. We investigated the effects of specific bacteria- and eukaryote-selective protein synthesis inhibitors on the recovery of acetylene (C2H2) inactivated NH3 oxidation in the marine AOA Nitrosopumilus maritimus and compared the results with recovery of the AOB Nitrosomonas europaea. Cycloheximide (CHX), but not kanamycin and gentamycin, inhibited the recovery of NH3-oxidizing activity in N.maritimus. CHX prevented the incorporation of 14CO2-labeling into cellular proteins providing further evidence that CHX acts as a protein synthesis inhibitor in N.maritimus. The combination of C2H2 inactivation followed by recovery of NH3 oxidation either in the presence of bacteria-selective protein synthesis inhibitors, or CHX, might be used to estimate the relative contributions of AOB and AOA to nitrification in natural environments.

Publications

Progress 09/01/10 to 08/31/11

Outputs
Target Audience: Scientific community Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One undergraduate student and one postdoctoral trainee. How have the results been disseminated to communities of interest? The results from this proposal were presented in the form of posters and invited talks to local, national, international scientific meetings. The results were disseminated through peer-reviewed publications. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? 1. Toward the elucidation of the metabolism of ammonia oxidation in AOA: Hydroxylamine oxidation and subsequent nitric oxide production by the archaeal ammonia oxidizer Nitrosopumilus maritimus. Although we were able to demonstrate that the stoichiometry of NO2- accumulation and O2 uptake during NH2OH oxidation by N. maritimus are coupled initially with longer incubation times, the rate of NH2OH oxidation in N.maritimus decreases gradually and the stoichiometry between NO2- accumulation and NH2OH consumption is lost. NH2OH rapidly disappears from the N. maritimus media, but the NO2- accumulation, O2 uptake do not follow suit. Interestingly, the NH2OH oxidation and NO2- accumulation resume when the N. maritimus cells are moved to fresh media containing NH2OH. Likely a by-product of NH2OH oxidation inhibits the further oxidation of NH2OH. To determine whether both NO and N2O are by-products of NH2OH oxidation and whether they could inhibit further oxidation of NH2OH, we monitored generation of 15NO and 15N2O during incubation of N. maritimus cells with 15N-labeled NH2OH. Our results indicated that NO is a direct by-product of NH2OH oxidation and is subsequently converted to N2O. 2. Toward the discrimination between the ammonia-oxidizing activities of AOA and AOB: Cycloheximide prevents the de novo polypeptide synthesis required to recover from acetylene inhibition in Nitrosopumilus maritimus. We developed a potential method to differentiate the relative contributions of ammonia-oxidizing thaumarchaea (AOA) and ammonia-oxidizing bacteria (AOB) to ammonia (NH3) oxidation. We investigated the effects of specific bacteria- and eukaryote-selective protein synthesis inhibitors on the recovery of acetylene (C2H2) inactivated NH3 oxidation in the marine AOA Nitrosopumilus maritimus and compared the results with recovery of the AOB Nitrosomonas europaea. Cycloheximide (CHX), but not kanamycin and gentamycin, inhibited the recovery of NH3-oxidizing activity in N.maritimus. CHX prevented the incorporation of 14CO2-labeling into cellular proteins providing further evidence that CHX acts as a protein synthesis inhibitor in N.maritimus. The combination of C2H2 inactivation followed by recovery of NH3 oxidation either in the presence of bacteria-selective protein synthesis inhibitors, or CHX, might be used to estimate the relative contributions of AOB and AOA to nitrification in natural environments.

Publications

• Type: Other Status: Accepted Year Published: 2012 Citation: Neeraja Vajrala, Luis A. Sayavedra-Soto, David A Stahl, Peter J Bottomley, Daniel J Arp Discriminatory inhibition of protein synthesis in ammonia-oxidizing bacteria and archaea At the Annual Meeting of the Center for Genome Research and Bioinformatics, OSU. Poster abstract 2012.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2012 Citation: " Anne E. Taylor, Neeraja Vajrala, Andrew T. Giguere, Luis Sayavedra-Soto, David D. Myrold, and Peter J. Bottomley. Discriminating between the contributions of ammonia oxidizing archaea and bacteria to soil nitrification. At the International Meeting ISME Aug 2012.
• Type: Conference Papers and Presentations Status: Accepted Year Published: 2011 Citation: Neeraja Vajrala, Willm Martens-Habbena, Luis A. Sayavedra-Soto,David A Stahl, and Daniel J Arp. Nitrosopumilus maritimus oxidizes hydroxylamine to nitrite. Oregon State University. Abstract-poster to the International Conference in Nitrification ICoN2 at the Netherlands. July 2011.

Progress 09/01/09 to 08/31/10

Outputs
OUTPUTS: We used N-alkynes (C2 to C10) on cultures of Nitrosomonas europaea (ammonia oxidizing Bacteria (AOB)) and Nitrosopumilus maritimus (ammonia oxidizing Archaea (AOA)) to test whether ammonia oxidation in AOB or AOA was affected differently. The C2 to C10 alkynes inhibited O2 consumption in N. europaea in a time-dependent fashion and were specific for ammonia oxidation. In this bacterium the higher alkynes (C6 to C10) inhibited O2 consumption, more potently than the lower alkynes. N. maritimus was unable to carry out ammonia oxidation in presence of C2-C5 n-alkynes, but unlike N. europaea, N. maritimus was able to carry out ammonia oxidation in presence of C6-C10 n-alkynes. While the O2 uptake activity of N. europaea was 88% inhibited in presence of 0.5 uM 1-Heptyne, N. maritimus O2 uptake was only 40% inhibited. We are also testing whether hydroxylamine can be used as a substrate by AOA. Hydroxylamine is an intermediate in the oxidation of ammonia to nitrite by ammonia oxidizing bacteria (AOB). This process is carried out by ammonia monooxygenase enzyme encoded by the amoABC genes and by the hydroxylamine oxidoreductase enzyme encoded by the hao gene. Although homologs for all three amo genes were identified in ammonia oxidizing archaea (AOA) such as N. maritimus, hao gene homolog has not been identified so far. The identification of amo gene sequences and the absence of hao suggest that the AOA's might use alternative mechanisms and/or intermediates for oxidation of ammonia to nitrite. N. europaea can oxidize hydroxylamine to nitrite either in the presence or absence of ammonia. N. maritimus (the AOA) can oxidize hydroxylamine to nitrite only at very low concentrations. Higher concentrations of hydroxylamine appear to inhibit nitrite formation in N. maritimus. Moreover, it appears that hydroxylamine completely inhibits N. maritimus capability to oxidize ammonia. N. maritimus ability to oxidize small amounts of hydroxylamine was inhibited by acetylene unlike in N. europaea. The ammonia oxidation in soils we have tested appears to be stimulated by hydroxylamine. Using molecular techniques, we have identified amoA gene products of AOB but not AOA in these soils. Our results indicate that bacterial ammonia oxidizer communities but not archaeal communities are stimulated by hydroxylamine. We now have a soil ammonia oxidizing enrichment that grows at 40C. Molecular techniques have demonstrated that this enrichment mostly contains ammonia oxidizing bacteria and little ammonia oxidizing archaea. This enrichment shows sensitivity to bacterial antibiotics such as kanamycin, gentamycin and neomycin but not to eukaryotic antibiotic such as cycloheximide that is shown to inhibit N. maritimus, an AOA. The ammonia oxidation activity of this enrichment is inhibited by acetylene and also recovers from acetylene inactivation once it is removed just like N. europaea and N. maritimus. PARTICIPANTS: Daniel J. Arp: principal investigator/project director. Luis A. Sayavedra-Soto: co-principal investigator. Neeraja Vajrala: post-doctoral fellow. Spencer Witter: undergraduate student. TARGET AUDIENCES: Not relevant to this project. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Our results indicated that the higher n-alkynes could estimate the nitrification potential of either bacteria or archaea in situ. These findings are being prepared for publication. We have several enrichments of AOA, some of which coexist with AOB and nitrite oxidizing bacteria (NOB). We have now a potential experimental system to probe for interaction among AOA, AOB and NOB during co-culture.

Publications

• No publications reported this period

Progress 09/01/08 to 08/31/09

Outputs
OUTPUTS: The laboratory maintains a culture of the chemolithoautotrophic ammonia oxidizing crenarchaeota Nitrosopumilus maritimus using Dr. Stahl's lab protocols. The cultures grow in filtered fresh synthetic crenarchaeota medium (1 mM ammonia; 30C; 10% inoculums) to a maximal cell density of 1.4 X 10E07 per ml with a generation time of ~24 h. We are using growth inhibitors on Nitrosomonas europaea (ammonia oxidizing Bacteria (AOB)) and N. maritimus (ammonia oxidizing Archaea (AOA)) to test whether the ammonia oxidation activities of AOB and AOA are affected differentially. N. maritimus tolerated up to 200 ug/ml sulfadiazine (SDZ) whereas N. europaea was inhibited by 2 ug/ml SDZ. Growth of N. maritimus was unaffected in up to 1000 ug/ml bacterial protein synthesis inhibitors (gentamycin, kanamycin, neomycin) whereas growth of N. europaea was inhibited by ~ 100 ug/ml of these protein synthesis inhibitors. Growth of N. maritimus was inhibited in presence of 200 ug/ml cycloheximide (eukaryotic protein synthesis inhibitor) whereas cycloheximide did not inhibit the growth of N. europaea at the concentrations tested. Acetylene inactivation and recovery experiments with N. maritimus cultures in presence or absence of bacterial and eukaryotic protein synthesis inhibitors are being carried out. Acetylene is a mechanism-based inactivator of ammonia monooxygenase (AMO) in N. europaea. Similarly 0.025% of acetylene in the headspace of N. maritimus cultures inhibited nitrite formation. Theses cultures recovered nitrite production ~2 h after acetylene removal. Acetylene apparently is a suicide substrate of the AMO of N. maritimus as well. No recovery was observed when 200 ug/ml cycloheximide was added to N. maritimus cultures after removal of acetylene indicating that de novo synthesis of AMO was required to oxidize ammonia after acetylene inactivation. As an autotrophic crenarchaeota, N. maritimus was also capable to incorporate Na14CO3 efficiently in overnight cultures. We tested the effect of methylhydrazine (a suicide inactivator of hydroxylamine oxidoreductase (HAO) of N. europaea) on growth of N. maritimus cultures. Growth of both N. europaea and N. maritimus cultures was inhibited by 5 uM methylhydrazine, indicating that methylhydrazine is not a suitable candidate to differentiate AOB and AOA. We are producing soil ammonia oxidizing archaeal enrichments. Molecular techniques have demonstrated that ammonia oxidizing archaea are prevalent in some soil environments. To date, there are only 2 marine AOA isolates. In an attempt to isolate soil AOA, we are screening enrichment cultures with 1 mM NH3 in presence and absence of bacterial antibiotics and SDZ using various soils. The enrichments are being screened by nitrite accumulation. Enrichment cultures with nitrite formation in presence of antibiotics were transferred (10%) into fresh NH3 media. After repeated transfers, enrichment cultures were screened for amoA gene by PCR using bacterial and crenarchaeal primers. To date we have one enrichment containing both AOA and AOB and is being transferred to 1mM NH3 media containing bacterial antibiotics in an attempt to isolate the AOA. PARTICIPANTS: The Investigators Arp D. and Sayavedra-Soto L. are engaged in all aspects of the project. Postdoctoral Associate Vajrala N. maintained the cultures, carried out the inhibition experiments and initiated the culture enrichments. Undergraduate Land M. during the Summer of 2009 participated in screening experiments to enrich AOA and in inhibition experiments as part of her training. TARGET AUDIENCES: Nothing significant to report during this reporting period. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Protein synthesis inhibitors, cycloheximide in combination with acetylene, and SDZ have potential to differentiate the activities of AOA and AOB in soils. Incorporation of radiolabeled C in N. maritimus can be used to detect de novo synthesized peptides after acetylene inactivation and recovery. We have a potential enrichment of soil AOA.

Publications

• No publications reported this period