GENETIC CHANGES AND ADAPTATION OF MICROBIAL COMMUNITIES TO THEIR ENVIRONMENT

• Sponsoring Institution: National Institute of Food and Agriculture
• Project Status: COMPLETE
• Funding Source: HATCH
• Reporting Frequency: Annual
• Accession No.: 0167668
• Project Start Date: Oct 1, 2011
• Project End Date: Sep 30, 2016
• Program Code: [(N/A)]- (N/A)

Recipient Organization
PURDUE UNIVERSITY
(N/A)
WEST LAFAYETTE,IN 47907

Performing Department
Agronomy

Non Technical Summary
Few if any investigations have directly coupled fundamental or "basic" molecular genetic research on microbial community dynamics to "mission-based" research on soil environmental problems. With the advancement of molecular genetic methods we can now begin to comprehensively study the dynamics and diversity of the resident soil microbial communities in order to advance our understanding of mechanisms involved in bacterial adaptation to the environment and ultimately optimize their management. In order to understand and manage obstacles of ecosystem productivity, we must first understand the ecology and community dynamics of the responsible biota. Efforts to study the relevant microorganisms controlling these processes have been severely hampered by a lack of methodology to directly profile microbial communities. Improved in-situ microbial characterization will provide more accurate assessment of the impact of anthropogenic technologies on indigenous microbial populations and the major functions they carry out.

Animal Health Component

(N/A)

Research Effort Categories

Basic

(N/A)

Applied

(N/A)

Developmental

(N/A)

Classification

Knowledge Area (KA)	Subject of Investigation (SOI)	Field of Science (FOS)	Percent
102	4099	1040	10%
102	4099	1070	10%
102	4099	1100	10%
133	4099	1040	10%
133	4099	1070	20%
133	4099	1100	10%
724	4099	1040	10%
724	4099	1070	10%
724	4099	1100	10%

Knowledge Area
724 - Healthy Lifestyle; 133 - Pollution Prevention and Mitigation; 102 - Soil, Plant, Water, Nutrient Relationships;

Subject Of Investigation
4099 - Microorganisms, general/other;

Field Of Science
1040 - Molecular biology; 1070 - Ecology; 1100 - Bacteriology;

Keywords

microbial community

16s rrna gene

bacteria

agricultural productivity

metal contamination

degradation of pollutants

soil

human/animal microbiome

microbe plant interactions

Goals / Objectives
The goals of this research are to determine and compare the structure, composition and adaptive mechanisms of microbial communities in perturbed environments. With the advancement of molecular genetic methods we are now beginning to comprehensively study the dynamics and diversity of microbial communities in order to advance our understanding of mechanisms involved in bacterial adaptation to their environment. Understanding of the physiological and genetic diversity of microbes has the potential to expand our view of the functional diversity of microbial communities. This knowledge can them be used to employ these bacteria for the benefit of humankind.

Project Methods
Methods that have been used previously will continued to be used in the renewal of this proposal. Changes in microbial community structure and composition resulting from exposure to a variety of perturbations are being studied using traditional microbiological and molecular genetics techniques. Changes in microbial community structure are being determined using genetic fingerprinting methods such as PCR-DGGE, whereas compositional changes are being determined using both low and high throughput 16S rRNA gene sequencing. Activity of specific functional genes will be quantified using quantitative PCR. To address ecologically significant questions a variety of statistical methods are being used to analyze and compare community structure and compositional data with environmental data. Specific populations that appear to be functionally important members of these perturbed community will be studied in greater detail. These specific populations will be cultivated (if possible) and studied using physiological and genetic approaches. Traditionally, only a fraction of microorganisms from the environment can be cultivated but some innovative approaches are now available that has greatly improved our success at "culturing the unculturable". Examples of perturbed ecosystems being studied are metal and/or hydrocarbon (pesticides and haloaromatic compounds) contaminated soils, environments exposed to human and animal waste, agronomically important crops, and human/animal microbiomes.

Progress 10/01/11 to 09/30/16

Outputs
Target Audience:Target audiences include all individuals and industries interested in the well-being of the environment and/or health of humans and animals. For example, researchers, industries or members of the public who are interested in using or understanding the role of microbial communities to maintain and contribute to a healthy environment. With respect to human or animal health, those interested in improving health or preventing disease through the consumption of healthy diets, especially diets that include microbially fermented fibers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I am co-advising one PhD level graduate student, who is just beginning her fourth year in the PULSe program. I am also co-mentoring a student in the newly established Post-baccalaureate Research and Education Program (PREP) at Purdue. Informal teaching and mentoring, mainly in microbial community analysis, was provided to graduate students and researchers from the programs of other collaborators. During the summer of 2016 worked on research planning, data analysis and manuscripts with researchers at the US-Geological Survey Lake Michigan Ecological Research Station. How have the results been disseminated to communities of interest?Dissemination of findings to the community includes publications in topic relevant journals, presentation at conferences and invited presentations given to academic, government and industrial research groups. What do you plan to do during the next reporting period to accomplish the goals?Continue on the same lines of research

Impacts
What was accomplished under these goals? Numerous approaches are being used to gain a better understanding of the role microorganisms in the environment. My laboratory has worked extensively to apply new molecular genetic tools for the interrogation of various environments. This has led to collaboration with researchers both within and outside of Purdue. For instance, in a publication with a faculty member at Clemson University we used a genetic fingerprinting method to investigate differences in microbial communities in soils following the invasion of Japanese knotweed (Polygonium cuspidatum) at locations in Eastern U.S and how it related to composition of carbon compounds excreted by the plant. I have continued research on the impact of invasive plant species on rhizospheres microbial communities with a new collaborator (Noel Palvolic) at the USGS. Also, a collaborative project with the water research group (led by Murulee Byappanahalli and Meredith Nevers) at the USGS was started. The objective of this project is to determine the impact of mitigations done on Lake Michigan to reduce beach closures due to water contamination. This work led to a poster of this study determining whether nearshore and adjacent beach microbial contamination was from riverine sources that was presented at the International Society for Microbial Ecology meeting. In addition to soil and water microbial community analysis my group has a number of different collaborations to determine the influence of diet/disease on intestinal microbiomes. In a collaborative project that I have continued with Yava Jones-Hall on the role of the microbiome in Crohn's Disease and we published a perspective on the research we have done thus far. Also funding from a industrial sponsor helps to continue a collaboration with and Connie Weaver in Nutrition Science on dietary fiber and Ca absorption.

Publications

• Type: Journal Articles Status: Published Year Published: 2016 Citation: Jones-Hall, Y.L. and C. H. Nakatsu. 2016. The intersection of TNF, IBD and the microbiome. Gut Microbes 7(1):58-62
• Type: Journal Articles Status: Published Year Published: 2016 Citation: Suseela, V., P. Alpert, C. H. Nakatsu, A. Armstrong and Tharayil, N. 2016. Plant-soil interactions regulate the identity of soil carbon in invaded ecosystems: implication for legacy effects. Functional Ecology 30:1227-1238.
• Type: Journal Articles Status: Accepted Year Published: 2016 Citation: Lu, H., H. Yan, R. Potu, M. G. Ward, C. C. Pelkman, O. Adeola, C. H. Nakatsu and K. M. Ajuwon. 2016. Effects of dietary resistant starch content on metabolic status, milk composition, and microbial profile in lactating sows and on offspring performance. Journal of Animal Physiology and Animal Nutrition
• Type: Books Status: Published Year Published: 2016 Citation: Yates, M.V., R. Miller, C.H. Nakatsu, and S. Pillai (editors). 2016. Manual for environmental microbiology, 4th Edition. ASM Press, Washington D. C.
• Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Nakatsu, C.H., M. Byappanahalli, and M. Nevers. Impact of rivers on near and offshore microbial communities. The 14th International Symposium on Microbial Ecology Abstracts. Montreal, Canada (August 2016)

Progress 10/01/14 to 09/30/15

Outputs
Target Audience:Target audiences include all individuals and industries interested in the well-being of the environment and/or health of humans and animals. For example, researchers, industries or members of the public who are interested in using or understanding the role of microbial communities to maintain and contribute to a healthy environment. With respect to human or animal health, those interested in improving health or preventing disease through the consumption of healthy diets, especially diets that include microbially fermented fibers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?I am co-advising one PhD level graduate student, who is just beginning her third year in the PULSe program. Informal teaching and mentoring, mainly in microbial community analysis, was provided to graduate students and researchers from the programs of other PIs. During the summer of 2015 worked on research planning, data analysis and manuscripts with a number of students and post doctoral researchers in the Key Laboratory of Alpine Ecology and Biodiversity, while visiting the Chinese Academy of Sciences in Beijing. Assistance in manuscript writing continued after returning to Purdue. How have the results been disseminated to communities of interest?Dissemination of findings to the community includes publications in topic relevant journals, presentation at conferences and invited presentations given to academic, government and industrial research groups What do you plan to do during the next reporting period to accomplish the goals?Continue on the same lines of research

Impacts
What was accomplished under these goals? Numerous approaches are being used to gain a better understanding of the role microorganisms in the environment. My laboratory has worked extensively to apply new molecular genetic tools for the interrogation of various environments. In addition to soil microbial community analysis my group has a number of different collaborations to determine the influence of diet on intestinal microorganisms. Published results from a collaboration with faculty/research scientists/students at Hokkaido University in Japan. Our main effort in this project has been placed on the influence of growth media and the addition of growth promoting factors on the novelty of bacteria that can be grown. I also continued research in the impact of diet on microbial communities in humans and animal model systems have continued and one manuscript from this project was published this year. This manuscript describes a clinical study testing the addition of dietary fiber product (soluble corn fiber) on relationship between gut microbiota composition and increased calcium absorption/retention in adolescents. This work also led to a joint patent between the industrial sponsor of this work (Tate & Lyle) and Purdue research leaders (Connie Weaver and Cindy Nakatsu). Began a new cooperation with a group in China this year in which we have begun determining the diversity of microbes in different alpine soil and water environments and factors affecting diversity and distribution.

Publications

• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Whisner, C.M., B.R. Martin, C.H. Nakatsu, and C.M. Weaver. The case of the non-responder in prebiotic-induced calcium absorption in adolescents. The 9th International Symposium on Nutritional Aspects of Osteoporosis. Montreal, Canada (June 2015).
• Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Kozik, A. J., Jones-Hall, Y. L. and C. H. Nakatsu. Tumor necrosis factor modulates the gut microbiota and drives colitis. Meeting of the Federation for American Society of Experimental Biology. FASEB J. Boston, MA (April 2015).
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Puspita, I.D., W. Kitagawa, Y, Kamagata, M. Tanaka and C. H. Nakatsu. 2015. Increase in bacterial colony formation from permafrost ice wedge dosed with a Tomitella biformata recombinant resuscitation promoting factor protein. Microbes Environ. 30(2)151-156.
• Type: Journal Articles Status: Published Year Published: 2015 Citation: Jones-Hall, Y. L., A. J. Kozik and C. H. Nakatsu. 2015. The effect of TNF on the colonic microbiome and acute colitis. PLoS one 10(3):e0119441.

Progress 10/01/13 to 09/30/14

Outputs
Target Audience: Target audiences include all individuals and industries interested in the well-being of the environment and/or health of humans and animals. For example, researchers, industries or members of the public who are interested in using or understanding methods to either degrade or immobilize pollutants to improve the environment. With respect to human or animal health, those interested in improving health or preventing disease through the consumption of healthy diets, especially diets that include microbially fermented fibers. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? I am co-advising one PhD level graduate student, who is just beginning her second year in the PULSe program. A “Methods in Biotechnology Workshop” that included lectures and laboratory practicals was given to Graduate Students and Staff at the Institut d’Economie Rurale, Centre Régional de Recherche Agronomique in Bamako, Mali. Informal teaching and mentoring, mainly in microbial community analysis, was provided to graduate students and researchers from the programs of other PIs. How have the results been disseminated to communities of interest? Dissemination of findings to the community includes publications in topic relevant journals, presentation at conferences and invited presentations given to academic, government and industrial research groups . What do you plan to do during the next reporting period to accomplish the goals? Continue on the same lines of research.

Impacts
What was accomplished under these goals? Numerous approaches are being used to gain a better understanding of the role microorganisms in the environment. My laboratory has worked extensively to apply new molecular genetic tools for the interrogation of various environments. In addition to soil microbial community analysis my group has a number of different collaborations to determine the influence of diet on intestinal microorganisms. In cooperation with a group in Japan this year we have been determining the diversity of microbes in different environments and compared them to the different types that can be cultivated. Our main efforts have been placed on the influence of growth media and the addition of growth promoting factors on the novelty of bacteria that can be grown. Also, along with a faculty member from University of Zambia we are publishing a manuscript that describes a test of a number of different growth media on the diversity of rock phosphate dissolving microorganisms (bacteria and fungi) obtained from soil that can be potentially used to improve phosphate availability to crops. Research in the impact of diet on microbial communities in humans and animal model systems have continued and two manuscripts have been published. One describes a clinical study testing the addition of dietary fiber product (soluble corn fiber) on relationship between gut microbiota composition and increased calcium absorption/retention in adolescents. The other describes the correlations between gut microbiota and soy metabolites produced after the addition of soy bars to the diet of post menopausal women.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2014 Citation: Whisner, C. M., B. R. Martin, C. H. Nakatsu, L. D. McCabe, G. P. McCabe, M. Peacock, C. M. Weaver. 2014. Soluble maize fibre affects short term calcium absorption in adolescent boys and girls: a randomized controlled trial using dual stable isotopic tracers. Brit. J. Nutri. 112:446-456.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Mayumi, D., J. Dolfing, S. Sakata, H. Maeda, Y. Miyagawa, M. Ikarashi, H. Tamaki, M. Takeuchi, C. H. Nakatsu, and Y. Kamagata. Impact of CO2 geological storage on the methanogenic activity and pathway in a high-temperature petroleum reservoir. The 13th International Symposium on Microbial Ecology Abstracts. Seoul, Korea (August 2014).
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Nakatsu, C.H., A. Armstrong, B. Martin, C. M. Weaver. Changes in human gut microbial communities with dietary fiber. The 13th International Symposium on Microbial Ecology Abstracts. Seoul, Korea (August 2014).
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Mayumi, D., J. Dolfing, S. Sakata, H. Maeda, Y. Miyagawa, M. Ikarashi, H. Tamaki, M. Takeuchi, C. H. Nakatsu, and Y. Kamagata. Impact of carbon capture and storage on the methanogenic activity and pathway in a high-temperature petroleum reservoir. Goldschmidt 2014, Sacramento, CA (June 2014)
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Jones-Hall, Y. L. and C. H. Nakatsu. The effect of TNF on the colonic microbiome and acute colitis. Meeting of the Federation for American Society of Experimental Biology. FASEB J. San Diego, CA (April 2014).
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Nakatsu, C. H. ,C. Whisner, B. Martin and C. M. Weaver. Effect of soluble corn fiber on gut microbiota, calcium absorption, and indices of bone health. 10th Vahouny Fiber Symposium. Bethesda, MA (March 2014).
• Type: Journal Articles Status: Published Year Published: 2014 Citation: Nakatsu, C.H., A. Armstrong, A.C. Clavijo, B. R. Martin, S. Barnes and C. M. Weaver. 2014. Fecal bacterial community changes associated with isoflavone metabolites in postmenopausal women after soy bar consumption. PLoS one 9(10): e108924.
• Type: Journal Articles Status: Awaiting Publication Year Published: 2014 Citation: Tanaka, T., K. Kawasaki, S. Daimon, W. Kitagawa, K. Yamamoto, H. Tamaki, M. Tanaka, C. H. Nakatsu, and Y. Kamagata. 2014. A hidden pitfall in agar media preparation undermines cultivability of microorganisms. Appl. Environ. Microbiol.
• Type: Journal Articles Status: Accepted Year Published: 2015 Citation: Mweetwa, A.M., E.A. Eckhardt, D.E. Stott, D. Schulze, G. Chilembo, A. Amstrong and C. H. Nakatsu. Isolation and characterization of Chilembwe and Sinda rock phosphate solubilizing soil microorganisms from Indiana soils. African J. Microbiol. Res.
• Type: Conference Papers and Presentations Status: Published Year Published: 2014 Citation: Daimon, S., T. Tanaka, W. Kitagawa, M. Tanaka, C. H. Nakatsu, and Y. Kamagata. What agar do bacteria like? The 13th International Symposium on Microbial Ecology Abstracts. Seoul, Korea (August 2014).

Progress 10/01/12 to 09/30/13

Outputs
Target Audience: Target audiences include all individuals and industries interested in well-being of the environment and health of humans and animals. For example, those interested improving human health through the consumption of healthy diets, especially diets that include fiber such as non-digestible oligosaccharides. Also, people or industries interested in using methods to either degrade or immobilize pollutants to improve the environment. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? One PhD level graduate student, Andrea Clavijo, successfully graduated in the spring of 2013 and manuscripts from her research are being written. Hui Yan a PhD student from Animal Science received training in my lab to use molecular microbial ecology methods in his research. There are opportunities for undergraduates to get research experience in the laboratory, this year Michael Allsop completed his undergraduate research project and successfully graduated from Purdue University with his B.S. How have the results been disseminated to communities of interest? In addition to the research publications efforts to disseminate information include formal and informal teaching of environmental microbiology. A graduate course in Molecular Microbial Ecology is given yearly. Also environmental microbiology lectures were given at the University of Zambia and Hokkaido University. Informal teaching mainly consists of mentoring of graduate students. What do you plan to do during the next reporting period to accomplish the goals? Continue on the current project.

Impacts
What was accomplished under these goals? Numerous approaches are being used to gain a better understanding of the role microorganisms in the environment. My laboratory has worked extensively to apply new molecular genetic tools for the interrogation of various environments. In addition to soil microbial community analysis my group has a number of different collaborations to determine the influence of diet on intestinal microorganisms. Previous research by my group on population differences in the common soil bacterium Arthrobacter has continued. This year we sequenced genomes of 6 of the 14 Arthrobacter strains being compared in this study. Preliminary comparisons of the genomes to the previously sequenced Arthrobacter genomes have begun. Research in the impact of diet on microbial communities in humans and animal model systems have continued. Publication of a clinical study of the effects dietary galactooligosaccharide (GOS) that found increased calcium absorption associated with changes in gut microflora in young premenarcheal girls. Along this line clinical tests of another fiber product, soluble corn fiber, is being tested in adolescents to determine if it can modulate the gut microbiota. Publication of a pig model system that was used to demonstrate strong interplay between dietary fat level and fiber type in determining susceptibility to obesity, and that fiber type had the most prominent role in determining the gut microbial community structure.

Publications

• Type: Journal Articles Status: Accepted Year Published: 2013 Citation: Nakatsu, C.H., R. Barabote, S. Thompson, D. Bruce, C. Detter, T. Brettin, C. Han, P. Richardson, F. Beasley, W. Chen, A. Konopka, and G. Xie. 2013. Complete genome sequence of Arthrobacter sp. strain FB24. Standards in Genomic Sciences 9:106-116.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Traor�, L., C. H. Nakatsu, A. DeLeon and D. E. Stott. 2013. Characterization of six phosphate-dissolving bacteria isolated from rhizospheric soils in Mali. African J. Microbiol. Res. 7(28): 3641-3650.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Mayumi, D., J. Dolfing, S. Sakata, H. Maeda, Y. Miyagawa, M. Ikarashi, H. Tamaki, M. Takeuchi, C. H. Nakatsu, and Y. Kamagata. 2013. CO2 concentration dictates alternative methanogenic pathways in oil reservoirs. Nature Communications 4:1998
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Whisner, C. M., B. R. Martin, E. G. H. M. van den Heuvel, C. H. Nakatsu, L. D. McCabe, G. P. McCabe, M. H. C. Schoterman, C. M. Weaver. 2013. Galacto-oligosaccharides increase calcium absorption and fecal content of bifidobacteria in young girls: A crossover trial. British Journal of Nutrition 110:1293-1303.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Yan, H., R. Potu, H. Lu, T. Stewart, A. Armstrong, O. Adeola, C. H. Nakatsu and K. M. Ajuwon. 2013. Dietary fat content and fiber type modulate hind gut microbial community and metabolic markers in the pig. PLoS one 8:e59581.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Puspita, I.D., M. Uehara, T. Katayama, Y. Kikuchi, W. Kitagawa, Y, Kamagata, , K. Asano, C. H. Nakatsu, and M. Tanaka. 2013. Resuscitation promoting factor (Rpf) from Tomitella biformata AHU 1821T promotes growth and resuscitates non-dividing cells. Microbes Environ. 28:58-64.
• Type: Journal Articles Status: Published Year Published: 2013 Citation: Puspita, I.D., Y, Kamagata, M. Tanaka, K. Asano, and C. H. Nakatsu 2012. Are uncultivated bacteria really uncultivable? Microbes Environ. 27:356-366.
• Type: Book Chapters Status: Published Year Published: 2013 Citation: Nakatsu, C. H. 2013. Microbial processes: Community analysis. In: S. Elias (ed.). 2013. Reference module in earth systems and environmental sciences. Elsevier Ltd., Oxford, U.K. online reference database. ISBN 978-0-12-409548-9
• Type: Book Chapters Status: Published Year Published: 2013 Citation: Whisner C.M., B. R. Martin, A. Clavijo, C. H. Nakatsu, G. P. McCabe, L. D. McCabe, E.G.H.M. van den Heuvel, M. H. C. Schoterman, and C. M. Weaver. 2013. Galactooligosaccharides: effects on calcium absorption and gut microflora in young premenarcheal girls. In Nutritional Influences on Bone Health 8th International Symposium. 2013. Burckhardt, P., B. Dawson-Hughes, C. M. Weaver, (eds.). Springer.

Progress 10/01/11 to 09/30/12

Outputs
OUTPUTS: Activities include conducting and analyzing experiments that assess and determine changes in the structure, composition and adaptive mechanisms of microbial communities in a variety of perturbed environments. Graduate students and a technician responsible for the research were mentored. Dissemination of findings to the community includes publications in topic relevant journals, presentation at conferences and invited presentations given to academic, government and industrial research groups PARTICIPANTS: For Individuals who worked on the project, describe the role the person played in the project and how this person participated in the project. Include: (1) principal investigator(s)/project director(s) (PIs/PDs); and (2) each person who has worked at least one person month per year on the project during the reporting period and received salary, wages, a stipend, or other support from the agency (a person month equals approximately 160 hours of effort). Include any that have been added to the project since initiation of the project or activity. Partner Organizations FrieslandCampina Domo who provided funding for the GOS project. Margriet H.C. Schoterman and Ellen G.H.M. van den Heuvel are both employed by them. Collaborators and contacts from within Purdue University are Connie Weaver, Berdine Martin and Corrie Whisner from the Department Nutrition Science and George McCabe and Linda McCabe from the Statistics Department. Joseph Irudayaraj from Agricultural and Biological Engineering . Training or professional development. Currently there is one PhD level graduate student, Andrea Clavijo, being trained in my laboratory. One MS student, Kate Stephen, successfully graduated in the summer of 2012 and had her MS research published in the Analyst. Graduate students from the PULSe program have been working in my lab for one of their rotations. In the spring of 2012 Melissa Mikolaj and this fall Morgan Teachey. There are opportunities for undergraduates to get research experience in the laboratory, Michael Allsop is working in the lab this fall. TARGET AUDIENCES: Target audiences include all individuals and industries interested in well-being of the environment and health of humans and animals. For example, those interested improving human health through the consumption of healthy diets, especially diets that include fiber such as non-digestible oligosaccharides. Also, those interested in improving the environment by either degrading or immobilizing pollutants. Efforts include formal and informal teaching of environmental microbiology. Informal teaching mainly consists of mentoring of graduate students. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Numerous approaches are being used to gain a better understanding of the role microorganisms in the environment. My laboratory has worked extensively to apply new molecular genetic tools for the interrogation of various environments. In addition to soil microbial community analysis my group has a number of different collaborations to determine the influence of diet on intestinal microorganisms. Extension of previous research by my group on population differences in the common soil bacterium Arthrobacter was conducted. In this new study Surface Enhanced Raman spectroscopy (SERS), a rapid and highly sensitive spectroscopic technique, was tested for its potential to measure chemical changes in bacterial cell surface properties in response to environmental changes. Fourteen closely related Arthrobacter strains, based on their 16S rRNA gene sequences, were used in this study. After performing principal component analysis in conjunction with linear discriminate analysis, we used a novel, adapted cross-validation method, which more faithfully models the classification of spectra. All fourteen strains could be classified with up to 97% accuracy. The hierarchical trees comparing SERS spectra from the liquid and solid media data sets were different. Additionally, hierarchical trees created from the Raman data were different from those obtained using 16S rRNA gene sequences (a phylogenetic measure). A single bacterial strain grown on solid media culture with three different chromate levels also showed significant spectral distinction at discrete points identified by the new Elastic Net regularized regression method demonstrating the ability of SERS to detect environmentally induced changes in cell surface composition. This study demonstrated that SERS is effective in distinguishing between a large numbers of very closely related Arthrobacter strains and could be a valuable tool for rapid monitoring and characterization of phenotypic variations in a single population in response to environmental conditions. Research in the impact of diet on microbial communities in humans and animal model systems have continued. A clinical part of a study of the effects dietary galactooligosaccharide (GOS) on calcium absorption and gut microflora in young premenarcheal girls was completed. Preliminary data have yielded results similar to our previous study using the rat animal model system. The proportion Bifidobacteria, a beneficial bacterium, was significantly greater when 5 g GOS per day was consumed as a part of the regular diet of adolescent girls.

Publications

• Nasuno, E., N. Kimura, M. J. Fujita, C. H. Nakatsu, Y. Kamagata, and S. Hanada. 2012. Phylogenetically novel LuxI/LuxR-type quorum sensing systems isolated using a metagenomic approach. Appl. Environ. Microbiol. 78:8067-8074.
• Stephen, K., D. Homrighausen, G. DePalma, C. H. Nakatsu, J. Irudayaraj. 2012. Surface Enhanced Raman Spectroscopy (SERS) for the discrimination of Arthrobacter strains based on variations in cell surface composition. Analyst 137:4280-4286.
• Johnson, A.J., and C. H. Nakatsu. Use of PCR-DGGE to characterize the distribution of bacterial populations in feces of Giraffes, African Elephants and White Rhinoceros. The 2012 AAZV Annual Conference. Oakland, California (October 2012). 
 Nakatsu, C. H., A. Clavijo, A. Armstrong, B. Martin, C. M. Weaver Impact of diet on human gut microbial communities. The 12th International Symposium on Microbial Ecology Abstracts. Copenhagen, Denmark (August 2012).
• Utami, N.A., M. Tanaka, T., Sone, C. H. Nakatsu, K. Asano. Feeding yacon tuber increases intestinal fermentation and alters microbial community profiles in rats. The 12th International Symposium on Microbial Ecology Abstracts. Copenhagen, Denmark (August 2012).
• Puspita, I. D., M. Uehara, T. Katayama, M. Tanaka, W. Kitagawa, C. H. Nakatsu, Y. Kamagata, and K. Asano. Rpf protein from Tomitella biformata AHU 1821T increase the number of cultivable bacteria from permafrost ice wedge. The 12th International Symposium on Microbial Ecology Abstracts. Copenhagen, Denmark (August 2012).
• Whisner C.M., B. R. Martin BR, A. Clavijo, C. H. Nakatsu, G. P. McCabe, L. D. McCabe, E.G.H.M. van den Heuvel, M. H. C. Schoterman, and C. M. Weaver. Galactooligosaccharides: effects on calcium absorption and gut microflora in young premenarcheal girls. Nutritional Influences on Bone Health. 8th International Symposium on Nutritional Aspects of Osteoporosis. Lausanne, Switzerland (May 2012).
• Whisner C.M., B. R. Martin BR, A. Clavijo, C. H. Nakatsu, G. P. McCabe, L. D. McCabe, E.G.H.M. van den Heuvel, M. H. C. Schoterman, and C. M. Weaver. Galactooligosaccharide effects on calcium absorption and gut microflora in young premenarcheal girls . Meeting of the Federation for American Society of Experimental Biology. San Diego, CA (April 2012).

Progress 10/01/10 to 09/30/11

Outputs
OUTPUTS: Activities include conducting and analyzing experiments that assess and determine changes in the structure, composition and adaptive mechanisms of microbial communities in a variety of perturbed environments. Graduate students and a technician responsible for the research were mentored. Dissemination of findings to the community includes publications in topic relevant journals, presentation at conferences and invited presentations giving to academic, government and industrial research groups PARTICIPANTS: Partner Organizations in this project is FrieslandCampina Domo who provided funding for the GOS project. Margriet H.C. Schoterman and Ellen G.H.M. van den Heuvel are both employed by them. Collaborators and contacts from within Purdue University are Connie Weaver, Berdine Martin and Dennis Saviano from the Department of Foods and Nutrition and George McCabe and Linda McCabe from the Statistics Department Training or professional development. Currently there are two PhD level graduate students, Andrea Clavijo and Kate Stephen, are being trained in my laboratory. Merlin Ariefjohan is a former student in the Department of Foods and Nutrition that graduated with her PhD in 2008. There are opportunities for undergraduates to get research experience in the laboratory, in the last year Aaron Geswein and Leslie Seals worked in the lab. TARGET AUDIENCES: Target audiences include all individuals and industries interested in well-being of the environment and health of humans and animals. For example, those interested improving human health through the consumption of healthy diets, especially diets that include fiber such as non-digestible oligosaccharides. Also, those interested in improving the environment by either degrading or immobilizing pollutants. Efforts include formal and informal teaching of environmental microbiology. Informal teaching mainly consists of mentoring of graduate students. PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Numerous approaches are being used to gain a better understanding of the role microorganisms in the environment. My laboratory has worked extensively to apply new molecular genetic tools for the interrogation of various environments. This year our major focus has been the influence of diet on intestinal microorganisms. To achieve this goal first culture-independent molecular fingerprinting methods such as of PCR and Denaturing Gradient Gel Electrophoresis (PCR-DGGE) were optimized to study microbial community structure in fecal samples. These protocols must be optimized prior to their application in order to ensure PCR efficiency and enhance the quality and accuracy of downstream analyses. The relative efficacy of four commercial DNA extraction kits (Mobio Ultra Clean Fecal DNA Kit, M; QIAamp DNA Mini Stool Kit, Q; FastDNA SPIN Kit, FSp; FastDNA SPIN Kit for Soil, FSo) were assessed and optimized for PCR-DGGE studies to test the efficacy of these methods to monitor variations in fecal communities. We found that regardless of kit, maximum DNA yield was obtained using 10 to 50 mg (wet wt) of feces and similar DGGE profiles were obtained. However, significantly greater amounts of DNA per g feces were obtained using kits FSp and FSo therefore we recommend these kits for future studies. This approach was then tested to determine if changes in microbial communities could be seen in rats fed different concentrations of galactooligosaccharides (GOS) in their diets. GOS is used as a pre-biotic non-digestible oligosaccharides, that can be fermented by gut microbes and potentially results in improving mineral balance and bone health. We specifically examined the dose-response effect of GOS supplementation on calcium and magnesium absorption, mineral retention, bone properties, and gut microbiota in growing rats. Dietary GOS significantly decreased cecal pH and increased cecal wall weight and content weight in a dose dependent manner (p<0.0001). Fingerprint patterns of 16S rRNA gene PCR-DGGE from fecal DNA indicated variance of bacterial communities structure, which was primarily explained by GOS treatments (p=0.0001). Quantitative PCR of the samples revealed an increase in relative proportion of bifidobacteria with GOS (p=0.0001). Regression modeling showed that GOS benefited calcium and magnesium utilization and vBMD through decreased cecal pH, and increased cecal wall and content weight, and increased proportion of bifidobacteria (a bacterium associated with good intestinal health).

Publications

• Weaver, C. M., B. R. Martin, C. H. Nakatsu, A. P.Armstrong, A. Clavijo, L. D. McCabe, G. P. McCabe, S. Duignan, M. H.C. Schoterman and E. G.H.M. van den Heuvel. 2011. Galactooligosaccharide supplementation improves mineral absorption and bone properties in growing rats through gut fermentation. Journal of Agriculture and Food Chemistry 59:6501-6510. (DOI: 10.1021/jf2009777)
• Ariefdjohan, M. W., D. A. Savaiano, and C. H. Nakatsu. 2010. Optimization of DNA extraction kits for PCR-DGGE analysis of human intestinal microbial communities from fecal specimens. Nutrition Journal 9:23.
• Henne, K. L., J. E. Turse, C. H. Nakatsu, and A. E. Konopka. 2011. Protein expression profile of an environmentally important bacterial strain: the Chromate response of Arthrobacter sp. strain FB24. In F. J. de Bruijn (ed.). Handbook of Molecular Microbial Ecology: Metagenomics and Complementary Approaches. Wiley/Blackwell Publ.
• Clavijo, A., B. Martin, C. M. Weaver, and C. H. Nakatsu. Effect of soy diet supplementation on bacterial communities in postmenopausal women. The 2010 SACNAS National Conference Abstracts. Anaheim, CA (October 2010)
• Clavijo, A., B. Martin, C. M. Weaver, and C. H. Nakatsu. Characterization of fecal bacterial communities in healthy postmenopausal women. The 11th International Symposium on Microbial Ecology Abstracts. Seattle WA (August 2010).

Progress 10/01/09 to 09/30/10

Outputs
OUTPUTS: The structure, composition and adaptive mechanisms of soil microbial communities in a variety of perturbed environments were determined. Commercial firms are using assessment tools developed in the lab to monitor impacted sites. Dissemination of findings to the community includes publications in topic relevant journals, presentation at conferences and invited presentations giving to academic, government and industrial research groups PARTICIPANTS: Weidong Kong is now a research scientist at UCSF TARGET AUDIENCES: Academics, industry, government PROJECT MODIFICATIONS: Nothing significant to report during this reporting period.

Impacts
Numerous approaches are being used to gain a better understanding of the role microorganisms in remediation of pollutants (both organic aromatic compounds and metals) in the environment. A real-time PCR method for quantification of a set of eight aromatic oxygenase genes that was developed in this lab using DNA as a template was extended to use RNA. First it was important to demonstrate that mRNA could be extracted from samples while maintaining its integrity for quantification. By adding mRNA to the method we were able to demonstrate the difference in monitoring metabolic activity through gene expression using mRNA template versus the use of DNA a better indicator of genetic potential in an ecosystem. Additionally, working as a member a subcommittee of the international Terregenome group a set of soil characters important for understanding soil biology and for interpreting metasequence data was established. The characters were based on surveys of the international soil biology community. The required and suggested metadata to submit with metagenomic sequence data has been accepted by the Genomic Standards Consortium (GSC) and is being harmonized with data standards for other environments for use by the global scientific community

Publications

• Kong, W. and C. H. Nakatsu. 2010. Optimization of RNA extraction for PCR quantification of aromatic compound degradation genes. Appl. Environ. Microbiol. 76:1282-1284
• Cole, J. R., D. D. Myrold, C. H. Nakatsu, P. R. Owens, G. Kowalchuk, C. Tebbe, and J. M. Tiedje. Development of soil metadata standards for international DNA sequence databases. 19th World Congress of Soil Science, Soil solutions for a changing world. Brisbane, Australia (August 2010).

Progress 10/01/08 to 09/30/09

Outputs
OUTPUTS: The structure, composition and adaptive mechanisms of soil microbial communities in a variety of perturbed environments were determined. Commercial firms are using assessment tools developed in the lab to monitor impacted sites. Dissemination of findings to the community includes publications in topic relevant journals, presentation at conferences and invited presentations giving to academic, government and industrial research groups. PARTICIPANTS: Brett Baldwin: Was a former student, now working for the commercial firm using the technology developed in the lab. Kristene Henne: Completed Ph.D. and now is a post doc at Argonne National Labs. Peter Koutev: Trained as a postdoctoral scientist and is now on faculty at Central Michigan University. Weidong Kong: Trained as a postdoctoral scientist. TARGET AUDIENCES: Government and academics PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
A number of different approaches were used to gain a better understanding of the role microorganisms play in remediation of pollutants in the environment. A real-time PCR method for quantification of a set of eight aromatic oxygenase genes that was developed in this lab was tested on two more field sites to further evaluate the practical application of this approach to monitor biodegradation. Typically chemical analysis of groundwater is used to assess pollutants such as, benzene, toluene, ethylbenzene, and xylene (BTEX), in groundwater. However, both chemical and biological evidence is needed to conclusively demonstrate bioremediation. We were able to show that there were increases in different oxygenase genes at sites using oxygen releasing compounds and multiphase extraction technologies to improve site conditions to enhance bioremediation. Although there were elevated levels of oxygenase genes that corresponded to decrease in petroleum hydrocarbon contamination in some of the wells the role played by additional treatments vs the microbes for site remediation was not clear. The method is being further developed in the lab to determine if monitoring metabolic activity through gene expression using mRNA is a more informative approach to site assessment as compared to DNA-based markers. Additionally, mechanisms for metal resistance by bacteria in contaminated soils was elucidated. Primarily the chromate resistance mechanism was determined for Arthrobacter, a common soil species.

Publications

• Henne, K. L., C. H. Nakatsu, , D. K. Thompson, and A. E. Konopka. 2009. Chromate resistance genes in Arthrobacter sp. strain FB24. BMC Microbiology 9:199.
• Kourtev, P.S., C.H. Nakatsu, and A. Konopka. 2009. Inhibition of nitrate reduction by chromium (VI) in anaerobic soil microcosms. Appl. Environ. Micro. 75:6249-6257.
• Henne, K. L., J. E. Turse, C. D. Nicora, M. S. Lipton, S. L. Tollaksen, C. Lindberg, G. Babbnig, C. S. Giometti, C. H. Nakatsu, D. K. Thompson, and A. E. Konopka. 2009. Global proteomic analysis of the chromate response in Arthrobacter sp strain FB24. J. Proteome Research 8:1704-1716
• Nebe, J., B. R. Baldwin, L. Nies, R. L. Kassab, and C. H. Nakatsu. 2009. Quantification of aromatic oxygenase genes to evaluate enhanced biodegradation by oxygen releasing materials at a gasoline-contaminated site. Environ. Sci. Tech. 43:2029-2034.
• Baldwin, B. R., C. H. Nakatsu, Nebe, J., G.S. Wickham, C. Parks, and L. Nies. 2009. Enumeration of aromatic oxygenase genes to evaluate biodegradation during multi-phase extraction at a gasoline-contaminated site. J. Hazardous Materials 63:524-530

Progress 10/01/07 to 09/30/08

Outputs
OUTPUTS: The structure, composition and adaptive mechanisms of soil microbial communities in a variety of perturbed environments were determined. Commercial firms are using assessment tools developed in the lab to monitor impacted sites. Dissemination of findings to the community includes publications in topic relevant journals, presentation at conferences and invited presentations giving to academic, government and industrial research groups. PARTICIPANTS: Kurt Jerke: Completed Ph.D., now is a research scientist for the US Army. Fred Beasley: Completed his M.S., now is pursuing his Ph.D. Brett Baldwin: Was a former student, now working for the commercial firm using the technology developed in the lab. Jin Liu: Completed Ph.D., now is a research scientist for Dupont. Corinne Ackerman: Completed Ph.D., now is a research soil scientist in California. Weidong Kong: Trained as a postdoctoral scientist. Collaborators on projects from Purdue: A. Konopka, L. Nies, L. Lee, J.K. Ghosh. J. Wilbur, collaborator at Worchester Polytechnical Institution. TARGET AUDIENCES: Soil scientist, environmental engineers, EPA in addition to state and local environmental agencies. PROJECT MODIFICATIONS: Not relevant to this project.

Impacts
A number of different approaches were used to gain a better understanding of the role microorganisms play in remediation of pollutants in the environment. Application of a real-time PCR method for quantification of a set of eight aromatic oxygenase genes that was developed in this lab was tested to evaluate the practical application of this approach to monitor natural attenuation (MNA). Typically chemical analysis of groundwater is used to assess pollutants such as, benzene, toluene, ethylbenzene, and xylene (BTEX), in groundwater. However, both chemical and biological evidence is needed to conclusively demonstrate bioremediation. We were able to show that different oxygenase genes, on the order of 106-109 copies L-1, were routinely detected in BTEX-impacted wells and not in sentinel wells outside the plume. Elevated levels of oxygenase genes corresponded to petroleum hydrocarbon contamination demonstrating the feasibility of using the method for MNA as a corrective measures. The method is being further developed in the lab to determine if monitoring metabolic activity through gene expression using mRNA is a more informative approach to site assessment as compared to DNA-based markers. Additionally, mechanisms for the spread of genes of environmental importance are being determined. Most recently we compared eight plasmids from five Arthrobacter strains in order to identify putative core genes involved in gene transfer. Despite the prevalence of Arthrobacter in soils little is known about their capacity to mediate horizontal gene transfer. These plasmids harbored a variety of genes that can play a role in the remediation of soils impacted by various pollutants, for example, heavy metals, pesticides, and petroleum products.

Publications

• Jerke, K., C. H. Nakatsu, F. Beasley and A. Konopka. 2008. Comparative analysis of eight Arthrobacter plasmids. Plasmid 59:73-85
• Baldwin, B., L. Nies, and C. H. Nakatsu. 2008. Detection and enumeration of aromatic oxygenase genes at gasoline-contaminated sites by real-time PCR. Water Research 42:723-731
• Park, J., J. D. Wilbur, J. K. Ghosh, C. H. Nakatsu and C. Ackerman. 2007. Selection of binary variables and classification by boosting. Communication in statistics simulation and computation 36:855-869
• Liu, J., L. S. Lee, L. F. Nies, C. H. Nakatsu and R. F. Turco. 2007. Biotransformation of 8:2 fluorotelomer alcohol in soil and by soil bacteria isolates. Env. Sci. Technol. 41:8024-8030.

Progress 10/01/06 to 09/30/07

Outputs
OUTPUTS: Yearly we have developed a deeper understanding of soil ecosystems. Using molecular methods we were able demonstrate how periodicity of feeding can impact microbial community structure. Sixteen replicate microcosms were inoculated with a mixed assemblage of heterotrophic bacteria and provided with discrete pulses of protein as carbon and energy source. The dynamics of community structure were monitored by 16S rRNA gene PCR-DGGE. The results were consistent with a strong role for biological interactions in maintaining diversity. Replicate microcosms developed different microbial communities. For systems exposed to nutrient pulses every 7 days, the number of phylotypes averaged 13 and the Dice similarity coefficient between pairs ranged from 0.08 to 0.67. In each of 16 systems provided protein once each day, there were dynamic changes over the first 30 days but community composition was stable over the next 20 days. However, most systems differed from each other; two-thirds of the pairwise comparisons had similarity coefficients in the range of 0.35 - 0.63. These 16 systems contained 10 phylotypes and in aggregate 34 phylotypes were found in the 16 systems. TARGET AUDIENCES: Soil Scientists

Impacts
The most biological diversity on this planet is likely harbored in soils. However, approximately 99% of bacterial cells from nature do not grow when traditional microbiological techniques are applied, hampering our ability to harness this vast resource for medical, environmental and industrial uses. Improved methodology to directly profile microbial communities and to cultivate previously difficult to cultivate bacteria is allowing us to gain a better understanding of indigenous microbial populations. In addition to the basic knowledge gained results from our research can be used for biotechnological applications.

Publications

• Konopka, A., M. Carrero-Colon, C. H. Nakatsu 2007. Community dynamics and heterogeneities in mixed bacterial communities subjected to nutrient periodicities. Environ. Microbiol. 9:1584-1590
• Nakatsu, C. H. 2007. The basics and application of denaturing gradient gel electrophoresis for soil microbial community analysis. Soil Sci. Soc. J. Am. 71:562-571
• Nakatsu, C. H. and T. L. Marsh. 2007. Analysis of microbial communities with denaturing gradient gel electrophoresis and terminal restriction fragment length polymorphism. in: C. A. Reddy, T.L. Beveridge, J.A. Breznak, G. A., Marzluf, T. M. Schmidt and L. R. Snyder (eds.) Methods for general and molecular bacteriology. ASM Press, Washington D. C. pp. 909-923.
• Chen, W., A. Konopka and C. H. Nakatsu. Physiological factors contributing to chromateresistance by Arthrobacter sp. FB24. American Society for Microbiology Annual Meeting Abstracts. Toronto, Canada. (May 2007).
• Chen, W., M. Kourteva, A. Konopka and C. H. Nakatsu. The limitation of carbon source utilization with metals in Arthrobacter sp. FB24. American Society of Agronomy Annual Meeting Abstracts, Indianapolis, IN. (November 2006)

Progress 10/01/05 to 09/30/06

Outputs
Using both modern molecular genetic based methods in combination with traditional microbiological approaches, we have begun to have a deeper understanding of soil ecosystems. Using molecular methods we were able demonstrate the greater complexity of microbial communities in agricultural ecosystems versus those highly stressed environments such as those exposed to pollutants. These methods could also be used to determine the spatial distribution of communities at scales ranging from meters to millimeters distance from each other. The results showed the true heterogeneous nature of soils and their indigenous microflora. Also, populations responding to soil perturbation such as heavy metals and organic pollutants have been identified to be both functionally and phylogenetically diverse despite their highly selective nature. To understand the interactions between relevant microbial populations in a community quantitative approaches are being used to evaluate their similarities and/or differences. Although progress has been tremendous, there are still many unanswered questions that must be addressed. Using a combination of molecular techniques with other approaches we now have the ability to understand the biotic component of one of the final frontiers on this planet, soil.

Impacts
The most biological diversity on this planet is likely harbored in soils. However, approximately 99% of bacterial cells from nature do not grow when traditional microbiological techniques are applied, hampering our ability to harness this vast resource for medical, environmental and industrial uses. Improved methodology to directly profile microbial communities and to cultivate previously difficult to cultivate bacteria is allowing us to gain a better understanding of indigenous microbial populations. In addition to the basic knowledge gained results from our research can be used for biotechnological applications.

Publications

• Nakatsu, C. H. 2005. Microbial Genetics. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.). Principles and applications of soil microbiology. Prentice Hall. New Jersey. pp. 85-98
• Konopka, A., M Carrerro-Colon, and C Nakatsu. Heterogeneous dynamics in microbial communities subjected to nutrient periodicities. The 10th International Symposium on Microbial Ecology Abstracts. Vienna, Austria. (Aug. 2006).
• Nebe, J. L., L. Nies, C. H. Nakatsu. Exploring the Relationship Between Cultivation and Non-Cultivation Based Methods for Bioremediation Assessment. The 10th International Symposium on Microbial Ecology Abstracts. Vienna, Austria. (Aug. 2006).
• Ariefdjohan, M. W., D. A. Savaiano, C. H. Nakatsu. Does the host genotype influence human intestinal microflora community? The 10th International Symposium on Microbial Ecology Abstracts. Vienna, Austria. (Aug. 2006).
• Jerke, K., C. Nakatsu, and A. Konopka. Analysis of the Lead Resistance Plasmid pSI-1 from Arthrobacter sp. SI-1, and its Metal Resistance Genes. American Society for Microbiology Annual Meeting Abstracts. Orlando, FL. (May 2006)
• Henne, K. L., S. L. Tollaksen, C. B. Lindberg, G. Babnigg, C. S. Giometti, C. Nakatsu, A. E. Konopka. Proteomic Analysis of the Chromate Response in Arthrobacter sp. FB24. American Society for Microbiology Annual Meeting Abstracts. Orlando, FL. (May 2006)
• Nakatsu, C. H. Soil community analysis using PCR of rDNA and DGGE. Symposium on Molecular Based Methods to Soil Microbiology. American Society of Agronomy Annual Meeting Abstracts, Salt Lake City, UT. (November 2005).
• Vargha, M., Z. Takats, A. Konopka, C. H. Nakatsu. 2006. Optimization of MALDI-TOF MS for strain level differentiation of Arthrobacter isolates. J. Microbiol. Meth. 66:399-409.
• Carrero-Colon, M., C. H. Nakatsu, and A. Konopka. 2006. Microbial community dynamics in nutrient-pulsed chemostat. FEMS Microbiol. Ecol. 57:1-8.
• Joynt, J., M. Bischoff, R. Turco, A. Konopka, and C. H. Nakatsu. 2006. Microbial community analysis of soils contaminated with lead, chromium and organic solvents. Microbial Ecology 51: 209-219.
• Kourtev, P. S., C. H. Nakatsu, and A. E. Konopka. 2006. Anaerobic bacterial community responses to organic C addition in chromium (VI) and iron (III) amended microcosms Appl. Env. Microbiol. 72: 628-637
• Nakatsu, C. H., N. Carmosini, B. Baldwin, F. Beasley, P. Kourtev, and A. Konopka. 2005. Soil microbial community responses to additions of organic carbon substrates and heavy metals (Pb and Cr). Appl. Environ. Microbiol. 71:7679-7689
• Carrero-Colon, M., C. H. Nakatsu, and A. Konopka. 2006. The effect of nutrient periodicity upon microbial community dynamics. Appl. Environ. Microbiol. 72: 3175-3183.
• Nakatsu, C. H. K. Hristova, S. Hanada, X.-Y. Meng, J. R. Hanson, K. M. Scow, and Y. Kamagata. 2006. Methylibium petroleiphilum gen. nov., sp. nov., a new methyl tert-butyl ether (MTBE)-degrading methylotroph of the beta-Proteobacteria. Internat. J. Syst. Evol. Microbiol. 56: 983-989.
• Becker, J. M., T. Parkin, C. H. Nakatsu, J. D. Wilbur, and A. Konopka. 2006. Bacterial activity, community structure, and cm-scale spatial heterogeneity in contaminated soil. Microbial Ecology 51: 220-231.

Progress 10/01/04 to 09/30/05

Outputs
The relationship between plant growth and members of the rhizosphere bacterial community has always been difficult to assess because of the limitations of cultivation and the need for high throughput analysis. Our goal was to test the efficacy of using a molecular community analysis method to identify bacterial populations that correlated with differences in plant growth. To simplify the system a single plant species, Zea mays (corn), was used. Corn was grown under four different agronomic treatments (combination of rotation with soybean or monoculture with plow or no-till) and sampled at different growth stages over three field seasons. Having used statistical tools (using principal components analysis, PCA, and analysis of variance, ANOVA) to identify significant bacterial populations within community fingerprints obtained by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene PCR products we worked this year to determine their significance. Corn growth in the presence of different Burkholderia sp. are currently being tested to verify if the potential growth promoters identified using this molecular/statistical approach does indeed influence corn growth. A second objective was to determine if mycorrhizal fungi were associated with the corn and/or soybean plants. Both direct analysis by microscopy and molecular methods were used. Three primer pairs were selected, LSU4Kf/LSU7Kr, Gig1/Gig2, Scut1/Scut2, which were designed to amplify 28S rRNA genes from three major genera of Glomineae: Glomus, Gigaspora and Scutellospora, respectively. Glomus-specific PCR indicated it was present in rhizosphere soil samples of corn and soybean at all the growth stages. PCR products of representative samples from four treatments were cloned and sequenced. Sequence analysis of 10 different inserts from four treatments matched two species in the GenBank database, G. intraradices, G. deserticola. Genus-specific PCR suggested Gigaspora and Scutellospora were also present in the rhizosphere soil samples. Microscopic observations showed the presence of mycorrhizal infection in all plants examined. These results indicate that the diversity of mycorrhizal associates of these crop plants is greater than we originally anticipated. The evidence is strong that the mycorrhizal fungi are common and likely essential associates of these crop plants. This is another important piece of the puzzle to understand the microbial communities associated with crop plants.

Impacts
The most biological diversity on this planet is likely harbored in soils. The microbiological component of soil has posed the greatest challenge until molecular biological approaches were applied to understand diversity and function. Improved methodology to directly profile microbial communities is allowing us to make a better assessment of the impact of anthropogenic technologies on indigenous microbial populations and in turn their plant associates. Results from our research have demonstrated that microbial communities are diverse but it is possible to start identifying some of the major players influencing plant development and productivity.

Publications

• Wang, J., C. Nie, S. Luo, and C. H. Nakatsu. Correlation between DIMBOA and Bt proteins concentrations among Bt and non-Bt corn hybrids. American Society of Agronomy Abstracts. Seattle, WA, (Nov. 2004).
• Maldonado, Y., J. C. Fiser, C. H. Nakatsu, and A. K. Bhunia. 2005. Cytotoxicity potential of Escherichia coli isolates from environmental and food sources. Appl. Environ. Microbiol. 71:1890-1898
• Lee, T. H., S. Kurata, C. H. Nakatsu, Y. Kamagata. 2005. Molecular analysis of bacterial community based on 16S rDNA and functional genes in activated sludge enriched with 2,4-dichlorophenolxyacetic acid (2,4-D) under different cultural conditions. Microb. Ecol. 48:151-162
• Nakatsu, C. H. and L. J. Forney. 2004. Parameters of nucleic acid hybridization experiments. in: G. A. Kowalchuk, F.J. de Bruijn, I. M. Head, A.D.L. Akkermans, and J.D. van Elsas (eds.). Molecular microbial ecology manual. Edition 2. Springer Publishers, Netherlands.
• Kourtev, P.S., A.E. Konopka, and C. Nakatsu. Bacterial community responsse to organic C addition in chromium (VI) and iron (III) amended anaerobic microcosms. American Society for Microbiology Annual Meeting Abstracts. Atlanta, GA. (June 2005).
• Konopka, A., J. Becker, C.H. Nakatsu, and R. Turco. High-efficiency cultivation of bacteria from soil habitats. Joint Meeting of the International Union of Microbiological Societies Abstracts. San Francisco, CA (July 2005).
• Sutton, B. D., J.Nebe, B. R. Baldwin, C. H. Nakatsu, and L. Nies. Validation of a real time PCR method for assessing biodegradation of aromatic hydrocarbon contaminated systems. Abstracts of the Battelle Bioremediation Conference in Orlando, FL (June, 2005)
• Nebe, J. L., B. R. Baldwin, L. Nies, and C. H. Nakatsu. Evaluation of ORC Enhanced BTEX Bioremediation through Enumeration of Aromatic Oxygenase Genes. American Society for Microbiology Annual Meeting Abstracts. Atlanta, GA. (June 2005).
• Henne, K. L., F. Beasley, C. Nakatsu, and A. Konopka. Chromium Resistance in Arthrobacter sp. FB24. American Society for Microbiology Annual Meeting Abstracts. Atlanta, GA. (June 2005).
• Ackerman, C. E., F. M. Wanjau, J. D. Wilbur, S. M. Brouder, R. W. Doerge, and C.H. Nakatsu. The Effects of Long Term Tillage and Rotational Treatments on the Rhizosphere Fungal Community Structure of Soybean. American Society of Agronomy Abstracts. Seattle, WA, (Nov. 2004).

Progress 10/01/03 to 09/29/04

Outputs
The relationship between plant growth and members of the rhizosphere bacterial community has always been difficult to assess because of the limitations of cultivation and the need for high throughput analysis. Our goal was to test the efficacy of using a molecular community analysis method to identify bacterial populations that correlated with differences in plant growth. To simplify the system a single plant species, Zea mays (corn), was used. Corn was grown under four different agronomic treatments (combination of rotation with soybean or monoculture with plow or no-till) and sampled at different growth stages over three field seasons. Community fingerprints were obtained by denaturing gradient gel electrophoresis (DGGE) of 16S rRNA gene PCR products from the rhizosphere soil of these plants. Principal components analysis (PCA) and analysis of variance (ANOVA) were used to identify bacterial populations that were statistically different in the rhizosphere of plants grown under different agronomic treatments. PCA revealed that in all three years, the bacterial community profiles grouped according to agronomic treatment. ANOVA revealed four bands that were consistently associated with specific plant growth conditions for all three seasons. Nucleotide sequence determination of two of these bands revealed that the closest matches were to Burkholderia sp. and Pseudomonas sp. Media specific for these organisms (PCAT and King's B, respectively) was used to cultivate them from the same soils. Combining a PCR-DGGE with PCA, ANOVA and cultivation appears to provide a stream-lined approach for the identification and isolation of bacterial populations potentially influencing corn growth. A second objective this year was to compare the fungal community structure of soybean rhizosphere over time and under different agronomic treatments. Treatments included: plow-monoculture soybean, plow-rotated soybean with corn, no-till monoculture soybean, and no-till soybean rotated with corn. Soybean plants were collected at specific growth stages throughout their lifecycle for two field seasons. Fungal community structure was determined by restriction fragment length polymorphism (RFLP) analysis of PCR amplified ITS1-5.8S-ITS2 gene fragments from rhizosphere soil extracted DNA. The presence of fungal DNA in the rhizosphere samples was indicated by PCR using universal primers for eukaryotes and subsequent sequencing, and the fungal specific ITS primers. Analysis of total eukaryotic rhizosphere community structure revealed limited diversity, especially when compared to the prokaryotic community. Research is ongoing to determine the diversity of the fungal rhizosphere community at different growth stages of soybean.

Impacts
In order to understand and manage obstacles of ecosystem productivity, we must first understand the ecology and community dynamics of the responsible biota. Efforts to study the relevant microorganisms controlling these processes have been severely hampered by a lack of methodology to directly profile microbial communities. Improved in-situ microbial characterization will provide more accurate assessment of the impact of anthropogenic technologies on indigenous microbial populations and the major functions they carry out. Results from our research have demonstrated that microbial community fingerprinting techniques can be used to show differences in the microbial community structure influenced by their environment. We have been studying the dynamics of resident microbial community or crop rhizospheres in order to advance our understanding of the influence of microbes in productivity of perturbed ecosystems such as agricultural cropping systems.

Publications

• Mesarch, M. B., C. H. Nakatsu, and L. Nies. 2004. Bench-scale and field-scale evaluation of catechol 2,3-dioxygenase specific primers for use in monitoring BTX bioremediation. Water Res. 38: 1281-1288.
• Nakatsu, C. H. 2004. Fundamentals of Microbial Genetics. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.). Principles and applications of soil microbiology. Prentice Hall. New Jersey.
• Nebe, J., L. Nies, C. Nakatsu, B. Baldwin. 2004. Molecular genetic detection and enumeration of aromatic oxygenase genes at gasoline-contaminated sites. The 9th International Symposium on Microbial Ecology Abstracts. Cancun, Mexico.
• Hristova, K., A. Chakicherla, S. Kane, P. Chain, C. Nakatsu, K. Scow. 2004. Metabolic diversity of Methylobium petroleophilum PM1, a methyl tert-butyl ether degrading bacterium present in California aquifers. The 9th International Symposium on Microbial Ecology Abstracts. Cancun, Mexico.
• Bulinski, D. A, J. D. Wilbur, S. M. Brouder, R. W. Doerge, and C. H. Nakatsu,. 2004. Microbial community analysis to identify bacterial populations influencing corn growth. American Society for Microbiology Annual Meeting Abstracts. New Orleans, LA.
• Vargha, M., F. Beasley, Z. Takats, C. H. Nakatsu. 2004. MALDI MS as a tool for differentiating Arthrobacter strains. American Society for Microbiology Annual Meeting Abstracts. New Orleans, LA.
• Nakatsu, C. H. 2004. Microbial community analysis. In D. Hillel, C. Rosenzweig, D. Powlson, K. Scow, M. Singer and D. Sparks (eds.). Encyclopedia of soils in the environment. Academic Press. London, U.K.
• Nakatsu, C. H. 2004. Fundamentals of Microbial Genetics. In D. M. Sylvia, J. J. Fuhrmann, P. G. Hartel, and D. A. Zuberer (ed.). Principles and applications of soil microbiology. Prentice Hall. New Jersey.
• Nakatsu, C. H. 2004. Microbial community analysis. In D. Hillel, C. Rosenzweig, D. Powlson, K. Scow, M. Singer and D. Sparks (eds.). Encyclopedia of soils in the environment. Academic Press. London, U.K.
• Vargha, M., A. Konopka, C. H. Nakatsu. 2004. Capture of chromate resistance by bacteria in heavy metal contaminated soil The 9th International Symposium on Microbial Ecology Abstracts. Cancun, Mexico.
• Jerke, K., C. Nakatsu, and A. Konopka. 2004. Sequence Analysis of a Heavy Metal Resistance Plasmid (pSI-1) from an Arthrobacter sp. American Society for Microbiology Annual Meeting Abstracts. New Orleans, LA.
• Becker, J., C.H. Nakatsu, R. Turco, and A. Konopka. 2004. Small scale spatial species biodiversity at a long-term contaminated site. American Society for Microbiology Annual Meeting Abstracts. New Orleans, LA.
• Beasley, F. C., C. H. Nakatsu, N. Carmosini, A. Konopka. 2003. Genotypic and phenotypic characterization of Arthrobacter isolates from chromate contaminated soil. American Society of Agronomy Abstracts. Denver, CO.
• Ackerman, C. E., F. M. Wanjau, J. D. Wilbur, S. M. Brouder, R. W. Doerge, and C.H. Nakatsu. 2003. The effects of long term tillage and rotational treatments on the rhizosphere microbial community structure of soybean. American Society of Agronomy Abstracts. Denver, CO.

Progress 10/01/02 to 09/30/03

Outputs
Efforts to study the relevant microorganisms controlling environmental processes were severely hampered in the past by a lack of methodology to directly profile microbial communities. Results from our research have demonstrated that microbial community fingerprinting techniques (16S rDNA PCR DGGE) can be used to show differences in the microbial community structure influenced by their environment. We have been studying the dynamics of resident microbial community in order to advance our understanding of the influence of microbes in productivity of perturbed ecosystems such as agricultural cropping systems and anthropogenetically contaminated soil. The results from our agronomic studies have revealed that the microbial rhizosphere community profiles mainly grouped according to agronomic treatment. This grouping was more evident in corn vs soybean rhizosphere. This indicates that agronomic treatment can influence the microbial ecology of the rhizosphere in corn and soybean plants. Potential populations contributing to differences in plant development have been isolated and are currently being studied. Using a similar approach in ecosystems contaminated with fossil fuels we have found a diverse community of microorganisms with the potential to remediate the sites. The successful bioremediation of sites contaminated with aromatic compounds typically relies on the presence and stimulation of aromatic hydrocarbon-degrading bacteria from the indigenous microbial population. Detecting strains capable of degrading these pollutants is therefore critical when evaluating bioremediation as a treatment alternative. A technique based on real time PCR amplification of aromatic oxygenase genes was developed to detect and quantify a number of aromatic catabolic genes in environmental samples. Preliminary examination of groundwater samples from gasoline-contaminated sites indicated that the method could predict the presence of gasoline degrading bacteria. Field verification of the method is now being tested on a greater number of sites over time to gain a greater understanding of the number of aromatic oxygenase genes detected in groundwater, concentrations of pollutants and the diversity of microorganisms carrying out the function.

Impacts
In order to understand and manage obstacles of ecosystem productivity, we must first understand the ecology and community dynamics of the responsible biota. Improved in-situ microbial characterization will provide more accurate assessment of the impact of anthropogenic technologies on indigenous microbial populations and the major functions they carry out.

Publications

• Baldwin, B., C. H. Nakatsu, and L. Nies. 2003. Detection and enumeration of aromatic oxygenase genes by multiplex and real-time PCR. Appl. Environ. Microbiol. 69:3350-3358.
• LaPara, T. M., T. Zakharova, C. H. Nakatsu, and A. Konopka. 2002. Functional and structural adaptations of bacterial communities growing on particulate substrates under stringent nutrient limitation. Microb. Ecol. 44: 317-326.
• Konopka, A., T. Zakharova, and C. H. Nakatsu. 2002. Effect of starvation length upon microbial activity in a biomass recycle reactor J. Indust. Microbiol. Biotech. 29: 286-291.
• Baldwin, B., L. Nies, C. Nakatsu, and J. Simonds. Molecular genetic detection and enumeration of aromatic oxygenase genes at gasoline-contaminated sites. Abstracts of the Battelle Bioremediation Conference in Orlando, FL (June 2-5, 2003)
• Kourtev, P. S., N. Carmosini, F. Beasley, C. Nakatsu, A. E. Konopka. Heavy metals modulate microbial community responses to inputs of organic carbon. American Society for Microbiology Annual Meeting Abstracts. Washington D.C. (May 2003).
• Carrero-Colon, M., C. Nakatsu, A. E. Konopka. Nutrient periodicity as a selective force in structuring microbial communities. American Society for Microbiology Annual Meeting Abstracts. Washington D.C. (May 2003).
• Jerke, K., C. H. Nakatsu, and A. Konopka. A physiological and genetic approach to studying lead resistance in Arthrobacter sp. VN23-1. American Society for Microbiology Annual Meeting Abstracts. Washington D.C. (May 2003).
• Hristova, K. R., B. Inceoglu, C. Naktsu. K. M. Scow. Characterization of enzymes involved in MTBE biodegradation in strain PM1. American Society for Microbiology Annual Meeting Abstracts. Washington D.C. (May 2003).
• Jerke, K., C. H. Nakatsu, and A. Konopka. A physiological and genetic approach to studying lead resistance in Arthrobacter sp. VN23-1. American Society of Agronomy Abstracts. Indianapolis IN, (Nov. 2002).
• Ackerman, C., J. Kerstiens, M. Hickman, S.M Brouder, and C.H. Nakatsu. The effects of cover crops on the rhizosphere bacterial community and development of the subsequent corn crop. American Society of Agronomy Abstracts. Indianapolis IN, (Nov. 2002).
• Becker, J., R. Turco, C. H. Nakatsu, and A. Konopka. Spatial heterogeneity of bacterial activity and community structure at a long-term contaminated site. American Society of Agronomy Abstracts. Indianapolis IN, (Nov. 2002).
• Beasley, F., C. Nakatsu, N. Caromosini, A. Konopka. American Society of Agronomy Abstracts. Indianapolis IN, (Nov. 2002).
• Bulinski, D. A, C. H. Nakatsu, J. D. Wilbur, S. M. Brouder, and R. W. Doerge. Identification of Bacteria Associated With Monoculture Yield Decline in Corn. American Society of Agronomy Abstracts. Indianapolis IN, (Nov. 2002).

Progress 10/01/01 to 09/30/02

Outputs
Fingerprints generated by PCR amplification of the rRNA gene from total soil DNA resolved by denaturing gradient gel electrophoresis (DGGE) are being used to compare the microbial community in the rhizosphere of crop plants. Statistical and visual analysis of the corn rhizosphere fingerprints collected over three years showed that the main differentiating treatment apparently was crop rotation, tillage and plant growth stage. Although there was some variation in the treatment effect between years, both plant and microbial community analysis showed there were differences associated with agronomic treatment most often monoculture corn. The same approach was used to compare the rhizosphere microbial community of soybean plants exposed to the same agronomic treatments. Plant measures and grain yields indicated that soybean plant productivity is not substantially affected by the agronomic treatments we tested. PCR-DGGE analysis also indicated that agronomic treatment did not affect the microbial community. Most differences were related to plant growth stage. Thus it appears that differences in microbial communities structure associated with plant productivity and agronomic treatments can be seen using this approach. The method was then expanded to determine the relationship between microbial community structure and physiology. Continuous-flow bioreactors with 100% biomass recycle were used to mimic substrate-limited conditions of soil. Cell activity (growth rate) is mainly coupled to an increase in total RNA (ribosomes), thus rRNA was tested as a template to differentiate active populations from those that were persistent (16S rDNA template). Results indicated that using RNA vs DNA showed greatest difference during physiological response to a transient change when there are differential changes in ribosome content among community members. However, under steady state conditions, analysis using either rDNA or rRNA templates provided similar information about the community dynamics. Using DGGE analysis of both rDNA and rRNA templates provides a more comprehensive view of community dynamics allowing us to not only understand the diversity of microorganisms in an ecosystem, but also differentiate those that are most active. Although this type of analysis has been possible for organisms with more complex structures, it has not been possible with bacteria due to their size and difficulty in cultivation. In order to understand the physiology of microbes in the environment we are also developing methods to quantify functional genes. Our present focus has been to quantify genes involved in the degradation of aromatic hydrocarbons. A technique based on real time PCR amplification of aromatic oxygenase genes was developed to detect and quantify these genes in environmental samples. The methods proved to be effective in laboratory enrichment microcosms and field sites being monitored for natural attenuation of contaminants. In addition to the basic knowledge gained in understanding microbial activity this method is invaluable as a means to document biodegradation potential and gain public and regulatory approval for monitored natural attenuation of pollutants.

Impacts
This project contributes significantly to the long-range improvement and sustainability of soils, and furthering our basic understanding of the immense diversity in biological processes for maintaining and improving soil and environmental quality. We have demonstrated the use of a genetic fingerprinting method to determine the impact of various agronomic management schemes on the soil microbial community in addition to the remediation of soils contaminated with organic pollutants and/or heavy metals.

Publications

• Beasley, F. C. and C. H. Nakatsu. 2002. Isolation and Characterization of Chromate Resistant Bacteria from Mixed Waste Contaminated Soil. American Society for Microbiology Annual Meeting Abstracts. Salt Lake City UT (May 2002) Abstract.
• Bulinski, D. A, C. H. Nakatsu, J. D. Wilbur, S. M. Brouder, and R. W. Doerge. 2001. Rhizosphere Microbial Population Dynamics Over Three Field Seasons. American Society of Agronomy Abstracts. Charlotte, NC, (Oct. 2001) Abstract.
• Baldwin, B. R., C. H., Nakatsu, and L. Nies. 2002. Detection and Enumeration of Aromatic Catabolic Oxygenase Genes from Environmental Samples. Bioremediation & Biodegradation Conference. Pacific Grove CA (June 2002) Abstract.
• Carrero-Colon, M., C. H. Nakatsu and A. Konopka,. 2002. Effects of Periodicity on Resource Supply upon Microbial Community Structure. American Society for Microbiology Annual Meeting Abstracts. Salt Lake City UT (May 2002) Abstract..
• Curtis, P., C. H. Nakatsu, and A. Konopka. 2002. Characterization of aciduric bacteria isolated from acid forest soils. Arch. Microbiol. 178:65-70.
• Wang, J.-W., S. M. Luo, Y.-J. Feng, and C. Nakatsu. 2002. Environmental fate and ecological effects of Bt toxin from transgenic Bt crops in soil. Acta Ecological Sinica (in Chinese).
• Wilbur, J. D., J. K. Ghosh, C. H. Nakatsu, S. M. Brouder, and R. W. Doerge. 2002. Variable selection in high-dimensional multivariate binary data with application to the analysis of microbial community DNA fingerprints. Biometrics 58: 378-386
• Morgan, C. A., A. Hudson, A. Konopka and C. H. Nakatsu. 2002. Analyses of microbial activity in biomass recycle reactors using denaturing gradient gel electrophoresis of 16S rDNA and 16S rRNA PCR products. Can. J. Microbiol. 48: 333-341.
• Ackerman, C. 2001. The effects of cover crops on the rhizosphere bacterial community and development of the subsequent corn crop. M. S. Thesis, Department of Agronomy, Purdue University, West Lafayette, IN.
• Wang, J. and C.H. Nakatsu. 2002. Bacillus thuringiensis (Bt) toxin released from root exudates of Bt corn has no apparent effect on rhizobacteria communities examined by 16S rDNA PCR-DGGE analysis. Abstracts of the 7th international Biosafety conference of GMO in Beijing.PRC (October 10-15, 2002) Abstract.
• Baldwin, B., C. Nakatsu, and L. Nies. 2002. Detection and Enumeration of Aromatic Catabolic Oxygenase Genes from Environmental Samples. in Midwestern States Risk Assessment Symposium Indianapolis IN (July 24-26, 2002) Abstract.
• Nakatsu, C. H., M. Carrero-Colon, and A. Konopka. 2002. Effects of substrate oscillations upon microbial community structure. BAGECO-7 Symposium on Bacterial Genetics and Ecology Bergen, Norway (June 2002) Abstract.
• Ovreas, L., C. Nakatsu, V. Torsvik, B. Baldwin, and P.J. Brandvik. 2002. Exploring the bacterial community diversity in an oiled sediment shoreline at Svalbard. BAGECO-7 Symposium on Bacterial Genetics and Ecology Bergen, Norway (June 2002) Abstract.

Progress 10/01/00 to 09/30/01

Outputs
Fingerprints generated by polymerase chain reaction (PCR) amplification of a region of the rRNA gene from total soil DNA, resolved by denaturing gradient gel electrophoresis (DGGE) are being used to compare the microbial community in the rhizosphere of crop plants (corn and soybean). The analysis of the corn rhizosphere fingerprint data collected over three field seasons is in progress. Statistical and visual analyses showed that the main treatment that appeared to be differentiating the fingerprint patterns was tillage, crop rotation and plant growth stage. The contribution of each treatment varied between years and was likely influenced by the weather. The same approach was used to compare the rhizosphere microbial community of corn planted after nine different winter cover crop mixtures. The corn rhizosphere community after cover crops of oat plus another plant was more alike, whereas the profiles from the other treatments could not be distinguished. Thus it appears that differences in microbial communities structure that correlate with different agronomic treatments can be seen using this approach. The impact on the microbial community of long-term exposure in the environment to metal and organic contamination was also investigated. Soil samples were collected along a transect dug in soils contaminated with road paint and paint solvents. Chemical analysis along the transect revealed a range from high to low concentrations of metals (lead and chromium) and organic solvent compounds. In this study PCR-DGGE showed fingerprint similarities that corresponded to contaminant levels. The results indicated that microbial communities have adapted to exist in a mixed waste contaminated site. The productivity of the community was not deterred by the contaminants but rather enhanced by the organic contaminants. This indicates that bioremediation is a viable means of cleaning up mixed waste contaminated sites.

Impacts
This project contributes significantly to the long-range improvement and sustainability of US agriculture by furthering our basic understanding of the immense diversity in biological processes for maintaining and improving soil and environmental quality. We have demonstrated the use of a genetic fingerprinting method to determine the impact of various agronomic management schemes on the soil microbial community in addition to the remediation of soils contaminated with organic pollutants and/or heavy metals.

Publications

• Sigler, W. V., C. H. Nakatsu, Z. J. Reicher, and R. F. Turco. 2001. Fate of the biologicl control agent Pseudomonas aureofaciens after application to turgrass. Appl. Environ. Microbiol. 67: 3542-3548.
• LaPara, T. M., C. H. Nakatsu, L. M. Pantea, and J. E. Alleman. 2001. Aerobic biological treatment of pharmaceutical wastewater: effect of temperature on COD removal and bacterial community development. Water Research. (in press).
• LaPara, T. M., A. Konopka, C. H. Nakatsu, and J. E. Alleman. 2001. Thermophilic aerobic wastewater treatment in a membrane-coupled bioreactor. J. Indust. Microbiol. Biotech. (in press).
• Huang, C., L. Lee, and C. H. Nakatsu. 2000. Impact of animal waste lagoon effluents on chlorpyrifos degradation. Environ. Toxicol. Chem. 66:2864-2870.
• Wilbur, J. D., C. H. Nakatsu, S. M. Brouder, J. K. Ghosh, R. W. Doerge. 2001. Statistical issues in the analysis of microbial communities in soil. in Proceedings of Conference on Applied Statistics in Agriculture. Kansas State University, Manhattan KS.
• Research Abstracts Wanjau, F. M., S.M Brouder, C.H. Nakatsu, and R.W. Doerge. 2000. Influence of crop rotation and tillage practices on root architecture of corn and soybean. in the American Society of Agronomy Abstracts.
• Ackerman, C., J. Kerstiens, M. Hickman, S.M Brouder, and C.H. Nakatsu. 2000. Corn rhizosphere bacterial community structure after various cover crop treatments. in the American Society of Agronomy Abstracts.
• Jerke, K., C. H. Nakatsu, and A. Konopka. 2001. Genetic and physiological analyses of lead resistance in Arthrobacter sp. isolated from contaminated soil. in the American Society for Microbiology Annual Meeting Abstracts.
• Nakatsu, C. H., M. Vargha, J. Steger, and A. Konopka. 2001. Chromium resistance by Arthrobacter sp. Cr15 isolated from metal contaminated soil. in the American Society for Microbiology Annual Meeting Abstracts. Carrero-Colon, M., C. A. Morgan, A. Konopka, T. P. Zakharova, and C. H. Nakatsu. 2001. Microbial community response to perturbation of a model wastewater treatment system. in the American Society for Microbiology Annual Meeting Abstracts.
• Curtis, P., C. H. Nakatsu, and A. Konopka. 2001. Acid tolerance of chemoheterotrophic bacteria isolated from an acidic (pH 3) soil. in the American Society for Microbiology Annual Meeting Abstracts.
• Becker, J., J. Joynt, A. Konopka and C. H. Nakatsu. 2001. Bacterial community diversity and activity at a mixed waste contaminated site. in the American Society for Microbiology Annual Meeting Abstracts.
• Wilbur, J. D., J. K. Ghosh, C. H. Nakatsu, S. M. Brouder, R. W. Doerge. 2001. A Bayesian approach to modeling and variable selection in high-dimensional multivariate binary data. in the 2001 Joint Statistical Meetings Abstract.
• Nakatsu, C. H., J. Joynt, and A. Konopka. 2001. Impact of metal and organic waste on a microbial community. in the 9th International Symposium on Microbial Ecology Abstracts.

Progress 10/01/99 to 09/30/00

Outputs
Fingerprints generated by polymerase chain reaction (PCR) amplification of a region of the rRNA gene from total soil DNA, resolved by denaturing gradient gel electrophoresis (DGGE) are being used to compare the microbial community in the rhizosphere of crop plants (corn and soybean). The third full season of sampling of the microbial community from soils exposed to different agronomic treatments (tillage and crop rotation) was completed. Statistical and visual analyses of rhizosphere fingerprint patterns showed that the main component differentiating the fingerprint patterns in the first year was the tillage treatment, followed by crop rotation then plant growth stage. In the second year the same components were found, however, crop rotation and plant growth stage appeared to have a greater contribution. Thus it appears that differences in microbial communities structure that correlate with different agronomic treatments can be seen using this approach. However, when the same approach was used to compare the rhizosphere microbial community of corn planted after different winter cover crops no contributing factors could be distinguished. The differences between the two studies are the agronomic treatments tested (tillage/rotation vs cover crop) and the length of the treatments (25 years for tillage/ rotation and 1 year for cover crop). This requires further investigation. At the gene scale, progress is being made on the investigation of gene transfer as a means of expanding the catabolic capabilities of environmental bacteria. A chemostat (continuous cultivation) was used in preliminary studies of gene transfer into bacterial community. The PCR-DGGE method was used to observe changes in the microbial community structure after exposure to a unique carbon source and the provision of genes required for its utilization. In this case the gene donor was unable to utilize the carbon source. The DGGE fingerprints revealed the disappearance of the donor strain and the persistence of strains that apparently acquired the degradation genes. Experimentation is continuing to determine factors that enhance and limit gene transfer.

Impacts
This project contributes significantly to the long-range improvement and sustainability of US agriculture by furthering our basic understanding of the immense diversity in biological processes for maintaining and improving soil and environmental quality. We have demonstrated the use of a genetic fingerprinting method to determine the impact of various agronomic management schemes on the soil microbial community. A long-term goal is to isolate organisms that can be used to enhance the growth of crops under conditions that would otherwise result in decreased crop yield.

Publications

• LaPara, T. M., C. H. Nakatsu, L. M. Pantea, and J. E. Alleman. 2000. Phylogenetic diversity of bacteria in mesophilic and thermophilic bioreactors treating pharmaceutical wastewater. Appl. Environ. Microbiol. 66:3951-3959.
• Nakatsu, C. H., V. Torsvik, and L. _vreOs. 2000. Soil community analysis using denaturing gradient gel electrophoresis (DGGE) profiles of 16S rDNA PCR products. Soil Sci. Soc. Amer. J. 64:1382-1388.
• LaPara, T. M., A. Konopka, C. H. Nakatsu, and J. E. Alleman. 2000. Thermophilic aerobic wastewater treatment in continuous-flow bioreactors. J. Environ. Eng. 126:739-744.
• Ramakrishnan, R., D. R. Suitor, C. H. Nakatsu, and G. W. Bennett. 2000. Feeding inhibition and mortality in Reticulitermes flavipes (Kollar) (Isoptera:Rhinotermitidae) following exposure to imidacloprid-treated soils. J. Econ. Entomol. 93:422-428.
• LaPara, T. M., A. Konopka, C. H. Nakatsu, and J. E. Alleman. 2000. Effects of elevated temperatures on bacterial community structure and function during aerobic biological wastewater treatment. J. Ind. Microbiol. Biotech. 24:140-145.
• Mesarch, M., C. H. Nakatsu, and L. Nies. 2000. Development of catechol 2,3-dioxygenase-specific primers for monitoring bioremediation by competitive quantitative PCR. Appl. Environ. Microbiol. 66: 678-683.
• Ramakrishnan, R., D. R. Suitor, C. H. Nakatsu, and G. W. Bennett. 1999. Imidacloprid-enhanced Reticulitermes flavipes (Kollar) (Isoptera: Rhinotermitidae) susceptibility to the entomopathogen Metarhizium anisopliae (Metschnikoff) Sorokin. J. Econ. Entomol. 92: 1125-1132.
• Joynt, J., A. Konopka, R. Turco, and C. Nakatsu. 2000. Bacterial community diversity at a mixed waste contaminated site. In Proceedings of International Conference on Heavy Metals in the Environment. Ann Arbor, MI (CD-ROM).
• Nakatsu, C. H., J. Steger, D. Petros and A. Konopka. 2000. Chromium resistance of bacteria from contaminated soil. In Proceedings of International Conference on Heavy Metals in the Environment. Ann Arbor, MI (CD-ROM).
• Wilbur, J. D., C H. Nakatsu, S. M. Brouder, and R. W. Doerge. 2000. Statistical analysis of microbial communities. In Proceedings of Interface 2000. The 32nd Symposium on the Interface: Computing science and statistics. New Orleans, LA (CD-ROM).
• Nakatsu, C H., S. M. Brouder, J. D. Wilbur, F. Wanjau, and R. W. Doerge. 2000. Impact of tillage and crop rotation on corn development and its associated microbial community. In Proceedings of 15th Conference of the International Soil Tillage Research Organization (ISTRO). Fort Worth, TX (CD-ROM).

Progress 10/01/98 to 09/30/99

Outputs
At the microbial community scale we are investigating the link, between microorganisms and monoculture yield decline when the same crop is grown continuously. Previously we showed that the dynamics among rhizosphere soil microbes and host plants are can be analyzed by comparing fingerprints generated by PCR amplification of rDNA sequences from total soil DNA, resolved by denaturing gradient gel electrophoresis (DGGE). The second full season of sampling and analysis of the microbial community from soils exposed to different agronomic treatments (tillage and crop rotation) was completed. Visual analysis of rhizosphere fingerprint patterns showed that there are bands common to specific agronomic during early corn growth. Statistical analysis showed that the main component differentiating the fingerprint patterns was the tillage treatment, followed by crop rotation then plant growth stage. Comparison of rhizosphere microbial fingerprint patterns from corn grown in the greenhouse versus the field revealed distinctly different patterns although soil from the field was used for greenhouse experiments. The results thus far show that the method chosen for this analysis does appear to show difference in microbial communities that correlate to different agronomic treatments. In order to demonstrate that this method gave consistent results in other ecosystems it was tested on metal contaminated soils, bioreactors and lakes. The results showed that the method was reproducible and that information can be gained on microbial community changes when exposed to different environmental perturbations. We also started a research project to determine if different winter cover crops can influence the rhizosphere microbial community of corn planted in the following season. At the gene scale, progress is being made in our investigation on the transfer mechanism of pesticide degradation genes into environmental bacteria. We have been studying an environmental soil bacterium, Ralstonia sp. TFD41, isolated because of its ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). Nucleotide sequence determination of the composite transposon has been completed. The element is flanked by one complete and one partial copy of IS1071. The intervening nucleotide sequence codes for the first gene in the 2,4-D degradation pathway and its regulatory gene, tfdA and tfdR and homologs of the remainder of the chlorophenol degradation pathway tfdBII, tfdCII, tfdDII, tfdEII and tfdFII. This genetic arrangement potentially provides a means for horizontal transfer of 2,4-D degradation genes. We believe this has contributed to the global distribution of these genes in soil bacteria. Ultimately this research will allow us to use IS1071 to transfer different degradation genes to assist microorganisms to remediate soils contaminated with more persistent chemicals such as, PCBs. This project is important not only for Indiana but has global significance because of the extensive use of synthetic chemicals throughout the world, and the need to prevent their accumulation.

Impacts
This project contributes significantly to the long-range improvement and sustainability of US agriculture by furthering our basic understanding of the immense diversity in biological processes for maintaining and improving soil and environmental quality. It contributes to our understanding of the interaction between plants and the microbial community as well as the capacity of natural soil biota to degrade, or transform agrichemicals and other pollutants.

Publications

• Konopka, A. E., T. Zakharova, M. Bischoff, L. Oliver, C. H. Nakatsu, and R. Turco. 1999. Microbial biomass and activity in lead contaminated soil. Appl. Environ. Microbiol. 65: 2256-2259.
• Konopka, A. E., T. Bercot, and C. H. Nakatsu. 1999. Bacterioplankton community diversity in a series of thermally stratified lakes. Microb. Ecol. 38:126-135.
• Soto, B. L., S. M. Brouder, and C. H. Nakatsu. 1999. Diversity of the rhizosphere bacterial community during early growth of corn. American Society for Microbiology Annual Meeting Abstracts. Chicago, IL, p. 490.
• Nakatsu, C. H., J. D. Wilbur, R. W. Doerge, and S. M. Brouder. 1999. Rhizosphere bacterial population dynamics and root architecture. American Society of Agronomy Abstracts. Salt Lake City, UT, p. 230.
• Brouder, S. M., C. H. Nakatsu, and R. W. Doerge. 1999. Microbial community dynamics in the rhizosphere of corn and soybean. American Society of Agronomy Abstracts. Salt Lake City, UT, p. 369.
• Bulinski, D. 1999. Nucleotide sequence analysis of TnTFD, a composite transposon containing 2,4-dichlorophenoxyacetic acid degradation genes. MS thesis, Purdue University, West Lafayette, IN. 114 pp

Progress 10/01/97 to 09/30/98

Outputs
At the microbial community scale we are investigating the link, between microorganisms and monoculture yield decline when the same crop is grown continuously. Dynamics among rhizosphere soil microbes and host plants are being analyzed by comparing fingerprints generated by PCR amplification of rDNA sequences from total soil DNA, resolved by denaturing gradient gel electrophoresis (DGGE). The first full season of sampling and analysis of the microbial community from soils exposed to different agronomic treatments (tillage and crop rotation) was completed. Whole growing corn and soybean plants and their rhizosphere were collected from the Long Term Tillage plots at the Purdue Agronomy Research Center. Our results show that distinct populations of bacteria dominate the rhizospheres of corn and soybean. There are a number of plant specific populations present in the rhizosphere that are observed during early plant growth. The populations that are represented by dominate bands in the fingerprints decrease with progressive stages of plant development. Differences in fingerprint were also observed when the various agronomic treatments (rotation and tillage) were compared. The differences were more evident in the corn rhizosphere compared to soybean. The results thus far show that the method chosen for this analysis does appear to be practical and can potentially show the contribution of the microbial community to crop productivity. At the gene scale, progress is being made in our investigation on the transfer mechanism of pesticide degradation genes into environmental bacteria. We have been studying an environmental soil bacterium, Ralstonia sp. TFD41, isolated because of its ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). In our research we have found the first gene in the of 2,4-D degradation pathway and its regulation gene, tfdA and tfdR respectively, are located within IS1071 containing composite transposons. A number of studies have shown that transposons delineated by the insertion element (a type of transposon), IS1071, contain many different degradation genes. This genetic arrangement potentially provides a means for horizontal gene transfer. We believe this has contributed to the global distribution of these genes in soil bacteria. Ultimately this research will allow us to use IS1071 to transfer different degradation genes to assist microorganisms to remediate soils contaminated with more persistent chemicals such as, PCBs. This project is important not only for Indiana but has global significance because of the extensive use of synthetic chemicals throughout the world, and the need to prevent their accumulation.

Impacts
(N/A)

Publications

• Nakatsu, C. H., R. Korona, R. E. Lenski, T. L. Marsh, F. J. DeBruijn, and L. J. Forney. 1998. Parallel and divergent genotypic evolution in experimental populations of Ralstonia sp. J. Bacteriol. 180: 4325-4331.
• Bulinski, D., and C. Nakatsu. 1998. Involvement of a transposon in the dissemination of 2,4-D catabolic genes. at the 8th International Symposium on Microbial Ecology in Halifax, Canada. (Aug. 1998).
• Nakatsu, C. H., S. M. Brouder, and J. Wells. 1998. Rhizosphere microbial ecology of corn and soybean: Relevance to system productivity. American Society of Agronomy Abstracts. Baltimore, MD, (Oct. 1998).

Progress 10/01/96 to 09/30/97

Outputs
At the microbial community scale we are investigating the link, if any, between microorganisms and monoculture yield decline when crops are grown continuously. Dynamics among rhizosphere soil microbes and host plants are being analyzed by comparing fingerprints generated by PCR amplification of rDNA sequences from total soil DNA, resolved by denaturing gradient gel electrophoresis (DGGE). Preliminary sampling and analysis of the microbial community from soils exposed to different agronomic treatments (tillage and crop rotation) have commenced. Samples have been collected from the rhizosphere of growing corn and soybean plants and from root exclusion zones within plant rows in the Long Term Tillage plots at the Purdue Agronomy Research Center. Our results show that distinct and different populations of bacteria dominate the rhizospheres of corn and soybean when these crops are grown in rotation with each other. These fingerprints which are host plant specific are easily differentiated from the non-specific smear generated by the bulk soil sample collected from a root exclusion zone within the treatment plot. The dominant plant specific populations also appear to change with different stages of development of their higher plant host. Samples from different treatments of the LTT field trial were also compared, and different fingerprint patterns were observed. Relating these fingerprints to root function and crop development will advance our understanding of agricultural productivity in rotational cropping systems and optimize their management. At the gene scale, progress is being made in our investigation on the transfer mechanism of pesticide degradation genes into environmental bacteria. A number of studies have shown that transposons delineated by the insertion element (a type of transposon), IS1071, contain many different degradation genes. We have been studying an environmental soil bacterium, Comamonas sp. TFD41, isolated because of its ability to degrade the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D). In our research we have found genes (tfdA and tfdR) required for the degradation of 2,4-D are located within IS1071 composite transposons. We believe this has contributed to the global distribution of these genes in soil bacteria. Ultimately this research will allow us to use IS1071 to transfer different degradation genes to assist microorganisms to remediate soils contaminated with more persistent chemicals such as, PCBs. This project is important not only for Indiana but has global significance because of the extensive use of synthetic chemicals throughout the world, and the need to prevent their accumulation.

Impacts
(N/A)

Publications

• Wells, J. E., S. M. Brouder, and C. H. Nakatsu. 1997. Soil Microbial Community Analysis Using Denaturing Gradient Gel Electrophoresis. American Society of Agronomy Abstracts. Anaheim, CA, (Oct. 26-30, 1997), pp. 213-214.
• Bulinski, D. A. and C. H. Nakatsu. 1997. Characterization of a Transposon Involved in Degradation of 2,4-Dichlorophenoxyacetic Acid. American Society of Microbiologist's Annual Meetings Abstracts. Miami Beach, FL, (May 4-8, 1997), p. 296.

Progress 10/01/95 to 09/30/96

Outputs
In agriculture, anthropogenic chemicals are extensively introduced into the environment in the form of pesticides. Although some compounds, such as 2,4-dichlorophenoxyacetic acid (2,4-D), have complex structures they are readily degraded in nature. We have preliminary evidence that 2,4-D catabolic genes are transferred into different bacteria on transposable elements. Transposable elements may be responsible for the rapid dissemination of the catabolic genes in a community resulting in the degradation of 2,4-D. A number of different genes are required for complete degradation of 2,4-D. Presently we have identified a tentative transposon carrying tfdA, the first gene in the 2,4-D degradation pathway. The mobility and distribution of this transposon in soil will be determined. Our work on whole microbial community analysis is just beginning and presently we have no results.

Impacts
(N/A)

Publications

Progress 10/01/94 to 09/30/95

Outputs
Laboratory equipment was purchased to set up a new laboratory. Specialized equipment for molecular genetic analysis of soil bacteria are required for the successful isolation of DNA from soil and their subsequent manipulation and transfer to other organisms. Equipment purchased are: a thermal cycler (PCR); and electrophoresis units for separation of small DNA fragments (horizontal sub electrophoresis units) and large fragments (CHEF Mapper).

Impacts
(N/A)

Publications

• NO PUBLICATIONS REPORTED THIS PERIOD.