1. Characterization of nitrogenase gene distribution and activity in WCA-2A periphyton Puja Jasrotia Image source: http://www-cyanosite.bio.purdue.edu

2. Why is Nitrogen important A major constituent of living cells. Nitrogen gas (N 2 ) makes up 79% of the atmosphere. Required by all living organisms, but only a few can fix it. Often the limiting nutrient in wetlands.

3. Biological Nitrogen fixation N 2 NH 4 + Catalyzed by nitrogenase enzyme Only prokaryotic organisms can fix nitrogen. Induced as a response to low conc. of fixed N 2. High conc. of O 2 and NH 4 + repress nitrogenase synthesis

4. VEGETATION: U3 Lake Okeechobee WCA-1 -3 Everglades Agricultural Area WCA-2A 012345 Km Everglades National Park Cattail Sawgrass/slough Cattail/sawgrass mix F1 F4 Nutrient Transect in WCA-2A Cattail ~ 1500 mg P/Kg Sawgrass ~ 500 mg P/Kg Florida Everglades

5. Periphyton processes Metabolically diverse microbial composition contributes to mat’s internal nutrient cycling. Important source of fixed N and C. Structural and functional changes indicative of nutrient enrichment. Contributes approx 10g N m -2 y in oligotrophic WCA2A (Inglett et al., 2004).

6. Lack of molecular characterization of diazotrophs. Identify diazotrophic groups for a broad understanding of nutrient limitation factors regulating growth indicator species of eutrophication and flow of N in periphyton

7. Objectives 1. Compositional analysis of diazotrophs along the nutrient gradient in WCA-2A based on nifH diversity. 2. nifH gene expression during a diel cycle. 3. Spatial distribution of pmoA defined diversity in floating periphyton along the nutrient gradient.

8. Hypotheses 1.Periphyton nifH composition differs between eutrophic and oligotrophic regions as a result of relative N limitation. 2.A shift in the most active nitrogen fixing groups will be observed throughout a diel cycle. 3.pmoA diversity will vary with nutrient concentrations.

9. Work plan Periphyton sampleEpiphyton sample Methane oxidation N 2 fixation Community assemblage composition analysis pmoA phylogenetic analysis by sequence analysis nifH phylogenetic analysis by sequence analysis N 2 fixation Functional gene Analysis RT-PCR - characterizing nifH transcripts

10. 1. Spatial variability of nifH diversity in periphyton

11. Eutrophic site Oligotrophic site Floating periphyton mats

12. Phylogenetic tree PeriphytonNucleic acid PCR amplification Cloning Sequencing Seq. Analysis RFLP Image source: www.rothamsted.bbsrc.ac.uk

13. Rarefaction analysis Rarefaction analysis of nifH clones from F1, F4 & U3.

14. Phylogenetic tree of cyanobacterial nifH clones from F1, F4 and U3. Heterocystous cluster Non- heterocystous unicellular cluster Non-heterocystous cluster Unidentified cluster Non-heterocystous cluster Unidentified cluster

15. Phylogenetic tree of proteobacterial nifH clones from F1, F4 & U3.  -proteobacteria  - proteobacteria  - proteobacteria

16. Distribution of nifH clones from F1, F4 & U3

17. Spatial and temporal interactions Image source: www.botany.hawaii.edu

18. Spatial and temporal interactions between oxygenic photosynthesis and N 2 fixation Night timeDaytime Mat Surface Anaerobic N 2 Fixation Oxygenic Photosynthesis Aerobic N 2 Fixation Anoxygenic Photosynthesis Anaerobic N 2 Fixation Organic Carbon Pool Oxic Anoxic Oxic Anoxic Organic C Fixed N After Paerl, et al., 1989 Fixed N

19. Consortial N 2 fixation between cyanobacteria and heterotrophic bacteria Cyanobacterium Heterotrophic Bacteria Organic Matter (C,N) Fixed N CO 2 PO 4 3- Vitamins, Chelators, Metals & Other Growth Factors After Steppe, et al., 1996

20. Conclusions - 1 Distinct shifts with nutrient enrichment in F1, F4, U3. Oligotrophic periphyton has diverse diazotrophs, including cyanobacteria,  -,  - &  -proteobacteria In eutrophic areas, diversity is limited, cyanobacterial species dominate, indicating a noticeable shift to bloom forming genera. Methanotrophs may be important groups of nitrogen fixers.

21. 2. nifH expression in epiphyton over a diel period

22. Epiphyton ‘sweaters’

23. Genomic DNA Nested PCR Sequence analysis Cloning and RFLP analysis Total RNA RT PCR Epiphyton sample Image source: www.dr-addie.com

24. Rarefaction & phylogenetic analyses of nifH DNA clones Cyanobacterial cluster  -Proteobacterial cluster

25. Distribution of clones

26. Phylogenetic analysis of RT-PCR nifH clones Cluster I Cluster II Cluster III Cluster IV Uncultured clone cluster Cluster V Cluster VI Cluster VII

29. Distribution of RT-PCR nifH clones

30. Conclusions - 2 Specific cyanobacterial groups express nitrogenase as a function of time. Unidentified clusters novel to oligotrophic Everglades epiphyton observed. Nitrogenase expression suggest nitrogen limitation. Presence of deep branching limits confidence in assigning diazotrophic groups.

31. 3. Methanotrophic diversity along the nutrient gradient in periphyton mats

32. Capable of growth on methane as sole source of carbon and energy Divided into 2 major phylogenetic groups: - Type I (  proteobacteria) - Type II (  proteobacteria) Methane oxidation - by methane monooxygenases (pMMO & sMMO) nifH characterization implicate methanotrophs as diazotrophs in periphyton. High methanogenesis in eutrophic sites Methanotrophic bacteria

33. Phylogenetic analysis of pmoA clones from F1, F4 & U3 Type I & X methanotrophs Unidentified clades Type II methanotrophs

34. Distribution of pmoA clones from F1, F4 & U3

35. Conclusions - 3 Type I methanotrophs dominate in three sites, suggesting N limitation. F4 harbor a greater diversity including type X and type II methanotrophs. F1 and U3 not diverse, and U3 represents single dominated unidentified clade. Presence in periphyton mats indicate methane oxidation.

36. Summary nifH diversity differs along the nutrient gradient Nitrogenase expression characterized as a function of time Expression patterns complex, suggests regulated diel nitrogen fixation Unique uncharacterized clades of diazotrophs and methanotrophs identified

37. Future Applications Genome characterization of nitrogen fixers. Assessment of environmental regulatory mechanisms. Mapping the flow of N in periphyton.

38. Acknowledgements Dr. Andrew V. Ogram Committee members: »Dr. Sue Newman »Dr. Edward Phlips National Science Foundation Lab members My family

39. “…Restoring the Everglades is not rocket science or brain Surgery. It’s much more complicated than that…” - Don Boesch University of Maryland