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Characterization of nitrogenase gene distribution and activity in WCA-2A periphyton

This study explores the composition and activity of nitrogenase genes in periphyton mats from WCA-2A in Everglades. It aims to identify diazotrophic groups and investigate their response to nutrient gradients. The study also investigates the diversity of pmoA genes involved in methane oxidation. The results provide insights into nutrient limitation and the flow of nitrogen in periphyton mats.

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Characterization of nitrogenase gene distribution and activity in WCA-2A periphyton

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  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 (N2) 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 • N2 NH4+ • Catalyzed bynitrogenase enzyme • Only prokaryotic organisms can fix nitrogen. • Induced as a response to low conc. of fixed N2. High conc. of O2 and NH4+ repress nitrogenase synthesis

  4. Nutrient Transect in WCA-2A Lake Okeechobee Everglades Agricultural Area WCA - 1 WCA - 2A WCA - 3 Cattail ~ 1500 mg P/Kg Everglades National Park F4 F1 ~ 500 mg P/Kg U3 VEGETATION : Cattail Cattail/ sawgrass mix 0 1 2 3 4 5 Sawgrass/slough Km Sawgrass 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-2y 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 Compositional analysis of diazotrophs along the nutrient gradient in WCA-2A based on nifH diversity. nifH gene expression during a diel cycle. Spatial distribution of pmoA defined diversity in floating periphyton along the nutrient gradient.

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

  9. N2 fixation Methane oxidation N2 fixation Community assemblage composition analysis Functional gene Analysis nifH phylogenetic analysis by sequence analysis pmoA phylogenetic analysis by sequence analysis RT-PCR - characterizing nifH transcripts Work plan Periphyton sample Epiphyton sample

  10. 1. Spatial variability of nifH diversity in periphyton

  11. Eutrophicsite Oligotrophicsite Floating periphyton mats

  12. Periphyton Nucleic acid Phylogenetic tree Cloning RFLP PCR amplification Seq. Analysis Sequencing 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. d-proteobacteria g-proteobacteria a-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 N2 fixation Daytime Night time Mat Surface Oxic Oxygenic Photosynthesis Anoxic Anaerobic N2 Fixation Organic C Fixed N Aerobic N2 Fixation Organic Carbon Pool Oxic Anoxic Anoxygenic Photosynthesis Organic C Fixed N Anaerobic N2 Fixation After Paerl, et al., 1989

  19. Consortial N2 fixation between cyanobacteria and heterotrophic bacteria Heterotrophic Bacteria Organic Matter (C,N) Fixed N Cyanobacterium CO2 Vitamins, Chelators, Metals & Other Growth Factors PO43- After Steppe, et al., 1996

  20. Conclusions - 1 • Distinct shifts with nutrient enrichment in F1, F4, U3. • Oligotrophic periphyton has diverse diazotrophs, including cyanobacteria, a-, g- & d-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 Total RNA Epiphyton sample Nested PCR RT PCR Cloning and RFLP analysis Sequence analysis Image source: www.dr-addie.com

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

  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

  27. Distribution of RT-PCR nifH clones

  28. 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.

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

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

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

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

  33. 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.

  34. 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

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

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

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

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