Towards ‘Environomics’ Uptake and Molecular Studies of Nitrate Assimilation

1. Towards ‘Environomics’ Uptake and Molecular Studies of Nitrate Assimilation by Marine Heterotrophic Bacteria Marc E. Frischer Skidaway Institute of Oceanography

2. Preparation 1985 A.B. Washington University in St. Louis (Microbial Genetics) 1985 - 1988 Protein Chemist - Sigma Chemical Co. 1988 - 1993 Ph .D. University of South Florida (Marine Science/Microbial Ecology) 1994 - 1996 Postdoc Rensselaer Polytechnic Institute (Molecular Microbial Ecology) 1996 - Present Skidaway Inst. Of Oceanography

3. Research Interests Exploration of microbial diversity and the elucidation of linkages between the diversity of microorganisms, the activity of microbial populations, and the role that microbial diversity plays in maintaining the stability and functioning of marine/aquatic ecosystems.

4. Aquatic Molecular Microbial Ecology Development and Use of Novel Molecular Techniques to Measure Microbial Diversity and Link These Parameters to the Functional Role of Microbes in Aquatic Systems. Applied Aquatic Molecular Planktonic Studies Application of Molecular Techniques for the Study of Eukaryotic Pathogens and Planktonic Bivalve Larvae

5. Linking N & C Cycles Role of Molecular Approaches in Biogeochemical Studies Case Study: Nitrate Assimilation by Heterotrophic Bacteria Because most marine environments are nitrogen limited, the nitrogen and carbon cycles are intimately linked

6. In particular, the pathway of nitrate assimilation into autotrophic or heterotrophic organisms can have a profound influence on carbon cycling N2 Denitrification Nitrogen Fixation NitrateAssimilation NO3- NH4+ Nitrification Decomposition Assimilation Organic N

7. CO2 CO2

8. Primary Questions Do Heterotrophic Marine Bacteria Assimilate Nitrate? If so, How Much? What are the Controls? Are they Competitive with Phytoplankton Can Molecular Tools (Gene Based) Be Used to Determine Who are the Nitrate Assimilators and What Controls Them? Are Gene Presence and Expression of nasA Quantitatively Linked to Nitrate Uptake Rates? CO2 Flux?

9. Are bacteria or the bacterial size class (<0.2µ) taking up a significant amount of NO3- in marine environments?

10. Mass Spectrometry Whole Sea Water > 0.8 µm filtered Sea Water < 0.8 µm filtered Sea Water 15N NO3 15N NH4 Filter onto 0.2 µm Silver Filters Incubate 1–3 hours Calculate NO3 & NH4 Uptake Rates By Size Class Estimation of Total and Bacterial N Uptake Rates Debbie Bronk - VIMS

11. ICE Open Water Barents Sea

12. Bacterial Uptake of DIN, Barents Sea June/July 1999 NCC North Atlantic Polar Front Drift Ice Pack Ice

13. Barents Sea Study

14. SkIO South Carolina Georgia Florida

15. South Atlantic Bight , April, 2000 Estuary Inner-Shelf Mid-Shelf

16. Bacteria Appear to Account for Significant NO3 Uptake and Utilization Up to 40% of Total NO3 Utilization May Be Due To Bacteria Under Some Circumstances, but 10-15% is Probably a More Reasonable Estimate However, Experimental Methods are Flawed, Manipulative and Laborious … Can Molecular Approaches be Useful?

17. Molecular Level Studies Cannot Provide Rate and Flux Estimates, but Can Provide Information Regarding • Genetic capability • Identification • Study Regulation: Transcription into mRNA • Study Regulation: Translation into protein, and post-translational modification • Protein characterization • Signal Transduction

18. narB narB nasA

20. Growth on NO3- As Sole N Source PCR + Growth + 16 16 Growth - 16 0 Presence of nasA = Ability to Assimilate NO3 (32 Isolates from the Barents Sea)

22. (45 clones, 2 isolates) 0.1 substitutions/site Marinobacter (10 clones, 1 isolate) Marinomonas 159 Clones 10 Clone Libraries (12 clones) Unknown (13 clones, 3 strains) Alpha (14 clones, 3 isolates) Vibrio (11 clones, 4 isolates) Alteromonas (43 clones) 100 Barents Sea Clones (3 clones) 100 Unknown SAB Clones 96 (1 clones, 1 isolate) Psychrobacter 100 (6 clones) Unknown SAB Clones 100 (2 clones) 100 Unknown Barents Sea Clones (2 strains) 100 Beta 72 100 (6 strains) Cyanobacteria Methanobacterium sp. (Formate Dehydrogenase

23. Doubling Time (hours) Yield (log Increase) Strain BS-25 5.16 3.13 BS-10 5.48 3.31 BS-4 3.78 3.60 BS-23 No Growth 0.59* BS-26 No Growth 0.47* All growth determination in NFG media (Tibbles and Rawlings, 1994) supplemented with 10 mM nitrate (KNO3) Are Genetic Differences Functionally Meaningful? Growth Characteristics

24. nasA expression regulation in Klebsiella oxytoca Are Genetic Differences Functionally Meaningful? 6 4.903 5 K. oxytoca NO3- to NH4+ 4 Gene Regulation K. oxytoca NH4+ to NO3- 3 O.D./ngTotalRNA used in 1st Rnd 1.878 2 1.2304 1.1806 1 0.119 0 Tp T0 T30 T60 Time 15NO3- uptake into Klebsiella oxytoca 35000 30000 K. oxytocanasA strictly 25000 regulated 20000 [NO3- ](µgatN/L/hr) by NO3- and NH4+ 15000 10000 5000 0 T0 Tp T60 Time

25. nasA expression regulation in Vibrio diazotrophicus Are Genetic Differences Functionally Meaningful? 60 51.36 50 40 V. diazotrophicus NO3- to NH4+ Gene Regulation V. diazotrophicus NH4+ to NO3- 30 OD/ngTotal RNA in 1st Rnd 20 10 1.134 1.015 1.043 1.082 0 0 0 0 15NO3- uptake in Vibrio diazotrophicus Tp T0 T30 T60 Time 2000.00 1500.00 1000.00 [NO3-](µgatN/L/hr) 500.00 Time 0.00 Tp T0 T60 V. diazotrophicus: nasA expression inhibited By NH4+, but not stimulated By NO3- However, NO3- uptake occurs In presence of NO3- (long lived transcripts?)

26. nasA expression regulation in Pseudoalteromonas citrea 12 P. citrea NO3- to NH4+ Are Genetic Differences Functionally Meaningful? P. citrea NO3- to NH4+ P. citrea NO3- to NH4+ P. citrea NH4+ to NO3- P. citrea NH4+ to NO3- P. citrea NH4+ to NO3- 10 9.7 8.697 8.509 8 7.312 OD/ngTotal RNA in 1st Round 6.612 6 Gene Regulation 4 1.9226 1.793 2 1.6198 1.312 1.2856 0 Tp T0 T15 T30 T60 Time 15NO3-uptake in Pseudoalteromonas citrea 3000 2500 2000 1500 [NO3-](µgatN/L/hr) 1000 500 0 Tp T0 T60 Time Pseudoaltermonas citrea: Inhibited by NH4, but not Stimulated by NO3

27. Does Genetic Identity Matter? • The nasA Gene is Regulated Differently in Different Bacteria • Growth Rates of Bacteria with Genetically Distinct nasA • Gene Sequences Differ Presumably These are Important Contributing Factors to The Ecology & Biogeochemistry of Nitrate Assimilation By Heterotrophic Bacteria in Nature

28. Molecular Field Ecology • Community Finger Printing – (TRFLP & RT-TRFLP) • Quantification – Q-PCR & QRT-PCR Is community composition of nasA containing bacteria correlated with nitrate parameters (NO3 concentration & NO3 uptake rates) and other biological/chemical parameters? Is nasA expression correlated with nitrate and other parameters?

29. Back to the Barents Sea Open Water (Station IV) Ice (Station I)

30. Barents Sea T-RFLP Patterns Cluster Analysis 57 Ice 62 79 80 Open Water 65 Principal Components Analysis Ice Open Water

31. NO3- Bac Abundance NO3- LV1, x & y: 14% & 10% Bac Prod % Active Cells Chl a NH4+ LV1, x & y: 56% & 11% PLS Model – Barents Sea July 1999 DNA TRFLP Fingerprints

32. Are nasA-encoding communities and nasA expressing communities the same? related? What factors are the diversity of each related to?

33. DK14 (14 sequences) GSD10 (12 sequences) DK18 TWS29 GSD10 (3 sequences) GSS39 TWS2100015 GSD16 (7 sequences) DK100013 DK3.5 DK2 GSS26 (2 sequences) TWS210007 (2 sequences) DK10003 TWS210008 TWS225 TWS210005 GSS4 (10 sequences) TWS1 (19 sequences) DK4 (4 sequences) TWS31 GSD11 (sequences) GSD19 GSD9 GSS33 TWS2100010 DK10006 TWS210003 (10 sequences) Expressed Sequences from Clone Library DK31 (5 sequences) DK100014 Expressed Sequences Detected by RT-TRFLP DK100012 TWS21006 GSD7 TWS216 TWS230 DK10007 DK3h22 DK4h19 DK29 DK3.11 DK3.1 GSD23 GSD26 GSS1 GSD27 GSS32 TWS2100014 TWS220 GSS12 GSS13 GSS24 GSS27 DK9 (5 sequences) GSD34 DK3.3 GSS34 DK100011 DK10009 (2 sequences) DK3.10 DK23 DK10005 DK35 Sequences derived from transcripts cluster together and distinctly from sequences derived from total community DNA

34. Sargasso DNA- and RNA-derived nasA (RT) TRFLP Studies 8 6 35d(DCM) 4 6-100r PC2 2 5d 800r 18r 82.9r(DCM) 85.9d(DCM) 0 450r 40r(DCM) 800d 82.9d(DCM) 40d(DCM) 450d -2 800d 82.9d(DCM) 100d -4 -6 -4 -2 0 2 4 PC1

35. Can we relate nasA expression measured with our PCR-based methods to 15N uptake or nutrient concentrations?

36. Quantification of nasA Transcripts (Skidaway River Estuary – 2001) 30 28 26 Y = -3.416 (log10X) + 36.25 r2 = 0.989 24 Cycle Threshold (Ct) 22 Standard 20 Unknown 18 16 14 12 2 3 4 5 6 7 Log10 Copy Number Real Time Q-PCR

37. 20 0.005 15 - N Bacterial NO Uptake (<0.8 um size-fraction) 3 SYBR Green Real-Time PCR 0.004 15 0.003 10 NO3 Uptake (nmole-N l-1 d-1) (< 0.8 µm) Marinobacter sp. nasA/16S rRNA Gene Copies 0.002 5 0.001 0 0.000 July '01 May '01 April '01 June '01 March '01 August '00 Ocotber '00 Janurary '01 Skidaway River Estuary

38. Skidaway River Estuary

39. Probably Dependent on Many Factors, Available Carbon, Community Composition, etc. Detection of mRNA transcripts may be transient nasA Gene Expression Sometimes Correlates with NO3 Concentration Barents Sea – Ice Stations South Atlantic Bight Other times with NO3 uptake Rates Skidaway River Estuary Barents Sea – Open Water Stations Sargasso Sea (sometimes) Sometimes Not With Either ???

40. Primary Questions & Conclusions Do Heterotrophic Marine Bacteria Assimilate Nitrate? YES – Varies in Space and Time But Can Account for a Significant Fraction of DIN Uptake Can Molecular Tools (Gene Based) Be Used to Determine Who are the Nitrate Assimilators and What Controls Them? Yes – Molecular Tools Provide Unique Insights and indicate that Genetic Identity Matters and Contributes to System Complexity Are Gene Presence and Expression of nasA Quantitatively Linked to Nitrate Uptake Rates? CO2 Flux? Sometimes, Incorporation into GCM Models Will Be Interesting!

41. 100’s - 1,000’s of genes per organism involved Multiple Regulation Pathways per Organism 1,000’s of organisms involved Lots of Signals But, Unsurprisingly, More Questions Than Answers … Complex Systems

42. So Where Do We Start??? Identification of and Focus on Simple But Relevant Systems and Primary Processes (e.g. Nitrogen Cycle) Focus on Key Functional Genes and Pathways (not just single genes) Simultaneous Analysis of Suites of Genes Combine Chemical, Nucleic Acid, and Protein Analyses HIGH THROUGPUT!!!!

43. rbcl Primary Production PEPcase Primary Production Photooxidation GDC nir, nos, nor Denitrification amoA Nitrification nifH Nitrogen Fixation dsrA Sulfate reduction nar, nasA Nitrate Assimilation pmo Methane Oxidation mcr Methanogenesis Microarray Development In Progress Jizhong Zhou (Joe) – Oakridge National Laboratory Function Gene

44. Chemical Stimuli Black Box Chemical Stimuli Biogeochemical Rates Chemical Stimuli Environomics ???

45. Combined Molecular & Chemical Approaches Are Complementary and Appear to be Leading to a More Complete Mechanistic Understanding of Bacterial Behavior … ENVIRONOMICS Chemical Stimuli Chemical Stimuli Gene Response Proteins Gene Expression Biogeochemical Rates Chemical Stimuli

46. Peter Verity (SkIO) Debbie Bronk (VIMS) Jon Zehr (UC Santa Cruz) Melissa Booth (SkIO/Roanoke) Department of Energy National Science Foundation Office of Naval Research Acknowledgements Andy Allen (Princeton Univ) Marta Sanderson Hendi Hendrickson Christina Archer Corina Knapp Sandra Walters