Why is there so much microbial diversity in NJ and Beyond? Rutgers University, Lee Kerkhof

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1 Institute of Marine and Coastal Sciences Why is there so much microbial diversity in NJ and Beyond? Rutgers University, Lee Kerkhof

2 Overview of the seminar Background on how we do oceanography and a small lecture Measurements of the coast of New Jersey Assessment of active bacteria in the Orinoco River Plume Determination of active bacteria in the subsurface Then, I get to revisit these sites again with a different methodology

3 Winch and A frame Text Text

4 Niskin Bottles to collect water samples at depth

5 Look at all the hard hats and safety gear Floats for equipment going over the side

6 Inside the ship in the science labs. All the space is used.

7 Why study bacteria in the ocean? ASM workshop--2000

How biologists understood the tree of life in 1866.

8 How biologists understood the tree of life in main groups of organisms Based primarily upon morphology

9 Anoxichypolimnionsample Microbial Life- 02 Perry et al.

10 Electron micrograph of bacteria collected off the coast of Hawaii

11 Viable counting of bacteria by serial dilution and plate count Microbial Life- 02 Perry et al.

12 Counting the number of viable cells by serial dilution and plate count Microbial Life- 02 Perry et al.

13 Results of bacterial counts with 6 methods Jannasch & Jones, 1959

14 What is Meant by Molecular Ecology? Using methodology that identifies specific molecules in a complex mixture to identify specific groups of microorganisms to address ecological questions Target molecules can be: Unique pigments or 2 o metabolites Lipids Proteins Nucleic Acids

15 What biomolecules can we work with? Microbial Life- 02 Perry et al.

16 Traditional Clone and Sequence Method

17 Bacteria Cyanobacteria The Ribosomal RNA SSU tree of life Proteobacteria Gram-Positive Bacteria Green Non-Sulfur Bacteria Archaea Methanococcus Eukarya Slime Molds Animals Methanobacterium Halobacterium Entamoebae Fungi Thermoplasma Plants Flavobacteria Thermotoga Aquifex Thermoproteus Thermococcus Sulfolobus Archaeoglobus Methanopyrus Ignicoccus Pyrodictium Ciliates Flagellates Microsporidia Diplomonads Universal Ancestor

18 Problem Why are there so many bacterial species in the ocean [or anywhere else]? There should be competitive exclusion, i.e. the best adapted organism out competes all the others. Hardin 1960

19 The solution to the Paradox of the Plankton Hutchinson 1961

20 The solution to the Paradox of the Plankton Hutchinson 1961

21 Stable Co-existence--resource partitioning Chesson 2000

22 Stable Co-existence--resource partitioning Chesson 2000

23 Stable Co-existence- [selective predation] Hutchinson 1961 Chesson 2000

24 Interesting question Do bacteria in the coastal ocean of New Jersey experience rapidly changing patchy niches that could account for microbial diversity?

25 Interesting question Do bacteria in the coastal ocean of New Jersey experience rapidly changing patchy niches that could account for microbial diversity? Test this with activity measurements of bacteria in the water column on a diel cycle.

26 1 Results from the GRIST Experiment The GRIST study aimed to target the active bacteria in the coastal ocean and assess the relationship between traditional rate measurements and gene-based methodologies for linking processes from the molecular to the global scale. LEO-15

27 Almost-diel study times indicated by the 2 orange boxes

28 Five approaches using RNA/DNA based methods to determine activity 1. mrna analysis 2. MICROFISH analysis 3. BrDU incorporation 4. Ribosome fingerprinting 5. Stable Isotope incorporation

29 What biomolecules can we work with? Microbial Life- 02 Perry et al.

30 Universal Ribosomal rrna content vs. Growth Rate 12 9 RNA/DNA Specific Growth Rate (h-1) 2.0 Kerkhof and Ward, 1993

31 What can we predict?

32 How does Terminal Restriction Fragment Length Polymorphism work? Sample Size separate on automated sequencer Gene 1 Gene 2 Gene 3 Gene 4 A B Purify DNA PCR with fluorescent primers Gene n Gene n+ 1 Restriction Enzyme Digest Only labeled fragments appear as peaks ABI Software Analysis

33 Ribosomal RNA fingerprints during the GRIST experiment Signal Strength TRFLP Size (in base pairs)

34 4 Incorporation of stable isotopes into DNA and separation by ultracentrifugation Radajewski et al 2000, Nature 403:

35 Semi-conservative replication of DNA 15N generations 14N Meselson and Stahl 1958, PNAS 44:

36 Map of Sample Sites

37 TRFLP fingerprints of nosz genes from benzoate fed slurries from Norfolk and LEO Signal Strength TRFLP Size (in base pairs) Gallagher et al., 2005 AEM 71:

38 Increase in TRFLP peak areas through time for LEO-15 and Norfolk incubations Gallagher et al., 2005 AEM 71:

39 7 Orinoco River Plume Puerto Rico 65 o W 60 o W Guadalupe Caribbean Basin 15 o N Satellite surface Chla 5-10 mg/m 3 St. Lucia 2-5 mg/m mg/m 3 Venezuela Trinidad Tobago 10 o N

40 Enrichment culture/ resource partitioning Wawrik et al., 2005

41 Carbon utilization patterns of bacteria in the plume L. Ke rkhof, C. Fe rraro, L. McGuinne ss Station 12 13C protein uptake 13C amino acid uptake 13C Pigment uptake 85 bp 112 bp 140 bp

42 Nitrogen utilization patterns of bacteria in the plume L. Ke rkhof, C. Fe rraro, L. McGuinne ss Far Plume Site

43 First Conclusions 1) Ribosome fingerprinting can distinguish active from resident bacteria. 2) Not every bacterial group is behaving uniformly in the field. But, there are some repeating patterns indicating non-randomness. 3) Roughly 25-50% of the microbial population are not growing in the Mid-Atlantic Bight and the Orinoco River Plume (i.e. DNA signal, no RNA signal). 4) So far, little correlation to measured environmental parameters.

44 Conclusions A) Whether there is resource partitioning among bacteria depends on your environment. B) The role of predation in structuring the community plays a more important role than typically appreciated. C) Elucidating the microbial food web using molecular tools is just in its infancy. There is so much more to learn!

MARLIA SINGGIH WIBOWO SCHOOL OF PHARMACY INSTITUT TEKNOLOGI BANDUNG

MARLIA SINGGIH WIBOWO SCHOOL OF PHARMACY INSTITUT TEKNOLOGI BANDUNG Farmakope Indonesia edisi IV, 1995. USP 32, 2009. Michael T Madigan, John M Martinko, Brock Biology of Microorganisms, 11 th edition,