East Coast MARE Ocean Lecture Mar 29, 2012 - Why is there so much microbial diversity in NJ &...

Why is there so much microbial diversity in NJ and Beyond?

Rutgers University, Lee Kerkhof

Institute of Marine and Coastal Sciences

Thursday, March 29, 12

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


TextText

Winch and “A” frame


Niskin Bottles to collect water samples at depth


Look at all the hard hats and safety gear

Floats for equipment going over the side


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


ASM workshop--2000

Why study bacteria in the ocean?


How biologists understood the tree of life in 1866.

3 main groups of organismsBased primarily upon morphology


Anoxic hypolimnion sample

Microbial Life-’02 Perry et al.Thursday, March 29, 12

Electron micrograph of bacteria collected off the coast of Hawaii


Viable counting of bacteria by serial dilution and plate count

Microbial Life-’02 Perry et al.


Counting the number of viable cells by serial dilution and plate count

Microbial Life-’02 Perry et al.Thursday, March 29, 12

Jannasch & Jones, 1959

Results of bacterial counts with 6 methods


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 2o metabolites•Lipids•Proteins•Nucleic Acids


What biomolecules can we work with?



Traditional Clone and Sequence Method


UniversalAncestor

BacteriaArchaea

Eukarya

Pyrodictium

Thermoproteus Thermococcus

Methanococcus

MethanobacteriumHalobacterium

Thermoplasma

Methanopyrus

Aquifex

Thermotoga

Flavobacteria

Cyanobacteria

ProteobacteriaGram-Positive Bacteria Entamoebae

Slime MoldsAnimals

Fungi

Plants

Ciliates

Flagellates

MicrosporidiaDiplomonads

Archaeoglobus

Green Non-Sulfur Bacteria

Sulfolobus

Ignicoccus

The Ribosomal RNA SSU tree of life


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


The solution to the Paradox of the PlanktonHutchinson 1961


The solution to the Paradox of the PlanktonHutchinson 1961


Stable Co-existence--resource partitioning

Chesson 2000


Stable Co-existence--resource partitioning

Chesson 2000


Stable Co-existence- [selective predation]

Chesson 2000

Hutchinson 1961


Interesting question

Do bacteria in the coastal ocean of New Jersey experience rapidly changing patchy niches that could account for

microbial diversity?


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.


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

1


Almost-diel study times indicated by the 2 orange boxes


Five approaches using RNA/DNA based methods to determine activity

1. mRNA analysis2. MICROFISH analysis3. BrDU incorporation4. Ribosome fingerprinting5. Stable Isotope incorporation


What biomolecules can we work with?



Universal Ribosomal rRNA content vs. Growth Rate

2.01.51.00.50.00

3

6

9

12

Specific Growth Rate (h-1)

RN

A/D

NA

Kerkhof and Ward, 1993Thursday, March 29, 12

What can we predict?


How does Terminal Restriction Fragment Length Polymorphism work?

Restriction Enzyme Digest

ABI Software AnalysisOnly labeled fragments appear as peaks

PCR with fluorescent primers

Gene 1

Gene 2

Gene 3

Gene 4

Gene n

Gene n+ 1

Size separate on automated sequencer

A B

Sample

Purify DNA


Ribosomal RNA fingerprints during the GRIST experiment

TRFLP Size (in base pairs)

Sign

al S

treng

th


Incorporation of stable isotopes into DNA and separation by ultracentrifugation

4

Radajewski et al 2000, Nature 403: 646–649Thursday, March 29, 12

Semi-conservative replication of DNA

Meselson and Stahl 1958, PNAS 44: 671-–649

generations15N

14NThursday, March 29, 12

Map of Sample Sites


TRFLP fingerprints of nosZ genes from benzoate fed slurries from Norfolk and LEO

Gallagher et al., 2005 AEM 71:5192-5196 TRFLP Size (in base pairs)

Sign

al S

treng

th


Increase in TRFLP peak areas through time for LEO-15 and Norfolk incubations

Gallagher et al., 2005 AEM 71:5192-5196 Thursday, March 29, 12

Venezuela

Caribbean Basin

Trinidad Tobago

St. Lucia

Puerto Rico

Guadalupe

Satellite surface Chla

5-10 mg/m3

2-5 mg/m3

0.5-1.5 mg/m3

15o N

65o W

10o N

60o W

Orinoco River Plume7


Enrichment culture/ resource partitioning

Wawrik et al., 2005Thursday, March 29, 12

13C amino acid uptake 13C Pigment uptake

112 bp85 bp 140 bp

13C protein uptakeStation 12

Carbon utilization patterns of bacteria in the plumeL. Kerkhof, C. Ferraro, L. McGuinness


Far Plume Site

L. Kerkhof, C. Ferraro, L. McGuinness

Nitrogen utilization patterns of bacteria in the plume


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.


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!


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East Coast MARE Ocean Lecture Mar 29, 2012 - Why is there so much microbial diversity in NJ &...

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