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

Institute of Marine and Coastal Sciences

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


Winch and “A” frame

Text
Text


Niskin
Bottles to
collect
water
samples at
depth


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


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


Why study bacteria in the ocean?

ASM workshop--2000


How biologists
understood the
tree of life in
1866.

3 main groups
of organisms
Based
primarily upon
morphology

Axhplmnap
ni yo n sm
oc i i l
o e

Microbial Life-’02 Perry et al.

Electron
micrograph of
bacteria
collected off
the coast of
Hawaii


Viable counting of bacteria by serial dilution and plate count


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


Results of bacterial counts with 6 methods

Jannasch & Jones, 1959

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


The Ribosomal RNA SSU tree of life

Bacteria Eukarya
Proteobacteria
Gram-Positive Bacteria
Archaea Slime Molds
Animals
Entamoebae
Green Non-Sulfur Bacteria
MethanobacteriumHalobacterium Fungi
Cyanobacteria Plants
Methanococcus Thermoplasma
Thermoproteus Thermococcus
Flavobacteria Ciliates
Sulfolobus Archaeoglobus
Methanopyrus
Ignicoccus Flagellates
Pyrodictium
Thermotoga Microsporidia
Diplomonads
Aquifex

Universal
Ancestor


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 Plankton
Hutchinson 1961


Stable Co-existence--resource partitioning

Chesson 2000


Stable Co-existence- [selective predation]

Hutchinson 1961

Chesson 2000


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.


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


Almost-diel study times indicated by the 2 orange boxes


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


Universal Ribosomal rRNA content vs. Growth Rate

12

9
RNA/DNA

6

3

0
0.0 0.5 1.0 1.5 2.0
Specific Growth Rate (h-1)
Kerkhof and Ward, 1993

What can we predict?


How does Terminal Restriction
Fragment Length Polymorphism work?

Sample Size separate on automated sequencer
Gene 1
A B
Gene 2
Gene 3
Gene 4

Gene n

Purify DNA Gene n+ 1

PCR with ﬂuorescent primers Restriction Enzyme Digest

Only labeled fragments appear as peaks ABI Software Analysis


Signal Strength Ribosomal RNA ﬁngerprints during the GRIST experiment

TRFLP Size (in base pairs)


4 Incorporation of stable isotopes into
DNA and separation by ultracentrifugation

Radajewski et al 2000, Nature 403: 646–649

Semi-conservative replication of DNA
15N generations

14N
Meselson and Stahl 1958, PNAS 44: 671-–649

Map of Sample Sites


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 71:5192-5196

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

Gallagher et al., 2005 AEM 71:5192-5196

7 Orinoco River Plume
Puerto Rico
65o W 60o W

Guadalupe

Caribbean Basin
15o N

Satellite surface Chla St. Lucia
5-10 mg/m3

2-5 mg/m3

0.5-1.5 mg/m3

Venezuela Trinidad
Tobago

10o N


Enrichment culture/ resource partitioning

Wawrik et al., 2005

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

Station 12 13C protein uptake 13C amino acid uptake 13C Pigment uptake

112 bp
85 bp 140 bp


Nitrogen utilization patterns of bacteria in the plume
L. Kerkhof, C. Ferraro, L. McGuinness

Far Plume
Site


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!


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

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