Transcript Looking at Black Sea Microbial Communities to Understand Ancient

Looking at Black Sea Microbial Communities to Understand Ancient Oceans
John
1
Kirkpatrick ,
Brian
2
Oakley ,
Clara
1School
1
Fuchsman ,
Sujatha
2
Srinivasan ,
2Department
James T.
of Oceanography and
of Microbiology
University of Washington, Seattle, WA
2
Staley ,
James W.
1
Murray
April, 2005
Abstract
Earth's oceans would have appeared foreign to the modern observer for
much of our planet's history, as evidence shows that the oceans had oxic
surface and anoxic deep layers from approximately 2 to 0.5 billion years ago
(cf. Canfield, 1998; Anbar and Knoll, 2002). This is much like the current
Fig. 1: The Black Sea
Black Sea, the world's largest anoxic basin. We are investigating the
anaerobic bacterial communities of the Black Sea in order to better understand
the ancient oceans. We are particularly interested in novel metabolisms, such
as anaerobic ammonium oxidizing (anammox) bacteria, chemoautotrophs
which can produce significant amounts of N2 gas.
Using samples collected from various depth horizons focused on the suboxic
zone, we have constructed and sequenced 16S rDNA clone libraries using
Planctomycetes-specific primers. This has allowed us to look at diversity
within the Planctomycetes group, with several interesting results.
•There are multiple unknown groups, many of which are highly divergent
from known Planctomycetes.
•One group of 16S rDNA environmental sequences (detected only with
Planctomycetes primers) branches separately from known groups and is
also found growing in a selective culture for anammox bacteria.
•In addition, there are several other environmental sequences similar to
known anammox bacteria.
•The upper depths of the suboxic zone have a relatively low diversity, with
the “basement” of the zone hosting a complex array of different groups.
This coincides with the potential for various S-based metabolisms.
Altogether, this raises interesting questions about the genetic diversity and
metabolism of the Planctomycetes in general and anammox bacteria in
particular. It also leads us to believe that there is much yet unknown about
As we are striving to better understand the different forms life has
the composition of early marine microbial communities, as well as their
taken on Earth, we are particularly interested in metabolic diversity.
interactions with and contributions to the planetary environment.
Since N- and S-based chemistry have a wide variety of lifesupporting reactions, we focused on a part of the Black Sea where
there is a large potential for “atypical” biogeochemistry. This is the
suboxic zone (cf. figure 2), a redoxcline spanning 10s of meters
Sampling focus: The Suboxic Zone
which is deficient in both oxygen and sulfide. Amongst others,
anaerobic ammonium oxidizing (anammox) bacteria are known to
exist here; these chemoautotrophs which can release significant
amounts of N2 gas (cf. figure 3).
Samples collected in 2003 on the R/V Knorr were analyzed to yield
depth-specific information on the chemistry and microbiology of the
water column. In order to characterize the microbial community,
Oxygen
and relate it to the chemistry of the system, we have utilized both
Sulfide
culture-independent and culture-based techniques.
DNA extraction, cloning, and sequencing has given us the basic
distributions and diversity of numerous bacteria. Here we have used
specific primers (58f and 926r) to focus on the Planctomycetes, an
unusual bacterial phylum characterized by intracellular membranes,
a complete lack of peptidoglycan, and a diverse distribution. The
Fig. 2: Strong water column stratification results in
results of these molecular studies are summarized in figures 4-6.
anoxic conditions with a well defined suboxic zone,
Enrichment cultures, designed to select for different metabolisms
where both O2 and H2S are absent. We consider this an
based on the media composition, have also yielded some successes.
ideal location to sample for microbes that have not been
Among these is anammox enrichment medium (sterile seawater with
well characterized (if at all).
NH4+ and NO2-) which has produced an unknown strain of
Planctomycetes (cf. figures 4,5).
Project Outline
Fig. 3: Black Sea Planctomycetes Diversity. DNA Samples from
various density layers were amplified with Planctomycetesspecific primers (58f and 926r), and the entire insert sequenced.
This phylogenetic tree, comparing our samples versus known
Planctomycetes, indicate a plethora of uncharacterized bacteria.
Density (depth)
dependence:
σ = 15.5
σ = 15.6
σ = 15.7
σ = 15.8
σ = 15.9
σ = 16.0
(Reference)
10%
16.0_JK219
15.5_BO698
15.7_JK707
75
100 15.8_JK523
15.9_JK445
100
15.6_JK871
15.5_BO699
70
15.9_JK487
15.9_JK312
15.7_JK727
15.7_JK728
100
15.8_JK617
100
7 15.7_JK721
77
15.6_JK826
4
Pirellula
15.7_JK742
94
100 16.0_JK235
15.9_JK415
Pirellula staleyi
80
15.8_JK613
70 16.0_JK221
96
74
100 15.9_JK416
15.6_JK843
15.5_BO719
15.5_BO681
Planctomyces sp.
15.5_BO704
89
100 15.5_BO726
15.5_BO715
79
15.5_BO720
100
15.6_JK852
100 15.5_BO694
73 15.9_JK448
16.0_JK211
15.7_JK791
97
Isosphaera
Gemmata
16.0_JK94f
15.9_JK522
15.9_JK512
16.0_JK238
97
16.0_JK236
15.9_JK460
99
15.7_JK697
15.7_JK776
100 100 15.6_JK846
15.5_BO684
87 100 16.0_JK81f
74
15.8_JK619
100 Anammox enrichment
15.5_BO703
15.8_JK602
76 15.9_JK485
75 15.7_JK701
15.6_JK831
15.6_JK798
100 15.5_BO708
15.9_JK417
96
"Kuenenia stuttgartiensis"
"Brocadia anammoxidans"
98
15.8_JK636
"Scalindua wagneri"
100
15.8_JK630
100 "Scalindua brodae"
15.9_JK461
97 15.6_JK854
16.0_JK215
"Scalindua sorokinii"
15.9_JK420
15.8_JK618
71
100 15.9_JK440
15.7_JK735
100
15.6_JK800
98
15.8_JK638
100 16.0_JK207
15.9_JK501
100
100 15.9_JK454
15.7_JK709
96
15.7_JK767
100 15.7_JK789
75 15.6_JK808
15.9_JK500
15.5_BO577
15.8_JK593
100 16.0_JK212
100
15.8_JK600
16.0_JK247
99
15.5_BO597
100
100 15.8_JK599
15.5_BO836
16.0_JK97
15.7_JK739
16.0_JK217
100
95
15.9_JK441
16.0_JK79
15.9_JK519
15.6_JK833
15.6_JK803
16.0_JK206
88
15.9_JK451
15.9_JK343
100
16.0_JK241
100
100 15.9_JK409
15.6_JK858
15.6_JK832
100 16.0_JK101
15.9_JK450
100 16.0_JK189
15.9_JK412
100
100 16.0_JK201
15.9_JK506
15.8_JK530
92
16.0_JK245
100 100 16.0_JK83f
15.9_JK419
86
100 16.0_JK102
15.9_JK442
Fig. 4: While anammox-type sequences were
detectable at all of the mid- and lower-depths,
they dominated clone libraries at σθ = 15.8.
This seems odd, because the anammox
reaction requires NH4+, which approaches zero
at σθ = 16.0.
86 15.8_JK624
Pirelulla-like
sequences: found
at all depths
15.8_JK616
15.8_JK632
15.8_JK625
15.8_JK528
15.8_JK588
15.8_JK622
15.8_JK584
15.8_JK587
15.8_JK594
15.8_JK609
15.8_JK583
15.8_JK598
15.8_JK614
15.8_JK639
15.8_JK597
15.8_JK615
15.8_JK529
15.8_JK586
10%
15.8_JK630
15.8_JK611
15.8_JK633
15.8_JK626
15.8_JK590
15.8_JK610
15.8_JK524
100
99 15.8_JK657
15.8_JK634
90 15.8_JK635
75 "Scalindua brodae"
"Scalindua sorokinii"
"Scalindua wagneri"
83
15.8_JK636
"Brocadia anammoxidans"
100
100
"Kuenenia stuttgartiensis"
15.8_JK613
100 15.8_JK638
15.8_JK618
99
15.8_JK629
100 15.8_JK593
90
15.8_JK591
15.8_JK595
78 Anammox enrichment
100 15.8_JK619
78 15.8_JK620
15.8_JK608
15.8_JK602
15.8_JK599
100
10015.8_JK600
15.8_JK596
Gemmata
98
Isosphaera
Planctomyces
sp.
100
93
Pirellula staleyi
Pirellula
15.8_JK617
15.8_JK523
15.8_JK530
Aquifex
Identified
anammox and
similar
sequences
Unknown group, showing
some genera-level
specificity to different
densities
100
88
Aquifex
Highly divergent group,
typically found at the base
of the suboxic zone.
Abstract #987
Fig. 5: Varying levels of diversity. This figure shows a
diversity index (Shannon-Weaver, normalized for all
depths by dividing by H’max); a higher number
indicates increased diversity. σ = 15.8, shown
above, is dominated by the anammox phylotype and
has low diversity. At deeper density interfaces,
approaching the bottom of the suboxic zone and the
onset of sulfide, Planctomycete diversity increases
dramatically. Further investigation will help us
determine what sort of N and / or S metabolisms are
related to these various groups of bacteria. (Note
that these results are PCR based, and so may reflect
the biases of that technique. Samples have all been
screened for chimeras using Bellerophon [see Huber
et al., 2004] and also RDP’s Chimera Check.)
H' / H'max
0
2
3
4
0.3
0.4
NO 3- mM
0
0.1
0.2
13.6
density, σ (depth proxy)
This graph shows the various
chemical distributions around
the suboxic zone. Ammonium
is produced at depth and is
consumed (along with nitrate)
in the suboxic zone; both reach
negligible values around σθ =
16.0. This depth corresponds
to an N2 gas maximum, relative
to saturation. Nitrite shows a
more complex profile, with
maxima at σθ = 15.0 and 15.9.
The suboxic zone is shaded
(cf. figure 2). Note the relative
scale bars.
1
NO 2- mM
14
Black Sea Central Gyre
NH4+
NO3NO2N2/Ar Sample/Saturation Ratio
Suboxic zone
14.4
14.8
15.2
15.6
16
16.4
16.8
0
10
20
30
40
1.015
1.02
NH 4+ mM
1
1.005
1.01
Conclusions
15.5
"Life as we know it" is defined not by 3 dimensions, but by 4. If we as Astrobiologists want
to overcome the inherent scientific difficulty of being in one place at one time, one way to
start is by thinking about not only other places, but other times. By considering the
unusual (yet historically important) environment of the Black Sea, we can gain insight not
only into Earth's past but also on the variety of forms and functions that life takes. Here
we have presented the results of a first-pass assessment of the microbes living in the Black
Sea. Among other observations, we can say that:
• There is a very large amount of diversity in the phylum Planctomycetes; more unknown
bacteria in this location, in fact, than previously known and characterized worldwide.
• The Planctomycetes of some depths (such as σ = 15.7, 15.8) appear to be dominated
by a few strains, possibly due to the favorability of the anammox reaction in certain
chemoclines.
• A few strains of Planctomycetes are ubiquitous throughout the suboxic zone. This
includes one type of bacteria, previously unknown, which grows in inorganic anammox
enrichment media.
• There appears to be increased diversity at the bottom of the suboxic zone; we
hypothesize that there might be a correlation to the viability of S-based metabolisms at
those depths.
0.4
0.6
0.8
15.6
Density, σ
Fig. 3: Chemical gradients and the suboxic zone
0.2
15.7
15.8
15.9
16.0
The R/V Endeavor.
Additional samples collected
3/26/05-4/5/05.
N2/Ar Sample/Saturation Ratio
Anammox bacteria are known to live in the suboxic zone, and survive autotrophically by
producing N2 gas. We are attempting to understand their importance in the complex
interplay of various N species (and their isotopes). The relevant reaction is:
Anammox Reaction: NH4+ + NO2-  N2(g) + H2O
Acknowledgements
Thanks to Billy Brazelton for all of his advice; and also to Mark Rider and
Audrey Harris for their laboratory assistance.
This work was funded by NSF Microbial Observatories 0132101 and NSFIGERT grant DGE-9870713.
Now that we have gathered essential 16S rDNA data on various depths of the Black Sea,
along with fresh samples, we can attempt to answer questions raised by our previous work.
These include:
 What are the dominant species? We are working on Fluorescent In-Situ Hybridization
(FISH) techniques to identify and count specific strains of bacteria.
 How active are these bacteria? We have incubated and collected samples spiked with
14C-bicarbonate in order to measure rates of chemosynthesis.
 Which species or groups are primary producers? We have collected samples for a
combined 14C and FISH analysis to determine which kinds of bacteria are fixing carbon.
 What are the bacteria doing? We have many samples growing in selective cultures,
including those for anammox, heterotrophic denitrification, thiodentrification, and sulfite
reduction, amongst others.
References
Anbar, A. D. , and A. H. Knoll. Proterozoic Ocean Chemistry and Evolution: A
Bioinorganic Bridge? Science 297, 1137-1142 (2002).
Canfield, D.E. A new model for Proterozoic ocean chemistry. Nature 396, 450-453
(1998).
Huber, T. , G. Faulkner and P. Hugenholtz. Bellerophon; a program to detect chimeric
sequences in multiple sequence alignments. Bioinformatics 20 2317-2319 (2004).