Looking at Black Sea Microbial Communities to Understand Ancient Oceans

1
Looking at Black Sea Microbial Communities to
Understand Ancient Oceans John Kirkpatrick1,
Brian Oakley2, Clara Fuchsman1, Sujatha
Srinivasan2, James T. Staley2, James W.
Murray1 1School of Oceanography and 2Department
of Microbiology University of Washington,
Seattle, WA April, 2005 Abstract 987

• 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 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 the composition
  of early marine microbial communities, as well as
  their interactions with and contributions to the
  planetary environment.

Fig. 4 While anammox-type sequences were
detectable at all of the mid- and lower-depths,
they dominated clone libraries at s? 15.8.
This seems odd, because the anammox reaction
requires NH4, which approaches zero at s? 16.0.
Project Outline As we are striving to better
understand the different forms life has taken on
Earth, we are particularly interested in
metabolic diversity. Since N- and S-based
chemistry have a wide variety of life-supporting
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
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,
and relate it to the chemistry of the system, we
have utilized both 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 results of these
molecular studies are summarized in figures 4-6.
Enrichment cultures, designed to select for
different metabolisms based on the media
composition, have also yielded some successes.
Among these is anammox enrichment medium (sterile
seawater with NH4 and NO2-) which has produced
an unknown strain of Planctomycetes (cf. figures
4,5).
Fig. 5 Varying levels of diversity. This figure
shows a diversity index (Shannon-Weaver,
normalized for all depths by dividing by Hmax)
a higher number indicates increased diversity. s
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 RDPs Chimera Check.)

• Conclusions
• "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 s
  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.
• 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.

Fig. 3 Chemical gradients and the suboxic zone
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 s? 16.0. This depth
corresponds to an N2 gas maximum, relative to
saturation. Nitrite shows a more complex
profile, with maxima at s? 15.0 and 15.9. The
suboxic zone is shaded (cf. figure 2). Note
the relative scale bars.
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
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).
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 NSF-IGERT grant
DGE-9870713.