Interactions of Chemosynthetic Bacteria with Mercury at Deepsea Hydrothermal Vents

1
Interactions of Chemosynthetic Bacteria with
Mercury at Deep-sea Hydrothermal Vents Melitza
Crespo-Medina1, Nicolas Bloom2, Aspassia
Chatziefthimiou1, John Reinfelder1, Costantino
Vetriani1 and Tamar Barkay1 1Cook College,
Rutgers University, New Brunswick, NJ, 2Studio
GeoChimica, Seattle, WA
ABSTRACT Microorganisms in geothermal
environments, such as deep-sea hydrothermal
vents, have likely evolved in presence of
elevated concentrations of mercury. Because this
mercury is likely complexed with sulfur,
chemolithoautotrophic microbes, that use reduced
sulfur and iron as a energy source are likely
exposed to the toxicity of mercury. To test this
hypothesis we collected water for chemical and
microbiological analyses at different distances
from diffuse flow vents at 9 ºN on the East
Pacific Rise (EPR), during oceanographic
expeditions in 2004 and 2005. Total mercury (THg)
concentrations in the 2004 samples ranged from
2.8 to 88.7 ng/L (methylmercury concentration 0.3 ng/L). Growth medium, designed to enrich for
chemolithoautotrophic thiosulfate oxidizing
microorganisms, was used to enumerate total and
mercury resistant (10 µM) microbes by the MPN
method. Mercury speciation in this medium,
determined with MINEQL (Version 4.5), showed the
exclusive presence of negatively charges -2
(61.3) and -4 (38.7) complexes of mercury
thiosulfate.  The bioavailability of these
complexes to microorganisms was determined using
mer-lux bio-reporters, which emit light as
mercury enters the cell. Results suggested an
enhanced bioavailability of mercury-thiosulfate
relative to mercuric-nitrate complexes. The
abundance of mercury resistant microbes was 6
and 13 of total thiosulfate oxidizing
chemolithoautotrophs in vent waters with THg
concentrations of 6.5 and 88.7 ng/L,
respectively. Thus, the number of resistant
microbes was directly related to the
concentration of THg in the water suggesting
acclimation of these microbes to life in the
presence of toxic mercury. Isolations from vent
water samples led to the identification of
mercury resistant bacteria belonging to several
genera, including, Thiomicrospira, Thioclava and
Halomonas whose mechanisms of mercury resistance
is currently being investigated. These initial
results begin to reveal the processes that
facilitate microbial life in metal rich
geothermal environment and will lead to an
understanding of the role of these microbes in
mercury biogeochemistry in these ecosystems.
MPN counts of chemosynthetic microbes in samples
collected on the EPR 9oN
RESULTS
Table 1. Selected chemical measurements of
diffuse flow waters from EPR 9ºN (Apr. 2004
expedition)
Table 3. Summary of MPN Results
1 MeHg concentration was

The percentage of resistant MPN counts was
between 0.1 and 13.9

The highest resistant MPN counts was found in the
sample with the highest mercury concentration

• HgT concentrations varied and were not always
  related to temperature and sulfide

Bioavailability of Hg(II) in medium 142-A
Figure 5. 16S rRNA gene phylogeny of isolates
from diffuse flow vents
I. MINEQL modeling of 142-A 10 µM HgCl2
Figure 2. Hg-thiosulfate speciation
OBJECTIVE Study the interactions between mercury
and chemosynthetic microorganisms isolated from
deep-sea hydrothermal vents

• All Hg(II) in 142-A with 10 µM HgCl2 was present
  as thiosulfate complexes, 61 as Hg(S2O3)2-2 and
  39 as Hg(S2O3)3-4

• METHODS
• Samples were collected on East the Pacific Rise
  9oN (Fig. 1), during DSV Alvin dives in April
  2004 and 2005. Samples were preserved on board of
  R/V Atlantis immediately upon retrieval
• Samples from the 2004 cruise were analyzed for
  chemical compositions at Frontier GeoScience Inc.
  (Seattle, WA)
• MINEQL Chemical Equilibrium Modeling System
  (Schecher et al., 1994), was used to determine
  mercury speciation in the growth (142-A) and the
  biosensor assay media
• mer-lux biosensor assays were done according to
  Barkay et al., (1998)
• The Most Probable Number (MPN) technique and
  growth medium 142-A were used to determine the
  number of chemosynthetic and chemosynthetic-mercur
  y (10 µM HgCl2) resistant bacteria. Total number
  of cells was determined by Acridine Orange Direct
  Counts.
• 16S rDNA gene phylogeny was obtained using
  Clustal X and NJ analysis

II. Hg speciation in the mer-lux biosensor assay
media
Table 2. The effect of increasing thiosulfate
concentrations on the speciation of Hg(II), added
as 10 nM Hg(NO3)2 to mer-lux biosensor assay
medium
mercury resistant strains

• A merA PCR fragment from EPR84 was sequenced and
  found most closely related to the sequence of
  merA of Tn501 and Tn21

• CONCLUSIONS
• Hydrothermal fluids from diffuse flow vents are
  enriched in mercury with concentrations ranging
  from 2.8 to 88.7 ng/mL (Table 1)
• Thiosulfate is the favored ligand of Hg in medium
  142-A, a growth medium formulated for
  chemolithoautotrophic marine bacteria (Fig. 2)
• The use of the biosensor assay in conjunction
  with MINEQL speciation is a useful approach to
  determine bioavailability of Hg(II) under
  specific conditions (Table 2 and Fig. 3)
• Thiosulfate reduces Hg(II) bioavailability, yet
  it does not abolish it
• The bioavailability of Hg(II) decreases with the
  increased negative charge of the Hg-thiosulfate
  complex (Fig. 3)
• In diffuse flow samples with higher mercury
  concentrations, the microbial community is likely
  to be adapted to the presence of mercury (Table
  3)
• Chemosynthetic bacteria from diffuse flow
  environments are distributed among ? and ?-
  Proteobacteria four of the isolates were
  resistant to 10 µM HgCl2 (Fig. 4)

• At 2 mM S2O3-2 Hg(II) speciation in mer-lux assay
  medium simulates that of 10 µM Hg(II) in 142-A
  growth medium

III. Bioavailability of varied Hg-S2O3 species
Figure 1. Study sites on the East Pacific Rise
9ºN 50N/104 17W
Figure 3. Induction of mer-lux decreased with
increasing concentrations of S2O3-2
LITERATURE CITED Barkay et al. 1998.
Lux-facilitated detection of mercury in natural
waters. in Bioluminescent Protocols, R. LaRossa
(ed). Humana Press, Inc. Totowa, NJ.pp.
231-246. Schecher et al. MINEQL A Chemical
Equilibrium Program for Personal Computers
Environmental Research Software Hallowell, ME,
1994.

• Induction of mer-lux decreased with S2O3-2 (A)
• At 2 mM S2O3-2 bioavailability decreased about
  55 relative to assay media devoid of thiosulfate
  (B)
• Bioavailability decreases in inverse proportion
  to an increase in the ratio of Hg(S2O3)3-4
  Hg(S2O3)-2 (B)

Acknowledgements This work was supported by a
National Science Foundation Graduate Research
Fellowship to MCM and by an NSF Grant OCE
03-27353 to C. Vetriani