Molecular Characterization of Microorganisms in Methylmercury Producing Adirondack Wetland

1
M-210
Molecular Characterization of Microorganisms in
Methylmercury Producing Adirondack Wetland
Sunday Lake
Ri-Qing Yu, Christopher DiPasquale, Tamar
Barkay Department of Biochemistry and
Microbiology, Rutgers University, New Brunswick,
New Jersey 08901. Email rqyu_at_eden.rutgers.edu
2. Classifying sulfate reducing bacteria and
their activities

• No specific SRB group either emerged or
  disappeared following enrichment with sulfate.
  The exception is a reduced abundance of group 5
  after enrichment in SURN samples.

ABSTRACT The microbial community in
riparian/limnic wetland soils that had been
identified as a source of methylmercury to Sunday
Lake, located in the western Adirondack, New
York, was characterized at the molecular level.
Small subunit ribosomal RNA gene-based cloning
and sequencing approach showed that lt 1 of the
microbes in the active soils belonged to groups
known to contain sulfate reducing prokaryotes,
the principle mercury methylators in many
environments. Subsequent attempts to detect dsrAB
genes, encoding for dissimilatory sulfite
reductase enzyme, detected signals of these genes
in DNA extracts of samples from bog, sedge and
floating mat. Because soil incubations that were
amended with sulfate showed enhanced dsrAB gene
abundance and production of methylmercury
relative to unamended controls, we concluded that
mercury methylation in the Adirondack soils was
carried out by a minor component of the resident
microbial community. We therefore increased the
sensitivity of our detection by employing a
nested PCR approach that distinguished 6
physiological groups of SRB and showed their
presence in most sampling sites within the lake
ecosystem. Three groups of incomplete oxidizers
(Desulfovibrio spp. (Group 6), Desulfotomaculum
spp. (Group 1), and Desulfobulbus spp. (Group 2))
and one group of complete oxidizers
(Desulfosarcina (Group 5)) SRB were present in
methylating samples, suggesting their involvement
in mercury methylation is in the floating mat and
riparian soils. This is the first investigation
of the role of specific physiological SRB groups
in mercury methylation in freshwater environments
and our preliminary results suggest that, unlike
previously described findings in saltmarsh
sediments, SRB that do not utilize acetate, i.e.,
the incomplete oxidizers, have a significant role
in this processes.
Fig. 4 PCR amplification of 16 rRNA genes using
SRB group-specific primer sets. Cheesequake
sample Saltmarsh control
3. Community profiling by DGGE of SRB-specific
PCR products from a floating mat sample
1 2 3 4 5 6 7 8 9 10
11 12 13 14 15 16 17 18 19 20
1 2 3 4 5 6 7 8 9 10 11
12 13 14 15 16 17 18 19 20
SRB 5
SRB 1
1 2 3 4 5 6 7 8
Fig. 5 Community profiling by DGGE of 16S rRNA
gene fragments using SRB-specific primers for
Floating Mat in Sunday Lake wetland. 1-4 SRB
group 1, 2, 5, 6 from Floating Mat, respectively
5-8 SRB group 1, 2, 5, 6 from Floating Mate
enriched with 2mM sulfate, respectively
700 bp
524 bp
SRB 2
SRB 6

• Many bands indicating a high diversity were
  present in preparation of all four groups.
• Significant change in SRB community structure did
  not occur during enrichment with sulfate with the
  exception of group 1 where new dominating bands
  appeared.
• The major bands in these DGGE profiles are
  currently sequenced to identify major SRB taxa in
  a Hg methylating Sunday Lake sample.

1120 bp
656 bp
Table 1 Characteristics of six groups of SRB
that were distinguished by PCR primer design
1. SURN-Top
8. Upland soil
15. G3 (D. autotrophicum) 2. SURN-Bottom
9. SURN-upper-SO42-
16. G4 (D.
curvatus) 3. SURF-Top
10. Bog-upper-SO42-
17. G5 (D. variabilis) 4. SURF-Bottom
11. Mat-SO42-
18. G6 (D.
desulfuricans) 5. Bog-Top
12. Size markers
19. Blank 6. Sedge
13. G1 (D.
nigrificans) 20.
Cheesequake (saltmarsh control) 7. Mat
14. G2
(D. propionicus)
SRB Group Terminal oxidation Common species Electron donor/ substrate Others
1 2 6 3 4 5 incomplete incomplete incomplete complete complete complete Desulfotomaculum spp. Desulfobulbus spp. Desulfovibrio spp. Desulfobacterium spp. Desulfobacter spp. Desulfosarcina spp. lactate, formate, H2, ethanol propionate, H2, lactate, ethanol lactate, pyruvate, ethanol, malate, H2 fatty acids, alcohols, dicarboxylic acids, H2 acetate lactate, formate, acetate, ethanol some thermophilic species no syntrophy to methanogens syntrophy brackish/marine brackish/marine freshwater or marine

• Presence/absence of 6 defined groups of SRB was
  revealed by use of group specific PCR primers.
• Three groups of incomplete oxidizers (SRB 1, 2,
  6) and one group of complete oxidizers (SRB 5)
  were detected SRB groups 1, 5, 6 seem to
  dominate in Sunday Lake.
• SRB groups 3 (Desulfobacterium spp.) and 4
  (Desulfobacter spp.) were not detected in any of
  our samples.

• INTRODUCTION
• Mercury contamination in the Adirondack
  watershed is due to atmospheric deposition and
  has a long historic record (Lorey and Driscoll
  1999). Two typical wetlands in this region,
  riparian wetlands and limnic wetlands, might play
  an important role in regulating the supply of
  MeHg to surface water. Studies showed that the
  riparian wetland in the Sunday Lake watershed is
  likely an important area of MeHg production
  (Driscoll et al. 1998). The characteristics of
  the soil microbial communities and how they
  relate to Hg methylation in this ecosystem is
  unknown.
• Sulfate reducing bacteria are the primary
  Hg(II) methylating microbes in anoxic sediments
  (Barkay and Wagner-Döbler, 2005). The purpose of
  this study is to investigate their role in Hg
  methylation Adirondack wetlands.

• CONCLUSIONS
• Permanently water saturated sites, floating mat
  and bog, harbored the most diverse SRB community
  (Fig. 4), carried dsrAB genes (Fig. 3), and were
  consistently found to methylate mercury (Adato
  and Hines poster).
• The addition of sulfate while stimulating
  methylation did not affect SRB community
  structure (Table 3), suggesting that all SRB
  groups present contributed to methylation.
• At this early stage it seems that incomplete
  oxidizers are the dominant methylators in the
  Adirondack wetland in contrast with their role in
  methylation in saltmarsh sediments (King et al.,
  2000).

• PCR primers for dsrAB genes were from Loy et
  al.(2004) PCR primers for 16S rRNA genes for
  each specific group of SRB and DGGE were from
  Daly et al. (2000) And Muyzer et al. (1993),
  respectively.
• Sulfate enrichments (2mM) were carried out by
  Adato and Hines (see their poster in this
  meeting).

RESULTS

1. Detection of dissimilatory disulfite reductase
(dsrAB) genes in Sunday Lake microbial biomass
MATERIALS AND METHODS

• Soil samples were collected from Sunday lake
  (44º20' N, 74º 18' W depth, 11 m surface area,
  4.0 ha vol., 21.9?104 m3), New York (Fig.1).
  Soil samples in sterile Falcon tubes were placed
  on dry ice and stored at -80?C. DNA/RNA
  extraction and later cloning and sequencing
  processes are depicted in Fig. 2.

REFERENCES

Barkay T, Wagner-Dobler I. 2005. Advances in
Applied Microbiology 57 1-52. Daly K, Sharp R J,
McCarthy A J. 2000. Microbiology 146
1693-1705. Dar S A, Kuenen J G, Muyzer G. 2005.
Appl. Environ. Microbiol. 71 2325-2330. Driscoll
C T, Holsapple J, Schofield C L, Munson R. 1998.
Biogeochemistry 40 137-146. Hurt R A, Qiu X, Wu
L, Roh Y, Palumbo A V, Tiedje J M, Zhou J. 2001.
Appl. Environ. Microbiol. 67 4494-4503. King J
K, Kostka J E, Frischer M E, Saunders F M. 2000.
Appl. Environ. Microbiol. 66 2430-2437. Lorey
P, Driscoll C T. 1999. Environ. Sci. Technol. 33
718-722. Loy A, Kusel K, Lehner A, Drake H L,
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G. 1993. Appl.Environ. Microbiol. 59 695700.

• The SRB community is more diverse in the mat and
  bog sample (stable wetting and methylation
  activities) than SURN, SURF and sedge samples.
  SRB2 (Desulfobulbus spp.) was only present in the
  bog and mat communities.

1.9 kb
New York State
Fig. 3 dsrAB gene PCR amplification products
from DNA extracts that were collected from Sunday
Lake in July 2005. Methods followed those of
Loy et al. (2004). Lanes marked as
1. SURN-Top 2. SURN-Bottom 3. SURF-Top 4.
SURF-Bottom 5. Bog-Top 6. Bog-Bottom 7. Sedge
8. Mat 9. Upland soil 10. SURN-top-SO42-
11. Bog-top-SO42- 12. Mat-SO42- 13. Cheesequake
(saltmarsh control) 14. Desulfovibrio
desulfurican (positive control) 15. Blank 16.
Size markers Top Sample collected at depth of
0-15 cm Bottom Sample collected at depth of
15-30 cm 2 mM Sulfate enrichment for the soil
samples
ACKNOWLEDGEMENTS
We appreciate Dr.Yanping Wang and Dr. Mark Hines
for their advice and valuable suggestions. This
project was supported by a National Science
Foundation Biocomplexity grant ATM0322022.

• dsrAB genes were detected in Sunday Lake wetland.
  
• Sulfate enrichment increased the dsrAB gene
  abundance in wetter sites such as Floating Mat
  and Bog but not the dry sites (e.g. SURN).

Fig. 1 Sampling sites in Sunday Lake, Adirondack
Mountains, New York. SURN Sunday Lake Riparian
Near SURF Sunday Lake Riparian Far BogBog
proximal to lake Sedge Sedge proximal to lake
Mat Floating mat near lake waterfront Soil
Upland soil