Molecular Characterization of Microorganisms in Methylmercury Producing Adirondack Wetland

Ri-Qing Yu, Christopher DiPasquale, Tamar BarkayDepartment of Biochemistry and Microbiology, Rutgers University, New Brunswick,

New Jersey 08901. Email: rqyu@eden.rutgers.edu

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 < 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.

MATERIALS AND METHODS

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, Wagner M. 2004. Appl. Environ. Microbiol. 70: 6998-7009. Muyzer G, de Waal E C, Uitterlinden A G. 1993. Appl.Environ. Microbiol. 59: 695–700.

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.

Fig. 4: PCR amplification of 16 rRNA genes using SRB group-specific primer sets. Cheesequake sample: Saltmarsh control

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.

• Soil samples were collected from Sunday lake (44º20' N, 74º 18' W; depth, 11 m; surface area, 4.0 ha; vol., 21.9104 m3), New York (Fig.1). Soil samples in sterile Falcon tubes were placed on dry ice and stored at -80C. DNA/RNA extraction and later cloning and sequencing processes are depicted in Fig. 2.

RESULTS

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

3. Community profiling by DGGE of SRB-specific PCR products from a floating mat sample

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).

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16

1.1. SURN-Top*; SURN-Top*; 2. 2. SURN-Bottom; SURN-Bottom; 3.3. SURF-Top; SURF-Top; 4. 4. SURF-Bottom; SURF-Bottom; 5. 5. Bog-Top; Bog-Top; 6.6. Bog-Bottom; Bog-Bottom; 7.7. Sedge; Sedge; 8.8. Mat; Mat; 9.9. Upland soil; Upland soil; 10.10. SURN-top-SO SURN-top-SO44

2-2-**; **; 11.11. Bog-top- Bog-top-

SOSO442-2-; ; 12.12. Mat-SO Mat-SO44

2-2-;; 13.13. Cheesequake (saltmarsh control); Cheesequake (saltmarsh control); 14.14. Desulfovibrio Desulfovibrio

desulfuricandesulfurican (positive control); (positive control); 15.15. Blank; Blank; 16.16. Size markers Size markers*Top: Sample collected at depth of 0-15 cm; Bottom: Sample collected at depth of 15-*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 samples30 cm; ** 2 mM Sulfate enrichment for the soil samples

• 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).

2. Classifying sulfate reducing bacteria and their activities

Sunday Lake

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

• 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.

1. SURN-Top 8. Upland soil 15. G3 (1. SURN-Top 8. Upland soil 15. G3 (D. autotrophicumD. autotrophicum))

2. SURN-Bottom 9. SURN-upper-SO2. SURN-Bottom 9. SURN-upper-SO442- 2- 16. G4 (16. G4 (D. curvatusD. curvatus))

3. SURF-Top 10. Bog-upper-3. SURF-Top 10. Bog-upper-SOSO442-2- 17. G5 ( 17. G5 (D. variabilisD. variabilis))

4. SURF-Bottom 11. 4. SURF-Bottom 11. Mat-SOMat-SO442- 2- 18. G6 (18. G6 (D. desulfuricansD. desulfuricans))

5. Bog-Top 12.5. Bog-Top 12. Size markers Size markers 19. Blank19. Blank

6. Sedge 13. G1 (6. Sedge 13. G1 (D. nigrificansD. nigrificans) 20. Cheesequake (saltmarsh control)) 20. Cheesequake (saltmarsh control)

7. Mat 14. G2 (7. Mat 14. G2 (D. propionicusD. propionicus))

700 bp

1120 bp

524 bp

656 bp

SRB 1

SRB 2SRB 6

SRB 5

• 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.

• 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.

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

1 2 3 4 5 6 7 8

• 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.

M-210

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:

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

Table 1: Characteristics of six groups of SRB that were distinguished by PCR primer design

• 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).

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

Table 2 Ecological niche distribution of SRB in Sunday Lake wetland during July 2005 sampling1 Niche SRB1 SRB2 SRB3 SRB4 SRB5 SRB6 SURN-top2 + - - - + + SURN-bottom + - - - + + SURF-top + - - - + + SURF-bottom + - - - + + Upland soil - - - - + - Bog-top + + - - + + Sedge + - - - + + Mat + + - - + + 1+ and – indicate presence and absence, respectively, of PCR products specific for the indicated group 2Samples were collected from the “top” 15 cm or the “bottom” 15 cm of the soil

Table 3 The effect of sulfate enrichment on SRB distribution in Sunday Lake soils1 Sample SRB1 SRB2 SRB3 SRB4 SRB5 SRB6 SURN + - - - + + SURN+SO4

2 + - - - +/- + Bog + + - - + + Bog+SO4 + + - - + + Mat + + - - + + Mat+SO4 + + - - + + 1+ and – indicate presence or absence, respectively, of the corresponding SRB group 2Sample to which sulfate (2 mM) was added followed by a three weeks incubation prior to methylation assays and DNA extraction

(1) a. Extract total DNA from soil (Hurt et al., 2001)

(2) Purify DNA from samples

(3) Touchdown PCR to amplify 16S rRNA genes with universal bacterial

primers (~1500 bp)

(4) PCR with specific primers for SRB 16S rRNA genes

Nested PCR

(6) Sequence DNA for identifying SRB species

(5) DGGE (Denaturing Gradient Gel Electrophoreses) separates the target SRB species

Gel purification

Gel electrophoresis to detectSRB signature in the samples

b. Extract total DNA from soil enriched with 2mM SO4

2-

Fig. 2: Protocols in molecular studies of Adirondack microbial communities

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