Microbial Pathways for the Reduction of Mercury inSaturated Subsurface Sediments

T. Barkay1 (PI), S.M. Ní Chadhain1,2, R. Theofanopoulos1, S. Hicks1, J. Schaefer1, H. Wiatrowski1,

G. J. Zylstra1,2, and L. Young2

1Dept. of Biochemistry and Microbiology, and 2Biotechnology Center for Agriculture and the Environment, RutgersUniversity, New Brunswick, NJ

Mercury in contaminated subsurface soils may be leached to

the saturated zone where its reduction to the elemental form,

Hg(0), may enhance its environmental mobilization.

Therefore, microbial transformations that reduce the mercuric

ion, Hg(II), to Hg(0) are of key importance to the remediation

of mercury in contaminated subsurface sediments. Microbes

may reduce Hg(II) by the activity of the inducible mercuric

reductase (MR), the gene product of merA, and this process is

well understood in a broad range of aerobic bacteria and

environments. Little is known about Hg(II) reduction in

anoxic environments. We therefore have initiated a study on

the presence of merA in microbial communities of anoxic

environments and the effect of anaerobic respiratory

pathways on MR expression and activities. PCR primers were

designed to span the known phylogenetic range of merA

genes of Gram-negative bacteria. In control experiments,

these primers successfully amplified a 288 bp region at the 3’

end of previously characterized merA genes from Shewanella

putrefaciens pMERPH, Acidithiobacillus ferrooxidans,

Pseudomonas stutzeri pPB, Tn5041, Pseudomonas sp. K-62,

and Serratia marcescens pDU1358. The abundance and

diversity of merA were assessed in anaerobic enrichments

from Berry’s Creek, a highly industrially contaminated site in

the Meadowlands, NJ, by sequencing a merA clone library

obtained by PCR from sediment DNA extracts. Anaerobic

sediment slurries were supplemented with two additions of 10

g Hg(II)/g and incubated for 3 weeks at which time the DNA

was isolated and PCR amplified. The amplicons were TOPO

cloned into pCR2.1 (Invitrogen) and sequenced. A total of

174 clones were sequenced of which 88 represented unique

amplicons. The sequences were aligned and a phylogenetic

tree was built. While many sequences aligned with previously

described merA genes from both Gram positive and Gram

negative isolates, four novel lineages (II through V), were

identified. These results reveal a previously unrecognized

diversity of merA and suggest that bacterial activities may

play a role in mercury reduction in anaerobic environments.

Anaerobic enrichments of Meadowlands sediments were set up

for the purpose of isolating pure cultures carrying novel merA

genes. Five oligoprobes specific for each of the novel merA

cluster were designed, tested, and hybridized with genomic

DNA of Hg(II) resistant isolates from the enrichments. Three

strains, a Bacillus sp. and a Streptomyces sp. with merA of

cluster V, and a Pseudomonas sp. with merA of cluster II,

were isolated from the fermentative enrichment. One

denitrifying isolate, a Paenibacillus sp. carried a cluster III

merA. The entire mercury resistance systems in these strains

are currently being examined genetically and biochemically to

fully characterize the mechanisms by which anaerobic bacteria

interact with mercury. Similar studies are being set up with

subsurface sediments from mercury contaminated and control

aquifers.

ABSTRACT

CONCLUSIONS

A PCR approach toencompass the known

diversity of MerA in gramnegative bacteria

Pseudomonas aeruginosa Tn501 (Z00027)

Pseudomonas stutzeri pPB20 (U80214)

Serratia marcesens pDU1358 (M15049)

Alcaligenes sp. pMER610 (Y08993)

Xanthomonas sp. Tn5053 (L40585)

Shigella flexneri Tn21 (NC002134)

Acidithiobacillus ferrooxidans (D90110)

Thiobacillus sp. T.2.3 (Y11706)

Pseudomonas sp. Tn5041 (X98999)

Xanthomonas campestris Tn5044 (Y17691)

Pseudomonas sp. ED23-33 (Y17897)

Pseudomonas sp. K-62 pMR26 (D83080)

Shewanella putrefaciens BW13 (Y17830)

Shewanella putrefaciens pMERPH (Z49196)

Pseudoalteromonas haloplanktis (AY005468)

Streptomyces lividans (X65467)

Streptomyces sp. CHR28 pRJ28 (AF222792)

Bacillus cereus RC607 (AF138877)

Bacillus megaterium MB1 (AB027307)

Staphylococcus aureus pI258 (L29436)

Sulfolobus solfataricus (AE006863)

100

100

100

59

89

100

100

100

100 100

84

100

90

100

98

75

69

95

0.1

Gram +

Gram -

Archaea

B

C

D

E

A

high G/C

low G/C

Lane designations

correspond to clades

in Figure 1. A,

pDU1358; B,

A c i d i t h i o b a c i l l u s

ferrooxidans; C,

Tn5041; D,

Pseudomonas sp. K-

62; E, pMERPH; –, no

DNA control; and L,

fX174 HaeIII (Fisher).

Arrow shows the

expected ~288 bp

product.

Figure 1: Neighbor-joining (NJ) tree showing the phylogenetic

relationship of complete MerA sequences from representative

Hg resistant microorganisms. Clades A–E within the Gram

negative bacteria are indicated on the tree. Bootstrap values

for the NJ tree are shown at each branch point. Nodes also

supported by parsimony analysis (heuristic search) with

bootstrap values >74 are shown as closed circles. Branch

points supported by NJ but not parsimony analysis are shown

as open circles. The bar represents 0.1 nucleotide substitution

per site. NCBI accession numbers are indicated in parentheses.

merA degenerate primers:Forward primer A1s-n.F: 5’-TCC GCA AGT NGC VAC BGT NGG-3’

Reverse primer A5-n.R: 5’-ACC ATC GTC AGR TAR GGR AAV A-3’

merA:

A1s-n.F

A5-n.R

288 bp PCR product

High diversity and novelclusters in a merA clone

library from mercurycontaminated sediments

+ 1 2 3

288 bp

Meadowlands, NJ

Sampling site is highly contaminated

with mercury

Water column concentrations of

mercury were:

HgT: 130 - 1360 ng/L

MeHg: 0.31 - 1.6 ng/L

Isolate DNA &

PCR

Sequence

TOPO clone

+ 10 g Hg(II)/g, 41

days at 28 oC and

anaerobic conditions

Experimental Approach

SBC281SBC287

SBC288pDU1358SBC282SBC293

SBC298SBC292

SBC284SBC305

SBC299

SBC300SBC302

SBC301

SBC303Tn501

SBC289Tn21

SBC283SBC280

SBC285SBC308

SBC333

SBC290SBC291

SBC297

SBC331Acidithiobacillus ferrooxidans

Tn5041SBC296

SBC304SBC295

SBC336SBC332

SBC286SBC294

SBC310SBC330

SBC318SBC345Xanthomonas campestris Tn5044Z

Pseudomonas K-62

SBC311SBC314

SBC315SBC335

SBC346SBC316

SBC337SBC309

SBC334SBC321

SBC326

SBC320SBC325

SBC319SBC322

SBC323SBC324

SBC340

SBC306SBC30

7 pMERPHSBC329

Shewanella BW13Pseudoalteromonas haloplanktis

SBC317SBC312

SBC313SBC344

SBC347Bacillus cereus

Bacillus megateriumStaphylococcus aureus

SBC339SBC341

SBC328SBC327

SBC343Streptomyces lividans

Streptomyces pRJ28Sulfolobus sulfataricus

0.1

A

B

C/D

E

II (34)

IV (7)

III (10)

V (4)

Figure 3: NJ tree of derived amino acid sequences from merA

PCR products (~244 bp) obtained from Berry’s Creek sediment.

Numbers in parentheses following clade designations indicate the

number of clones within that clade. Unique clades (II – V)

detected in this library are outlined in boxes.

2B3

Snc38

2A2Tn501

1A12

Snc77

Snc44

Snc44

Snc38

Snc38

1 gsnc38

100ngsnc38

50ngsnc38

10ngsnc38

Figure 4: DNA hybridization signals with biotin-labeled probes

for unique merA clades (A) Specificity of the probe to clade V.

(B) Identification of enrichment culture 1A12 as belonging to

merA clade II. (C) Identification of enrichment cultures 2B1

and 2B3 as belonging to merA clade V.

Tn501

2B1

(A) (B) (C)

Enrichment culturesbelonging to novel merA

clades were isolated

• Pure cultures of Hg(II) resistant bacteria wereisolated from Meadowlands sediment enrichments underfermentative or denitrfication conditions• Genomic DNA was hybridized to oligoprobes designedfor unique clades II - V• mer operons from strains belonging to merA clades II,III, and V are currently analyzed

97%III Paenibacillus sp. N10

98%V Streptomyces sp. 2B3

97%V Bacillus sp 2B1

94%II Pseudomonas sp. 1A12

16SrRNA

similarity

CladeTaxon of closest relative

Cultures currently under study are:

Figure 2: Gel electrophoresis showing merA amplification

products from reference strains.

• 174 merA clones

sequenced• 88 unique phylotypes

• 55 clones belong

to four novel clusters

• Three of the clusters

branch within

the

lowG+C

Gram +bacteria

• An experimental approach andmethods for the study of the diversityof genes encoding for mercuricreductase enzymes in environmentalmicrobial biomaass are available

• A high, and hitherto unrecognized,diversity of merA was found in themicrobial community of acontaminated anoxic sediment

• This study will be expanded to themicrobial biomass of subsurfacesediments