Post on 26-Dec-2019
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