RESEARCH ARTICLE

Mercuric reductase genes (merA) and mercury resistance plasmids in High Arctic snow, freshwater and sea-ice brine Annette K. Møller1, Tamar Barkay1,2, Martin A. Hansen3, Anders Norman3, Lars H. Hansen3, Søren J. Sørensen3, Eric S. Boyd4 & Niels Kroer1 1

Department of Environmental Science, Aarhus University, Roskilde, Denmark; 2Department of Biochemistry and Microbiology, Rutgers, The State University of New Jersey, New Brunswick, NJ, USA; 3Department of Biology, University of Copenhagen, Copenhagen, Denmark; and 4 Department of Chemistry and Biochemistry, Montana State University, Bozeman, MT, USA

Correspondence: Niels Kroer, Department of Environmental Science, Aarhus University, Frederiksborgvej 399, DK-4000 Roskilde, Denmark. Tel.: +45 87 15 87 01; fax: +45 87 15 50 10; e-mail: [email protected] Received 19 March 2013; revised 18 July 2013; accepted 29 July 2013. Final version published online 23 August 2013.

MICROBIOLOGY ECOLOGY

DOI: 10.1111/1574-6941.12189 Editor: Cindy Nakatsu Keywords High Arctic; merA; mercury resistance; plasmids; horizontal transfer.

Abstract Bacterial reduction in Hg2+ to Hg0, mediated by the mercuric reductase (MerA), is important in the biogeochemical cycling of Hg in temperate environments. Little is known about the occurrence and diversity of merA in the Arctic. Seven merA determinants were identified among bacterial isolates from High Arctic snow, freshwater and sea-ice brine. Three determinants in Bacteriodetes, Firmicutes and Actinobacteria showed < 92% (amino acid) sequence similarity to known merA, while one merA homologue in Alphaproteobacteria and 3 homologues from Betaproteobacteria and Gammaproteobacteria were > 99% similar to known merA’s. Phylogenetic analysis showed the Bacteroidetes merA to be part of an early lineage in the mer phylogeny, whereas the Betaproteobacteria and Gammaproteobacteria merA appeared to have evolved recently. Several isolates, in which merA was not detected, were able to reduce Hg2+, suggesting presence of unidentified merA genes. About 25% of the isolates contained plasmids, two of which encoded mer operons. One plasmid was a broad host-range IncP-a plasmid. No known incompatibility group could be assigned to the others. The presence of conjugative plasmids, and an incongruent distribution of merA within the taxonomic groups, suggests horizontal transfer of merA as a likely mechanism for High Arctic microbial communities to adapt to changing mercury concentration.

Introduction Mercury (Hg) is a toxic heavy metal that occurs naturally in elemental (Hg0), oxidized (Hg2+) and organic forms. The toxicity is due to a strong affinity of especially monomethyl-Hg and Hg2+ to sulphur atoms in cysteine residues and, hence, interference with protein structure and function (Carty & Malone, 1979; Philbert et al., 2000). Arctic food webs are becoming increasingly contaminated with Hg (Muir et al., 1999; Braune et al., 2005) resulting in elevated exposure levels of indigenous human populations (Van Oostdam et al., 2005). Following natural and anthropogenic emissions, Hg is transported as gaseous elemental mercury (GEM) to the Arctic (Skov et al., 2004; Pirrone et al., 2010). Asia is the dominant source of GEM (Durnford et al., 2010) rendering the Arctic region particularly vulnerable as emissions from Asia ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

are expected to increase in future (Streets et al., 2009). Modelling has estimated that more than 300 tons of Hg is deposited annually in the Arctic (Ariya et al., 2004). Much of this deposition occurs during the polar sunrise due to oxidation of Hg0 in the atmosphere by reactive halogen radicals from sea salt aerosols (Skov et al., 2004; Steffen et al., 2008). Bacteria may respond to the toxicity of Hg2+ by transforming it to gaseous Hg0 resulting in a detoxification of the immediate environment, by synthesis of thiols binding Hg2+, or by changed membrane permeability (Robinson & Tuovinen, 1984). The most common resistance mechanism is the enzymatic reduction in Hg2+ to Hg0 by the mercuric reductase (MerA) encoded by the merA gene. merA is part of the mer operon that encodes a group of proteins involved in the detection, scavenging, transport and reduction in Hg (Barkay et al., 2003). FEMS Microbiol Ecol 87 (2014) 52–63

53

merA genes in the High Arctic

In contrast to temperate regions, little is known about the occurrence, diversity and distribution of merA among arctic bacterial communities. Similarly, the knowledge on the potential for bacterial reduction in Hg2+ is limited. We have previously isolated and characterized Hg-resistant bacteria in snow, freshwater and sea-ice brine in the High Arctic (Møller et al., 2011) and shown that 0–31% of the culturable bacteria were resistant to Hg (10 lM HgCl2) and calculated that up to 6% of the total reduction in Hg2+ in snow could be attributed to the activity of mercury-resistant bacteria. Because bacterial reduction in Hg2+ leads to the formation of volatile Hg0, mercuryresistant bacteria may act as re-emitters of Hg to the atmosphere and, hence, contribute to the detoxification of the contaminated environment. Furthermore, as Hg2+ is the substrate of methylation reactions, the mercuryresistant bacteria may also play a role in lowering the supply of substrate for the methylation processes (Møller et al., 2011). Due to this potentially central role of merA in the cycling of Hg in the Arctic, it is important to not only assess the occurrence and diversity of this gene, but also understand its taxonomic and environmental distribution and evolutionary history. Here, we report the identification of novel and globally distributed merA gene sequences among Hg–resistant bacteria from the High Arctic. A cultivation-based approach was applied in order to link the merA phylogeny with the phylogeny of the bacterial hosts. The merA genes were identified by PCR using primers specific for merA. In one case, however, it was not possible by PCR to amplify merA. Hence, whole-genome sequencing was applied. Furthermore, as in temperate regions, the mer operon is often found on mobile genetic elements (Barkay et al., 2003), we isolated and sequenced plasmids from some of the isolates to assess the potential for horizontal transfer of merA. The data suggested that the pool of merA among the bacterial assemblages was diverse, mobile and represent both evolutionary recent and ancient lineages in the merA phylogeny.

Material and methods Isolation and identification of mercuryresistant bacteria

Snow, freshwater and sea-ice brine water were sampled at Station Nord in Northeastern Greenland (81°36′N; 16°40′ W). The snow and brine samples were collected in Dagmar Sund, while the freshwater samples were taken from a small ice and snow-covered lake 2 km south of Station Nord. Mercury-resistant bacteria isolated in a previous study (Møller et al., 2011) were used. Briefly, bacteria were isolated by (1) direct plating on 10% strength tryptic soy FEMS Microbiol Ecol 87 (2014) 52–63

agar (TSA) (Difco) and (2) by pregrowth to microcolony-size under simulated natural conditions before plating on 10% TSA (supplemented with 4.5% sea salts (Sigma-Aldrich) in the case of brine isolates). The latter approach was included as it has been shown to induce growth of hard-to-culture mercury-resistant soil bacteria (Rasmussen et al., 2008). Plates and filters were incubated at 4–10 °C. Mercury resistance was tested by streaking on 10% TSA plates supplemented with 10 lM HgCl2 and resistant isolates identified by partial 16S rRNA gene sequencing (Møller et al., 2011). Liquid cultivation of isolates was performed at room temperature in 10% tryptic soy broth or, in the case of three Bacteroidetes isolates (SOK29, SOK62 and SOK79), at 15 °C in PYG medium (5 g polypeptone, 5 g tryptone, 10 g yeast extract, 10 g glucose and 40 mL salt solution in 1 L. The salt solution, pH 7.2, contained per L: 0.2 g CaCl2, 0.4 g MgSO4
7H2O, 1.0 g K2HPO4, 1.0 g KH2PO4, 10.0 g NaHCO3 and 2.0 g NaCl. Both media were supplemented with 10 lM HgCl2. merA sequencing and phylogeny

DNA was extracted from the mercury-resistant bacteria by boiling as previously described (Fricker et al., 2007). For some of the isolates (8D5s, 8D7, 8D12, 8D12b, SOK1b, SOK 15, SOK17a, SOK17b, SOK19, SOK19y, SOK27, SOK32, SOK33, SOK35, SOK38, SOK43, SOK48, SOK52, SOK57), extraction by boiling was not effective. Instead, the PowerMax DNA soil kit (MoBio Laboratories, Inc.) was used to extract DNA following the manufacturer’s instructions. All DNA preparations were stored at 20 °C prior to sequence analysis. Detection of merA was carried out by PCR using 10 phylum-specific merA primer sets. Primers, primer targets, annealing temperatures, PCR extension times and product sizes are listed in the Supporting Information, (Table S1). The PCR conditions were 94 °C for 2 min followed by 35 cycles of 94 °C for 1 min, 50–64 °C (Table S1) for 30 s, 72 °C for 1–3 min (Table S1) and one cycle of final extension at 72 °C for 10 min. PCR mixtures contained 0.4 lM of each primer, 400 lM dNTP, 2.5 mM MgCl2 and 2 U Taq DNA polymerase (Fermentas). PCR products were gel-purified using Qiagen Gel Extraction Kit. Sequencing of PCR products from both the 5′ and 3′ directions was performed by GENEWIZ (USA) or Eurofins MWG Operon (Germany) using the same primers as those listed for the initial PCR. For some isolates, no PCR products were obtained. In the case of isolate SOK62, the merA sequence was retrieved by whole-genome sequencing (see below). Sequences were assembled in BioEdit (http://www. mbio.ncsu.edu/BioEdit/bioedit.html), manually trimmed ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

54

to remove primer sequences and blasted in NCBI GenBank using BLASTN and BLASTX [DNA sequences translated with the translate tool at Swiss Institute of BioInformatics, http://www.expasy.org/tools/dna.html (Gasteiger et al., 2003)]. Sequences were also aligned in BioEdit and 99% identical sequences grouped. One representative of each group, together with the most closely related database sequence and selected representative MerA protein sequences, was aligned using the Gonnet substitution matrix and default settings within the program CLUSTALX (version 2.0) (Larkin et al., 2007). The NmerA extension was trimmed and the resulting alignment was subjected to evolutionary model prediction using ProtTest (version 2.4) (Abascal et al., 2005). The evolutionary history was inferred by maximum likelihood with the Whelan and Goldman with fixed amino acid frequencies (F) and gamma-distributed (4 categories, k = 1.8967) rate variation with a proportion of invariable sites (proportion = 0.028634), as recommended by ProtTest (version 2.4) (Abascal et al., 2005). The tree was drawn to scale, with branch lengths measured in the number of substitutions per site. All ambiguous positions were removed for each sequence pair. There were a total of 514 positions in the final data set. Evolutionary analyses were conducted in MEGA5 (Tamura et al., 2011). Plasmid DNA preparation and sequencing

Plasmid DNA was isolated from freshly grown colonies using QIAprep Spin Miniprep Kit (Qiagen). Cells of isolates belonging to the Firmicutes, Actinobacteria and Bacteriodetes were treated with lysozyme for 1 h at room temperature prior to the plasmid DNA isolation. Plasmid preparations were gel-electrophoresed (0.8% agarose) and grouped according to their migration patterns. Representative plasmids from each of the five observed groups were sequenced. Plasmid DNA was prepared from exponentially growing cells of isolates SOK1b, SOK15, SOK19, SOK65 and SOK71 using QIAGEN Midi Prep Kit. Isolate SOK1b (Bacillus sp.) was treated with lysozyme for 1 h prior to DNA isolation. Plasmid DNA was treated with Plasmid-SafeTM ATP-Dependent DNase (Epicentre Biotechnologies) overnight at 37 °C to digest any contaminating chromosomal DNA and subsequently ethanol precipitated. Between 2 and 5 lg of plasmid, DNA was fragmented by nebulization and shotgun libraries built according to the standard protocol (Roche) except for preparations of SOK15. Here, an additional DNA amplification was performed using REPLI-g UltraFast Mini Kit (Qiagen) to increase the input DNA prior to library construction. Each library was tagged with a standard MID-tag (FLX MID’s 1–5 as provided by supplier) ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

A.K. Møller et al.

to allow mixing of the different libraries in one region of the picotiter plate. Following establishment of libraries, the DNA was amplified by PCR using FLX-fw: GCCTCCCTCGCGCCATCAG and FLX-rev: GCCTTGCC AGCCCGCTCAG. Amplified DNA fragments were then separated by gel electrophoresis and bands between 350 and 650 bp were cut from the gel and purified using QIAEX II kit (Qiagen). The purified DNA was quantified using a QubitTM fluorometer (Invitrogen) and pooled in equimolar concentrations. Sequencing of the pooled libraries was performed on a Genome Sequencer FLX instrument using an emPCR kit II (Amplicon A, Paired End) and a standard FLX sequencing Kit. The depth values from the 454 Alignmentinfo.tsv file, generated by the Newbler assembler software (version 2.0.01.14) of the Genome Sequencer FLX instrument, were used to calculate the average sequencing coverage (i.e. the average number of times a given DNA nucleotide was represented in the sequence reads) of each contig. Based on a frequency distribution of the depth values, a minimum coverage for each assembled library was set to distinguish between plasmid and chromosomal contigs originating from incomplete exonuclease degradation. Thus, contigs having an average coverage between 40 and 200X were considered to be part of the plasmid(s), while contigs with an average coverage < 20X were considered to be chromosomal and excluded from the assembly. The low-coverage contigs were checked by BLAST analysis to confirm chromosomal-specific sequences (e.g. 16S rRNA gene). Open reading frames of contigs were determined with the Prodigal gene-finding program (Hyatt et al., 2010) and loaded into the Biopieces bioinformatics tool (www. biopieces.org) for further analysis. Here, contigs were blasted against the merA sequences identified in this study as well as with all known bacterial MerA proteins sequences. Both MerA and MerP contain a conserved heavy-metal-associated domain; thus, merP was also identified in the MerA blast. Contigs were also blasted against several plasmid replication or plasmid transfer-specific genes [IncN: rep, kikA, oriT; IncP: oriT, trfA1, trfA2, korA, traG; IncQ: repB, oriV, oriT; IncW: oriV, oriT, trwAB (G€ otz et al., 1996)] to identify the Inc group of the plasmids. Whole-genome sequencing of isolate SOK62

DNA for whole-genome sequencing was prepared from exponentially growing cells with DNeasy Blood and Tissue Kit (Qiagen). A paired-end library with an insert size of 500 bp was prepared from 5 lg of high-molecular weight DNA from isolate SOK62 according to the Illumina GA II paired-end library preparation protocol. Sequencing on the FEMS Microbiol Ecol 87 (2014) 52–63

55

merA genes in the High Arctic

lllumina GA II instrument for 2 9 32 cycles using the paired-end settings resulted in 17.3 million paired-end reads. Sequence assembly was performed using Velvet version 0.7.59 (Zerbino & Birney, 2008) with parameters obtained using VelvetOptimizer (http://bioinformatics.net. au/software.velvetoptimiser.shtml). These were as follows: velveth: 23 -fastq –shortPaired; velvetg: -ins_length 500 -exp_cov auto -min_contig_lgth 500 –cov cut-off 35.9458254230799. Automatic annotation was performed using the RAST annotation system (Aziz et al., 2008). merA sequence accession numbers

Representative merA sequences have been submitted to GenBank and given accession numbers JX415486 – JX415511 for SOK43, SOK44, SOK50, SOK52, SOK54, SOK59, SOK41, SOK33, SOK32, SOK19y, SOK19, SOK15, SOK5, SOK1b, 8D5s, SOK84, SOK80, SOK75, SOK73, SOK89, SOK13, SOK70, SOK68, SOK65 and SOK62, respectively.

Results Diversity of mercury-resistant bacteria

Among a total of 791 bacterial isolates, 71 were Hg resistant as indicated by growth on 10% strength TSA amended with 10 lM HgCl2. The majority of the resistant isolates (63%) were from the freshwater lake (Table 1). Isolates were affiliated with the Alphaproteobacteria (6 isolates), Betaproteobacteria (9 isolates), Gammaproteobacteria (37 isolates), Flavobacteria (4 isolates), Sphingobacteria (2 isolates), Actinobacteria (9 isolates) and Bacilli (4 isolates). Within each phylum/class, several subgroups with nearly identical (97% sequence similarity) partial 16S rRNA gene sequences were observed (Table 1). Diversity of merA

merA was detected in 38% of the mercury-resistant isolates representing all four of the observed bacterial phyla

Table 1. Distribution of merA and plasmids in mercury-resistant isolates

Phylum

Subgroup

Isolate*

merA†

Plasmid‡

Plasmid group§

Proteobacteria

Alpha I Alpha II Beta I Beta II Beta III Gamma I

SOK5, SOK19, SOK19y, 8D1 30D12, 8D12b SOK35, SOK17a, SOK17b, SOK18t, SOK48, SOK51, SOK57 SOK15 8D36 SOK32, SOK33, SOK43, SOK52 SOK61, SOK67, SOK71 SOK70 SOK80 8D32, 8D45, 8D48b, 8D48s SOK41, SOK44, SOK50, SOK54, SOK59, SOK65, SOK68, SOK73, SOK75 SOK84, SOK85, SOK89 SOK19w, SOK84s, SOK90, SOK12, 8D21, 8D26, 8D41, 8D55s, 8D55t, 8D56, 8D64b SOK13 SOK18b SOK62 SOK29, SOK79 SOK70s, 8D64s 8D5s 8D5b, 8D7, 8D8, 8D10, 8D11, 8D12, 8D13 68F56 SOK1b SOK27 SOK38 BD41

+

+

III

+

+

II

+ + + + +

V V IV IV IV

+

I

Gamma II

Bacteroidetes

Actinobacteria

Firmicutes

Gamma III Flavo I Flavo II Sphingo Actino I Actino II Bacilli I Bacilli II Bacilli III Bacilli IV

+ + + + + + +

+

+

*The SOK preface indicates that the isolate originated from freshwater; notations prefaced by 8, 30,and 68 indicate that the isolate came from snow; the BD preface indicates the isolate origin is brine. † + indicates it was possible to detect merA by PCR (or whole-genome sequencing for isolate SOK62) while– indicates that merA PCR was performed but with negative result. ‡ + indicate that if was possible to isolate plasmid(s); indicates that plasmid isolation was attempted but with negative result. § Plasmid groups based on gel-migration patterns (see Fig. 2).

FEMS Microbiol Ecol 87 (2014) 52–63

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

56

A.K. Møller et al.

(Table 1). No merA were detected in the remaining 62% of the isolates, 13 of which were positive for Hg volatilization (Møller et al., 2011). The majority (76%) of the merA sequences were found in isolates belonging to the Gammaproteobacteria, while 12% belonged to the Alphaproteobacteria and 4% to each of the Betaproteobacteria, Firmicutes and Actinobacteria. In some phylogenetic subgroups (e.g. Beta I), merA could not be detected, while in other subgroups, merA was detected only in some members. In subgroup Gamma II, for instance, merA was detected in only 44% of the isolates (Table 1). Sequencing of the merA amplicons revealed 7 unique loci (‘merA types’) as defined by a 99% sequence similarity within each type. merA types 1 and 3 were observed in several isolates (Table 2). Among the isolates of

subgroup Gamma I, two merA types (3 and 4) were observed, while in all other taxonomic subgroups, only one merA type was detected (Table 2). merA types 3 and 4 from the Gammaproteobacteria were 99–100% identical at the amino acid level to merA of Tn5042 and Tn5041, respectively (Fig. 1 and Table 2). merA type 2 was only found in one isolate belonging to the Betaproteobacteria, but was identical to a merA previously found in a Gammaproteobacterium (Fig. 1 and Table 2). Three type 1 merA sequences from isolates belonging to the Alphaproteobacteria were 100% identical at the amino acid level to a previously described alphaprotebacterial merA (Table 2) and a hypothetical protein in the genome of the filamentous fungus, Sordaria macrospore (Table 2) having a 19 aa deletion at the MerA redox active site.

Table 2. Similarities of partial merA gene fragments of isolated bacteria to sequences reported in the literature BLAST

Size of merA fragment (bp)

results

Similarity (%) AA†

99

100

Sphingobium sp. SYK6 (YP_004831132.1); Sordaria macrospora (XP_003342866.1)

100

100

99

99

Pseudomonas sp.K-62 pMR26 (BAA20337) P. fluorescens SBW25 pQBR103 Tn5042 (YP_001202176)

99–100

99–100

Isolate

Subgroup

1

SOK5 SOK19 SOK 19y SOK15

Alpha I

725–743

Beta II

245

Gamma I

1140–1154

Gamma II

185–1156

3

SOK32 SOK33 SOK43 SOK52 SOK80 SOK41 SOK44 SOK50 SOK54 SOK59 SOK65 SOK68 SOK73 SOK75 SOK84 SOK85 SOK89 SOK13

Gamma III

680

100

100

4

SOK70

Gamma I

1032

100

100

5

SOK62

Flavo II

1659

NH

84

6

8D5s

Actino I

1095

80

92

7

SOK1b

Bacilli

411

83

91

2 3

3

Most closely related MerA (protein accession number)

DNA*

merA type

P. fluorescens SBW25 pQBR103 Tn5042 (YP_001202176)

P. fluorescens SBW25 pQBR103 Tn5042 (YP_001202176) Pseudomonas sp Tn5041 (CAA67451) Sphingobacterium spiritivorum ATCC 33300 (ZP_03969449.1) Corynebacterium genitalium ATCC 33030 (ZP_05707785) Clostridium butyricum (BAA86102.1)

*BLASTN. † BLASTX. NH, no hits.

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

FEMS Microbiol Ecol 87 (2014) 52–63

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merA genes in the High Arctic

Tn5042 (YP_001202176) Tn5041 (CAA67451)

Gammaproteobacteria

Proteobacteria

Alphaproteobacteria

Actinobacteria

Firmicutes

Bacteroidetes

Aquificae

Archaea

Fig. 1. Maximum-likelihood phylogenetic tree showing the phylogeny of representatives of the merA types (translated nucleotide sequence) identified among the Arctic mercury-resistant isolates (shown in bold). The tree is rooted with the paralog enzyme dihydrolipamide dehydrogenase. Bootstrap support is indicated for each node, with those with < 50% bootstrap support collapsed. Numbers in parentheses indicate the number of merA sequences within each type (99% sequence similarity).

FEMS Microbiol Ecol 87 (2014) 52–63

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

58

The predicted MerA proteins from the Bacteroidetes, Actinobacteria and Firmicutes isolates had lower degrees of similarity (84–92%) to MerA sequences in databases as compared with those found in the Proteobacteria (99–100%) (Table 2). At the amino acid level, type 5 MerA from strain SOK62 was 82% similar to the most closely related database sequence, while no hits came up in the DNA blast (BLASTN). Type 5 merA was identified by whole-genome sequencing as it could not be amplified using degenerate primers or primers designed on basis of the putative merA of the Flavobacterium Loewenhokiella blandensis (Table S1). The low similarity between the merA locus of SOK62 and that of L. blandensis, or any other merA loci, probably explains the negative PCR result. Type 6 merA, representing nine Actinobacteria isolates (taxonomic subgroup Actino I), was 80% and 92% similar to DNA and protein sequences in the databases, respectively. Finally, the type 7 merA sequence of an isolate belonging to the Firmicutes was 83% similar to a merA gene from an Enterococcus faecium isolate and 91% similar at the amino acid level to a merA from Clostridium butyricum. Sequencing of plasmids and identification of mer-operon genes on plasmid contigs

Plasmids were found in 24 of the 71 (34%) mercuryresistant isolates. However, among the isolates where merA was detected, the plasmid incidence was 68% (Table 1). Five putative plasmid groups were identified based on agarose gel-migration patterns (Fig. 2). Sequencing of plasmid DNA from isolates representing each of the different patterns resulted in several contigs. Based on the average depth of coverage of the individual contigs, they were classified as either plasmid DNA or contaminating chromosomal DNA. Also depending on the sequence coverage, the plasmid DNA contigs were determined to belong to one or more plasmids. Plasmids from Firmicutes isolate SOK1b (plasmid group I) resulted in 6 contigs and a total of 39 798 bases. The coverage of the contigs suggested presence of two plasmids with sizes of ~23.7 kb (3 contigs) and ~16.1 kb (3 contigs). On the largest contig (16.1 kb) of the 23.7-kb plasmid, a merA sequence was identified along with merB and 3 regulator sequences (merR, merR2 and an arsR family regulator) (Fig. 3). Several homologues of genes involved in transposition were also present (Fig. 3). Five contigs of a total of 87.2 kb from the Gammaproteobacterium isolate SOK65 (plasmid group IV) were identified. The sequence coverage of the contigs suggested the presence of only one plasmid. On a 6.9-kb contig, a mer operon containing merA, merB, merC, merP, merT and merR was present (Fig. 3). In addition, three transposases were present. ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

A.K. Møller et al.

I

II

III

IV

V

23130 bp 9416 bp 6557 bp 4361 bp

2322 bp 2027 bp

564 bp

Fig. 2. Agarose gel of representative plasmid profiles from Hgresistant Arctic bacterial isolates. Lanes are labelled with the name of the isolate from which the plasmids were extracted. Roman letters indicate the grouping of the plasmids.

No merA sequences were seen on group V plasmids isolated from the Gammaproteobacterium isolate SOK71 (6 contigs, total of 22.8-kb, one 14.2-kb and one 8.6-kb plasmid predicted). However, 3 other mer genes, merR, merT and merP, were identified on a 6.3-kb contig belonging to the 14.2-kb plasmid (Fig. 3), a pattern previously associated with hypersensitivity to Hg (Hamlett et al., 1992). Because SOK71 is highly resistant to and reduces Hg (Møller et al., 2011), a merA gene is most likely present elsewhere in the genome of the strain. However, we could not amplify this locus using any of 10 merA-specific PCR primer sets (Table S1). In proximity to the incomplete mer operon, 2 transposases and a resolvase were observed. None of the 9 contigs (total of 57.5 kb, 2 plasmids of 48.6 kb and 8.9 kb) originating from the plasmid DNA of the Betaproteobacterium isolate SOK15 (plasmid group II) contained mer genes. However, on two small contigs, merA and other mer genes (merP, merB, merD and merE) were present (Fig. 3). Given that these contigs had a relatively low-sequencing coverage, it is uncertain if they originated from plasmid or chromosomal DNA. No merA on any of the contigs from plasmids isolated from the Alphaproteobacterium isolate SOK19 (plasmid group III, 16 contigs, total of 100.3 kb, 3 plasmids of 20.5 kb, 51.7 kb and 28.1 kb) were observed. All contigs were searched for DNA sequences specific to incompatibility groups IncP, IncN, IncW and IncQ. Four of the 5 contigs of the 48.6-kb plasmid of SOK15 FEMS Microbiol Ecol 87 (2014) 52–63

59

merA genes in the High Arctic

Plasmid pattern I (Firmicutes isolate SOK1b) R B R2 a b A

c

d

e

f

a – ArsR family transcriptional regulator, b – cytochrome C biogenesis protein, c – Transposase, d – Relaxase, e – Mobilization protein, f – Transposase, g - Resolvase

g 16120 bp

Plasmid pattern IV (γ-Proteobacterium isolate SOK65) a b c B A C P T R a - Transposase, b – Transposase, c - Transposase

6915 bp

Plasmid pattern V (γ-Proteobacterium isolate SOK71) R T P a b c a - Transposase, b – Transposase, c - Resolvase

6257 bp

Plasmid pattern II (β-Proteobacterium isolate SOK15) aE D

B A 1826 bp

A

P

1325 bp

a - TniM Fig. 3. Genetic organization of mer-operon genes on plasmid contigs. Open reading frames are shown as box arrows, with the pointed end indicating the direction of transcription. Black reading frames indicate genes encoding mer proteins. Grey striped reading frames indicate genes encoding proteins involved in plasmid transfer or transposition. Grey reading frames indicate other genes encoding other proteins, and white reading frames indicate genes encoding hypothetical proteins or proteins with unknown function.

(plasmid group II) contained sequences identical to traG, trfA1, trfA2, korA and oriT from IncP-a (G€ otz et al., 1996). No known Inc specific gene sequences were found in any of the contigs from the other plasmid groups. Whole-genome sequencing of isolate SOK62 and identification of mer-operon genes

The whole genome of isolate SOK62 (taxonomic subgroup Flavo II) was sequenced as it did not contain plasmids (Table 1), and it was not possible by PCR to amplify merA despite the fact that all isolates of this subgroup previously were shown to volatilize ionic mercury (Møller et al., 2011). Sequencing resulted in 17.3 million paired-end reads. The assembly yielded a total of 48 contigs (longest contig 505 321 nucleotides, n50 = 193 975) indicating a chromosome size of approximately 3.9 Mbp with an average GC content of 34%. Annotation identified 257 subsystems (network of metabolites and enzymes that comprise, for example, a metabolic or signal transduction pathway), 3 502 coding sequences and 46 RNAs. An open reading frame with similarity to merA was identified on a 275-kb contig by blasting with merA from Tn501 and 4 putative MerA sequences from Bacteroidetes isolates in the databases. The BLAST search produced several hits, but only one open reading frame could produce a protein (553 amino acids) with conserved amino acid FEMS Microbiol Ecol 87 (2014) 52–63

motifs characteristic of mercury reductases, including the redox active site, a C-terminal vicinal cysteine pair and the N terminus NmerA domain (Barkay et al., 2010). The genetic organization of part of the 275-kb contig with merA and surrounding genes is shown in Fig. 4. Upstream to merA are two gene homologues that together with merA may constitute a primitive mer operon. The first gene, arsR, is a transcriptional regulator while the second gene is a putative mercuric transport protein. Other genes proximal to the putative mer operon include genes involved in metal metabolism such as a multicopperoxidase, a high affinity Fe2+/Pb2+ permease and a heavy-metal transporting ATPase.

Discussion Seven different merA determinants were identified among the 71 mercury-resistant bacterial isolates from High Arctic snow, freshwater and sea-ice brine. Three of the determinants, found in isolates of the Bacteriodetes, Firmicutes and Actinobacteria phyla, were novel showing low amino acid sequence similarity (< 92%) to known MerA’s, while one MerA observed among the Alphaproteobacteria isolates was identical to a previously described alphaprotebacterial MerA gene (Table 2) and a hypothetical protein in an filamentous fungus, whose function as a reductase is questioned by a deletion at the redox active site. The ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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A.K. Møller et al.

b

c

d

a

e

T

A

l

f

SOK62 g

a

h

T

i

B

A

j

T

h

B

A

j

Sphingobacterium spiritivorum g

h

k

a

Chryseobacterium gleum 1000 bases

a – ArsR family transcriptional regulator, T – Mercury transport protein, B – Mercurial lyase (MerB), A – Mercuric reductase (MerA), l – Heavy metal transporting ATPase b – Multicopperoxidase, c – Ferrodoxin, d – Universal stress protein, e - High affinity Fe2+/Pb2+ permease, f – Protein with DNA binding domain excisionase, g – Protein found in conjugative transposon, h – TraF, i – Coppertranslocating P family ATPase, j – Protein found in conjugative transposon, k – TraE, Fig. 4. Genetic organization of Bacteroidetes mer operons. Included are Flavobacterium sp. SOK62, Sphingobacterium spiritvorum and Chryseobacterium gleum. Open reading frames are shown as box arrows, with the pointed end indicating the direction of transcription. Black reading frames indicate genes encoding proteins assumed to be involved in mercury resistance. Grey and striped reading frames indicate genes encoding proteins involved in conjugation or transposition. Grey reading frames indicate genes encoding other proteins. White reading frames indicate genes encoding hypothetical or conserved proteins.

remaining 3 MerA exhibited > 99% identity with sequences from transposons and plasmids previously known from other environments. Thus, the pool of merA among the arctic mercury-resistant isolates was diverse, representing both novel homologues as well as homologues that appear to be globally distributed. The fraction of isolates identified to contain merA was relatively small (38%) considering that the mer system is the most common mode of bacterial mercury resistance (Osborn et al., 1997). Even more striking is the fact that we previously demonstrated that 18 of the resistant isolates were capable of reducing Hg2+ to Hg0 (Møller et al., 2011), yet in only 5 of these isolates was merA detected. This suggests that in addition to the 3 novel merA determinants, still unidentified merA genes may be discovered in the Arctic environment by use of, for example, metagenomic-enabled approaches or additional PCR primer sets. Only one study has previously examined the presence and diversity of merA in polar regions (Poulain et al., 2007). More than 100 partial (291 nucleotides) merA sequences were obtained from DNA and RNA extracts of algal biofilms from sea ice and a coastal lagoon in the Canadian High Arctic. The majority of the sequences were closely related to proteobacterial merA. In contrast, a much higher diversity of merA was obtained in the present study by targeting cultured bacteria. A higher diversity of merA from cultured bacteria is unexpected as cultivation-based methods generally underestimate species diversity compared with DNA-based techniques. Pyrosequencing of 16S rRNA genes from the snow and freshwater environments previously demonstrated a high species ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

diversity, especially in the snow pack (Møller et al., 2013). Poulain et al. (2007) did not study the bacterial community structure of the algal biofilms, but the higher merA diversity in our study may possibly be attributed to differences in bacterial community structures. In the phylum Bacteroidetes, 10 putative MerA proteins are available in the databases. BLASTX analysis against nonredundant proteins showed that the MerA of SOK62 was only partially similar (42–84%) to the putative MerA loci in Sphingobacterium spiritivorum ATCC 33300, Chryseobacterium gleum ATCC 35910, Loewenhokiella blandensis and Rhodothermus marinus DSM 4252. As shown in Fig. 1, the Bacteroidetes MerA sequences form a cluster basal to most bacterial clusters. The deep branching position of the Bacteroidetes MerA lineage clearly indicates that these proteins represent an ancient lineage in the MerA phylogeny. On the other hand, type 2, 3 and 4 MerA sequences cluster at the crown of the MerA tree (Fig. 1) in the most recently branching lineage and with a high similarity to MerA in the Alpha- and Betaproteobacteria (Table 2). Thus, MerA from the High Arctic represents both more recent and more ancestral lineages of this locus. The merA of SOK62 was a part of an operon consisting of the merA homologue and homologues of an ArsR-like regulator and a mercury-transport protein. The putative mer operon of SOK62 shares some characteristics with the operons of S. spiritivorum and C. gleum (Fig. 4) in that the ArsR-like regulator and the mercury-transport protein gene are located upstream of merA and all are transcribed in the same direction. However, contrary to FEMS Microbiol Ecol 87 (2014) 52–63

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the operon in SOK62, the other two operons contain a merB homologue upstream of merA. It should also be noted that in S. spiritivorum and C. gleum, the operons are surrounded by several genes involved in transposition while in SOK62, some of the functions encoded by genes proximal to the mer system are associated with metal transformation. We did not identify merR, the universal regulator of the mer operon, in any of the Bacteroidetes operons. Rather, the ArsR family regulator was the first gene encoded in all the Bacteroidetes operons. The MerR and ArsR families represent two general classes of metal-binding transcriptional regulatory proteins responding to heavy-metal stress and toxicity (Brown et al., 2003; Busenlehner et al., 2003). ArsR acts exclusively as a transcriptional repressor that dissociates from the DNA upon interaction with metal ligands (Xu et al., 1996; Chen & Rosen, 1997), while MerR-like regulators repress transcription in the absence of the effector and induce transcription in its presence (Brown et al., 2003). Thereby, MerR may facilitate a finer regulation of mer-operon expression as compared with ArsR. ArsR regulators are often associated with early MerA lineages and are the only regulators observed in archaeal mer operons and in some mer operons in Actinobacteria and Firmicutes (Barkay et al., 2010). Thus, it is possible that merR is a later development in the evolution of the mer operon replacing the ArsR regulator genes and resulting in more efficient mer systems. If so, the presence of ArsR-like regulators emphasizes the ancestral position of the Bacteroidetes mer system among the Eubacteria. Regardless, the Hg-resistant Flavobacterium SOK62 showed high level of resistance with a minimal inhibitory mercury concentration of 50 lM (Møller et al., 2011), illustrating that an evolutionary ancient mer operon may be highly efficient. The mer operon is often found on mobile genetic elements (Barkay et al., 2003) and merA homologues known from transposons such as Tn5042, Tn5058 and Tn4051 have been found in environments as diverse as sugar beet leaves in the UK (Tett et al., 2007), Hg-contaminated soil in Tennessee, USA (Oregaard & Sørensen, 2007), 120 000year-old Siberian permafrost samples (Mindlin et al., 2005), a mercury mine in central Asia (Kholodii et al., 1997), a phenylmercury-polluted soil (Kiyono et al., 1997) and mercury-polluted river sediment in Kasakhstan (Smalla et al., 2006). Horizontal transfer alone probably cannot explain the global distribution of almost identical MerA proteins; however, it supports the hypothesis of Osborn et al. (Osborn et al., 1997) who suggested that the distribution of MerA is the result of a combination of highly conserved proteins, localized selective pressure and horizontal transfers. Several lines of indirect evidence suggest horizontal transfer of merA among the mercury-resistant bacteria in FEMS Microbiol Ecol 87 (2014) 52–63

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the investigated High Arctic environments. For instance, the occurrence of the same merA type in different taxonomic subgroups (i.e. type 3 in Gamma I-III), the presence of mer operons on plasmids and in proximity to genes specifying transposition functions (Figs 3 and 4), the apparent lack of merA in several of the members of the Gamma I and II subgroup bacteria, and the observation that merA types 1 and 2 were most similar to homologues previously found in other taxonomic groups collectively indicate a potential location of the merA’s on mobile genetic elements. To assess the potential for horizontal transfer of merA, plasmids from the resistant bacteria were isolated. About two-thirds of the isolates with merA also contained one or more plasmids (Table 1). Sequencing of the different plasmids demonstrated that one plasmid carried merA (and merB), another the entire mer operon, while a third plasmid carried mer genes other than merA (Fig. 3). The plasmid isolated from strain SOK15 belonged to IncP-a, a group of plasmids known to have a broad host range (Thomas & Smith, 1987). Even though the merA in SOK15 probably was located on the chromosome, the IncP-a plasmid may have been the shuttle for a transposon carrying merA. Indeed, a high similarity (99–100%) of the chromosomal merA in SOK15 to merA of plasmid pMR26 (Kiyono et al., 1997) and transposon Tn5058 (Smalla et al., 2006) was noted. Considering the broad host range of IncP plasmids (Thomas & Smith, 1987; Musovic et al., 2006), the presence of these plasmids in the arctic microbial communities emphasizes that horizontal transfer of mercury resistance genes might be a mechanism for the microbial communities to rapidly adapt to increasing mercury concentrations, as for example, during spring-time atmospheric mercury-depletion events. Plasmid-carrying bacteria have previously been demonstrated in both Arctic (Miteva et al., 2004) and Antarctic environments (Kobori et al., 1984; Miller et al., 2009), and at least one arctic plasmid has been shown to have a broad host range (Miteva et al., 2008). Only one of our plasmids could be affiliated with a known incompatibility group. Therefore, the other plasmids may be unknown broad host-range conjugative plasmids with the potential of acting as vectors for the spreading of merA. In conclusion, our study suggested that the pool of merA loci among the arctic bacterial assemblages was diverse, mobile and represents both evolutionary recent and ancient lineages in the MerA phylogeny. The SOK62 merA gene was found to be part of an early mer operon in an isolate of the Bacteroidetes, a phylum where mercury resistance has only recently been described (Allen et al., 2013). A phylogenetic analysis of this sequence together with other putative merA loci from Bacteroidetes ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved

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suggested these loci to represent an early lineage in the mer phylogeny. In addition to the 7 identified merA types, several undescribed merA loci, that could not be amplified using primers designed using known sequences, were most likely present as indicated by their Hg2+-reducing activity. Our results also demonstrated that some of the merA loci were plasmidborne and that horizontal transfer of merA is a likely mechanism for the adaptation of arctic microbial communities to changes in the mercury concentration. Altogether, our data suggest a role of Arctic environments in shaping the evolutionary history of merA and that High Arctic microbial communities have the capacity to play an important part in the transformation of Hg.

Acknowledgements We thank Karin Vestbjerg for excellent technical assistance. The work was funded by the Danish Agency of Science (Grant # 645-06-0233), the U.S. National Science Foundation (EAR-0433793 and EAR1123689), the U.S. Department of Energy (DE-FG02-05ER63969) and the NASA Exobiology and Evolutionary Biology Program (NNX10AT31G).

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Supporting Information Additional Supporting Information may be found in the online version of this article: Table S1. Primers used for amplifying merA.

ª 2013 Federation of European Microbiological Societies. Published by John Wiley & Sons Ltd. All rights reserved