High diversity of bacterial mercuric reductase genes from surface and sub-surface floodplain soil

15 Pages·2007·0.2 MB·English

TheISMEJournal(2007)1,453–467
&2007InternationalSocietyforMicrobialEcology Allrightsreserved1751-7362/07$30.00
www.nature.com/ismej
ORIGINAL ARTICLE
High diversity of bacterial mercuric
reductase genes from surface
and sub-surface floodplain soil
(Oak Ridge, USA)
Gunnar Oregaard1 and Søren J Sørensen2
1Chr. Hansen—Molecular Microbiology, Hoersholm, Denmark and 2Department of Microbiology, Institute
of Biology, University of Copenhagen, Copenhagen K, Denmark
DNAwasextractedfromdifferentdepthsoils(0–5,45–55and90–100cmbelowsurface)sampledat
Lower East Fork Poplar Creek floodplain (LEFPCF), Oak Ridge (TN, USA). The presence of merA
genes, encoding the mercuric reductase, the key enzyme in detoxification of mercury in bacteria,
wasexaminedbyPCRtargetingActinobacteria,Firmicutesorb/c-Proteobacteria.b/c-Proteobacteria
merAgenesweresuccessfullyamplifiedfromallsoils,whereasActinobacteriawereamplifiedonly
from surface soil. merA clone libraries were constructed and sequenced. b/c-Proteobacteria
sequences revealed high diversity in all soils, but limited vertical similarity. Less than 20% of the
operational taxonomic units (OTU) (DNA sequences X95% identical) were shared between the
differentsoils.Onlyoneofthe62OTUwasX95%identicaltoaGenBanksequence,highlightingthat
cultivated bacteria are not representative of what is found in nature. Fewer merA sequences were
obtainedfromtheActinobacteria,butthesewerealsodiverse,andallweredifferentfromGenBank
sequences. A single clone was most closely related to merA of a-Proteobacteria. An alignment of
putativemerAgenesofgenomesequencedmainlymarinea-Proteobacteriawasusedfordesignof
merAprimers.PCRamplificationofsoila-Proteobacteriaisolatesandsequencingrevealedthatthey
wereverydifferentfromthegenome-sequencedbacteria(only62%–66%identicalattheamino-acid
level), although internally similar. In light of the high functional diversity of mercury resistance
genes and the limited vertical distribution of shared OTU, we discuss the role of horizontal gene
transferasamechanism ofbacterial adaptationto mercury.
TheISME Journal(2007)1, 453–467; doi:10.1038/ismej.2007.56;published online19July 2007
SubjectCategory: microbial ecology and functional diversity of natural habitats
Keywords: mercuric reductase; mercury resistant bacteria; merA diversity; soil bacteria; subsurface
soil; Oak Ridge
Introduction activities account for 70% of the last 100 years
mercury emissions (Schuster et al., 2002). The
Of all the heavy metals, mercury (Hg) is the most
atmospheric mercury is transported globally, but
toxic and with no biological function (Nies, 1999).
deposited locally, and pose a threat to organisms at
Mercuryisnotabundantinthesoilcrust,butcanbe
the top of food webs, due to bioaccumulation of
found in ores, the dominant being cinnabar (HgS).
methyl mercury. However, severe mercury contam-
Mercury is emitted to the atmosphere by natural
ination of natural environments can often be
sources, for example, soil erosions and volcanic
attributed to inappropriate human activities. Exam-
eruptions, and by anthropogenic sources, for exam-
ples include the Minamata Bay in Japan, where the
ple, coal-fueled power plants (Nriagu and Pacyna,
local fish-consuming villagers were exposed and
1988). Glacial ice core studies have shown that
poisoned by high levels of methylmercury in their
during the timescale of industrialization in the
seafood due to contamination by a nearby acetalde-
western world, mercury has increased approxi-
hydeproductionfacility(Eto,2000;Tomiyasuetal.,
mately 20-fold in the ice cores, and anthropogenic
2006).
The Y12 nuclear weapons plant facility at Oak
Correspondence: SJ Sørensen, Department of Microbiology, Ridge (TN, USA) used mercury for lithium isotope
Institute of Biology, University of Copenhagen, Solvgade 83H, separation during the 1950s and 1960s, and this
1307CopenhagenK,Denmark. resulted in an estimated 75–150tonnes of mercury
E-mail:[email protected]
inadvertently being released to the local environ-
Received28February2007;revised6June2007;accepted11June
2007;publishedonline19July2007 ment, including the East Fork Poplar Creek (Han
Bacterialmercuricreductasegenesfromsoil
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454
et al., 2006). Examination of the lower East Fork mercury and furthermore that two tyrosine residues
Poplar Creek floodplain (LEFPCF) soil revealed are important for optimal functionality (Moore and
total mercury concentrations ranging from 42 to Walsh, 1989; Distefano et al., 1990; Schiering et al.,
2400mgHgg(cid:1)1 dry weight (Barnett et al., 1995). In 1991; Moore et al., 1992). Some mercuric reductase
the LEFPCF studies, the largest fraction of the proteins contain a mercury binding N-terminal
mercury was estimated to be immobilized as HgS domain, which gives better protection against
(Revis et al., 1989). Although several studies have oxidized mercury (Ledwidge et al., 2005), but this
been conducted at Oak Ridge dealing with mercury is not essential, as it is not found in the mercuric
effects on higher trophic level organisms like fish reductase enzymes of, for example, Actinobacteria
(BurgerandCampbell,2004)andmammals(Stevens and Archaea.
et al., 1997), little has been done to elucidate the If there has been long-term selective pressure for
effects of mercury on the microbial community in mercury-resistant bacteria, all individual bacteria
the soil. will probably be resistant, and the resistance trait
Three mechanisms for microbial adaptation to (the merA protein) will probably have evolved
environmentalstresshavebeensuggested(reviewed differentlyindifferentbacteria,thusleadingtohigh
in Sørensen et al., 2002): (i) enrichment of popula- functional diversity, closing in on 16S rRNA
tions that carry the required resistance/tolerance diversity. Alternatively, if the selective pressure by
traits, (ii) induction of enzymes involved in the mercury is of a newer date, only resistant bacteria
detoxification or resistance mechanisms, and (iii) and those bacteria that could adapt to the new
genetic adaptation. The first two are manifested by selective pressure by, for example, horizontal gene
previously existing sub-populations of resistant transfer thus receiving the required resistance traits
strains, while the process of genetic adaptation would proliferate. In the latter case scenario, the
creates changes in the existing genetic pool of the community diversity would decrease somewhat,
microbialcommunityandthustheevolutionofnew and the functional diversity would be low.
capabilities and an increase in the functional Most of the studies dealing with heavy metal
diversity of the microbial community. Horizontal contamination in soil have been conducted with
gene transfer is a mechanism by which the genetic surface soils (Rasmussen and Sørensen, 2001;
diversity among bacteria may be enhanced. Thus, it Muller et al., 2001a; Muller et al., 2001b; Ellis
may play an important role in the adaptation and et al., 2003), whereas only few studies have looked
evolutionofmicrobialcommunities(Sørensenetal., at sub-surface soils, and most of these have dealt
2005). Metal-resistance genes are often carried on with other metals, for example, uranium (North
plasmids and other mobile genetic elements (Silver et al., 2004; Abulencia et al., 2006) or organic
and Phung, 1996) and therefore, exchange of these pollutants (Lowe et al., 2002; Zhou et al., 2004).
mobile elements through metal-impacted commu- The purpose of the present study was to examine
nities may facilitate rapid alleviation of metal the diversity of merA genes from surface and sub-
toxicity. This is supported by the higher incidence surface soil, exposed to mercury for decades, and
of plasmid DNA among bacteria isolated from thus address the role of horizontal gene transfer in
polluted environments (Rasmussen and Sørensen, bacterial adaptation. It is well known that most soil
1998) and by increased degradation by indigenous bacteria are fastidious with regards to cultivation
bacteria after plasmid transfer with relevant cata- media(Janssen,2006),andithasbeenestimatedthat
bolic genes (Top et al., 2002). in1gofsoil,theremightbeasmanyas106different
Mercury resistance is found in both Bacteria bacterial species (Gans et al., 2005). With the
(Barkay et al., 2003) and Archaea (Schelert et al., obvious limitations of cultivation of soil bacteria,
2004).MercuryresistanceinBacteriaisconferredby molecular approaches may assist in deciphering
the mer operon. Several mer operon-encoded pro- bacterial functional diversity. In the present study,
teinsareinvolvedintransportofinorganicoxidized we used a cultivation-independent approach, based
mercury into the cytosol, where the merA-encoded on PCR amplification, cloning and partial sequen-
mercuric reductase protein, in a NAD(P)H-depen- cing of the merA gene, enabling determination of
dent manner, reduces Hg2þ (aq) to volatile, less merA diversity in soil. Bacterial isolates from
reactiveelementalmercury(Hg0(g))(mercuryinthe LEFPCF (Oregaard et al., 2007) were also examined
environment and bacterial resistance is thoroughly with regards to the presence of merA genes.
reviewed in Barkay etal., 2003).Mercury resistance
has been recognized in many different phyla,
including Firmicutes, Actinobacteria and Proteo- Materials and methods
bacteria, of both clinical and environmental origin
and it is considered to be an ancient resistance merA primer design
mechanism (Osborn et al., 1997). Biochemical and Mercuricreductase(merA)sequenceswereobtained
structural studies of the homodimeric mercuric by searching the GenBank nucleotide database at
oxidoreductase have shown that there are four NCBI.Putativemercuricreductasegeneswerefound
cysteine residues in each monomer, that are essen- in the following phyla: Firmicutes, Actinobacteria,
tial for reducing oxidized mercury to elemental Deinococcus-Thermus,Bacteroidetes,Proteobacteria
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Table1 merA-specificprimersfordifferentbacterialphyla
Phyluma Primer Primersequenceb Degeneracyc Reference
Act Act-Fw 50-CSGAVTTCGTSTACGTCGC-30 12 Thisstudy
Act Act-Rv 50-GCCATGAGGTASGGG-30 2 Thisstudy
Firm Fir-Fw 50-GTTTATGTWGCWGCYTATGAAGG-30 8 Thisstudy
Firm Fir-Rv 50-CCTTCWGCCATYGTTARATAWGG-30 16 Thisstudy
1832
Firm Fir-Rv 50-CCTGCACARCAAGATAATTTBGA-30 6 Thisstudy
1892
a Al-Fw 50-TCCAAGGCGMTGATCCGCGC-30 2 Thisstudy
a Al-Rv 50-TAGGCGGCCATGTAGACGAACTGGTC-30 1 Thisstudy
b/g BG-Fw 50-CCATCGGCGGCACCTGCGT-30 1 Thisstudy
349
b/g BG-Fw 50-TCGAYCGCTGCYTVRTCGCCAC-30 24 Thisstudy
698
b/g #91(A7s-n.F) 50-CGATCCGCAAGTGGCIACBGT-30 3 Schaeferd
b/g #54(A5-n.R) 50-ACCATCGTCAGRTARGGRAAVA-30 24 Schaefer
b/g BG-Rv e 50-CAVGTIGCCACTTGCGGATCG-30 3 Thisstudy
1349
aTheprimersweredesignedtobeselectiveformerAofparticularphylaAct,Actinobacteria;Firm,Firmicutes;aandb/g,Proteobacteriasub-
phyla.
bThedegeneratenucleotidesare:S:G/C;V:G/C/A;W:A/T;Y:C/T;R:A/G;B:G/T/C;I:Inosine;M:A/C.
cDegeneracy:thenumberindicateshowmanyprimersarepresentintheprimersolution.
dSchaeferetal.,2004.
e#91reverseandcomplimentary.
(a-, b-, g- and d-) and in Crenarchaeota and supplemented with 10mM HgCl , with High Pure
2
Euryarcheota, the latter two belonging to Archaea. PCR Template Preparation Kit (Roche Diagnostics
The DNA sequences were prepared in FASTA GmbH, Mannheim, Germany) according to the
format and subjected to alignment by RevTrans manual.
(http://www.cbs.dtu.dk/services/RevTrans/; Wer-
nerssonandPedersen,2003).Inshort,thesequences
Soil
wereinsilicotranslatedtoaminoacids,alignedand
The soil came from LEFPCF, which has a history
then converted back to DNA sequences, but accord-
of mercury contamination. The soil samples were
ing to the amino-acid alignment. These multiple
obtained from three depths at the same site: 0–5cm
DNA alignments were examined manually using
(surface,soilB),45–55cmbelowsurface(soilC)and
clustalX software (Chenna et al., 2003). Primers
90–100cm below surface (soil D). The soil was
weremanuallydesigned,placingtheminconserved
stored at 41C after initial pre-sieving (2mm mesh).
regions ofthemultiplealignments.The merAofthe
different phyla were too diverse for primers encom-
passingthemall. Primers intended tobe specificfor
Soil pre-treatments
thefollowingphylaweredesigned;a-Proteobacteria,
Thesoilswerepre-treatedwithmercury(orwaterin
additional b/g-Proteobacteria primers, Firmicutes
control treatments) and set up in plastic bags with
and Actinobacteria (Table 1).
the equivalent of 20g dry soil. All pre-treatments
The primer pairs were tested in the laboratory on
were done in triplicate. Pre-treatment with mercury
available merA genes from g-Proteobacteria (merA
was performed to selectively enrich the soils with
fromSerratiamarcessensplasmidpDU1358,cloned
mercury-resistant bacteria. A volume of 1ml auto-
into pHG103 (Griffin et al., 1987), kind gift of Dr
claved distilled H O was added to the control soils
Tamar Barkay, Rutgers University) and Firmicutes 2
(w). The mercury pre-treatments (Hg) included
merA genes. Three different merA genes originating
HgCl , in a final volume of 1ml. The amount of
from Staphylococcus aureus pI258 (Laddaga et al., 2
mercury was adjusted for each soil type to obtain a
1987), Bacillus sp. RC607 (Wang et al., 1987) and
start mercury concentration of 10mgg(cid:1)1 soil–water.
Bacillus megaterium MB1 (Huang et al., 1999), all Thesoilmicrocosmswerestoredat251Cinthedark
clonedintoEscherichiacolivectors(pRAL2,pYW40 for 7 days, after which the soil was stored at(cid:1)801C.
and pGB3A, respectively, kind gift of Dr Tamar
Barkay, Rutgers University) were used. Primers
targeting Actinobacteria or a-Proteobacteria were merA clone libraries
tested on mercury-resistant isolates from Oregaard Soil DNAwas extracted from control and mercury-
et al. (2007). The plasmids containing merA stimulated soil with Fast DNA Spin for Soil kit BIO
genes were extracted from their E. coli host cells 101 Systems (http://www.qbiogene.com).
with QIAprep Spin Miniprep Kit (Qiagen, Hilden, The b/g-Proteobacteria PCR reactions were per-
Germany) after overnight growth in LB broth. formed with 2ml soil DNA, primers #91 and #54
Genomic DNA of uncharacterized isolates was (0.4mM final concentration) and water in a total
extracted from colonies grown on dilute media volume of 25ml with PuReTaq Ready-To-Go PCR
(Oregaard et al., 2007) or in 10% tryptic soy broth beads(GEHealthcare,Hillerød,Denmark).Expected
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456
amplicon size was 288bp. The PCR reaction con- three steps: 941C/20s, 501C/15s and 601C/60s.
sisted of an initial denaturing step at 951C/3min; Subsequently, 30 cycles were done in the same
then 45 cycles with two steps: 951C/10s and 601C/ manner but with elongation occurring for 120s.
1min. Final extension was at 721C for 2min. Finally,therewasasinglestepat601Cfor5min.The
TheActinobacteriaPCRreactionsweredonewith sequence reaction mix was purified with a Sepha-
Phusion High-Fidelity DNA Polymerase (Finn- dex G-50 plate (96 wells), and the 96-well plates
zymes, Espoo, Finland). The PCR reaction mix were sequenced on a MegaBACE 1000 Sequenator
consisted of 2ml soil DNA, Act-Fw and Act-Rv (Amersham Biotech, Amersham, UK).
primers (0.5mM final concentration), 4ml HF buffer, The sequences were manually trimmed for
dNTP (200mM final concentration), Phusion DNA vector and primer sequences. Blastn (http://
polymerase(0.02Uml)andwaterinatotalvolumeof www.ncbi.nlm.nih.gov/BLAST/) (Altschul et al.,
20ml. Expected amplicon size was 391–397bp. The 1997) was used to detect probable gap positions in
PCR reaction consisted of an initial denaturing step the clone sequences, and in silico translation of
at981C/2min;then35cycleswiththreesteps:981C/ sequences into functional mercuric reductase pro-
10s,621C/10sand721C/20s.Finalextensionwasat tein sequences was done to confirm that the
721C for 2min. obtained DNA sequence resembled mercuric reduc-
The Firmicutes PCR reactions were performed tase enzymes. The sequences were subjected to
with Phusion polymerase. The PCR reaction mix RevTrans alignment as above. The multiply aligned
consisted of 1ml soil DNA, Fir-Fw and Fir-Rv DNA sequences were subjected to DNA distance
1892
primers (0.5mM final concentration), 2ml HF buffer, matrix calculations with the DNADIST of the
dNTP (200mM final concentration), Phusion DNA Phylogeny Inference Package (Phylip, version 3.65,
polymerase(0.02Uml)andwaterinatotalvolumeof Joseph Felsenstein, http://www.evolution.genetics.
10ml. Expected amplicon size was 455bp. The PCR washington.edu/phylip.html).TheDOTURsoftware
reaction consisted of an initial denaturing step at program (Schloss and Handelsman, 2005) was fed
981C/2min; then 35 cycles with three steps: 981C/ with the DNA distance matrices for determination
10s,641C/10sand721C/20s.Finalextensionwasat of operational taxonomic units (OTUs) and calcu-
721C for 2min. lation of different diversity measures, including
ThetotalPCRreactionvolumefromeachreaction Shannon–Weaver diversity index and Chao1 rich-
was loaded onto 1.2% agarose gels, gel electro- ness estimator. Library coverage was calculated as:
phoresed and stained with ethidium bromide. If C¼[1(cid:1)(n/N)]100%, where n is number of OTUs
merAbandsofcorrectsizewereobserved,theywere and N the number of sequences.
cut out and purified with QIAEX II Gel Extraction
Kit(Qiagen).Theextractedampliconsobtainedwith
the same primers were pooled before cloning (for Sequences
example, soil B, stimulated with water, three The sequences obtained in this study have been
replicates, all mixed and aliquot of this used for deposited at NCBI. The merA clones of b- and g-
cloning).ThemerAampliconswereclonedwiththe Proteobacteria and Actinobacteria are associated
TOPO TA cloning kit (Invitrogen, Paisley, UK). A with accession numbers EF460128–EF460310. The
volume of 2–4ml merA amplicon extracted from gel merA sequences from the isolates are associated
was mixed with 1ml salt solution and 1ml pCR4- with accession numbers EF455060–EF455079.
TOPO vector, and water to a final volume of 6ml.
Thecloningandtransformationwasdoneaccording
to the manual,using competent TOP10 E. coli cells. Results
The transformants were grown overnight at 371C
on LB agar with Ampicillin (100mgml(cid:1)1) and re- Full-lengthmercuricreductasesequencesdeposited
streaked onto LB agar with Kanamycin (50mgml(cid:1)1). at GenBank (NCBI) were retrieved and analyzed.
The four essential cysteine residues (positions 207,
212, 628 and 629 in Bacillus cereus RC607 merA
merA sequencing, analysis and phylogeny enzyme, BAB62433) along with the two important
The transformants were grown overnight and the tyrosine residues(positions 264and605)were used
cloning vectors (with merA inserts) were extracted to discriminate mercuric reductase sequences from
with Qiaprep Spin Miniprep Kit (Qiagen). The other similar disulfide oxidoreductases like dihy-
plasmid DNA concentration was determined spec- drolipoamide dehydrogenases. Using these amino
trophotometrically (A /A ). The sequence reac- acidsascriteriaforacceptingsequencesasmercuric
260 280
tion mix consisted of 4ml DYEnamic ET Dye reductases, along with general similarity to merA
Terminator mix (MegaBACE, Amersham, UK), 1ml sequences found with blastn/blastp (Altschul et al.,
M13 forward primer (10mM), water and 200–400ng 1997; Schaffer et al., 2001), merA of different phyla
plasmidDNAinafinalvolumeof10ml.Thedideoxy wereidentified(Table2).Itwasapparent,thatmany
nucleotide sequencing (Sanger sequencing) reaction sequences annotated as mercuric reductase encod-
wasdoneinthefollowingway:initialdenaturingat ing genes, were probably wrongly annotated, when
941C for 3min followed by 30 cycles consisting of using these criteria, since they lacked the two
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Table2 Presenceofparticularaminoacids,essentialformercuricreductaseactivity
Phyluma No.ofsequencesb Essentialaminoacidsinmercuricreductaseenzymes
Cys207c Cys212 Tyr264 Tyr605 Cyr628 Cyr629
a 14 + + + + + +
b/g 30 + + +d + + +
d 4 + + + + + +
Dein-Therm 2 + + + + + +
Bacteroidetes 1 + + + + + +
Firmicutes 11 + + + + + +
Actinobacteria 9 + + + + + +
Archaea 12 + + + Phee + +
Outgroup 4 + + (cid:1) +f (cid:1) (cid:1)
aa,b/gandd,sup-phylaofProteobacteria;Dein-Therm,Deinococcus-Thermus.SamesequencesasusedforFigure1.
bNumberofsequencesretrievedfromGenBankandexaminedforconservedaminoacids.
cThenumberingisfromtheBacillusspRC607mercuricreductaseprotein(BAB62433).
dIsoleucineinCAC80079.
ePhenylalanineinposition605ofallArchaea.
fPhenylalanineinYP_628559.
30
14*
8 Acti. (68.1 - 71.8)
1 Acti. (R. xylanophilus)
11 Firm. (38.8 - 46.8)
2* D.-T. (73.1 - 73.5)
4*
1* Bact. (34.8)
12 Arch. (40.9 - 52.5)
4 Out
0.05
Figure1 Neighbour-joiningtreeofmercuricreductaseproteinsequences.ThesequenceswereobtainedfromGenBankatNCBI.The
boxesreflectphylawithmorethanonesequence.Thegrayboxesrepresentphylawithphenotypicmercuryresistance,basedonmercuric
reductaseproteins.Theasterisk(*)indicatesphylaorisolatesinwhichphenotypicmercuryresistancehasnotbeenrecognized.The
horizontallengthofaboxisequivalenttothemaximumlengthfromnodetotipofaparticularsequenceinthatphylum.Thenumberat
theright-handcorneroftheboxreflectsthenumberofproteinsincludedinthealignment.ThevalueinparenthesisistherangeofGC
contentofthemerAgeneoftheparticularphylum.Blackandwhitecirclesatnodesreflectbootstrapvalueshigherthan95and50%,
respectively(outof1000bootstraps).Thebaratthebottomreflects5%differenceattheamino-acidlevel.Theaccessionnumbersofthe
proteins used for the neighbour-joining tree were as follows Archaea; NP_376996, NP_147957, CAF18529, NP_560142, NP_344015,
YP_256307, NP_110770, YP_023042, ZP_00610004, YP_134400, NP_394797 and ZP_00610602; d-Proteobacteria: NP_954464,
ZP_01140457, ZP_01388909 and YP_383148; Outgroup: ABC45686, NP_952368, YP_628559 and YP_010258; Bacteroidetes:
ZP_01060916; Deinococcus-Thermus: YP_144419and YP_004762. Theb/g-Proteobacteria and Actinobacteria merA proteinaccession
numbersareshowninFigures2and5,respectively.a-ProteobacteriaandFirmicutesaccessionnumbersareshowninFigure4.
terminal cysteines, Cys628 and Cys629 (data not experimentally characterized as conferring mercury
shown). All mercuric reductase proteins used for resistance to their host. A neighbour joining tree
primer design by multiple alignments contained of merA proteins of different phyla is shown in
all four cysteines and both tyrosines, except one Figure1.Theoutgroupsequenceswerefourproteins
Pseudomonas sequence in which Tyr264 had annotated as mercuric reductases, but without the
been substituted with isoleucine (CAC80079). signature Tyr264, Cys628 and Cys629 amino acids
All the Archaea sequences had a phenylalanine (Table 2).
residue at position 605, in contrast to tyrosine in The multiple alignments were used to design
bacteria (Table 2). None of the Archaea sequences primer pairs targeting merA from Firmicutes, Acti-
from the NCBI database used here have been nobacteria and a-Proteobacteria, and additional
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primers for b/g-Proteobacteria (Table 1). In silico the Actinobacteria primers, which also seemed to
PCR reactions with the primersagainstthe different equally well amplify b/g-Proteobacteria merA.
phylarevealedthattheygaveampliconsofexpected Three different merA genes originating from S.
size and were specific to their phyla, except for aureus (Laddaga et al., 1987), Bacillus sp. RC607
AAN62178 Pseudomonas aeruginosa
YP_113802 Methylococcus capsulatus
AAC33905 Tn21 (Shigella flexneri)
O-36-B-18
ZP_01663300 Ralstonia pickettii
O-50-C-20
O-51-C-22
P17239 Acidithiobacillus ferrooxidans
O-61-D-08
12 0-16 (Ba)
O-44-Ba-34
CAC69251 Acidithiobacillus ferrooxidans Tn5037
CAA72398 Thiobacillus sp.
5 O-07 (Ba, pKris-2, CAD91352 Pseudomonas Tn5042)
CAA67451 Pseudomonas sp. Tn5041
CAB65705 Xanthomonas campestris pKLH443
CAC80079 Pseudomonas sp. Tn5046
O-56-C-42
3 O-11 (C,D)
3 O-03 (B, C, AAR91471 similar to pDU1358)
CAC80891 Acinetobacter sp. pKLH208B
AAC38220 Pseudomonas stutzeri pPB
O-58-C-45
AAP88282 Delftia acidovorans pMC1 (b)
CAD31047 Acinetobacter sp. pKLH205
Q52109 Acinetobacter calcoaceticus pKLH201
CAE51647 Serratia marcesens pR478
NP_361073 pSB102
3 O-14 (pKris-1, pKris-4, AAR31066 pJP4)
O-74-pKris-3
17 O-08 (C,D)
3O-23 (C)
NP_840913 Nitrosomonas europaea
O-67-D-37
5+1 O-18 (D) + O-72 (D)
O-63-D-18
3 O-21 (B)
O-60-C-48
CAC86905 Pseudomonas putida Tn5041D
BAA20337 Pseudomonas sp. K-62 pMR26
O-47-C-02
O-52-C-24
O-33-B-14
4
O-02 (B, C, D)
O-37-B-23
2
O-28 (D)
O-66-D-36
O-71-D-50
O-53-C-25
6
O-10 (C,D)
8 O-01 (B,C,D)
4 O-20 (D)
O-64-D-28
2 O-30 (D)
2 O-12 (C,D)
2O-29 (D)
O-48-D-04
O-41-B-53
2O-13 (C,D)
O-59-C-47
O-32-B-08
0.05
Figure2 Neighbour-joiningtreeofmerADNAsequences.Thesequenceswereobtainedfromb/g-typemerAclonelibraries(B,C,Dand
Ba)orfromexogenouslyisolatedplasmids(pKris).TheboxesreflectOTUswithmorethanonesequence.Thehorizontallengthofabox
isequivalenttothemaximumlengthfromnodetotipoftheparticularOTU.TheOTUsweredefinedbyDOTUR.Blackcirclesatnodes
reflectbootstrapvalueshigherthan90%(outof1000bootstraps).Lettersinparenthesisreflectthesoiltype.Thenumberinsidethebox,
or at the upper left-hand corner of the box reflects the number of sequences within the particular OTU. Archaea merA gene from
Haloarculamarismortuiwasusedasoutgroup.Thebaratthebottomreflects5%difference.
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12 O-17 (B)
2 O-24 (B)
4 O-04 (B,D)
O-57-C-43
O-42-B-59
O-65-D-29
4 O-05 (B,C)
O-73-C-29
O-39-B-30
O-35-B-17
O-45-Ba-38
2
O-25 (Ba)
O-62-D-17
2
O-26 (C)
O-70-D-48
O-49-C-08
O-54-C-26
7+1 O-09 (C,D) + O-68 (D)
O-38-B-24
O-31-B-06
AAM08005 Providencia rettgeri
CAC14715
3O-22 (C)
O-40-B-52
YP-315099 Thiobacillus denitrificans
O-46-Ba-45
YP_344737 Nitrosococcus oceani
YP_957199
YP-156028
YP_957535 Marinobacter aquaeolei
4 O-19 (B)
2 O-27 (C)
O-43-B-60
O-55-C-31
YP-134400 Haloarcula marismortui (Archaea)
0.05
Figure2 Continued.
(Wangetal.,1987)andB.megaterium(Huangetal., merA genes were only amplified in surface soil
1999)confirmedthatFirmicutestypemerAprimers, (in both control and mercury-stimulated soil). The
Fi-Fw/Fi-Rv1832 and Fi-Fw/Fi-Rv1892, resulted in Firmicutes-type merAwas not detected in any soil.
ampliconsofapproximately395and455bp,respec- The merA amplicons of the b/g and the Actinobac-
tively (data not shown). The b/g-type new primers teria were cloned and transformed. Cloned partial
worked with pDU1358 merA (Griffin et al., 1987) b/g merA genes, mainly from control soils stimu-
(data not shown). To test the applicability of the lated with water were sequenced, resulting in 43
Actinobacteria primers, genomic DNA from the sequences from surface soil (B), 49 sequences from
mercury-resistant Streptomyces isolate (Is-BDOE1) sub-surface soil (C) and 51 sequences from deeper
from soil B (Oregaard et al., 2007) was used in PCR sub-surface soil (D). Furthermore, of the 40 se-
reactions. The amplicon was approximately 400bp, quences amplified with Actinobacteria primers, 20
corresponding to the expected 391–397bp (data not merA sequences turned out to be b/g-Proteobacteria
shown). in origin (Ba). All b/g-Proteobacteria merA clone
In the present study, we were interested in sequences (B, C, D and Ba) were aligned along with
describing the diversity of merA genes in surface 30referenceb/gmerAsequencesfromGenBank.The
and sub-surface soils with a history of mercury multiple alignment was subjected to DOTUR analy-
contamination. Primers targeting b/g-Proteobacteria sis (Schloss and Handelsman, 2005), allowing
(#91 and #54), Actinobacteria (Act-Fw and Act-Rv) determination of OTUs and diversity estimates of
and Firmicutes (Fi-Fw and Fi-Rv ) (Table 1) were the different soils. When discriminating bacterial
1892
used in PCR reactions with DNA extracted from species from one another based on 16S rRNA
soils pre-treated with mercury or control soils pre- sequences, a threshold of 3% is typically used,
treated with water. The b/g PCR reactions gave and for genus separation, 5% are used (Schloss and
partial merA amplicons in surface and both sub- Handelsman, 2005). A threshold of 5% difference
surface soils, and this was seen for both stimulated was used in the analysis, obtaining more conserva-
and control soils. In contrast, partial Actinobacteria tive estimates of diversity, compared to 3% thresh-
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460
old. With the 5% threshold for definition of OTUs, Estimates of merA diversity in the three different
the163b/gmerAclonesfellinto70OTUs,ofwhich soilmicrocosms,basedonthesequencedclones,are
28 contained more than one sequence. The phylo- shown in Table 3. The diversity estimates were
geny of the b/g merA sequences from the different similar for all three soils. Although all three soils
soils is shown in Figure 2 along with reference were very similar in merA diversity, the actual
sequences from GenBank. Since the cloned se- cloned sequences were generally different as is
quences were onlyaround244bpinlength(exclud- shown in the Venn diagram in Figure 3. The circles
ing the primers), all the reference sequences and represent different soil clones and plasmids from
plasmids were also truncated, to only contain the these soils, as indicated by letters on their side,
fragment obtained with primers #91 and #54 and reference sequences from GenBank, with the
primers. All the cloned sequences showed highest
similarity to other b/g merA genes at GenBank, as
P
determined by blastn or blastp search, except clone
1
C-31,seebelow.ItisapparentfromFigure2thatthe
1
b/g merA gene is highly diverse. Since all partial Ba
1
merA clone sequences were most similar to other 5
merA sequences at GenBank, it is unlikely that the
high diversity observed was due to non-specific
24
amplificationofgenesotherthanmerA.Ifsuchnon- B
specific amplification occurred, it would be most
probablethattheampliconshaddifferentsizesthan 16
the merA fragments. Interestingly, all the clones 1
were more than 5% different from the reference
sequences,exceptOTU-03(B-63andC-37)grouping 1
with merA from pDU1358, and OTU-07 (Ba-39, C
Ba-44 and Ba-47) grouping with merA of plasmid
19 2 1
pKris-2 and a merA gene from a Pseudomonas
isolate (Figure 2).
Exogenous plasmid isolation had been conducted
on soils B, C and D, resulting in four different
D
plasmids (pKris-1 to -4), as determined by plasmid
6
restriction enzyme analysis (Lipthay et al., 2007). 16
All plasmids conferred mercury resistance to their
host(E.coliorPseudomonasputida).Inthepresent
study, the merA gene was amplified with b/g
primersBG-Fw and#54andsequenced,obtaining Figure 3 AVenn diagram representing shared and unique b/g-
349 typemerAOTUs.TheOTUsweredefinedasbeingnolessthan
over 1100 nucleotides from each merA gene. The
95%identicalattheDNAlevel.SharedOTUsareinterconnected
phylogenetic relationship of these merA genes is as between the relevant circles. The area of the circles reflects the
mentioned above,shown in Figure 2.When looking numberofOTUsbelongingtothese.Atotalof198sequenceswere
at the 244bp fragment obtained with primers #91 includedintheanalysis,ofwhich30camefromNCBI(27OTUs),
foursequencesfromexogenouslyisolatedplasmids(threeOTUs),
and #54 (excluding primers), the four plasmids
20 Ba sequences (six OTUs), 43 B sequences (21 OTUs), 49 C
fell into three different OTUs (X95% identity)
sequences (29 OTUs) and 51 D sequences (25 OTUs). OTUs,
(Figure 2). operationaltaxonomicunits.
Table3 Diversityofclonedb/g-ProteobacteriamerAsequences
Soil No.ofsequences EstimatesbyDOTUR Coverage%c
OTU(5%)a Chao1b Shannon–Weaverdiversityindex
B 43 21 52(29–133)d 2.6(2.3–3.0) 51
C 49 29 58(38–117) 3.2(2.9–3.4) 41
D 51 25 43(30–85) 2.9(2.6–3.2) 51
Bae 20 6 8(6–21) 1.3(0.8–1.7) 70
B-C-D-Ba 163 68 143(101–239) 3.8(3.6–3.9) 52
aOTU,operationaltaxonomicunit(DNAX95%identical).
bChao1:biascorrectedChoa1richnessestimator.
cCoverage:{1(cid:1)[OTU/N]}100%;N¼no.ofsequences.
dValuesinparenthesis:95%confidenceinterval.
eClonesfromsoilB,amplifiedwithActinobacteriaprimersAct-FwandAct-Rv,butshowinghighestsimilaritytob/g-ProteobacteriamerADNA.
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461
Haloarcula marismortui (YP_134400)
Rubrobacter xylanophilus (YP_644538)
Acidothermus cellulolyticus (YP_872449)
Streptomyces lividans (CAA46460)
Streptomycessp. CHR28 (AAF64138)
Is-BDOE1
OTU-83
OTU-91
OTU-84
OTU-81 (3)
OTU-85
Arthrobacter aurescens (YP_950110)
OTU-88
OTU-90
Arthrobacter sp. FB24 (YP_829216)
OTU-87
Nocardioides sp. JS614 (YP_919388)
Mycobacterium flavescens (ZP_01195643)
Corynebacterium glutamicum (BAD83994)
OTU-82 (2)
OTU-80 (7)
0.05
Boostrap value higher than 95 %
Figure 4 Neighbour-joining tree of Actinobacteria merA DNA clone sequences, isolate Is-BDOE1 and reference sequences from
GenBank.TheboxesreflectOTUswithmorethanonesequence.Thehorizontallengthofaboxisequivalenttothemaximumlengthfrom
nodetotipoftheparticularOTU.TheOTUsweredefinedbyDOTUR(X95%similar).Blackcirclesatnodesreflectbootstrapvalues
higherthan95%(outof1000bootstraps).ValuesinparenthesisreflectthenumberofsequencesintheparticularOTU.ArchaeamerA
genefromHaloarculamarismortuiwasusedasoutgroup.Thebaratthebottomreflects5%difference.OTU,operationaltaxonomicunit.
numbersinsideindicatingthenumberofOTUs.The (59 identical amino acids (72%)) (Figure 5). The
smaller interconnecting circles show how many neighbour-joining tree in Figure 5 includes nine
OTUs were shared between the connected circles partial merA sequences obtained from anoxic river
(environments). Only two OTUs contained se- sediments, contaminated with mercury and ob-
quences from all three soils (OTU-01 and OTU-02, tained from PCR reactions using similar primers as
in total 12 sequences) (Figure 2). The OTUs with inthepresentstudy(NiChadhainetal.,2006).Note
representatives of different soils seemed fairly how two of the sequences (SBC-049 and SBC-183)
equally distributed between B/C (four OTUs), B/D are most closely related to other a-Proteobacteria
(three OTUs) and C/D (eight OTUs) (Figure 3). Note merAsequences.TheothersevenSBCsequencesall
that only three OTUs were connected to GenBank seem closer related to a-Proteobacteria than to the
sequences. Gram-positive Firmicutes-type merA genes.
The Actinobacteria merA PCR and cloning re- Several isolates obtained from the same soils as
sulted in 20 sequences showing highest similarity used in this study (Oregaard et al., 2007) were
to Actinobacteria merA proteins. One of these subjected to merA-specific PCR with the primers
was discarded, as it seemed chimeric, by high showninTable1,andsomewerepartiallysequenced
N-terminal similarity to b/g merA, whereas the (Table 4). The Streptomyces (Is-BDOE1) isolate
C-terminal protein region was most similar to seemed to contain a merA sequence diverging
Actinobacteria. The remaining 19 sequences were from its closest relative by approximately 27% at
alignedagainstnineActinobacteriamerAreferences the amino-acid level (Table 4). The merA gene of
and a DNA Neighbour-Joining tree was calculated Is-BDOE1 was amplified on two occasions and
(Figure 4). The 19 sequences fell into 10 OTUs sequenced with both primers (Act-Fw and Act-Rv)
(5%threshold),ofwhichsevenOTUscontainedone but only the forward primer resulted in acceptable
sequence. It is noteworthy that most of the cloned sequence data. The sequences obtained with the
sequences are quite divergent from the sequences forward primer were identical. The Dyella-like iso-
deposited at GenBank. lateshadmerAgenesthatdifferedsomewhatfromthe
The C-31 clone sequence mentioned above and closest match, pSB102, a full-sequenced plasmid
obtained with b/g-type primers did not show from Alfalfa rhizosphere (Schneiker et al., 2001).
similarity to other merA genes by blastn. When the Partial a-Proteobacteria merA DNA sequences of
C-31clonesequencewasvirtuallytranslatedinto81 several different isolates were subjected to both
amino acids, the protein sequence showed similar- blastn and blastp search. Blastn did not reveal any
ity to other putative merA proteins from a-Proteo- relatedness to other merA genes and did not result
bacteria, for example, Xanthobacter autotrophicus in any meaningful hits. Blastp search showed
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Bacterialmercuricreductasegenesfromsoil
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Haloarcula marismortui (YP_134400)
SBC-164
SBC-126 I
SBC-108
647 SBC-107
SBC-171
II
SBC-176
SBC-158
771 SBC-049
Maricaulis maris (YP_757559)
999
708 Oceanicola batsensis (ZP_01001238)
939 Rhodobacterales bacterium (ZP_01015197)
Sulfitobacter sp. (ZP_00964369)
390
Parvularcula bermudensis (ZP_01018332)
Roseovarius sp. (ZP_01036716)
361 Aurantimonas sp. (ZP_01227191)
Oceanicola granulosus (ZP_01156286)
Oceanicola batsensis (ZP_00997524)
901 Sulfitobacter sp. (ZP_00955861)
950 715 SBC-183
Xanthobacter autotrophicus (ZP_01199704)
918
Xanthobacter autotrophicus (ZP_01199483)
355 C-31 (present study)
Sphingopyxis alaskensis (YP_617552)
Geobacillus kaustophilus (YP_148949)
Bacillus licheniformis (CAC14663)
Bacillus cereus (CAA70224)
Bacillus macroides (CAA71038)
Streptococcus oralis (CAE46804) es
Bacillus RC607 pYW40 (AAA83977) cut
BEacnitlelurso cmoeccguaste fraiuemci u(mCA (AA7A1R014004)25) Firmi
Streptococcus agalactiae (NP_688256)
Staphylococcus aureus pI258 (AAA98245)
0.05 Alicyclobacillus vulcanalis (ABB00295)
Figure5 Neighbour-joiningtreeofa-ProteobacteriamerAclonesequencesobtainedinthepresentstudy(C-31)andinastudyofanoxic
sediments (SBC-prefix), see Discussion for details. All the reference sequences from a-Proteobacteria are putative merA genes.
Firmicutes-type merA DNA is also included. I and II are clades defined in the originalpaper describing these clones, obtained with
similarprimersasusedfortheb/g-typemerAclonelibraryofthepresentstudy.Thevaluestotheleftatsomeofthenodesreflectthe
bootstrapvalues(outof1000).ArchaeamerAgenefromHaloarculamarismortuiwasusedasoutgroup.Thebaratthebottomreflects5%
difference.
Table4 LEFPCFsoilisolatesfromwhichpartialmerAsequenceswereobtained
Isolatea Genus Phylumb DNAsequencesc GC% Blastp
Ident.aa(%)d Accessione Genus
Is-BDOE1 Strepytomyces Actino. 252 69.4 73 AAF64138 StreptomycesspCHR28
Is-D308 Bradyrhizobium a 785 63.8 66 ZP_01199704 Xanthobacter
Is-B203a Rhizobium a 781 64.0 66 ZP_01199704 Xanthobacter
Is-C091 Sphingomonas a 783 63.9 66 ZP_01199704 Xanthobacter
Is-B040 Rhizobiales-like a 719 63.5 62 ZP_01199704 Xanthobacter
Is-D310 Cupriavidus b 537 65.9 100 CAC86905 Pseudomonasputida
Is-C065 Ralstonia b 683 64.8 94 CAA70190 Alcaligenessp
Is-D282 Variovorax b 396 64.3 99 CAD10785 Pseudomonasputida
Is-D134 Dyella g 435 59.7 92 NP_361073 pSB102
Is-D136 Dyella g 771 64.2 85 NP_361073 pSB102
Is-BDOE2 Pseudomonas g 673 66.8 97 ZP_01294748 Pseudomonasaeruginosa
pKris-1(plasmid) 1165 64.8 97 CAC80891 Acinetobactersp
pKris-2(plasmid) 1135 65.9 96 CAD91352 Pseudomonasfluorescens
pKris-3(plasmid) 1150 65.1 96 AAR91471 Klebsiellapneumoniae
pKris-4(plasmid) 1159 66.0 99 AAR31066 pJP4
aIsolatesandplasmidswereobtainedinpreviousstudies.
bPhylumofisolates,Actino.,Actinobacteria;a,b,gsub-phylumwithinProteobacteria.Theplamidswereexogenouslyextractedfromthesoil
withg-Proteobacteria.
cThelengthofthesequences(notfull-lengthmerA).
dIdenticalaminoacidsinpercentage(thesequenceswerevirtuallytranslatedtoaminoacidsandsubjectedtoblastpsearch).
eAccessionnumbersofclosestrelatedmerAprotein.
similarity to all the putative a-Proteobacteria merA theBradyrhizobium,RhizobiumandSphingomonas
protein sequences used in the phylogenetic tree in isolates (Table 4) were identical. The merA of the
Figure 5. The a-Proteobacteria merA fragments of Rhizobiales-like isolate was somewhat different.
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