FEMS Microbiology Ecology, 91, 2015, fiv011

doi: 10.1093/femsec/fiv011
Advance Access Publication Date: 28 January 2015
Research Article

RESEARCH ARTICLE

Metagenomic analysis reveals adaptations to

a cold-adapted lifestyle in a low-temperature
acid mine drainage stream

Sweden, 2 Center for Bioinformatics and

Department of Molecular Biology, Umea University, S-901 87 Umea,
Ciencia & Vida and Depto. de Ciencias Biologicas,

Genome Biology, Fundacion

Facultad de Ciencias Biologicas,
3
Bello, Santiago 7780272, Chile, Bio-Computing and Applied Genetics Division, Fraunhofer
Universidad Andres
Chile Research Foundation, Center for Systems Biotechnology, Santiago, Piso 14, 7550296, Chile and 4 Centre
for Ecology and Evolution in Microbial Model Systems (EEMiS), Linnaeus University, 392 31 Kalmar, Sweden
Corresponding author: Centre for Ecology and Evolution in Microbial Model Systems (EEMiS), Department of Biology & Environmental Sciences,

Linnaeus University, Landgangen

3, 392 31 Kalmar, Sweden. Tel: +46-0-480-447334; E-mail: mark.dopson@lnu.se
#
M.L., F.J.O. and C.G. contributed equally to this work.

Present addresses: Sukithar Rajan, Department of Biological and Environmental Science, Jyvaskyl
a University, Finland; Adam Stell, Department of
Clinical Genetics, Maastricht University, Maastricht, the Netherlands.
One sentence summary: This study identifies the dominant microorganisms present in a low temperature acid mine drainage microbial community,
describes potential adaptations to the low temperature, and reconstructs the function of community members.
Editor: Cindy Nakatsu

ABSTRACT
An acid mine drainage (pH 2.52.7) stream biofilm situated 250 m below ground in the low-temperature (610 C)
Kristineberg mine, northern Sweden, contained a microbial community equipped for growth at low temperature and acidic
pH. Metagenomic sequencing of the biofilm and planktonic fractions identified the most abundant microorganism to be
similar to the psychrotolerant acidophile, Acidithiobacillus ferrivorans. In addition, metagenome contigs were most similar to
other Acidithiobacillus species, an Acidobacteria-like species, and a Gallionellaceae-like species. Analyses of the metagenomes
indicated functional characteristics previously characterized as related to growth at low temperature including cold-shock
proteins, several pathways for the production of compatible solutes and an anti-freeze protein. In addition, genes were
predicted to encode functions related to pH homeostasis and metal resistance related to growth in the acidic
metal-containing mine water. Metagenome analyses identified microorganisms capable of nitrogen fixation and exhibiting
a primarily autotrophic lifestyle driven by the oxidation of the ferrous iron and inorganic sulfur compounds contained in the
sulfidic mine waters. The study identified a low diversity of abundant microorganisms adapted to a low-temperature acidic
environment as well as identifying some of the strategies the microorganisms employ to grow in this extreme environment.
Keywords: metagenome; acid mine drainage; psychrotolerant; Acidithiobacillus ferrivorans; low temperature

Received: 11 November 2014; Accepted: 26 January 2015


C FEMS 2015. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.

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2,3,#

Maria Liljeqvist1,# , Francisco J. Ossandon2,# , Carolina Gonzalez

,
4,
4,
3
2
Sukithar Rajan , Adam Stell , Jorge Valdes , David S. Holmes
and Mark Dopson1,4,

FEMS Microbiology Ecology, 2015, Vol. 91, No. 4

INTRODUCTION

MATERIALS AND METHODS

Mine site, sampling and colony isolation
Kristineberg mine is a multimetal sulfide mineral mine located
in northern Sweden (65.064325N, 18.5651833E) from which Zn,
Cu, Pb, Au and Ag are extracted (File 1, Supplementary Information). Measurements for annual temperature fluctuations in the
mine were based upon multiple readings at two distinct sampling times, in April 2010 and November 2011 when the temperature was 6 C and 10 C, respectively. In April 2010, a total of 50 L
of mixed AMD liquid and biofilm was removed from a stream
flowing along a tunnel at 250 m below the surface. The mixed
liquid and biofilm were stored in sterile containers and transported to the laboratory where they were immediately processed
as described below (processing took place 3 h after sampling).
From the same sample, liquid from the stream was inoculated onto plates containing 5 mM tetrathionate and tetrathionate plus 0.02% yeast extract at pH 2.5 and incubated at 4 C
until colonies were observed. A further sampling was carried
out in November 2011 to carry out a second isolation of pure
cultures from both the planktonic and biofilm fractions utilizing overlay plates containing Fe2+ /tetrathionate/tryptone soya
broth, Fe2+ alone and yeast extract (Johnson and Hallberg 2007).
The plates were incubated at either 12 or 22 C until colonies
were observed. The incubation temperatures were chosen to be
close to the temperature in the mine stream at time of the first
sampling (4 C), while the second sampling for overlay plates
experiments were incubated at a temperature similar to the
stream temperature at time of sampling (i.e. 11 C) and 22 C
to encourage potential psychrotolerant microorganisms present
in the mine water to grow at a higher rate. Other measured
parameters were redox potential (Pt electrode against an Ag0
AgCl; 3.5 M KCl), soluble Fe2+ by titration with ceric sulfate, and
soluble total iron and copper by atomic adsorption spectroscopy

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Sulfidic mine sites are characterized by high concentrations of

iron and sulfur that are utilized as both electron donors and acceptors by acidophilic microorganisms (Bonnefoy and Holmes
2012; Dopson and Johnson 2012). The final product of S0 and inorganic sulfur compound (ISC) oxidation is sulfuric acid, which
generates the low pH in which acidophilic microorganisms optimally grow. The oxidation of Fe2+ and ISCs by acidophiles is exploited in the biotechnological process of biomining, whereby
the microorganisms catalyze sulfide mineral dissolution and
release soluble metals (Vera, Schippers and Sand 2013). However, uncontrolled discharge of acidic, metal-laden solutions
from sulfide mineral mines, termed acid mine drainage (AMD),
can cause catastrophic environmental damage. Although several studies address AMD generation mediated by mesophilic
microorganisms (e.g. Tyson et al., 2004), knowledge of the microbial community that catalyzes AMD generation in the cold is
lacking.
The majority of the earths surface is cold and these milieus include the deep oceans, polar ice, the mesosphere and
stratosphere, and permafrost. Psychrophiles are defined as having a temperature optimum for growth <15 C (maximum growth
temperature of 20 C) while microorganisms able to grow at
<0 C but having a temperature optimum of 2025 C are defined as psychrotolerant. Psychrotolerant species are more likely
to be isolated from land environments that are prone to seasonal changes in temperature (Helmke and Weyland 2004). Coldadapted microorganisms are found in all three domains of life
and they are able to grow by utilizing a range of metabolic strategies in diverse environments. The ability to adapt to low temperature requires the microorganisms to sense a decrease in
temperature that induces upregulation of cold-associated genes
(Shivaji and Prakash 2010). Major prokaryotic processes that are
either affected by the cold or cold induced include: (i) transcription and translation (Noon et al., 2003); (ii) adaptation of
proteins to allow them more flexibility (Grzymski et al., 2006);
(iii) increased membrane fluidity (Chintalapati, Kiran and
Shivaji 2004); (iv) cryoprotectants such as compatible solutes
and anti-freeze proteins to protect against freezing (Gilbert
et al., 2004); (v) production of stress proteins (Weber and Marahiel
2002); (vi) mechanisms to reduce reactive oxygen species damage caused by increased solubility of gases at low temperature
(Methe et al., 2005) and (vii) production of large amounts of
exopolymeric substances in aquatic environments (Krembs
and Deming 2008). In addition to the cold, low-temperature
habitats can have multiple stresses including limited water
availability, oligotrophy, high salinity, UV irradiation and high
pressure (Tehei and Zaccai 2005). For example, microorganisms
in the stratosphere and mesosphere are challenged by temperatures as low as 100 C, UV radiation, desiccation and poor nutrient availability (reviewed in Margesin and Miteva 2011). Acidophile responses to abiotic stresses related to growth in an
AMD environment, such as production of oxygen radicals by
Fe2+ and high osmolarity due to ISC oxidation to sulfate may
also protect against the cold.
Only a few acidophiles that are metabolically active at low
temperature have been described. These include Acidithiobacillus (At.) ferrivorans (Hallberg, Gonzalez-Toril and Johnson 2010),
Ferrovum (F.) myxofaciens (Johnson, Hallberg and Hedrich 2014),
and an Acidiphilium-like bacteria (Berthelot, Leduc and Ferroni
1993, 1994). An 8.5 C underground AMD community in Cae
Coch, Wales is dominated in various parts of the mine by At.
ferrivorans and F. myxofaciens (Kimura et al., 2011; Kay et al., 2013;

Johnson, Hallberg and Hedrich 2014). Acidithiobacillus ferrivorans

oxidizes Fe2+ and ISCs (Kupka et al., 2007, 2009), catalyzes sulfide mineral dissolution at 6 C (Dopson et al., 2007) and has been
utilized to remove ISCs from mining process waters (Liljeqvist
et al., 2011a). F. myxofaciens is an obligate aerobe that oxidizes
Fe2+ and can fix CO2 and N2 (Kay et al., 2013).
Metagenomics is the isolation, sequencing and characterization of microbial community DNA and has allowed
elucidation of the phylogeny and metabolic potential of mixed
populations (Sharon and Banfield 2013). The technique has been
applied to acidic environments such as Iron Mountain, California that is characterized by extremely low pH and a temperature of 3545 C (Tyson et al., 2004; Dick et al., 2009; Goltsman
` France that
et al., 2009); an arsenic-rich ecosystem in Carnoules,
has a temperature in May of 15.1 C (Bertin et al., 2011); and a
snottite biofilm fed by H2 S in the Frasassi cave system, Italy
that has a constant temperature of 13 C (Jones et al., 2012).
Despite the relatively low temperature of the arsenic-rich
` and snottite biofilm, none of the cited
ecosystem in Carnoules
acidic pH environment metagenome studies focused on temperature adaptations.
In this study, community DNA was analyzed from a low temperature (610 C) underground AMD stream (pH 2.52.7) situated
at a depth of 250 m below the surface in the Kristineberg mine,
northern Sweden. The Kristineberg multimetal sulfide mine has
been in operation since 1940 and is worked at a maximum depth
of 1300 m below the surface. The analyses provide insights into
how acidophiles are able to grow at low temperatures.

Liljeqvist et al.

(Dopson and Lindstrom

1999). Metal concentrations were analyzed from two water samples with technical replicates making
a total of five replicates.

DNA preparation and metagenome sequencing

Metagenome bioinformatic analysis

The biofilm and planktonic reads were assembled separately
using the Newbler Assembler 2.3 (454 Life Science) using the
default parameters. 16S rRNA genes were identified by comparing both metagenomes against the GREENGENES (DeSantis
et al., 2006), RDP (Cole et al., 2009) and SILVA (Pruesse et al., 2007)
databases using an E-value threshold of 1e5 . Contig sequences (>500 bp) from the two separate planktonic and biofilm
metagenomes were binned to taxonomic groups using a number
of algorithms including: MEGAN v4.60.1 (Mitra, Stark and Huson
2011) using a bit score threshold of 35; RAIphy (Nalbantoglu
et al., 2011) using a custom-built database containing complete and draft genomes from acidophile representatives (File 2,
Supplementary Information) and the default RAIphy database;
WEBCARMA (Gerlach and Stoye 2011) and SOrt-ITEMS (Monzoorul et al., 2009). The results from these different techniques were similar and MEGAN results were chosen as representative for further analysis. In addition, TBlastX (Altschul
et al., 1997) searches were performed against all the RefSeq
genomes (Pruitt et al., 2012) for all contigs with lengths 180 nu-

cleotides. Custom-made Perl scripts were used to align each contig nucleotide sequence against RefSeq database genomes using
TBlastX the results were ordered by E-value first, then query coverage (percentage of the contig participating in the alignment)
and finally, alignment similarity and the best 10 genomes were
retained. The top hit with the best statistics was then assigned to
the contig as Best Species. Contigs associated with more than
one taxonomic group were classified as unassigned.
In addition, the two metagenomes were assembled as a
single metagenome. Combined contigs were annotated using
Glimmer (Kelley et al., 2012) in default mode and Prodigal (Hyatt
et al., 2010) in metagenomic mode, using a minimum length of
100 nucleotides. After manually comparing the performance of
both tools, Prodigal results were selected for further analysis because they predicted more open reading frames (ORFs). MEGAN
was used to identify the operational taxonomic units present in
the reassembly of both metagenomes.
Predicted coding sequences (>60 amino acids) were selected to perform Blastp searches against the NCBI-NR and
SWISSPROT protein databases, while domain searches were also
performed against the Conserved Domain Database (MarchlerBauer et al., 2013) using RPS-Blast, which includes COG (Tatusov
et al., 2001), Pfam (Punta et al., 2012), PRK (Klimke et al., 2009) and
TIGRfam (Haft et al., 2013); for all searches, an E-value cut-off of
105 was used. The NCBI accession number of the sequence data
is AOMP00000000-AOMQ00000000.

De-novo assembly of metagenome samples

For metabolic reconstruction and curation of pathways, the two
Kristineberg metagenome samples were pooled together and reassembled as a new, single assembly using the CLC Genomics
Workbench (version 6.5.2; http://www.clcbio.com). ORFs were
predicted and annotated as described for the individual planktonic and biofilm metagenomes. Metabolic reconstruction and
curation of specific pathways was performed on using the PRIAM
(Claudel-Renard et al., 2003) and Ptools (Karp, Paley and Romero
2002) software.

Comparison of metagenomes in this and other studies

The planktonic and biofilms metagenomes (this study) were
compared to the Iron Mountain (Tyson et al., 2004; Dick
`
et al., 2009; Goltsman et al., 2009; Yelton et al., 2013), Carnoules
(Bertin et al., 2011) and Frasassi cave system (Jones et al., 2012)
metagenomes. First, a cold-resistance Blast database was made
with amino acid sequences of all the trehalose pathway
genes (Avonce et al., 2006). Then, amino acid sequences of
all metagenome annotated genes were compared with the
database using Blastp with an E-value cut-off of 105 , and the
best database hit for each gene was registered. Second, and complementary to the first approach, a word search was also done
using a word list crafted for that purpose that included coldresistance gene acronyms, gene descriptions and terms related
to cold resistance.

Molecular phylogenetic analysis of isolates grown on

overlay plates
16S rRNA gene copies from colonies isolated on the overlay plates were PCR amplified using illustra PuReTaq ReadyTo-Go PCR Beads and the 16S rRNA gene primers GM5F and
907R (Morales et al., 2005). PCR products were purified using a PCR Clean-up Kit (Qiagen), quantified and submitted to

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The biofilm and planktonic cell fractions were separated by removing the solid biofilm before pelleting the planktonic cells
by centrifugation (10 000 g for 10 min). The biofilm fraction was resuspended in mineral salts medium (Dopson and

Lindstrom
1999) pH 2.5 and homogenized by a two-step beadbeating method: the biofilm was roughly dispersed using 5 mm
beads for 10 min and then divided into smaller aliquots and further homogenized for 2 min using 1 mm glass beads. Cells from
both the biofilm and planktonic fractions were resuspended
in lysis buffer (100 mM Tris-HCl and 1 mM EDTA, pH 8) and
treated with 20 mg mL1 lysozyme (Sigma) for 30 min and 400
g mL1 proteinase K (Sigma) for 20 min. SDS (2% wt vol1 ; final concentration) was added to enhance cell lysis. DNA was extracted using phenol/chloroform/isoamyl alcohol (ratio 25:24:1),
washed once with 70% ethanol, and precipitated with 100%
ethanol and 3 M sodium acetate pH 5.2. To remove small DNA
fragments from the biofilm preparation, high molecular weight
DNA (40 kDa) was extracted from agarose gels with GELaseTM
agarose gel-digesting system (Epicentre Technologies Corporation) according to the manufacturers instructions. DNA was
sent to Mats ohf, Reykjavik, Iceland for sequencing. First, a
DNA library consisting of 8001000 bp single-stranded fragments was constructed using the GS FLX Titanium General
Library Preparation Kit (Roche, 454 Life Sciences). Shotgun sequencing of the DNA library was carried out using the GS FLX
Titanium reagents as described by the manufacturer (Roche, 454
Life Sciences). This involved hybridization of the purified singlestranded DNA fragments onto DNA capture beads and amplification of separate fragments by emulsion PCR. Beads containing amplified DNA were deposited onto a 75 75 mm Titanium
PicoTiterPlate equipped with a two-lane gasket and the pyrosequencing was performed in a single run. A single titanium plate
was equally split between the biofilm and planktonic DNA samples. Generated sequence data were assembled using the GS
De Novo Assembler software, version 2.6, from Roche, 454 Life
Sciences with the default stringency settings.

FEMS Microbiology Ecology, 2015, Vol. 91, No. 4

Macrogen (Netherlands) for capillary Sanger sequencing. Phylogenetic analysis was carried out by removing primer sequences
and aligning the isolate 16S rRNA gene sequences with representative acidophiles (File 3, Supplementary Information) using
Muscle (Edgar 2004). The maximum number of iterations was
set to 100 and poorly aligned and divergent regions were eliminated using Gblocks (Castresana 2000). The maximum number
of contiguous non-conserved positions was set to 8 and intermediate gap positions were allowed. This resulted in 676 positions in the alignment that were used to generate maximumlikelihood trees using PhyML (Guindon and Gascuel 2003). The
phylogenetic tree was assessed using 1000 bootstrap replicates.

RESULTS AND DISCUSSION

Environmental conditions and sampling

Overview of the metagenomic data set

Metagenome sequencing generated 14 279 423 (19 051 contigs) and 8 006 447 (8115 contigs) nucleotides in the planktonic
and biofilm libraries, respectively (File 4, Supplementary Information). A total of 20 606 and 10 694 ORFs from planktonic
and biofilm datasets were predicted, respectively, from which
16 271 (78%) and 8230 (77%) had significant similarity to sequences available in public databases. A normalized comparison of the COG classifications (against the total number of
coding sequences) from both communities (File 5, Supplementary Information) demonstrated essentially similar percentages
of protein-encoding sequences assigned to the COG classifications. The total reads for COG classifications RNA processing
and modification (A) and chromatin structure and dynamics
(B) were <0.03% in both metagenomes indicating either a low
incidence of eukaryotic sequences or a bias in the method for
DNA preparation that preferentially lysed prokaryotes. However,
of the coding sequences matching COGs A and B, nearly 2-fold
normalized sequences were present in the biofilm fraction. This
suggested that eukaryotes, such as the identified acidophilic
yeast (see below), were more prevalent in the biofilm where a
higher concentration of organic carbon was likely available compared to typically oligotrophic AMD waters.

Molecular phylogeny of the biofilm and planktonic

species
A phylogenetic tree was constructed using 16S rRNA gene
sequences from isolated colonies and the Kristineberg mine
metagenome (File 6, Supplementary Information). All 27 of the
16S rRNA gene sequences derived from the overlay plates incubated at 22 and 12o C (13 and 14 colonies, respectively) aligned
with the psychrotolerant acidophile, At. ferrivorans (for clarity,

Species identification and binning of the metagenomic

sequences
Binning of the metagenome sequences was carried out with
MEGAN (Fig. 1 & File 7, Supplementary Information) and
other methods (File 8, Supplementary Information). The binning methods consistently demonstrated that species from
the Acidithiobacillus genus and, in particular, an At. ferrivoranslike species dominated both the biofilm and planktonic
communities.
Based upon the number of reads against the RefSeq genomes,
the dominant species in both the planktonic (307 185 of assigned
639 459 reads; 48.0%) and biofilm (430 290 of assigned 672 719
reads; 64.0%) fractions was At. ferrivorans-like (File 8, Supplementary Information). The number of planktonic and biofilm
contigs assigned to the At. ferrivorans-like species were 5574
(constituting 90% of the genome) and 2631 (also constituting 90%
of the genome), respectively. The next most abundant strains
were binned as a Fe2+ and ISC oxidizing At. ferrooxidans-like
strain (both ATCC 23270 and ATCC 53993) with a total of 9.9%
and 8.7% in the planktonic and biofilm metagenomes, respectively followed by an ISC oxidizing At. caldus-like strain (3.6% and
2.7%, respectively). In addition, a Gallionella-like sp. (G. capsiferriformans; 1.0% and 0.2%, respectively) that is a neutrophilic ferrous oxidizer capable of growth at 6 C but not 30 C (Emerson and
Moyer 1997) and an Acidobacterium-like sp. (Ac. capsulatum; 1.0%
and 0.1%, respectively) that is a Gram-negative, organotrophic
acidophile that grows at 25 C (Kishimoto, Kosako and Tano 1991)
were also identified. The presence of contigs attributed to relatives of mesophilic and moderately thermophilic species is most
likely explained by these being the only sequenced genomes
available to which metagenome contigs can be matched. A low
species diversity dominated by just two (Tyson et al., 2004), seven
(Bertin et al., 2011) and one (Jones et al., 2012) strain(s) appears
to be a typical trait of acidic environments (Hallberg et al., 2006),
where primary producers able to fix carbon constitute the majority of the sampled microbial population.
Small differences in binning were observed between the
two metagenomes with a higher percentage of contigs assigned

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The AMD stream contained large streamer-type biofilms consisting of chains of cells twisted together (File 1, Supplementary Information). When the samples for the metagenomes were
taken in April 2010 the stream had in situ characteristics of: 6o C,
pH 2.7, an oxidation reduction potential versus the standard hydrogen potential of 640 mV and 16.8 mM soluble Fe2+ . During
a subsequent visit in November 2011, the stream was 11o C, pH
2.5 and had soluble iron (both Fe2+ and Fe3+ ) and copper concentrations of 39 0.9 and 5 0.1 mM, respectively (number of
replicates = 5). This suggests that while the pH did not significantly change, small variations in temperature occurred potentially due to infiltration of surface waters.

only two representative sequences are presented). The presence

of an At. ferrivorans-like strain on the enrichment plates agreed
with previous data showing a low-temperature bioreactor for ISC
removal inoculated with Kristineberg AMD water contained At.
ferrivorans (Liljeqvist et al., 2011a). In addition, gene sequences
identified from the metagenomes included: two 16S rRNA gene
copies phylogenetically most related to At. ferrivorans; three 16S
rRNA gene copies most related to Gram-positive, Fe2+ oxidizing acidophilic heterotrophs of the genera Acidimicrobium and
Ferrimicrobium (Wisotzkey et al., 1992; Johnson et al., 2009); two
16S rRNA gene copies most related to Alicyclobacillus acidocaldarius (Wisotzkey et al., 1992); one 16S rRNA gene copy most related
to the Fe2+ oxidizing neutrophile Gallionella (G.) capsiferriformans
(Emerson et al., 2007); and two 16S rRNA gene copies most related to the low-temperature species Terriglobus (Te.) saanensis

(Mannist
o et al., 2011) and Acidobacterium (Ac.) capsulatum (Kishimoto, Kosako and Tano 1991). Two colonies from plates incubated at 4o C were found to be yeast species related to Cryptococcus sp. (data not shown). Acidophilic yeasts from AMD environments have been previously reported (Gadanho and Sampaio
2006) and likely metabolize organic carbon produced by the autotrophic acidophiles. However, the lack of 18S rRNA genes and
other eukaryotic related species in the metagenome suggests
that they constituted a minor portion of the mixed population.

Liljeqvist et al.

to the Acidithiobacillus genus in the biofilm fraction (25.5%)

compared to the planktonic fraction (19.2%); At. ferrivoranslike species (24.5% and 14.6%, respectively); At. ferrooxidanslike species (7.8% and 6.3%, respectively); and At. caldus-like
species (2.8% and 2.6%, respectively) (File 8, Supplementary
Information). In contrast, more contigs were assigned to the
Acidobacteria-like species in the planktonic fraction (5.4%)
compared to the biofilm (0.9%) and the Gallionellaceae-like
species (3.9% and 0.2%, respectively) (File 8, Supplementary
Information).
Although the environment was dominated by a few species,
there was also a significant tail of low-abundance species.
These included contigs that were assigned by sequence similarity to: (i) an Acidimicrobium-like spp. (Acidimicrobium ferrooxidans), a Gram-positive moderately thermophilic ferrous iron oxidizer (Clark and Norris 1996); (ii) a Te. saanensis-like sp. which
is an organotrophic Acidobacterium capable of growth at pH

4.57.5 and 430 C (Mannist

o et al., 2011); (iii) a Sideroxydans
lithotrophicus-like sp., an acid-tolerant ferrous oxidizer (Ludecke
et al., 2010); (iv) a Rhodoferax ferrireducens-like sp., a psychrotolerant iron reducer that grows at 430 C (Finneran, Johnsen and
Lovley 2003) and (v) a Leptospirillum ferrodiazotrophum-like sp., an
acidophilic obligate ferrous oxidizer (Goltsman et al., 2009).

Cold-shock and cold acclimation strategies

Microorganisms undergo a cold shock when their external temperature drops below its optimal range. The response to cold
shock in the psychrophilic Psychrobacter arcticus included downregulation of genes associated with energy metabolism and substrate incorporation and upregulation of genes for membrane
maintenance and a DEAD-box RNA helicase protein A (Kuhn
2012). In contrast, genes for RNA and protein chaperones were
not upregulated under the tested conditions (Kuhn 2012). The
response to cold between, for example, mesophilic and psychrophilic microorganisms can vary as a cold-shock gene in
a mesophile can act as a cold acclimation protein in a psychrophile. For example, CspA was the most abundant protein
in Escherichia coli upon downshift from 37 to 10 C (Goldstein,
Pollitt and Inouye 1990) while it acted as a cold acclimation

protein in the psychrotolerant species, Arthrobacter globiformis

(Berger et al., 1996). It is unclear if the genes identified in this
study were cold-shock or acclimation proteins.
An increase in membrane rigidity as a response to cold shock
is sensed by a membrane-associated two-component system
in Bacillus subtilis (Aguilar et al., 2001). Also, methyl-accepting
chemotaxis proteins (Maeda and Imae 1979) and 3-oxoacyl-(acyl
carrier protein) synthase II (Garwin and Cronan 1980) play an
important regulatory role in sensing temperature changes in
E. coli. A total of 34 membrane-associated two-component systems, 26 methyl-accepting chemotaxis systems and 6 copies of
3-oxoacyl-(acyl carrier protein) synthase II were found in the
metagenomes. These proteins could be used to sense a wide variety of environmental signals and a potential role in sensing
varying temperatures remains to be investigated.
Increased membrane fluidity can be achieved by changes in
fatty acid isomerization, changing the composition of sterol-like
compounds, and adjusting the levels of saturated and unsaturated fatty acids (Russell 2008). Fatty acid desaturases not only
modulate the fluidity of the membrane but also protect against
increased concentrations of reactive oxygen species. Oxidative
stress proteins are especially important at low temperature due
to the increased solubility of gases. Two types of desaturases
can be found in the metagenome, fatty acid desaturase and
stearoyl-CoA 9-desaturase. In addition, several species in the
metagenome contained genes for catalase, superoxide dismutase, peroxiredoxin and alkyl hydroperoxide reductase as well
as several dioxygenases of various functions that protect against
reactive oxygen species. Oxidative stress defense strategies are
common in AMD environments due to the presence of Fe2+ . For
example, peroxiredoxins are upregulated in Ferroplasma acidarmanus in response to the presence of Fe2+ (Dopson, Baker-Austin
and Bond 2005). A requirement for these systems at low temperature may be increased due to the higher solubility of gases
contributing to increased oxidative stress.
Cold sensing occurs via DNA super coiling at low temperature and by the ability of some RNA molecules to alter their secondary structure as a result of a temperature shift (reviewed in
Shivaji and Prakash 2010). The most studied response is generally referred to as the cold-shock response. The cold-shock

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Figure 1. Pie charts of relative species composition between the planktonic (A) and biofilm (B) contigs binned by MEGAN. Sequences labeled Acidithiobacillus could be
assigned to the Acidithiobacillus core genome but further classification could not be made.

FEMS Microbiology Ecology, 2015, Vol. 91, No. 4

response is characterized by the induction of small DNA/RNAbinding cold-shock proteins (CSPs), cold-shock RNA helicases
and several proteins involved in replication, transcription and
translation. The majority of cold-induced RNA helicases contain
the DEAD/H-box motif (reviewed in Owttrim 2006). The biofilm
and planktonic metagenomes contained 5 and 6 CSPs and 11
and 26 DEAD/H-box helicases, respectively. However, some of
the CSPs have significant similarity to CspD that is upregulated
in stationary phase and under nutrient starvation, rather than
being associated with cold shock (Yamanaka and Inouye 1997).
Many microorganisms accumulate compatible solutes such
as trehalose, glycine-betaine, choline and sucrose that along
with other functions are involved in the cold-stress response
(Kawahara 2008). Trehalose appears to be common in AMD environments (Yelton et al., 2013) and its other functions include mitigating osmotic stress, carbon storage and heat shock (Martins
et al., 1997). The metagenome contains genes encoding five pathways for microbial trehalose production (Avonce et al., 2006;
Fig. 2 and File 9, Supplementary Information). Functional redundancy for trehalose biosynthesis has been suggested to be
due to the need to accumulate trehalose under changing environmental conditions (Reina-Bueno et al., 2012) and the presence of multiple pathways may reflect its relative importance
in this milieu. Binning of the sequences demonstrated that
the At. ferrivorans-like species had complete trehalose synthase
(TS), TreYZ and TreT pathways as well as the gene-encoding
trehalose-6-phosphate synthase (TPS) from the TPS/trehalose6-phosphate phosphatase (TPP) pathway; the Acidobacteria-like
species had the TS and TreP pathways; and the At. ferrooxidanslike species the TreT pathway as well as a gene-encoding TreY.
It was interesting that contigs assigned to the psychrotolerant
At. ferrivorans-like species encoded three separate trehalose syn-

thesis pathways and that the At. ferrooxidans-like species had an

additional partial trehalose synthesis pathway compared to the
two sequenced At. ferrooxidans genomes. The multiple synthesis pathways suggest the importance of trehalose as a potential
response to low temperature in this environment.
In addition, the biofilm metagenome contained predictions
for genes involved in the production of other compatible solutes and cryoprotectants. As for trehalose, the compatible
solutes glycine betaine and sucrose also have other functions and their role in cold adaptation has not been confirmed. Genes associated with cryoprotection included glycine
betaine synthesis including the betaine transcriptional regulator (betI) and two copies of the gene-encoding choline
dehydrogenase (betA), although the second enzyme in the
pathway, betaine aldehyde dehydrogenase (betB), was not detected. The genes encoding sucrose synthase and sucrosephosphate synthase involved in sucrose synthesis from UDPglucose were also predicted in both fractions. Anti-freeze proteins are ice-binding proteins that induce thermal hysteresis and thereby, inhibit the growth of ice crystals (Kawahara
2008). The planktonic fraction contained a gene predicted to
encode a Type II anti-freeze protein previously identified in Te.
saanensis (Rawat, Mannisto and Starovoytov 2012). A final investigated signature for low-temperature adaptation was for
the presence of de novo adenosine 5-monophosphate synthetic
enzymes (also involved in purine biosynthesis) that are enriched in low-temperature-adapted microorganisms compared
to mesophiles (Parry and Shain 2011). Four and five gene
copies encoding adenylosuccinate synthetase (purA) were identified in the biofilm and planktonic metagenomes while five
and two copies of adenylosuccinate lyase (purB) were found,
respectively.

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Figure 2. The various pathways for trehalose production in bacterial acidophiles. Figure adapted from Liljeqvist (2012).

Liljeqvist et al.

Biofilm formation

genes has not been confirmed. Acidophile sulfur oxidation models have been suggested for At. ferrooxidans (Quatrini et al., 2009)
and At. caldus (Mangold et al., 2011; Chen et al., 2012). Although
a model was not generated, genes encoded by the At. ferrivorans genome have also been assigned to ISC oxidation (Liljeqvist,
Rzhepishevska and Dopson 2013). Based upon the earlier studies, an ISC oxidation pathway is proposed for the At. ferrivoranslike species (File 12, Supplementary Information). Other identified genes annotated as involved in ISC oxidation included
components of heterodisulfide reductase from At. ferrooxidanslike, At. caldus-like, Ac. ferrooxidans-like and an Acidobacteria-like
species.
Genes encoding electron transport components were
identified in the At. ferrivorans-like species. These included
cytochrome bo terminal oxidase (cyoABCD), an aa3 -type
cytochrome c oxidase (coxABCD) and subunits of a cytochrome
c oxidase. Additionally, two cbb3 genes (ccoN & ctaG) and a cytochrome bd ubiquinol oxidase gene (cydA) were also identified.
All subunits from a respiratory nitrate reductase (narGHI) were
annotated to the At. ferrivorans-like strain suggesting that it
may utilize nitrate as a terminal electron acceptor.

Carbon dioxide and nitrogen fixation

Substrate oxidation and energy conservation
Of the acidophiles, At. ferrooxidans has the best characterized
Fe2+ oxidation system (Quatrini et al., 2009). All components of
this system were detected in the metagenomes (File 11, Supplementary Information) with orthologs found in both the At.
ferrivorans-like and At. ferrooxidans-like contigs. This suggests
that these species may oxidize Fe2+ , although expression of the

Genes predicted to encode the two unique enzymes involved

in carbon assimilation via the CalvinBensonBassham cycle, namely ribulose-1,5-bisphosphate carboxylase/oxygenase
(RuBisCO) and phosphoribulokinase (CbbP), were found in At.
ferrivorans-like and At. ferrooxidans-like contigs (File 13, Supplementary Information). This is in agreement with published
data that these microorganisms fix carbon (Esparza et al., 2010;

Table 1. Genes associated with a low-temperature lifestyle identified in the Kristineberg metagenome (this study) compared to Iron Mountain,
` France (Bertin et al., 2011) and the Frasassi
California (Tyson et al., 2004; Dick et al., 2009; Goltsman et al., 2009; Yelton et al., 2013); Carnoules,
cave system, Italy (Jones et al., 2012)

Temperature

Kristineberg

Iron Mountain

`
Carnoules

Frasassi cave

611 C

35 45 C

15.1 C

13 C

SCD

SCD; DesA

TS; TPS; TPP; TreY; TreZ;

TreT; TreP
BetA; BetB; Gbu

TPS; TreZ

Response to low temperature

Fatty acid desaturase;
Membrane fluiditya
SCD
Compatible solutes and antifreeze proteins
TS; TPS; TPP; TreY; TreZ;
Trehalose biosynthesisb
TreT; TreP
BetA; BetI
Glycine betainec
Anti-freeze proteins
Type II
Oxidative stress and low-temperature response
CSP; CsbD; GyrA;DeaD;
Cold-shock responsed
RecA; PpiC; SurA; RbfA;
Pnpase; Pnp; InfB; NusA;
ClpB
Grx (& GrxB); Ahp; SOD;
Oxidative stresse
Prx; CcpA; Cat

TS; TPS; TPP; TreY; TreZ

BetB

GyrA; DeaD; RecA; RbfA;

Pnp; InfB; NusA

CsbD; GyrA; DeaD; RecA;

PpiC; RbfA; Pnpase; Pnp;
InfB; ClpB

Grx; Prx; Ahp

Grx; Ahp; SOD; Prx (&

Bcp); CcpA; Cat; BtuE

BetB

Grx; AhP; SOD; Prx; Cat

SCD, Stearoyl-CoA 9-desaturase; DesA, 12 acyl-lipid desaturase.

Description of genes given in File 9 (Supplementary Information).
c
BetA, choline dehydrogenase; BetB, betaine aldehyde dehydrogenase; BetI, BetI DNA-binding transcriptional repressor; Gbu; glycine betaine ABC transporter.
d
CSP, DNA binding cold-shock protein; CsbD, cold-induced stress protein; GyrA, DNA gyrase; DeaD, DEAD-box RNA helicase; PpiC, peptidyl-prolyl cis-trans isomerase;
SurA, survival protein; RecA, DNA strand exchange and recombination protein; RbfA, ribosome binding factor; PNPase, polynucleotide phosphorylase/polyadenylase;
Pnp, polyribonucleotide nucleotidyltransferase; InfB, protein chain initiation factor IF2; NusA, transcription termination factor; ClpB, ATP-dependent chaperone.
e
Grx, glutaredoxin; GrxB, reduced glutaredoxin 2; Ahp, alkyl hydroperoxide reductase; SOD, superoxide dismutase; Prx, peroxiredoxin; CcpA, cytochrome c peroxidase;
Cat, catalase; BtuE, glutathione peroxidase.
a

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Genes were predicted for all stages in biofilm formation in the

At. ferrivorans-like DNA sequences (File 10, Supplementary Information). Genes predicted to encode pili formation and a
suite of genes for exopolysaccharide formation and export was
also present in the At. ferrooxidans-like DNA sequences but they
lacked any evidence for genes involved in the Lux quorum sensing system, flagella formation, che signaling pathway quorum
sensing genes and the diguanylate cyclase signaling system.
The At. caldus-like strain contained predicted genes for chemotaxis, pilus formation and flagella formation. This attribution
of predicted genes and functions to specific reconstructed microbial species in the metagenome sequences is consistent
with their respective individual genomes (Valdes et al., 2008;
Valdes et al., 2009; Liljeqvist et al., 2011b) and supported with
experimental (Ruiz et al., 2008) and additional bioinformatic
evidence (Cardenas et al., 2010). Evidence was found for the
ability to form a biofilm by the Gallionellaceae-like (principally Gallionella-like and Sideroxydans-like) and Acidobacterialike metagenome bins. This includes pili formation in both
species as well as genes coding for EPS biosynthesis in the
Acidimicrobium-like strain.

FEMS Microbiology Ecology, 2015, Vol. 91, No. 4

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Figure 3. Model of the Kristineberg low-temperature AMD microbial community showing the major functions of the identified Acidithiobacillus-like spp., an unidentified
Acidobacteria-like species and an unidentified Gallionellaceae-like species. The figure was adapted from Valdes et al. (2008).

Liljeqvist et al.

A cold-adapted metagenome
A search of the metagenome sequences identified unique genes
assigned to the At. ferrivorans-like strain (but not to the other
characterized Acidithiobacillus spp.) suggesting it was adapted to
low temperature (File 15, Supplementary Information). These
genes included a trehalose synthase, stressing its potential importance for adaptation to low temperature; a multisensor hybrid histidine kinase that acts as a cold sensor (Phadtare 2004);
helicase containing proteins; phosphoadenosine phosphosulfate reductase previously shown to be upregulated at low temperature (Topanurak et al., 2005); the 30S ribosomal protein S21
involved in adaptation to low temperature (Sato et al., 1997); oxidative stress proteins; CsbD involved in survival at low temperature (Raengpradub, Wiedmann and Boor 2008) and genes encoding for biofilm formation. The presence of these genes supports
that the At. ferrivorans-like strain is adapted to the low temperature. However, without isolation of the other species detected
it is impossible to determine if they are psychrophilic, psychrotolerant or mesophilic.
A comparison of the genes identified in this study to the
Iron Mountain (Tyson et al., 2004; Dick et al., 2009; Goltsman
` (Bertin et al., 2011)
et al., 2009; Yelton et al., 2013), Carnoules
and Frasassi cave system (Jones et al., 2012) metagenomes also
suggested adaptations to a low-temperature lifestyle (Table 1).
This comparison must be taken with care as it will be affected
by the number of species in the community, with a less diverse community likely to have a smaller pan genome as well
as the depth of sequencing. However, a higher incidence of
genes suggested to contribute to life in the cold was evident in
` AMD
the Kristineberg metagenome followed by the Carnoules
stream that had a temperature in May of 15.1 C. These differences were particularly evident for genes assigned as encoding
for compatible solutes and antifreeze proteins as well as genes
associated with oxidative stress associated due to the higher solubility of gases at low temperature (Table 1). We hypothesize
that the higher degree of similarity between the Kristineberg and
` AMD stream metagenomes was likely due to adapCarnoules
tations to the temperature rather than the presence of similar
species. This is supported by more genes suggesting adaptations
` AMD stream despite that
to a low temperature in the Carnoules

the Frasassi cave system contained a similar microbial population to the Kristineberg community.

Model of the Kristineberg low-temperature AMD

microbial community
The metagenome binning was summarized to provide
the attributes of the dominating Acidithiobacillus-like spp.,
Acidobacteria-like sp. and Gallionellaceae-like sp. (File 16, Supplementary Information). These attributes were used to
create a model of the Kristineberg low-temperature AMD
community (Fig. 3). All analyses suggested a predominantly
chemolithotrophic lifestyle with the At. ferrivorans-like species
fixing carbon dioxide. The other identified species are suggested
to play a lesser role such as via metabolizing organic carbon that
is toxic to chemolithoautotrophic acidophiles and potentially
contributing to biofilm formation.

SUPPLEMENTARY DATA
Supplementary data is available at FEMSEC online.

ACKNOWLEDGEMENTS
The authors thank Professor Barrie Johnson for isolation of pure
cultures and Rodrigo Ortiz for technical assistance.

FUNDING
MD would like to thank the JC Kempe Stiftelserna and Swedish

Research Council (Vetenskapsradet

contract number 621-20073537) for financial support. DH acknowledges Fondecyt 1130683.
JV was supported by grants from FONDECYT (11110434) and
InnovaChile-CORFO (FCR-CSB 09CEII-6991).
Conflict of interest. None declared.

REFERENCES
Aguilar PS, Hernandez-Arriaga AM, Cybulski LE, et al. Molecular
basis of thermosensing: a two-component signal transduction thermometer in Bacillus subtilis. EMBO J 2001;20:168191.
Altschul SF, Madden TL, Schaffer AA, et al. Gapped BLAST and
PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 1997;25:3389402.
Avonce N, Mendoza-Vargas A, Morett E, et al. Insights on the
evolution of trehalose biosynthesis. BMC Evol Biol 2006;6:109.
Berger F, Morellet N, Menu F, et al. Cold shock and cold acclimation proteins in the psychrotrophic bacterium Arthrobacter globiformis SI55. J Bacteriol 1996;178:29993007.
Berthelot D, Leduc LG, Ferroni GD. Temperature studies of ironoxidizing autotrophs and acidophilic heterotrophs isolated
from uranium mines. Can J Microbiol 1993;39:3848.
Berthelot D, Leduc LG, Ferroni GD. The absence of psychrophilic
Thiobacillus ferrooxidans and acidophilic heterotrophic
bacteria in cold tailings effluents from a uranium mine. Can
J Microbiol 1994;40:603.
Bertin PN, Heinrich-Salmeron A, Pelletier E, et al. Metabolic diversity among main microorganisms inside an arsenic-rich
ecosystem revealed by meta- and proteo-genomics. ISME J
2011;5:173547.
Bonnefoy V, Holmes DS. Genomic insights into microbial oxidation and iron homeostasis in extremely acidic environments.
Environ Microbiol 2012;14:1597611.

Downloaded from http://femsec.oxfordjournals.org/ by guest on March 2, 2016

Hallberg, Gonzalez-Toril and Johnson 2010). There was no genetic evidence for CO2 fixation by other known pathways. Evidence of heterotrophic lifestyles included candidate genes encoding licheninase, neopullulanase, dextranase and other proteins involved in sugar biodegradation and transport. Due to
the typically low availability of organic carbon in AMD environments, primary producers are likely dominant and the relatively
low number of heterotrophs in the biofilm was expected. The
likely role of the heterotrophs is to metabolize organic carbon
that can be toxic to autotrophic acidophiles (Nancucheo and
Johnson 2010).
Genes annotated for ammonium and nitrate uptake systems
in the At. ferrivorans-like species were present together with nitrate and nitrite reductases, suggesting that nitrogen was assimilated from different sources (File 14, Supplementary Information). Nitrate is a potential nitrogen source from explosives used for mining in an otherwise oligotrophic environment. In addition, nitrogen fixation genes were identified in the
Gallionellaceae-like species. There was no genetic evidence for
ammonia or ammonium oxidation, nitrification or denitrification (nitric oxide reductases and nitrous oxide reductases catalyzing the last two steps of denitrification were not found).

10

FEMS Microbiology Ecology, 2015, Vol. 91, No. 4

Garwin JL, Cronan JE. Thermal modulation of fatty acid synthesis

in Escherichia coli does not involve de novo enzyme synthesis.
J Bacteriol 1980;141:14579.
Gerlach W, Stoye J. Taxonomic classification of metagenomic shotgun sequences with CARMA3. Nucleic Acids Res
2011;39:e91.
Gilbert JA, Hill PJ, Dodd CE, et al. Demonstration of
antifreeze protein activity in Antarctic lake bacteria.
Microbiology 2004;150:17180.
Goldstein J, Pollitt NS, Inouye M. Major cold shock protein of
Escherichia coli. P Natl Acad Sci USA 1990;87:2837.
Goltsman DS, Denef VJ, Singer SW, et al. Community genomic
and proteomic analyses of chemoautotrophic iron-oxidizing
Leptospirillum rubarum (Group II) and Leptospirillum ferrodiazotrophum (Group III) bacteria in acid mine drainage biofilms.
Appl Environ Microb 2009;75:4599615.
Grzymski JJ, Carter BJ, DeLong EF, et al. Comparative genomics of
DNA fragments from six Antarctic marine planktonic bacteria. Appl Environ Microb 2006;72:153241.
Guindon S, Gascuel O. A simple, fast, and accurate algorithm to
estimate large phylogenies by maximum likelihood. Syst Biol
2003;52:696704.
Haft DH, Selengut JD, Richter RA, et al. TIGRFAMs and genome
properties in 2013. Nucleic Acids Res 2013;41:D38795.
Hallberg KB, Coupland K, Kimura S, et al. Macroscopic streamer
growths in acidic, metal-rich mine waters in north Wales
consist of novel and remarkably simple bacterial communities. Appl Environ Microb 2006;72:202230.
Hallberg KB, Gonzalez-Toril E, Johnson DB. Acidithiobacillus ferrivorans, sp. nov.; facultatively anaerobic, psychrotolerant
iron-, and sulfur-oxidizing acidophiles isolated from metal
mine-impacted environments. Extremophiles 2010;14:919.
Helmke E, Weyland H. Psychrophilic versus psychrotolerant
bacteria-occurrence and significance in polar and temperate
marine habitats. Cell Mol Biol 2004;50:55361.
Hyatt D, Chen GL, LoCascio PF, et al. Prodigal: prokaryotic gene
recognition and translation initiation site identification. BMC
Bioinformatics 2010;11:119.
Johnson DB, Bacelar-Nicolau P, Okibe N, et al. Ferrimicrobium
acidiphilum gen. nov., sp. nov. and Ferrithrix thermotolerans gen.
nov., sp. nov.: heterotrophic, iron-oxidizing, extremely acidophilic actinobacteria. Int J Syst Evol Micr 2009;59:10829.
Johnson DB, Hallberg KB. Techniques for detecting and identifying acidophilic mineral-oxidizing microorganisms. In: Rawlings DE, Johnson DB (eds). Biomining. Berlin: Springer-Verlag,
2007.
Johnson DB, Hallberg KB, Hedrich S. Uncovering a microbial
enigma: isolation and characterization of the streamergenerating, iron-oxidizing, acidophilic bacterium Ferrovum
myxofaciens. Appl Environ Microb 2014;80:67280.
Jones DS, Albrecht HL, Dawson KS, et al. Community genomic
analysis of an extremely acidophilic sulfur-oxidizing biofilm.
ISME J 2012;6:15870.
Karp PD, Paley S, Romero P. The Pathway Tools software. Bioinformatics 2002;18:S22532.
Kawahara H. Cryoprotectants and ice-binding proteins. In: Margesin R, Schinner F, Marx JC, et al. (eds). Psychrophiles:
From Biodiversity to Biotechnology. Berlin: Springer, 2008,
22946.
Kay CM, Rowe OF, Rocchetti L, et al. Evolution of microbial streamer growths in an acidic, metal-contaminated
stream draining an abandoned underground copper mine.
Life 2013;3:189210.

Downloaded from http://femsec.oxfordjournals.org/ by guest on March 2, 2016

Cardenas JP, Valdes J, Quatrini R, et al. Lessons from the genomes

of extremely acidophilic bacteria and archaea with special
emphasis on bioleaching microorganisms. Appl Microbiol Biot
2010;88:60520.
Castresana J. Selection of conserved blocks from multiple alignments for their use in phylogenetic analysis. Mol Biol Evol
2000;17:54052.
Chen L, Ren Y, Lin J, et al. Acidithiobacillus caldus sulfur oxidation
model based on transcriptome analysis between the wild
type and sulfur oxygenase reductase defective mutant. PLoS
One 2012;7:e39470.
Chintalapati S, Kiran MD, Shivaji S. Role of membrane lipid fatty
acids in cold adaptation. Cell Mol Biol 2004;50:63142.
Clark DA, Norris PR. Acidimicrobium ferrooxidans gen. nov., sp.
nov.: mixed-culture ferrous iron oxidation with Sulfobacillus
species. Microbiology 1996;142:78590.
Claudel-Renard C, Chevalet C, Faraut T, et al. Enzyme-specific
profiles for genome annotation: PRIAM. Nucleic Acids Res
2003;31:66339.
Cole JR, Wang Q, Cardenas E, et al. The Ribosomal Database
Project: improved alignments and new tools for rRNA analysis. Nucleic Acids Res 2009;37:D1415.
DeSantis TZ, Hugenholtz P, Larsen N, et al. Greengenes,
a chimera-checked 16S rRNA gene database and workbench compatible with ARB. Appl Environ Microb 2006;72:
506972.
Dick GJ, Andersson AF, Baker BJ, et al. Community-wide analysis of microbial genome sequence signatures. Genome Biol
2009;10:R85.
Dopson M, Baker-Austin C, Bond PL. Analysis of differential protein expression during growth states of Ferroplasma strains
and insights into electron transport for iron oxidation. Microbiology 2005;151:412737.
Dopson M, Halinen AK, Rahunen N, et al. Mineral and iron oxidation at low temperatures by pure and mixed cultures of
acidophilic microorganisms. Biotechnol Bioeng 2007;97:1205
15.
Dopson M, Johnson DB. Biodiversity, metabolism and applications of acidophilic sulfur- metabolizing micro-organisms.
Environ Microbiol 2012;14:262031.
EB. Potential role of Thiobacillus caldus in
Dopson M, Lindstrom
arsenopyrite bioleaching. Appl Environ Microb 1999;65:3640.
Edgar RC. MUSCLE: multiple sequence alignment with high accuracy and high throughput. Nucleic Acids Res 2004;32:17927.
Emerson D, Moyer C. Isolation and characterization of novel
iron-oxidizing bacteria that grow at circumneutral pH. Appl
Environ Microb 1997;63:478492.
Emerson D, Rentz JA, Lilburn TG, et al. A novel lineage of Proteobacteria involved in formation of marine Fe-oxidizing microbial mat communities. PLoS One 2007;2:e667.
Esparza M, Cardenas JP, Bowien B, et al. Genes and pathways for CO2 fixation in the obligate, chemolithoautotrophic acidophile, Acidithiobacillus ferrooxidans. BMC Microbiol 2010;10:229.
Finneran KT, Johnsen CV, Lovley DR. Rhodoferax ferrireducens sp.
nov., a psychrotolerant, facultatively anaerobic bacterium
that oxidizes acetate with the reduction of Fe(III). Int J Syst
Evol Micr 2003;53:66973.
Gadanho M, Sampaio JP. Microeukaryotic diversity in the extreme environments of the Iberian Pyrite Belt: a comparison
between universal and fungi-specific primer sets, temperature gradient gel electrophoresis and cloning. FEMS Microbiol
Ecol 2006;57:13948.

Liljeqvist et al.

Mitra S, Stark M, Huson DH. Analysis of 16S rRNA environmental

sequences using MEGAN. BMC Genomics 2011;12:S17.
Monzoorul HM, Ghosh TS, Komanduri D, et al. SOrt-ITEMS: Sequence orthology based approach for binning and improved
taxonomic estimation of metagenomic sequences. Bioinformatics 2009;25:172230.
Morales TA, Dopson M, Athar R, et al. Analysis of bacterial diversity in acidic pond water and compost after treatment of
artificial acid mine drainage for metal removal. Biotechnol Bioeng 2005;90:54351.
Nalbantoglu OU, Way SF, Hinrichs SH, et al. RAIphy: phylogenetic classification of metagenomics samples using iterative
refinement of relative abundance index profiles. BMC Bioinformatics 2011;12:41.
Nancucheo I, Johnson DB. Production of glycolic acid by
chemolithotrophic iron- and sulfur-oxidizing bacteria and
its role in delineating and sustaining acidophilic sulfide
mineral-oxidizing consortia. Appl Environ Microb 2010;76:
4617.
Noon KR, Guymon R, Crain PF, et al. Influence of temperature
on tRNA modification in archaea: Methanococcoides burtonii
(optimum growth temperature [Topt], 23o C) and Stetteria hydrogenophila (Topt, 95o C). J Bacteriol 2003;185:548390.
Owttrim GW. RNA helicases and abiotic stress. Nucleic Acids Res
2006;34:322030.
Parry BR, Shain DH. Manipulations of AMP metabolic genes
increase growth rate and cold tolerance in Escherichia
coli: implications for psychrophilic evolution. Mol Biol Evol
2011;28:213945.
Phadtare S. Recent developments in bacterial cold-shock response. Curr Issues Mol Biol 2004;6:12536.
Pruesse E, Quast C, Knittel K, et al. SILVA: a comprehensive
online resource for quality checked and aligned ribosomal
RNA sequence data compatible with ARB. Nucleic Acids Res
2007;35:718896.
Pruitt KD, Tatusova T, Brown GR, et al. NCBI Reference Sequences
(RefSeq): current status, new features and genome annotation policy. Nucleic Acids Res 2012;40:D1305.
Punta M, Coggill PC, Eberhardt RY, et al. The Pfam protein families database. Nucleic Acids Res 2012;40:D290301.
Quatrini R, Appia-Ayme C, Denis Y, et al. Extending the models for iron and sulfur oxidation in the extreme acidophile Acidithiobacillus ferrooxidans. BMC Genomics 2009;
10:394.
Raengpradub S, Wiedmann M, Boor KJ. Comparative analysis of
the B -dependent stress responses in Listeria monocytogenes
and Listeria innocua strains exposed to selected stress conditions. Appl Environ Microb 2008;74:15871.
Rawat SR, Mannisto MK, Starovoytov V, et al. Complete genome
sequence of Terriglobus saanensis type strain SP1PR4T , an Acidobacteria from tundra soil. Stand Genomic Sci 2012;7:5969.
M, Salvador M, et al. Role of treReina-Bueno M, Argandona
halose in salinity and temperature tolerance in the model
halophilic bacterium Chromohalobacter salexigens. PLoS One
2012;7:e33587.
Ruiz LM, Valenzuela S, Castro M, et al. AHL communication is a
widespread phenomenon in biomining bacteria and seems
to be involved in mineral-adhesion efficiency. Hydrometallurgy 2008;94:1337.
Russell NJ. Membrane components and cold sensing. In: Margesin R, Schinner F, Marx JC, et al. (eds). Psychrophiles: From
Biodiversity to Biotechnology. Berlin: Springer-Verlag, 2008,
17790.

Downloaded from http://femsec.oxfordjournals.org/ by guest on March 2, 2016

Kelley DR, Liu B, Delcher AL, et al. Gene prediction with Glimmer
for metagenomic sequences augmented by classification and
clustering. Nucleic Acids Res 2012;40:e9.
Kimura S, Bryan CG, Hallberg KB, et al. Biodiversity and geochemistry of an extremely acidic, low-temperature subterranean
environment sustained by chemolithotrophy. Environ Microbiol 2011;13:2092104.
Kishimoto N, Kosako Y, Tano T. Acidobacterium capsulatum; gen.
nov., sp. nov.: An acidophilic chemoorganotrophic bacterium
containing menaquinone from acidic mineral environment.
Curr Microbiol 1991;22:17.
Klimke W, Agarwala R, Badretdin A, et al. The national center for
biotechnology informations protein clusters database. Nucleic Acids Res 2009;37:D21623.
Krembs C, Deming JW. The role of exopolymers in microbial
adaptation to sea ice. In: Margesin R, Schinner F, Marx JC,
et al. (eds). Psychrophiles: From Biodiversity to Biotechnology.
Berlin: Springer, 2008, 24764.
Kuhn E. Toward understanding life under subzero conditions:
the significance of exploring psychrophilic cold-shock proteins. Astrobiology 2012;12:107886.
Kupka D, Liljeqvist M, Nurmi P, et al. Oxidation of elemental sulfur, tetrathionate, and ferrous iron by the psychrotolerant
Acidithiobacillus strain SS3. Res Microbiol 2009;160:76774.
Kupka D, Rzhepishevska OI, Dopson M, et al. Bacterial oxidation of ferrous iron at low temperatures. Biotechnol Bioeng
2007;97:14708.
Liljeqvist M. Genomics, physiology and applications of cold tolerant
acidophiles. PhD thesis, Umea University, Sweden, 2012.
Liljeqvist M, Rzhepishevska OI, Dopson M. Gene identification and substrate regulation provides insights into sulfur accumulation during bioleaching with the psychrotolerant acidophile Acidithiobacillus ferrivorans. Appl Environ Microb
2013;71:9517.
Liljeqvist M, Sundkvist J-E, Saleh A, et al. Low temperature removal of inorganic sulfur compounds from mining process
waters. Biotechnol Bioeng 2011a;108:12519.
Liljeqvist M, Valdes J, Holmes DS, et al. Draft genome of the psychrotolerant acidophile Acidithiobacillus ferrivorans SS3. J Bacteriol 2011b;193:43045.
Ludecke C, Reiche M, Eusterhues K, et al. Acid-tolerant
microaerophilic Fe(II)-oxidizing bacteria promote Fe(III)accumulation in a fen. Environ Microbiol 2010;12:281425.
Maeda K, Imae Y. Thermosensory transduction in Escherichia coli:
inhibition of the thermoresponse by L-serine. Proc Natl Acad
Sci USA 1979;76:915.
Mangold S, Valdes J, Holmes DS, et al. Sulfur metabolism in
the extreme acidophile Acidithiobacillus caldus. Front Microbiol
2011;2:17.

Mannist
o MK, Rawat S, Starovoytov V, et al. Terriglobus saanensis
sp. nov., an acidobacterium isolated from tundra soil. Int J
Syst Bacteriol 2011;61:18238.
Marchler-Bauer A, Zheng C, Chitsaz F, et al. CDD: conserved
domains and protein three-dimensional structure. Nucleic
Acids Res 2013;41:D34852.
Margesin R, Miteva V. Diversity and ecology of psychrophilic
microorganisms. Res Microbiol 2011;162:34661.
Martins LO, Huber R, Huber H, et al. Organic solutes in hyperthermophilic Archaea. Appl Environ Microb 1997;63:896902.
Methe BA, Nelson KE, Deming JW, et al. The psychrophilic
lifestyle as revealed by the genome sequence of Colwellia psychrerythraea 34H through genomic and proteomic analyses.
Proc Natl Acad Sci USA. 2005;102:109138.

11

12

FEMS Microbiology Ecology, 2015, Vol. 91, No. 4

Valdes J, Quatrini R, Hallberg K, et al. Draft genome sequence

of the extremely acidophilic bacterium Acidithiobacillus caldus ATCC 51756 reveals metabolic versatility in the genus Acidithiobacillus. J Bacteriol 2009;191:
58778.
Vera M, Schippers A, Sand W. Progress in bioleaching:
fundamentals and mechanisms of bacterial metal
sulfide oxidationpart A. Appl Microbiol Biot 2013;97:
752941.
Weber MHW, Marahiel MA. Coping with the cold: the cold shock
response in the soil bacterium Bacillus subtilis. Philos T R Soc
B 2002;357:895907.
Wisotzkey JD, Jurtshuk P, Fox GE, et al. Comparative sequence
analyses on the 16S Ribosomal-RNA (RDNA) of Bacillus acidocaldarius, Bacillus acidoterrestris, and Bacillus cycloheptanicus
and proposal for creation of a new genus, Alicyclobacillus gen.
nov. Int J Syst Bacteriol 1992;42:2639.
Yamanaka K, Inouye M. Growth-phase-dependent expression of
cspD, encoding a member of the CspA family in Escherichia
coli. J Bacteriol 1997;179:512630.
Yelton AP, Comolli LR, Justice NB, et al. Comparative genomics in
acid mine drainage biofilm communities reveals metabolic
and structural differentiation of co-occurring archaea. BMC
Genomics 2013;14:485.

Downloaded from http://femsec.oxfordjournals.org/ by guest on March 2, 2016

Sato N, Tachikawa T, Wada A, et al. Temperature-dependent

regulation of the ribosomal small-subunit protein S21
in the cyanobacterium Anabaena variabilis M3. J Bacteriol
1997;179:706371.
Sharon I, Banfield JF. Genomes from metagenomics. Science
2013;342:10578.
Shivaji S, Prakash JSS. How do bacteria sense and respond to low
temperature? Arch Microbiol 2010;192:8595.
Tatusov RL, Natale DA, Garkavtsev IV, et al. The COG database:
new developments in phylogenetic classification of proteins
from complete genomes. Nucleic Acids Res 2001;29:228.
Tehei M, Zaccai G. Adaptation to extreme environments: macromolecular dynamics in complex systems. Biochim Biophys
Acta 2005;1724:40410.
Topanurak S, Sinchaikul S, Sookkheo B, et al. Functional proteomics and correlated signaling pathway of the thermophilic bacterium I TLS33 under cold-shock stress. Proteomics 2005;5:445671.
Tyson GW, Chapman J, Hugenholtz P, et al. Community structure and metabolism through reconstruction of microbial
genomes from the environment. Nature 2004;428:3743.
Valdes J, Pedroso I, Quatrini R, et al. Acidithiobacillus ferrooxidans
metabolism: from genome sequence to industrial applications. BMC Genomics 2008;9:597.