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Environmental Microbiology Reports (2012)
doi:10.1111/j.1758-2229.2012.00389.x
The genome and transcriptome of a newly described
psychrophilic archaeon, Methanolobus psychrophilus
R15, reveal its cold adaptive characteristics
Zijuan Chen,1† Haiying Yu,2† Lingyan Li,1
Songnian Hu2 and Xiuzhu Dong1*
1
State Key Laboratory of Microbial Resources, Institute
of Microbiology, Chinese Academy of Sciences. No. 1
West Beichen Road, Beijing 100101, China.
2
Key Laboratory of Genome Science and Information,
Beijing Institute of Genomics, Chinese Academy of
Sciences, Beijing 100029, China.
Summary
We analysed the cold-responsive gene repertoire for
a psychrophilic methanogen, Methanolobus psychrophilus R15 through genomic and RNA-seq
assayed transcriptomic comparisons for cultures at
18°C (optimal temperature) versus 4°C. The differences found by RNA-seq analysis were verified using
quantitative real time-PCR assay. The results showed
that as in the Antarctic methanogen, Methanococcoides burtonii, genes for methanogenesis, biosynthesis and protein synthesis were all downregulated
by the cold in R15. However, the RNA polymerase
complex was upregulated at cold, as well as a
gene cluster for a putative exosome complex, suggesting that exosome-mediated RNA decay may be
cold-accelerated. Unexpectedly, the chaperonin
genes for both thermosome and GroES/EL were all
upregulated at 4°C. Strain R15 possessed eight
protein families for oxygen detoxification, including
both anaerobe-specific superoxide reductase (SOR)
and the aerobe-typical superoxide dismutase (SOD)catalase oxidant-removing system, implying the
higher oxidative tolerance. Compared with a mesophilic methanogen, R15 survived in higher paraquat,
a redox-cycling drug. Moreover, 71 one-component
systems and 50 two-component systems for signal
transduction ranked strain R15, together with M. burtonii, as being highly adaptive among archaea. Most
of them exhibited cold-enhanced expression, indicating their involvement in cold adaptation. This study
Received 29 October, 2011; revised 30 June, 2012; accepted 23
August, 2012. *For correspondence. E-mail [email protected]; Tel.
(+86) 10 6480 7413; Fax (+86) 10 6480 7429. †Z.C. and H.Y. contributed equally to this work.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd
has added new perspectives on the cold adaptation
of methanogenic archaea.
Introduction
Though far fewer archaeal species have been cultured
compared with bacteria, they are ubiquitous on Earth
(Schleper et al., 2005). The significant environmental
impacts of Archaea, such as in driving biogeochemical
processes and recycling environmental materials, can be
predicted based on their abundance in diverse environments. Cold regions, like the polar and alpine regions,
constitute about 75% of our planet’s environments (Cavicchioli, 2006), and cold-adapted microorganisms are predicted to play pivotal roles in material recycling processes
in cold environments.
Microbial cold adaptive mechanisms not only provide
insights into how life survives (or bio-macromolecules
implement cellular processes) below the optimal physiological temperatures, even in a water-frozen environment, but also provide diverse applications of
psychrophile-based biotechnology, such as cold active
enzymes (Cavicchioli et al., 2002). What we know about
cold adaptive mechanisms comes mainly from bacteria,
as the scarcity of cultured psychrophilic archaea (Cavicchioli, 2006). The few cultured psychrophilic archaeal
strains are mainly methanogens, because they are amenable to laboratory cultivation. This makes methanogens
a good model for the study of archaeal cold adaptive
mechanisms. Using Methanococcoides burtonii, a methanogen isolated from Ace Lake in Antarctica (Franzmann
et al., 1992), as a model archaeal psychrophile, extensive
studies on the cold adaptive characteristics have been
carried out (Thomas et al., 2000; Saunders et al., 2003;
Nichols et al., 2004; Allen et al., 2009; Williams et al.,
2010; Campanaro et al., 2011). Similar to bacteria, the
cold adaptive mechanisms of M. burtonii belong to the
following cellular processes: cellular membrane-lipid
unsaturation to increase the membrane fluidity (Nichols
et al., 2004); protein translation apparatus (Thomas et al.,
2001); TRAM-domain protein in assistance of RNA folding
at low temperature (Giaquinto et al., 2007), and modification of tRNA (Noon et al., 2003).
In this study, we present the cold adaptation-related
gene repertoire and the regulation of expression for
2
Z. Chen et al.
another methanogenic archaeon, Methanolobus psychrophilus R15 (JCM 14818T). Strain R15 was isolated
from a freshwater wetland: the Zoige wetland of the Tibetan
Plateau (Zhang et al., 2008). It grows at 0–25°C, with
highest growth rate at 18°C. Phylogenetically, strain R15 is
a member of the genus Methanolobus (Zhang et al., 2008),
while M. burtonii belongs to the genus Methanococcoides,
both are affiliated with the family Methanosarcinaceae.
Physiologically, R15 resembles M. burtonii in performing
obligately methylotrophic methanogenesis. On the basis of
the genomic and transcriptomic data, we found that strain
R15 exhibited similar cold adaptive mechanisms to M. burtonii in terms of the general cold-responsive cellular components and fundamental cellular processes. However, in
addition, some different cold-related protein categories
were observed for R15, such as the possible involvement
of chaperonins, and diverse and redundant protein families
for antioxidation, which may be involved in the coldadaptation of this methanogen.
Results and discussion
Overview of the genome and cold responsive
gene expression
Methanolobus psychrophilus R15 is one of the few psychrophilic methanogens to be cultured to date, enabling a
comprehensive study of its genome with the aim of understanding the archaeal cold adaptation mechanisms at the
genetic level. As shown in Table S1, the genome of Methanolobus psychrophilus R15 is composed of a circular
chromosome of 3 072 769 bp, with a GC content of 44.6%.
Open reading frames (ORFs) were identified using
Glimmer 3.02 and GeneMark, then the auto-annotation
was manually revised from the output of the NCBI Microbial
Genome Submission Check website (http://www.ncbi.nlm.
nih.gov/genomes/frameshifts/frameshifts.cgi), eventually
four genes were identified as pseudogenes. A total of 3167
ORFs were predicted in the genome. Based on the transcriptional single nucleotide coverage data, the start
codons of 13 genes were modified. The protein sequences
were used for BLAST searched against the non-redundant
protein database (nr) at NCBI and Clusters of Orthologous
Groups (COGs) database. The highest scoring alignment
with the E-value < 10-5 was selected as significant match.
In total, 2611 ORFs had significant matches in the nonredundant protein database, 556 ORFs were predicted to
be unknown function. Metabolic pathways were constructed using the KEGG database. The sequence data
have been submitted to the GenBank under accession
number CP003083.
Global transcriptome analysis confirmed that more than
90% of the ORFs of strain R15 were expressed under the
growth conditions in this study. The transcriptome data
also revealed that the actual translation start site of 13
ORFs were located downstream of the predicted translation start site. Using operon prediction tools and a contiguous sequence coverage obtained from RNA-seq, 458
operons comprising 1697 genes were identified in the
genome (Table S2). In total, 1295 genes were expressed
significantly differentially (by over twofold) in cultures at
4°C versus 18°C, of which 944 genes were upregulated
and 351 were downregulated (Table S1). Furthermore, we
used relative quantitative real-time PCR (qRT-PCR)
assay to verify the 36 differential expressed genes (fourfold to 10-fold changes) found by RNA-seq. The assay for
two batches of R15 cultured at 4°C and 18°C indicated
that 30 out of the 36 genes were shown the different
expressions consistent with those detected by RNA-seq
(Fig. S1, Table S3). The correlation coefficient (r-value)
between RNA-seq and qRT-PCR data are calculated as
0.80, while the qRT-PCR data from the two biological
replicates are 0.89 (Fig. S1). This indicates the high reliability of the RNA-seq data.
Figure 1 showed the depicted cold-responsive gene
categories that essentially function for cellular processes,
those were discussed in the following sections.
Metabolism and biosynthesis
As shown in Table S4, except for a few of corrinoid proteins, the majority of the genes for methylotrophic methanogenesis showed reduced expression at 4°C, including
those involved in both the methylotrophic pathway and its
oxidative branch. Methylotrophic corrinoid proteins have
been reported in response to oxidation (Ferguson et al.,
2009), the upregulation of them can be an indicative that
strain R15 was under a higher oxidative stress at lower
temperature as mentioned in antioxidation genes section.
Genes for electron transfer, such as the fpo operon and
the rnf operon, were also concomitantly downregulated in
cold conditions. The reductive acetyl-CoA pathway is
commonly used by methanogens for biosynthesis, and
the genes involved in this pathway all exhibited significantly decreased expression at 4°C (Table S4). This is in
agreement with the reduced growth as well as decreased
methanogenesis of strain R15 at lower temperatures
(Zhang et al., 2008).
Protein synthesis and post-translational processing
As in the cold-adaptive methanogen M. burtonii (Thomas
et al., 2000), most components of the protein translation
apparatus in strain R15, such as the super operon
Mpsy_1124–1146 encoding 23 subunits of ribosome,
were downregulated by cold conditions, except for some
translation initiator factors (Table S5). Protein degradation
process seemed to be cold-accelerated in strain R15.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
Cold adaptation of a psychrophilic methanogenic archaeon
3
Fig. 1. Depicted cold-responsive gene categories that essentially function for cellular processing. Icons for genes in red and blue refer to
those upregulated and downregulated at 4°C respectively. Gene expression levels were calculated by the RPKM function [(1e + 9)*Exon#/
ExonLen*Total Unique Mapped Reads Number], and normalized by the total number of unique mapped reads. Differentially expressed genes
were identified using DEG-seq software (Wang et al., 2010). More details were in supplementary methods. TMA, trimethylamine; CODH/ACS,
CO dehydrogenase/acetyl-CoA synthase; Ths, thermosome; RNAP, RNA polymerase; ROS, reactive oxygen species; Anx, antioxidant; HK,
histidine kinase; RR, response regulator.
Genes both for the energy-dependent 20S proteasome
(Mpsy_1075, 2726, 2881) and membrane-associated Lon
proteases (Mpsy_0309, 0809, 3037) all displayed coldenhanced expression. This suggests that protein misfolding may also occur in the cold. As expected, the genes for
HtpX-like protease (Mpsy_0743 and Mpsy_0744), which
act as heat shock protein, did not show a cold responsive
expression.
Sixteen protein chaperone-encoding genes were found
in the R15 genome, including one copy of group I chaperonin complex (GroES/EL, Mpsy_2746, 2747) and three
copies of group II chaperonin (thermosome, Mpsy_1969,
2247, 3167); three copies of heat shock protein Hsp20
(Mpsy_0075, 0869, 2176) and a-, b-prefoldins (co-factor
of thermosome, Mpsy_0490, 2735); and two copies of
chaperone DnaJ (Mpsy_2251, 2842), one copy of DnaK
(Mpsy_2550), and chaperone ClpB (Mpsy_2749).
Surprisingly, most of the chaperone genes in R15 were
upregulated at 4°C (Table S5), including DnaK, DnaJ,
GroES/EL, thermosome, prefoldin, ClpB and many small
heat shock proteins. The cold enhanced expression
of the three copies of thermosome and one copy of
GroEL were also confirmed through qRT-PCR assays.
Cold enhanced expression of GroES/EL genes is also
found in a marine bacterium Sphingopyxis alaskensis
(Ting et al., 2010). Protein folding seems as a ratelimiting step for the growth of psychrophiles, and overexpression of GroES/EL genes from Oleispira antarctica
RB8T in E. coli made it grow well at 8°C (Piette et al.,
2011). Therefore, the R15 chaperones are predicted to
play a role in assisting protein-folding at low temperatures. In comparison, M. burtonii does not possess
GroES/EL genes, while its chaperone genes, such as
DnaK, DnaJ and thermosome, are all downregulated
(Williams et al., 2011).
It was also found that psychrophilic bacteria use different protein chaperones in responding to cold, e.g. when
growing in cold, Pseudoalteromonas haloplanktis overexpressed trigger factors, the chaperones binding to
nascent peptides, whereas repressed other chaperones
(such as GroES/EL), which binding to the proteins had
been folded by trigger factors (Piette et al., 2011).
However, in Psychrobacter arcticus 273–4, GroES/EL
exhibited higher expression, while the trigger factors
decreased the expression by the cold. In contrast, all the
protein chaperones in R15 showed higher expression by
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
4 Z. Chen et al.
the cold, implying a distinct way in protein folding in
responding to cold.
RNA transcription and processing
As in the marine bacterium Sphingopyxis alaskensis (Ting
et al., 2010), but in contrast to M. burtonii (Williams et al.,
2011), most of the genes for RNA polymerase (RNAP)
subunits of R15 (Mpsy_0880, 0971, 1005–1009, 2259,
2331, 2732, 2774) were upregulated at 4°C (Table S6),
except for RNAP subunit D (Mpsy_1111) and subunit N
(Mpsy_0879). However, subunit D was shown upregulated at cold in qRT-PCR assay. R15 encodes one copy of
transcription initiation factor B (TFB) (Mpsy_0890), TATA
box binding protein (TBP) (Mpsy_1517), and homologues
of bacterial transcription elongation factors NusA
(Mpsy_1011) and NusG (Mpsy_1497), and they all
showed cold-enhanced expression. Bell and Jackson
(2001) reported that the archaeal TFB, TBP and RNAP
efficiently initiated the transcription of a wide range of
promoters in vitro. qRT-PCR also verified the upregulation
of the genes for NusG, TBP, and most subunits of RNAP
(Table S6) in R15. The TBP expression of M. burtonii was
also enhanced at cold (Williams et al., 2011). The coldenhanced expression of RNAP genes could be explained
by that more RNA synthase are produced to compensate
for the lower enzymatic activities in cold.
In addition, genes for a putative exosome complex
(Mpsy_2728–2730) in R15 were upregulated at 4°C
(Table S6), resembling M. burtonii under cold stress at
-2°C (Williams et al., 2011). Polynucleotide phosphorylase (PNPase), the exosome homologue of Escherichia
coli and Bacillus subtilis, also showed a cold-induced
expression; while the pnp deletion strain was sensitive to
cold, hence determined that PNPase was essential for the
organisms to grow in low temperatures (Luttinger et al.,
1996; Awano et al., 2008). Further research indicated that
the cold-sensitive phenotype was related to the RNase
activity of PNPase. These suggest that exosomemediated RNA decay in R15 is accelerated at low temperature; or more exosomes are needed to compensate
for the reduced enzymatic activities at cold.
DEAD-box RNA helicases act in unwinding and clamping duplex RNAs, and they have also been shown to aid
bacterial survival at low temperatures by removing unnecessary secondary structures of RNA to facilitate translation (Jones et al., 1996). Four putative DEAD-box RNA
helicase genes were found in the genome of strain R15,
and three of them were up regulated at 4°C (Table S6),
implying their involvement in the cold response of R15.
Evguenieva-Hackenberg and colleagues (2003) reported
that a DEAD-box RNA helicase was found to be
co-purified with the exosome subunits in a member of the
Crenarchaeota, Sulfolobus solfataricus, implying that the
DEAD-box RNA helicases have a role in RNA decay in
synergy with the RNA degradation machinery. Lorentzen
and Conti (2006) reported that archaeal exosome
complex only degraded the RNAs without secondary
structure, because of the narrow channel (about 8–10 Å)
of the catalytic centre. Hence, it is reasonable to predict
the DEAD-box RNA helicases in assisting the decay of
aberrant RNAs by unwinding RNA, making it enter the
catalytic centre of the exosome. In addition, a pair of
GroES/EL genes (Mpsy_2746 and Mpsy_2747) are clustered with a putative DEAD-box RNA helicase gene
(Mpsy_2748) in R15, and they were equivalently upregulated at 4°C. Thus suggests that the chaperonin of strain
R15 may also act as the RNA chaperones in cooperation
with DEAD box RNA helicases in removing the aberrant
RNAs when cells are under cold stress. Chaperones work
as RNA-binding proteins have been reported in Sulfolobus solfataricus (Ruggero et al., 1998), and GroEL was
also found as a component of an mRNA protection
complex in E. coli (Georgellis et al., 1995).
We did not find any cold shock protein (Csp) or cold
shock domain containing protein in the genome of R15,
but four TRAM-domain proteins. Three of the TRAMdomain proteins (Mpsy_0643, Mpsy_3043, Mpsy_3066)
showed obviously upregulation at cold. Williams and colleagues (2010; 2011) found that three proteins, each with
a single TRAM domain were upregulated at cold. Anantharaman and colleagues (2001) reported that TRAM as a
predicted RNA-binding domain commonly presents in
RNA-modifying enzymes and other proteins associated
with translation machinery. In addition, the low molecular
weights of the R15 TRAM-domain proteins were in the
accordance of that of the bacterial Csp proteins. Therefore, it is reasonable to hypothesize that TRAM domain
proteins function as a kind of RNA chaperone analogue to
bacterial Csps.
Antioxidation genes
Methanogens are categorized as oxygen-hypersensitive
organisms and do not use oxygen molecules as the terminal electron acceptor for respiration; therefore, no
metabolic reactive oxygen species (ROS) would be generated. However, those living in the cold conditions may
encounter chemical ROS, because more dissolved O2
can be anticipated at low temperatures, as oxygen
content is about 2.55 ml l-1 higher at 4°C than at 18°C in
pure water under one atmosphere (Weiss, 1970). In addition, reduced respiration of the coexisted aerobes also
leads to the increase of oxygen in the cold environments;
because of reduced ATP demand at low temperature,
electron accumulation in respiration chain may also
increase the possibility of ROS production (Williams
et al., 2011). In conclusion, psychrophilic methanogens
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
Cold adaptation of a psychrophilic methanogenic archaeon
have to develop more antioxidant pathways to cope with
the higher oxygen. As seen in Table S7, compared with
the most of thermophilic methanogens, R15 and M. burtonii (Williams et al., 2011) possess diverse variety and
redundancy of proteins involved in antioxidation, which
includes eight and six different families of proteins
respectively.
Remarkably, in addition to the superoxide reductase
(SOR)-dependent anaerobic oxides-decomposition pathway, strain R15 carries an aerobe-characteristic ROSremoving mechanism, which comprises one copy of superoxide dismutase (SOD) (Mpsy_1546) and three copies of
catalase (Mpsy_0021, 0349 and 1225) (Table S8), while
M. burtonii is absence of SOD gene and presence of
only one copy of catalase. Except for Mpsy_0021, the
remaining two catalase genes and one SOD gene were
apparently upregulated (by twofold to fourfold) at 4°C.
The upregulation of these genes were confirmed by qRTPCR assays (Table S3).We did detect marked catalase
activity for strain R15, but for a comparison, not for Methanosaeta hurandinaceae 6Ac, a mesophilic methanogen
isolated from a waste water treatment reactor. Superoxide
reductase (SOR) is a type of ROS decomposing protein
that is ubiquitously present in anaerobes (Jenney et al.,
1999). Rubrerythrin, a non-haem peroxidase, performs
multiple functions, such as a rubredoxin: peroxide oxidoreductase in concert with the action of SOR. Strain R15
possesses two SOR genes (Mpsy_1707 and Mpsy_2711)
and three rubrerythrin genes (Mpsy_1053, Mpsy_1872
and Mpsy_2023), and they were all upregulated (by
twofold to sevenfold change) at 4°C (Table S8), suggesting
that the methanogen is with a high potential to cope with
the increased oxidative stress in cold.
By chelating Fe2+, the protein bacterioferritin and DNAbinding proteins from starved cells (Dps) prevent Fenton
reaction, a Fe2+-facilitated chemical reaction to convert
H2O2 to •OH (Zhao et al., 2002). One of the two bacterioferritin genes (Mpsy_1055) of R15 was dramatically
upregulated (13.64 times) at 4°C, providing another clue
that this methanogen may encounter oxidative stress in
the cold.
An F420H2 oxidase-encoding gene (Mpsy_2473) was
found in the R15 genome, which exhibits the highest
amino acid sequence similarity to that of the rhizospheric
methanogen, RC-I (Erkel et al., 2006). Mpsy_2473 displayed 11.41-fold increased expression at 4°C compared
with that at 18°C, implying that it plays a role in coping
with O2 or responding to lower temperature. As F420H2
was only found in methanogens and sulfate-reducing
archaea up to date (Seedorf et al., 2007), F420H2 oxidase
can be regarded as the archaeon-specific oxygen detoxification pathway. Genes encoding haemethrythrin of
R15 (Mpsy_0022, Mpsy_2017, Mpsy_2467), an O2 carrier
protein (Stenkamp, 1994), also showed remarkably
5
upregulation at cold. In comparison, M. burtonii contains
less oxygen detoxification genes and without those for
SOD or F420H2 oxidase. The difference in oxygen detoxification gene categories and redundancy between the two
psychrophilic methanogens can be attributed to their habitats. M. burtonii inhabits the methane-saturated Ace Lake,
whereas strain R15 lives in the air-infiltrated rhizosphere.
The multiple antioxidative pathways in R15 were summarized as depicted in Fig. 2A. Furthermore, ROS-stress
assay showed that M. psychrophilus R15 survived up to
0.8 mM paraquat (Fig. 2B), a redox-cycling drug. In parallel, M. harundinacea 6Ac was tolerant to as low as
0.05 mM paraquat, thus supporting the hypothesis of the
genetically endowed antioxidation capability of R15.
Diverse signal transduction systems
Bacteria have evolved multiple signal transduction
systems in response to environmental cues, and the
number or fraction of them in the genome can be a
measure of a bacterial IQ, i.e. their ability to adapt to their
environment (Galperin, 2005). To investigate the correlation of the abundance of signal transduction systems with
the methanogens response to temperature change, we
performed an exhausted survey of the available genome
sequences for the archaea growing at various temperatures, including 50 strains from 50 genera; those comprise
psychrophiles (2 strains), mesophiles (20 strains), thermophiles (9 strains) and hyperthermophiles (19 strains).
Figure 3 and Table S9 shows that, while the proportion of
one-component systems (OCS) are about the same
among the genomes of all the archaea (mean values and
standard deviations are as follows: psychrophiles,
2.5% ⫾ 0.004; mesophiles, 3.3% ⫾ 0.007; thermophiles,
3.0% ⫾ 0.005; hyperthermophiles, 2.7% ⫾ 0.006), the
two-component system (TCS) show different situation.
Generally, a higher ratio of TCS genes is present in the
archaeal genomes of psychrophiles (1.8% ⫾ 0.002) and
mesophiles (1.4% ⫾ 0.017). None or very few TCSs were
found in the 28 thermophilic or hyperthermophilic archaea
except for two strains, Methanothermobacter marburgensis str. Marburg and Archaeoglobus fulgidus DSM 4304.
TCSs were abundantly present in the two psychrophilic
methanogens, by M. burtonii having 45 TCSs (2.0% of
total genes) (Allen et al., 2009), and strain R15 containing
50 TCSs [40 histidine kinases and 10 response regulators
(RRs), 1.6% of total genes]. This manifests the psychrophiles being a higher IQ archaeal group. Transcriptome analysis (Table S10) showed that the majority OCS
genes (68.9%) of R15 showed higher expression at 4°C.
Similarly, 64.7% (30) of the TCS genes of R15 were
upregulated at cold as well. These results suggested that
both OCSs and TCSs are involved in the cold adaptation
of strain R15.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
6
Z. Chen et al.
Fig. 2. Three pathways for detoxification of oxygen predicted for Methanolobus psychrophilus R15 (A) and its survived paraquat levels (B).
Paraquat was added to a 24 h culture of strain R15 at 18°C and 6Ac at 37°C to the indicated final concentrations. The cultures continued to
be incubated at 18°C and 37°C. CH4 yields were measured at intervals of 24 h until the late exponential growth phase. Each experiment was
performed in triplicate for two batches of cultures. ROS, reactive oxygen species; SOR, superoxide reductase; Prx, peroxidase; SOD,
superoxide dismutase; CAT, catalase; •OH, hydroxyl radical; O2-, superoxide radical; FprA, F420H2 oxidase; Rbr, rubrerythrin; BFR,
bacterioferritin; FR, ferritin; Dps protein, DNA-binding proteins from starved cells; Ahp, alkylhydroperoxidase like protein.
Fig. 3. Abundance of signal transduction systems distributed in the genomes of the archaea growing at various temperatures. OCS,
one-component system; TCS, two-component system; The genes encoding signal transduction systems in strain R15 were identified based on
domain analysis by InterProScan. The categorization of the histidine kinase, response regulator and one-component system was described in
supplementary methods (Supporting information). The signal transduction systems of other archaea, those used in comparison with R15, were
cited from http://mistdb.com/.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
Cold adaptation of a psychrophilic methanogenic archaeon
To gain an insight into the diversity of OCSs and TCSs
in the R15 genome, we performed a thorough analysis of
their domain architectures. As shown in Table S10 that the
OCSs have more diverse regulator domain types than the
TCSs in R15. The 21 varieties of DNA binding domains of
the OCSs are mainly categorized to those families containing the following DNA binding domains: AsnC_trans_
reg (an autogenously regulated activator of asparagine
synthetase A), HTH_3 (mainly falling in the XRE family
transcriptional regulators), HTH_5 (mainly falling in ArsR
family), MarR (multiple antibiotic resistance regulator),
TrmB (falling in sugar-specific regulator for trehalose ABC
transporter and GntR family) and other winged helix DNAbinding domains.
Fifty TCSs were screened in the R15 genome
(Table S10), of which 10 were hybrid TCSs (mostly cytoplasmic proteins) and 30 were sensor histidine kinases
(sHK). However, only 10 RRs were found, similar to the
ratios of sHK to RR in other archaeal genomes. Notably,
the CheY-like domain is the only type of RR of the TCSs.
The characteristic structures of the sHK proteins of R15
indicate that most of them are large, with hybrid domains,
a hint of the ‘promiscuity’ of the archaeal proteins. The
majorities of sHK proteins contain a PAS/PAC-GAF-PYPlike domain sensor, and exhibit cold-responsive expression. The second most abundant dominant sensors are
the Cache and CHASE4-domain sensors. No GGDEF
domain or EAL domain phosphodiesterase was found in
the genome of R15; hence the secondary signal di-c-GMP
is not predicted.
Conclusion
This study provides an overall genomic view on how the
psychrophilic methanogen, Methanolobus psychrophilus
R15, responds to the low temperatures. As in M. burtonii,
methanogenesis and biosynthesis are all downregulated
by the cold in R15 as well. Other pathways involved in
the cold adaptation of R15 include diverse pathways and
abundant genes for detoxification of oxygen, specially
the aerobe-characteristic SOD and catalase genes, and
the relevant functions are experimentally demonstrated
for R15. Abundant TCS genes are another feature for
the psychrophiles and most of them exhibit coldenhanced expression. Genes for transcription machinery
are upregulated at cold; based on the decreased growth
and metabolism at the cold, this is assumed that more
enzyme proteins are needed to compensate the low
activities at low temperature. Genes for exosome, which
involve in RNA decay, are also upregulated at the low
temperature, suggesting an increased RNA degradation in the cold. Interestingly, we find that a set of chaperones genes, including GroES/EL complex and thermosomes, are upregulated at 4°C, implying their roles in
7
the cold adaptation, but the mechanisms remain to be
revealed.
Acknowledgements
This work was supported by the National Natural Science
foundation of China under Grant No. 30621005, 30830007
and 31000011.
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Supporting information
Additional Supporting Information may be found in the online
version of this article:
Fig. S1. Correlation analysis of RNA-seq and qRT-PCR.
A. correlation coefficient of fold changes between RNA-seq
and qRT-PCR.
B. correlation coefficient of fold changes between two
batches of qRT-PCR.
Table S1. Overview of the genome of R15 and the expression profiles of the genes.
Table S2. Operon map of Methanolobus psychrophilus R15.
Table S3. Verification of the transcriptomic data of 16 genes
using qRT-PCR.
Table S4. Differential transcript abundance of the genes for
methanogenesis and biosynthesis from the cultures growing
at 4°C versus 18°C.
Table S5. Differential transcript abundance of genes for
protein translation and processing from the cultures growing
at 4°C versus 18°C.
Table S6. Differential transcript abundance of genes for transcription and RNA processing from the cultures growing at
4°C versus 18°C.
Table S7. Distribution of the oxygen detoxification genes in
the available methanogen genomes.
Table S8. Differential transcript abundance of the genes for
antioxidation from the cultures growing at 4°C versus 18°C.
© 2012 Society for Applied Microbiology and Blackwell Publishing Ltd, Environmental Microbiology Reports
Cold adaptation of a psychrophilic methanogenic archaeon
Table S9. Signal transduction systems distributed in the
genomes of the archaea growing at various temperatures.
Table S10. Signal transduction systems in the genome of
strain R15 and their temperature-responsive expression.
9
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