Abstract
Acidithiobacillus ferrivorans is an acidophile that often occurs in low
temperature acid mine drainage, e.g., that located at high altitude.
Being able to inhabit the extreme environment, the bacterium must
possess strategies to copy with the survival stress. Nonetheless,
information on the strategies is in demand. Here, genomic and
transcriptomic assays were performed to illuminate the adaptation
mechanisms of an A. ferrivorans strain YL15, to the alpine acid mine
drainage environment in Yulong copper mine in southwest China. Genomic
analysis revealed that strain has a gene repertoire for
metal-resistance, e.g., genes coding for the mer operon and a variety
of transporters/efflux proteins, and for low pH adaptation, such as
genes for hopanoid-synthesis and the sodium:proton antiporter. Genes
for various DNA repair enzymes and synthesis of UV-absorbing
mycosporine-like amino acids precursor indicated hypothetical UV
radiation—resistance mechanisms in strain YL15. In addition, it has two
types of the acquired immune system–type III-B and type I-F CRISPR/Cas
modules against invasion of foreign genetic elements. RNA-seq based
analysis uncovered that strain YL15 uses a set of mechanisms to adapt
to low temperature. Genes involved in protein synthesis, transmembrane
transport, energy metabolism and chemotaxis showed increased levels of
RNA transcripts. Furthermore, a bacterioferritin Dps gene had higher
RNA transcript counts at 6°C, possibly implicated in protecting DNA
against oxidative stress at low temperature. The study represents the
first to comprehensively unveil the adaptation mechanisms of an
acidophilic bacterium to the acid mine drainage in alpine regions.
Introduction
Acid mine drainage as a typical extreme environment is associated with
metal or coal mines and derelict mines. It is often highly acidic
(typically pH<3) and usually contain elevated concentrations of zinc,
copper and a variety of other heavy metals [[48]1]. Acid mine drainage
is common on the earth. It is distributed among mine sites with
distinct climatic conditions, even those mainly characterized by low
temperatures. For instance, a number of mine sites are located at high
altitudes and latitudes, of which the temperatures are below 10°C for
the majority of the year [[49]2,[50]3].
The harsh conditions in low temperature acid mine drainage inhibit
growth of most organisms. Despite the harshness, some microorganisms
can survive in this extreme environment, one of which is the
gammaproteobacterium Acidithiobacillus ferrivorans [[51]4]. The
bacterium accounts for a considerable part in cold mine-affected water
bodies. The species can grow at subzero temperatures and has a fastest
growth temperature around 30°C. According to a recent conception, all
microorganisms that are indigenous to cold environments are
psychrophiles [[52]5]. Therefore, the species A. ferrivorans should be
psychrophilic rather than psychrotolerant as it was formerly considered
to be [[53]4].The species is an iron- and sulfur-oxidizing,
diazotrophic, obligate chemoautotroph. It was once regarded as
cold-adapted A. ferrooxidans, however, it differs from A. ferrooxidans
in their cell motility, tolerance to low temperature and responses to
pH [[54]4]. Apart from the physiological aspects, its ferrous and
reduced inorganic sulfur compounds oxidation pathways have been well
discussed [[55]6,[56]7]. The species is implicated in biomining for the
recovery of metals from sulfide minerals at low temperatures and this
has been studied [[57]8]. Christel et al. [[58]9] found that when using
potassium tetrathionate as an energy source, the A. ferrivorans strain
SS3 had little RNA transcript response related to cold stress and thus
it was concluded that the strain is adapted to growth at 8^°C.
Though there are some studies on the microbial adaptation mechanisms to
acid mine drainage, e.g. reports by Hua et al. [[59]10] and Liljeqvist
et al. [[60]11], few has been conducted on microorganisms in acid mine
drainage environments in alpine regions at a strain-level, e.g. those
located in High Andes in Chile [[61]3] and Tibet Plateau in China,
which is characterized by low temperature and elevated UV radiation
(UVR). Microbial adaptation to environment associates with a variety of
biological processes. A large number of genes are implicated in these
processes. Therefore, it is difficult to barely explain the mechanisms
by traditional culture-dependent or modern molecular biological
approaches. The advent of high-throughput next-generation sequencing
(NGS) technology now affords new opportunities to address the knowledge
gaps by comprehensively characterizing the genes and processes that are
involved in the adaptation by microorganisms to their habitats. For
instance, comparative transcriptomic analysis reveals the adaptation of
microbial communities to acid mine drainage in south China [[62]12]. We
presented here the adaptation-related gene repertoire of an A.
ferrivorans strain YL15 isolated from acid mine drainage in an alpine
copper mine, using genomic and transcriptomic methods. In particular,
we focused on the potential strategies the strain uses to cope with the
abiotic and biotic constraints of its natural habitats including
extremely acidic pH, high metal ion concentrations, UVR, low
temperature and intrusion of extraneous genetic elements.
Materials and methods
Strain and culture conditions
Strain YL15 was isolated from acid mine drainage in Yulong copper mine
in Tibet, China. The sampling site was located at an altitude of about
4,600 meters (31°21’34'‘N, 97°46’57”E), and the physicochemical
properties of the acid mine drainage was listed in [63]S1 Table. No
specific permissions were required for activities in Yulong copper
mine, because the mine is available to the public. The sampling did not
have any impact on the local environment and this field study did not
involve endangered or protected species. The strain was isolated using
FeTSB solid medium as described previously [[64]13]. It has a fastest
growth at 28°C when ferrous sulfate is used as an energy source. It was
grown routinely at pH of 2.0 and temperatures of 28°C and 6°C in shake
flasks at 160 rpm. In this study, we selected 6°C as the low
temperature because it is a typical temperature occurred in Yulong
copper mine. The culture medium was 9K [[65]14] and filtration
sterilized ferrous sulfate was supplied at a concentration of 50 mM.
Nucleic acid extraction
For DNA extraction, strain was cultured at 28°C until it entered the
mid logarithmic phase. Bacterial cells were harvested by centrifugation
at 10,000x g for 10 min. The pelleted cells were washed twice using
diluted sulphuric acid (pH 2.0). Genomic DNA was extracted and purified
from the washed cells using TIANamp Bacteria DNA kit (TIANGEN, Beijing,
China) as per the manufacturer’s instructions and finally suspended in
TE buffer. The genomic DNA was quantified by ethidium bromide-UV
detection on an agarose gel and stored in -80°C until used for genome
sequencing.
As for RNA extraction, strain was respectively cultured at 28°C and 6°C
(designated as S28 and S6). When cells entered the mid logarithmic
phase at 42h (for S28) and 144h (for S6) respectively ([66]S1 Fig), the
cultures were rapidly cooled and harvested by centrifugation at 10,000x
g for 5 min at 4°C. Total RNA was extracted by using Total RNApure kit
(Zoman, Beijing, China) according to the manufacturer’s instructions.
Trace genomic DNA was digested using DNase I. The quality of total
extracted RNA was confirmed by 1% agarose gel electrophoresis and
quantified using a NanoDrop ND-1000 Spectrophotometer (NanoDrop
Technologies, Wilmington, USA).
Genome sequencing, assembly and annotation
The purified genomic DNA sample was used to construct a shotgun library
with an average insert size of ~300 bp. The tagging and fragmentation
of genomic sample, indexing and PCR amplification, PCR clean-up,
library normalization and pooling were conducted using the Illumina
Nextera XT DNA Sample Preparation Kit (Illumina, California, USA) as
per the manufacturer’s instructions. The library was then sequenced
(250 bp paired-end reads) using the Illumina MiSeq sequencing platform
(Illumina, California, USA). The raw reads were assembled into contigs
using SOAPdenovo2 package [[67]15]. The genome completeness was
estimated using the program CheckM [[68]16]. Coding sequences were
predicted with the ORF finders Glimmer [[69]17] and GeneMark [[70]18].
All CDSs were manually verified by alignment against the NCBI
non-redundant [[71]19] and COG databases [[72]20] using the BLAST
software [[73]21]. In particular, the cusCBA gene clusters were
identified by method described by González et al [[74]22]. Clustered
regularly interspaced short palindromic repeats (CRISPRs) loci were
identified using the web server CRISPRFinder [[75]23]. The tRNA and
rRNA genes were identified using the software tRNAscan-SE [[76]24] and
the webserver RNAmmer [[77]25], respectively. This Whole Genome Shotgun
project has been deposited at DDBJ/ENA/GenBank under the accession
[78]MASQ00000000. The version described in this paper is version
[79]MASQ01000000.
Genome homology and synteny analyses
The average nucleotide identity (ANI) between genomes of strain YL15
and A. ferrooxidans ATCC 23270, A. thiooxidans ATCC 19377, and the
other two A. ferrivorans strains SS3 [[80]26] and CF27 [[81]27], was
calculated using OrthoANI [[82]28]. Genome synteny analysis between
genomes of strain YL15 and A. ferrivorans strain SS3 and A.
ferrooxidans ATCC 23270 was performed using the Nucmer program in the
MUMmer package using the default parameters [[83]29].
RNA-seq and analysis of genes with significantly different RNA transcript
counts
For sequencing of YL15 mRNA, rRNA was removed from total RNA using the
Ribo-Zero Magnetic Kit (Bacteria, Epicentre Biotechnologies, Wisconsin,
USA), then the remaining RNA was employed for library construction
using the Illumina TruSeq RNA Sample Preparation Kit (Illumina,
California, USA) according to the manufacturer’s instructions. Briefly,
mRNA was fragmented into small pieces using divalent cations under
elevated temperature. The cleaved RNA fragments were then copied into
first strand cDNA using random primers and reverse transcriptase.
Second strand cDNA synthesis followed, using RNase H and DNA
polymerase. The cDNA fragments then went through an end repair process,
the addition of a single ‘A’ base to 3’ ends, and then ligation of the
adapters. The products are then purified and enriched with PCR to
create the final cDNA library. The library was then sequenced (75 bp
paired-end reads) using the Illumina MiSeq sequencing platform
(Illumina, California, USA). The raw sequencing data was deposited at
Sequence Read Archive under the accession SRP091325.
Clean data were obtained from raw data by removing reads that
containing low quality reads, adapter, and poly-N. Computational
processing and analysis of qualified reads were conducted through the
pipeline using TopHat and Cufflinks packages for identification of
genes with significantly different RNA transcript counts [[84]30]. The
qualified reads for each condition were mapped to the YL15 genome with
TopHat. The resulting alignment files were provided to Cufflinks to
generate a transcriptome assembly for each condition. Cuffmerge
program, which is included in the Cufflinks package, was used to merged
together the assemblies. Cuffdiff, another utility in the Cufflinks
package, calculated transcript expression levels, performed
differential analysis and tested the statistical significance of
observed changes (p-value). Significantly differentially expressed
genes were determined with a selection threshold of adjusted p-value ≤
0.05 and fold change of RNA transcripts ≥ 2.0 (up-regulation) or ≤ 0.5
(down-regulation). Gene ontology (GO) was implemented using TBtools
([85]https://github.com/CJ-Chen/TBtools) and WEGO [[86]31] and KOBAS
2.0 was employed to conduct KEGG pathway mapping analysis against the
KEGG background [[87]32].
Quantitative real-time PCR (qRT-PCR) to verify RNA-seq data
The extracted RNA was first retro-transcribed into cDNA with Reverse
Transcriptase (Zoman, Beijing, China) and random primers following the
manufacturer’s instructions. Real-time PCR was carried out with the
iCycler iQ Real-time PCR detection system (Bio-Rad Laboratories, USA)
as previously reported [[88]33]. Primers for selected genes were listed
in [89]S2 Table. The absolute quantification of each gene was carried
out by making standard curves. All tests were carried out in
triplicate.
Results and discussion
Genomic analysis of strain YL15
Genomic features
The draft genome sequence of strain YL15 has a total length of
2,996,582 bp, with a GC content of 56.6%. After assembly, a total of
190 contigs was created, ranging from 200 bp to 105,515 bp. Given a
99.03% genome completeness provided by CheckM, and a 123x genome
coverage, it is reasonable to infer that the majority of genes in
genome of strain YL15 were included in the current draft. Comparing to
the other two A. ferrivorans strain SS3 and CF27, the genome of YL15 is
smaller, and in particular, 12.7% shorter than the genome of CF27 in
length ([90]https://www.ncbi.nlm.nih.gov/genome). The genome has 43
tRNA genes and 2,798 protein-coding sequences, of which 1,852 were
assigned as proteins with known functions, while the rest 946 were
regarded as hypothetical proteins.
The genome has one rRNA operon, and the similarities of 16S rDNA to
those of other A. ferrivorans strains are over 99%. The ANI values
calculated by OrthoANI for strains YL15 and SS3, CF27, A. ferrooxidans
ATCC 23270 and A. thiooxidans ATCC 19377 were 96.90%, 98.42%, 84.03%
and 73.84%, respectively. According to the ANI threshold of 95% which
has been proposed for species demarcation, YL15 is affiliated to A.
ferrivorans [[91]34]. The results were consistent with that of synteny
analysis. Genome of strain YL15 has a high degree of synteny with that
of strain SS3, while few synteny regions were observed between the YL15
genome and that of A. ferrooxidans ATCC 23270 ([92]Fig 1).
Fig 1. Dot plots for synteny of YL15, A. ferrivorans SS3 and A. ferrooxidans
ATCC 23270 genomes.
[93]Fig 1
[94]Open in a new tab
In the plots, every dot indicates a match between the two genomes being
compared, with forward matches colored in red and reverse matches
colored in blue.
Metal resistance
Elevated concentrations of metal ions especially heavy metals are toxic
to microbial cells, mainly as a result of their ability to denature
protein molecules [[95]35]. In response to the toxic assault,
microorganisms have developed a set of resistance mechanisms. In
general, these mechanisms include: (i) converting the ions to less
toxic forms and then pump them out of cells; (ii) exporting the metal
ions to the periplasm and reduce them to lower oxidative or decreased
soluble states; (3) exporting the ions out of the cell entirely
[[96]36].
A large number of genes that are predicted to be involved in metal
resistance were identified in genome of strain YL15, including: i) mer
operon for mercuric resistance and regulation ([97]S3 Table); ii) genes
for arsenic resistance: an arsenate reductase (BBC27_RS06310), an
ATP-dependent chaperone gene clpB (BBC27_RS08660) and an arsRCDA operon
([98]S3 Table), Interestingly, the gene for arsenical efflux pump
membrane protein ArsB (BBC27_RS09700) is located separately from the
arsRCDA operon, which is different from the arsRDABC operon in E.coli
[[99]37]. A set of recently illustrated arsenic resistance genes
retrieved from functional metagenomic approaches were also identified
[[100]38], e.g. a phospholipid metabolism-associated gene and three
genes coding for RNA-modification enzymes ([101]S3 Table); iii) genes
for copper resistance: a gene (BBC27_RS13630) coding for a
copper-translocating P-type ATPase (CopB) related to the transport of
copper from the cytoplasm to the periplasmic space, and four clusters
of putative cusCBA genes coding for the Cus systems which transfers
copper directly to the extracellular space [[102]22,[103]39]; iv) genes
associated with major facilitator family (MFS) / multidrug /
resistance-nodulation-cell division (RND) transporters and efflux
protein ([104]S3 Table). These genes are mainly for removal of ions
like Mg^2+, Co^2+, Cd^2+ and Zn^2+. The genes for Mg^2+ efflux are
overrepresented in genome of strain YL15. This is in accordance with
the fact that the concentration of Mg^2+ in the acid mine drainage
where strain YL15 inhabits achieves as high as 249 mg·l^-, which is
much higher than that in other minesites, e.g. three copper mines in
central Norway [[105]40].
Adaptation to low pH
Owing to the natural proton concentration gradient across the membrane
in the acid mine drainage, if uncontrolled, influx of protons may lead
to drastic disturbances of the intracellular pH homeostasis. In order
to grow at low pH environments, acidophilic microorganisms have to
maintain a pH gradient of several pH units across the cellular membrane
[[106]41]. Acidophiles achieve this via several ways, which mainly
include: (i) generate a reversed membrane potential to inhibit the
influx of protons via active influx of K^+ or other cations; (ii)
develop highly impermeable cell membranes to limit the influx of
protons into cells; (iii) carry protons out of cells via various
transporters and (iv) employ chemicals as buffer to bind and sequester
protons [[107]42].
Genome of strain YL15 harbors genes coding for a kdp-type potassium
uptake ATPase system (kdpEFABC, [108]S3 Table). By this means, cells of
YL15 are capable of partially deflecting the inward flow of protons
[[109]41]; Membrane lipid components are known to maintain pH
homeostasis in acidophiles. Hopanoid, a type of bacterial membrane
lipid structures, is regarded as a critical strategy for microbial
survival in extremely acidic environments [[110]43]. A cluster of
hopanoid-synthesis genes were identified in genome of YL15, e.g. a
squalene/phytoene synthase gene (BBC27_RS14705), a squalene-hopene
cyclase coding gene (SHC, BBC27_RS14700) and a number of genes coding
for hopanoid-associated proteins (HpnAIJKNHM, [111]S3 Table); Besides,
several genes for producing of buffer molecules, e.g. genes for
arginine and glutamate decarboxylase Adi and GadB (BBC27_RS02605 and
04705), exist in the genome of strain YL15. Furthermore, the existence
of sodium:proton antiporter genes (BBC27_RS09220 and 12540) confirmed
that cells can export excess protons and simultaneously uptake Na^+ to
cope with an increase of the intracellular proton concentration.
Some recently illustrated acid resistance genes, such as the ClpXP gene
(BBC27_RS08380 and 08385) coding for an ATP-dependent Clp protease and
the lexA (BBC27_RS08345) gene for a repressor protein, were found in
YL15 genome [[112]44]. It is noted that the clpB gene and the
RNA-modification enzyme genes, which are involved in arsenic
resistance, were also proved to confer acid resistance to microbes
[[113]38]. ClpB proteins are also known to be crucial in microbial
adaptation to oxidative stress, suggesting its versatility in cell
survival [[114]45,[115]46].
Resistance to elevated UVR
Environments in high altitude regions are typically characterized by
elevated UVR. Excessive or intense exposure to UVR is detrimental to
organisms [[116]47]. Some microorganisms survive under radiation due to
defensive mechanisms provided by a variety of UV-absorbing substances,
e.g. mycosporine-like amino acids (MAAs), which are the secondary
metabolic products in many organisms. The precursor of MAAs,
3-dehydroquinate, is formed during the early stages of the shikimate
pathway [[117]47]. Strain YL15 is presumably to produce MAAs to combat
UVR, as the genes for 3-dehydroquinate synthesis are found in the
genome of strain YL15. The genes are BBC27_RS08435 and 13320 (for
3-deoxy-7-phosphoheptulonate synthase) and BBC27_RS09460 (for
3-dehydroquinate synthase).
UVR leads to the production of reactive oxygen species (ROS), therefore
the ROS-scavenging metabolite and/or enzymes are supposed to function
in UVR-resistance. Except for MAAs, superoxide dismutase has been known
to link to survival of organisms under radiation [[118]48]. In genome
of strain YL15, one copy of gene for superoxide dismutase
(BBC27_RS13900) was identified. UVR causes mutagenic and cytotoxic DNA
lesions [[119]49]. Strain YL15 has dozens of DNA repair-associated
genes, many of which have been substantiated to be induced by UVR, e.g.
the UVr ABC system and a variety of Rec proteins ([120]S3 Table). In
addition, some other proteins, for instance, the histone-like DNA
binding protein HU and Hsp70 protein (dnaK), have also been supposed to
confer resistance to radiation [[121]48], and their coding-genes are
also identified in genome of strain YL15 ([122]S3 Table).
Clustered regularly interspaced short palindromic repeats
(CRISPR)/CRISPR-associated (Cas) gene systems
The CRISPR/Cas (clustered regularly interspaced short palindromic
repeats/CRISPR-associated genes) systems are adaptive immunity systems
that are developed by prokaryotes to protect cells against foreign
genetic elements such as viruses and plasmids [[123]50]. The genome of
YL15 has 2 CRISPR/Cas loci, both of which have 70 spacers ([124]Fig 2A,
20,113 bp, [125]Fig 2B, 13,095 bp). To the best of our knowledge, the
repeats/spacers outnumber most of the known acidophilic bacteria
([126]S4 Table). In order to identify the types of CRISPR/Cas systems,
we annotated the Cas genes using Hmmscan program of HMMER package 3.1
[[127]51] against the Pfam database 30.0 [[128]52] and BLASTP program
against the nr database. According to the classification and
nomenclature of CRISPR-associated genes, the two CRISPR/Cas systems are
presumed to be affiliated to type III-B targeting DNA and I-F targeting
both DNA and RNA, respectively [[129]53]. Interestingly, the type III-B
system has a gene (csx1) for a Cas-NE0113 family protein which has been
reported in type III-U system and two copies of genes for Cas1/Cas2
proteins ([130]Fig 2 and [131]S5 Table).
Fig 2. Proposed CRISPR/Cas systems in genome of YL15.
[132]Fig 2
[133]Open in a new tab
The genome of YL15 harbors (a) a putative type III-B and (b) a putative
type I-F CRISPR/Cas system. The type III-B system has a cluster of
genes for repeat-associated mysterious proteins (RAMPs, Cmr—Cmr6, with
Cmr2/Cas10 as a signature protein), a putative Cas protein and a Cas6
protein which is involved in CRISPR transcript processing. The type I-F
CRISPR/Cas system has genes encoding for a Cas1 protein, three csy
proteins and a Cas3” protein which has only the HD domain of Cas3
protein. Both of the systems have genes coding for two hypothetical
proteins (hyp) with unknown functions.
CRISPR/Cas systems are considered to function via a RNA-silencing-like
mechanism, and the spacer sequences are often found to share high
similarities with virus or plasmid sequences [[134]54]. However, our
BLASTN results showed that except for one spacer sequence (spacer 39,
CCTATCAACGATTCGCCAATACTATCGATGTG) in the type I-F system has a
similarity of 93% to a fraction of the Sulfuricurvum kujiense DSM 16994
plasmid pSULKU02 sequence (90% coverage), there were no other exact or
full-length matches to any known phage or plasmid sequences. This may
be due to that our knowledge of phage or plasmid in the acid mine
drainage environments was limited and only a small fraction of their
sequences have been deposited in the databases. Bacteriophages are the
most abundant forms of life on the Earth, and the phage abundance is
estimated to be about 5–10 times more than that of bacteria in the
ocean [[135]55]. The large number of spacers in the two CRISPR/Cas
modules indicates that strain YL15 may encounter a complicated
biological context. Intrusions of bacteriophage and plasmid elements
are common and often lethal [[136]56]. YL15’ s CRISPR/Cas systems help
to defend against phage and plasmid invasions and thus are
indispensable to its survival in the acid mine drainage environment.
RNA-seq to reveal cold adaptation mechanisms of strain YL15
Overview of RNA-seq data and genes with significantly different RNA
transcript counts at 28°C and 6°C
In order to gain deep insights into the cold adaptation mechanisms of
strain YL15, we performed RNA-seq analysis from two biological
replicates for cells grown at 28°C (S28_rep1 and S28_rep2) and at 6°C
(S6_rep1 and S6_rep2). A total of 5.21 Gb clean data was created after
removing reads with adapter, poly-N and low quality reads ([137]Table
1). More than 91% of all reads were mapped to the genome of YL15
([138]Table 1). Overall differences in RNA transcript counts were
observed between S28 and S6. A total of 372 genes with significantly
different RNA transcript counts were identified, of which 199 and 173
had higher and lower RNA transcript counts at 6°C, respectively
([139]S6 Table).
Table 1. Overview of Illumina RNA-seq data quality.
Clean data was obtained from raw data by removing reads containing
adapter, poly-N and low quality reads.
Sample name Raw data (Gb) Clean data (Gb) Clean reads Percent of reads
mapped
S28_rep1 1.41 1.33 3451903 92.4
S28_rep2 1.20 1.14 2971511 92.9
S6_rep1 1.37 1.30 3380233 93.1
S6_rep2 1.53 1.44 3748436 91.1
[140]Open in a new tab
To examine the expression of genes identified from RNA-seq analysis,
quantitative real-time PCR (qRT-PCR) was performed on 25 selected
genes. Glyceraldehyde-3-phosphate dehydrogenase gene (gapdh,
BBC27_RS12850) was used as a reference since changes of the gene’s RNA
transcripts were very small at 6°C and 28°C. It was shown that the
correlation coefficient (r-value) between RNA-seq and qRT-PCR data was
calculated as 0.84 ([141]S2 Fig). This indicates the suitable quality
of the RNA-seq data.
For the purpose of acquiring the functional classification of the
identified genes, gene ontology (GO) and KEGG pathway enrichment
analyses were performed. The genes with significantly different RNA
transcript counts were assigned to 29 functional categories by GO
enrichment analysis. Among the three main GO categories, namely
cellular component, molecular function and biological process, “cell”
and “cell part”, ‘‘binding” and “catalytic”, “cellular process” and
“metabolic process” were the most dominant subcategories, respectively.
It was also found that most of the subcategories in the cellular
component and biological process are up-regulated at 6°C ([142]Fig 3).
KEGG pathway enrichment analysis showed that the most dominant pathways
were ribosome, oxidative phosphorylation and carbon metabolism
([143]Fig 4).
Fig 3. GO enrichment analysis of the differential expressed genes between S28
and S6.
[144]Fig 3
[145]Open in a new tab
The numbers in red and black on the right represent the number of genes
with higher and lower RNA transcripts at 6^°C, respectively.
Fig 4. KEGG pathway enrichment analysis of differentially expressed genes
between S28 and S6.
[146]Fig 4
[147]Open in a new tab
Enrichment factor is calculated as followed:
[MATH: Enrichmentfactor=Genehits/GenepathwayHitstotal/Genetotal :MATH]
Gene hits is the number of hits in the selected pathway; Gene[pathway]
is the number of genes in the selected pathway of KEGG background;
Hits[total] represents the number of total hits in all pathways; Gene
[total] is the number of total genes in all pathways of KEGG
background.
Transcription
A number of genes involved in transcription showed increased number of
RNA transcript counts in cold condition compared to those in mesophilic
condition. At 6°C, most of the genes for RNA polymerase complex core
enzyme subunits (alpha, beta and beta’) had higher RNA transcripts. In
particular, the beta subunit coding gene had 5.44- fold more RNA
transcript counts. The up-regulation of RNA polymerase has been
observed in some other microorganisms, e.g. a marine bacterium
Sphingopyxis alaskensis [[148]57] and a methanogenic archaeum
Methanolobus psychrophilus R15 [[149]58] and also in A. ferrivorans
strain SS3 [[150]9]. Besides, the gene coding for RNA polymerase sigma
factor RpoD was also induced by cold. Several transcription factor
genes showed cold-enhanced RNA transcript counts, including those
coding for a transcriptional initiation protein Tat, a transcription
elongation factor GreA, a transcription termination factor Rho and
transcription-repair coupling factor. Moreover, the YL15 genome has
three genes coding for transcription antitermination protein, and two
of them (nusA and nusG) had higher RNA transcript levels at 6°C. The
elevated RNA transcript levels of cellular components of the
transcriptional machinery at low temperature, together with the
induction of genes involved in the transcriptional processes by cold,
demonstrated that transcriptional regulation is central to cold
adaptation in strain YL15.
Translation and post-translational processing
A large quantities of genes that are involved in translation and
post-translational processing had greater number of RNA transcript
levels at 6°C. Genes for 37 ribosome proteins, including genes for 24
large subunit and 13 small subunit proteins, and a ribosome maturation
factor showed increased RNA transcript counts at 6°C. In addition, the
translation initiation factors (IFs) have been found to be related to
translation of cold-induced genes [[151]59]. The genes for the
translation initiation factors, IF-1 and IF-3, had 3.59 and 5.06-fold
more RNA transcript counts at 6°C, respectively. Besides, a gene coding
for the translation elongation factor G, which catalyzes the
translocation of the tRNA and mRNA down the ribosome at the end of each
round of polypeptide elongation, also had elevated RNA transcript
counts at 6°C.
The rise of RNA transcript counts for genes involved in translation may
reflect a requirement for more proteins to cope with the cold-stress
conditions. Nevertheless, it has been speculated that some ribosomal
proteins may have other functions apart from protein synthesis, such as
acting as a temperature sensor to cold stress [[152]60]. Moreover,
ribosomal proteins have also been found to be bacterial surface and
secreted proteins, thus it was inferred that some ribosomal proteins
may be secreted to the surface of the cells or out of cells as a
defensive mechanism in response to external environmental changes
[[153]61].
RNA transcript counts for some genes encoding for factors involved in
posttranslational processing were affected by changes of temperature. A
dozen of chaperone-encoding genes were found in the YL15 genome,
including the group I chaperonin complex (GroEL/ES) coding genes and
genes for molecular chaperone HtpG, dnaK and dnaJ. RNA transcript count
for the HtpG-coding genes was elevated at 6°C. Another chaperone gene,
coding for a peptidylprolyl isomerase (PPIase) was also induced by
temperature downshift. It has been found that PPIase enhances protein
folding by catalyzing the rate-limiting cis/trans isomerization of
peptidyl-prolyl bonds in polypeptides, and some PPIases are also
capable of refolding unfolded proteins [[154]62].
Transmembrane transport
Numerous ABC transporter-associated genes were identified in the genome
of strain YL15, 4 of which showed increased number of RNA transcript
counts in the cold. Notably, genes (pstSCA) for the high-affinity
ABC-type phosphate uptake system in the genome had greater number of
RNA transcript counts at 6°C. The increases in the RNA transcript
counts for this high-affinity system suggest an urge demand for
maintaining a sufficient supply of phosphate for use in central
metabolic actions at low-temperatures (e.g. DNA replication and protein
synthesis). This may also reflect a decline in transport efficiency and
that the increased transcript levels of these genes might compensate
for reduced enzyme activity at low temperatures.
Proteins can be exported out of the cytoplasmic membrane via different
pathways, e.g., the Sec protein-translocation pathway. The Sec
translocases offer a major pathway of protein translocation from the
cytoplasm across the cell membrane in bacteria [[155]63]. Genome of
YL15 has genes for Sec pathway (secYABDEFGyajC), and four of them
(secYEFyajC) showed greater RNA transcript counts at 6°C. It has been
found that a large number of surface proteins including secreted
proteins were more abundant at 4°C than at 23°C in a psychrophilic
archaeum Methanococcoides burtonii [[156]64]. The roles that the
secreted proteins play remains obscure, they may facilitate
intercellular interactions that promote nutrient exchange under
unfavorable environments, improve the stability of the cell membrane,
and/or act as a part of extracellular polymeric substances (EPS) to
cope with the low temperatures.
Energy metabolism
Previous studies have shown that genes involved in energy metabolism
are induced by cold [[157]65]. In A. ferrivorans strain SS3, eight
genes involved in inorganic sulfur compounds oxidation and 20 electron
transport genes had higher RNA transcript counts at 8^°C [[158]9]. In
contrast, in strain YL15, most of the genes for F[0]F[1] ATP synthase
had higher RNA transcript levels at low temperature. It was found that
the genes for F[0]F[1] ATP synthase subunit A, B, C, alpha, gamma and
delta had increased number of RNA transcript counts at 6°C. In A.
ferrivorans, the synthesis of ATP via F[0]F[1] ATP synthase is coupled
with the ferrous iron pathway. The downhill pathway of ferrous iron
oxidation can consume protons entering the cells via the ATP synthase
complex and drive ATP synthesis [[159]6,[160]66]. It was observed that
the gene coding for the key enzyme short-chain dehydrogenase in the
iron-oxidation pathway, showed a rise in RNA transcript counts at low
temperature. These results indicate that strain YL15 has higher energy
demands at low temperatures, to fuel the increased production of
specific proteins and enhanced metabolic processes required to deal
with cold conditions.
Chemotaxis and motility
Bacterial chemotaxis is expected to enable cells to move towards
favorable environments and evade unfavorable conditions. Three genes
involving in chemotaxis, namely chemotaxis phosphatase gene CheZ, and a
gene for a methyl-accepting chemotaxis protein, had more RNA transcript
counts at 6^°C. The induction of chemotaxis genes upon long-term
adaptation to low temperature has been found in the human pathogen
Yersinia enterocolitica [[161]67]. It can be inferred that cell
motility may also be cold-enhanced since bacterial chemotaxis relies on
cell motility. The genome of YL15 has a set of genes for assembly and
function of the microbial motility organelle–flagellum. The bacterial
proteins MotA and MotB are required for the rotation of the flagellar
motor [[162]68,[163]69]. The genome of YL15 has five and three copies
of genes coding for MotA and MotB proteins, respectively. Two of the
MotA gene (BBC27_RS01105, 02275) had higher RNA transcript levels at
low temperature. Particularly, one of the MotA gene (BBC27_RS01105) had
7.25-fold rise in RNA transcript counts at 6°C. In addition, except for
one gene (FlhB, BBC27_RS10385), the other genes for flagellum assembly
had no significantly difference in RNA transcript counts at 6°C and
28°C. These results indicated that the Mot genes may play a critical
role and flagella in cells of YL15 may function with a unique mechanism
in response to cold.
Other mechanisms
Cold shock proteins (Csps) function in bacterial survival in various
adverse conditions including rapid temperature downshifts
[[164]70,[165]71]. Csps are thought to function by serving as RNA
chaperons that may prevent the formation of mRNA secondary structures
at low temperatures and thus facilitate translation [[166]72]. We found
that one Csp gene (BBC27_RS12050) had an elevated RNA transcript level
at 6°C. The strain YL15 has been acclimated for months before our
experiment, therefore, the Csp gene is also a cold acclimation protein
(Cap) gene, which was induced during balanced growth at low
temperatures [[167]73]. Moreover, the gene had high RNA transcript
counts at both 6°C and 28°C, suggesting that the gene was responsible
for not only cold adaptation but also survival of strain YL15 at
mesophilic temperature.
It is noted that a gene coding for a bacterioferritin (also known as
DNA binding proteins from starved cells, Dps) had a 10-fold increase in
RNA transcript counts at 6°C. Dps is thought to protect DNA against
oxidative stress mediated by H[2]O[2]. H[2]O[2] is involved in Fenton
reaction, a Fe^2+-facilitated chemical reaction to generate hydroxyl
radical, which is a type of reactive oxygen species (ROS) that are
detrimental to organisms [[168]74,[169]75]. Elevated concentration of
H[2]O[2] can be anticipated due to increased solubility of oxygen at
low temperatures [[170]11,[171]74]. This indicates that Dps in strain
YL15 may aid cells in protecting DNA from oxidative damages.
In summary, this report represents the first comprehensive study of the
microbial adaptation mechanisms to the alpine acid mine drainage
environments at a strain-level. Microorganisms in the acid mine
drainage environments are faced with severe survival threats either
abiotic or biotic. The main survival pressures include resistance to
metal ions, maintaining a near-neutral intracellular pH and precluding
invasion of extraneous nucleic acid substances. However, for strains
like YL15 that inhabit acid mine drainage in alpine regions, additional
vital challenges are adaptation to low temperatures and resistance to
UVR. Genomic analysis illustrated the metal-tolerance, pH homeostasis
and UVR-resistance mechanisms and the acquired immune system—CRISPR/Cas
modules of strain YL15. For the first time, we characterized the
potential UVR-resistance mechanisms and the CRISPR/Cas systems with a
large number of repeats/spacers in an acidophile. Transcriptomic assays
revealed the cold adaptation mechanisms of the strain. The results also
demonstrated that although our results are consistent with those of
Christel et al. [[172]9] in some aspects, e.g. changes in transcript
counts for genes for RNA polymerase complex and translation regulation,
considerable differences lie in variation of transcript levels for
genes involved in ribosomal proteins, energy metabolism, chemotaxis and
motility and biofilm formation ([173]S7 Table). These differences may
be the results of differences in the energy sources, the temperatures
and / or the strains adopted. On the basis of our analyses, a schematic
model for selected gene products and / or processes involved in
adaptation of strain YL15 to the alpine acid mine drainage environment
was proposed ([174]Fig 5). This study adds to our knowledge on the
mechanisms by psychrophilic acidophiles to deal with the mine-affected
water bodies and also sheds light on the adaptation mechanisms of other
microorganisms to the extreme environments.
Fig 5. Proposed model for processes involved in adaptation of YL15 to the
alpine acid mine drainage.
[175]Fig 5
[176]Open in a new tab
The structure of hopanoid was shown in detail on the right. The numbers
in parentheses represent the number of genes with higher RNA
transcripts at 6°C versus 28°C.
Supporting information
S1 Fig. Growth curves of A. ferrivorans YL15 at 6°C and 28°C.
Filtration sterilized ferrous sulfate was used as an energy source.
(TIF)
[177]Click here for additional data file.^ (715.2KB, tif)
S2 Fig. Correlation analysis of RNA-seq and qRT-PCR.
(TIF)
[178]Click here for additional data file.^ (111.3KB, tif)
S1 Table. Physicochemical properties of the acid mine drainage sample
for strain YL15.
(DOCX)
[179]Click here for additional data file.^ (16.6KB, docx)
S2 Table. Selected genes and primers for quantitative real-time PCR.
(DOCX)
[180]Click here for additional data file.^ (22.2KB, docx)
S3 Table. Genes predicted to involved in metal resistance, pH
homeostasis and UVR-resistance.
The symbol ‘/’means the genes has no specific functions.
(DOCX)
[181]Click here for additional data file.^ (23.6KB, docx)
S4 Table. Comparison of CRISPRs loci among acidophilic bacteria.
CRISPRs loci of YL15 were annotated using CRISPRFinder while those of
other strains were retrieved from CRISPRs database.
(DOCX)
[182]Click here for additional data file.^ (16.9KB, docx)
S5 Table. Identification of CRISPR-associated (Cas) proteins in strain
YL15.
(DOCX)
[183]Click here for additional data file.^ (19.1KB, docx)
S6 Table. Functional genes with differential transcritpts based on
analysis by TopHat and Cufflinks packages.
There were 199 and 173 genes with significantly higher and lower RNA
transcripts out of the total 2,798 protein-coding genes in genome of
strain YL15. Classification of protein functions was based on KEGG
annotation.
(DOCX)
[184]Click here for additional data file.^ (52KB, docx)
S7 Table. Comparison of number of genes in main categories with higher
transcripts levels at low temperatures between results from Christel et
al. and this study.
The bold and regular letters represent categories and sub-categories,
respectively. Sub-categories were based on classification of protein
functions as listed in [185]S6 Table.
(DOCX)
[186]Click here for additional data file.^ (17.3KB, docx)
Acknowledgments