Journal of Proteomics xxx (xxxx) xxx–xxx

Contents lists available at ScienceDirect

Journal of Proteomics
journal homepage: www.elsevier.com/locate/jprot

Response of the biomining Acidithiobacillus ferrooxidans to high cadmium

concentrations☆
Javiera Ramos-Zúñigaa,1, Sebastián Gallardoa,1, Cristóbal Martínez-Busseniusa,
⁎
Rodrigo Norambuenaa, Claudio A. Navarroa, Alberto Paradelab, Carlos A. Jereza,
a
Laboratory of Molecular Microbiology and Biotechnology, Department of Biology, Faculty of Sciences, University of Chile, Santiago, Chile
b
Proteomics Laboratory, National Biotechnology Center, CSIC, Madrid, Spain

A B S T R A C T

Cadmium is a heavy metal present in contaminated soils. It has no biological role but when entering cells generates DNA damage, overexpression of stress response
proteins and misfolded proteins, amongst other deleterious effects. Acidithiobacillus ferrooxidans is an acidophilic bacterium resisting high concentrations of heavy
metals such as cadmium. This is important for industrial bioleaching processes where Cd+2 concentrations can be 5–100 mM. Cadmium resistance mechanisms in
these microorganisms have not been fully characterized. A. ferrooxidans ATCC 53993 contains genes coding for possible metal resistance determinants such as efflux
systems: P-type ATPases, RND transporters and cation diffusion facilitators. In addition, it has extra copies of these genes in its exclusive genomic island (GI). Several
of these putative genes were characterized in the present report by determining their transcriptional expression profiles and functionality. Moreover, an iTRAQ
proteomic analysis was carried out to explore new cadmium resistance determinants in this bacterium. Changes in iron oxidation components, upregulation of
transport proteins and variations in ribosomal protein levels were seen. Finally, increased concentrations of exclusive putative cadmium ATPases present in strain
ATCC 53993 GI and other non-identified proteins such as Lferr_0210, forming part of a possible operon, could explain its extreme cadmium resistance.
Significance: Cadmium is a very toxic heavy metal present in mining operations and contaminated environments, it can affect all living organisms, including humans.
Therefore, it is important to know the resistance mechanisms of bacteria highly resistant to this metal. These microorganisms in turn, can be used to bioremediate
more efficiently environments highly polluted with metals. The results obtained suggest A. ferrooxidans strain ATCC 53993 can be an efficient bacterium to remove
cadmium, copper and other metals from contaminated sites.

1. Introduction redox agents or stabilizers of proteins structure [8]. Cadmium (Cd) is

Heavy metal pollution in the environment is triggered by natural
and industrial or antrophogenic causes [1]. Pesticides, mining, waste
from mining, metal plating, industrial and domestic wastes, amongst
others, could generate heavy metals pollution [2]. Bioremediation use
biological mechanisms to remove or prevent contamination caused by
heavy metals using bacteria, fungi, yeast, algae and plants [3,4]. This
practice to control heavy metal contamination is important, because is
efficient and environmental-friendly [5]. In general, bacteria associated
with this processess are able to mobilize metals by leaching, alkylation
and metals reduction; or immobilize the toxic elements by biosorption
and metal-binding molecules, amongst other specific mechanisms [6,7].
Microbial cells require several metals, which are essential for many
biochemical processes, participating as catalysts, enzymatic co-factors,
one of the heavy metals present in contaminated soils and apparently it
has no biological role. In bacteria, this metal is able to enter the cyto-
plasm via transport systems for divalent cations such as Mn and Zn [9].
Once in the cytoplasm, Cd can alter a series of cellular functions due to
its chemical behavior very similar to Zn and it can be toxic by its in-
teraction with nucleic acids. Additionally, Cd binds to thiol groups and
displaces Ca and Zn from proteins [10,11]. For these reasons, bacteria
have developed mechanisms to decrease levels of cadmium in the cy-
toplasm, based mainly on efflux systems [12]. These systems belong to
3 main families: P-type ATPases, RND transporters and cation diffusion
facilitators (CDF). P-type ATPases cross the inner membrane and use
ATP to pump metal ions from cytoplasm to periplasm [13]. On the
other hand, RND transporters are 3-component pumps inserted through
the inner and outer membranes, acting as chemostatic antiporters that

☆
Journal of Proteomics 10th Anniversary
It is a pleasure for the authors to contribute with this original research article and become part of the special celebration for the 10th year anniversary of Journal of
Proteomics. Since its creation, Journal of Proteomics has published a great number of excellent articles related to proteomics and its applications in all biological
areas. Its wide world distribution no doubt, has been very important for science development.
⁎
Corresponding author.
E-mail address: cjerez@uchile.cl (C.A. Jerez).
1
JR* and SG* contributed equally to this work.

https://doi.org/10.1016/j.jprot.2018.12.013
Received 30 September 2018; Received in revised form 11 December 2018; Accepted 12 December 2018
1874-3919/ © 2018 Elsevier B.V. All rights reserved.

Please cite this article as: Ramos-Zúñiga, J., Journal of Proteomics, https://doi.org/10.1016/j.jprot.2018.12.013
J. Ramos-Zúñiga et al.
export metals from the cytoplasm and periplasm to the external en-
vironment [14]. Finally, CDF proteins are a family of transporters that
act as chemostatic ion-proton exchangers, which transport divalent
metals from cytoplasm to periplasm by using the proton-motive force
[15,16]. There are well characterized proteins from each of these
transporter families. For example, a P-type ATPase CadA from Staphy-
lococcus aureus encoded in a plasmid conferring antibiotic resistance
has been described [13]. Also, a CzcCBA complex from Ralstonia me-
tallidurans CH34 was reported [17]. The first described example of a
CDF type transporter was CzcD, an efflux system for Cd and Zn from R.
metallidurans [18]. Beyond the existence of these systems, in general
bacteria have a low resistance to cadmium, and small amounts of it is
enough to cause death of cells. An exception to this situation is Acid-
ithiobacillus ferrooxidans, a Gram-negative, acidophilic and chemo-
lithoautotrophic bacteria, widely used in mineral bioleaching. In these
industrial processes, some strains maintain metabolic activity in
80–500 mM Cd [19]. Although mechanisms of resistance to cadmium in
acidophiles are not well known, bioinformatic approaches have been
used to search for genes involved in resistance to this metal. Two of the
more studied strains of A. ferrooxidans are ATCC 23270 and ATCC
53993. These strains are 80.5% similar in their genomes and differ
mainly in their respective exclusive genomic islands (GI) [20]. Speci-
fically, strain ATCC 53993 GI contains putative genes for resistance to
metals, including Cu and Cd [21,22]. A. ferrooxidans has been widely
studied to find the molecular elements conferring its high resistance to
copper. Different types of proteins are induced due to this metal ex-
posure [21–28]. In addition, a general mechanism for metals resistance,
based in the use of inorganic polyphosphate (PolyP), to extrude the
toxic metal from cells has been proposed initially for E. coli [29,30] and
also for biomining acidophiles [31,32]. Due to the environment in
which A. ferrooxidans and other acidophiles grow, they should have
efficient mechanisms to avoid Cd toxicity, making them highly resistant
to the heavy metal. Previous studies in A. ferrooxidans ATCC 23270
have found some proteins upregulated in presence of cadmium, in-
cluding a transcriptional regulator CmtR [33], genes coding for heavy
metal efflux proteins (czcA1, czcA2, czcB1 and czcC1) and also for pu-
tative cation channel proteins (cadA1 and cadB1) [34].
In this study, a global quantitative proteomic analysis was carried
out to further explore the existence of new possible cadmium resistance
determinants in A. ferrooxidans ATCC 53993. Additionally, bioinfor-
matic analyses of this microorganism's genome allowed identification of
a large number of candidate genes possibly related to Cd resistance, and
several of these putative genes were characterized in the present report
by measuring their transcriptional expression profiles and functionality.
2. Materials and methods
2.1. Bacterial strains and growth conditions
A. ferrooxidans strain ATCC 53993 was grown at 30 °C in 9 K
medium containing ferrous sulfate (33.33 g/L) with an initial pH of
1.45 [35] in absence or presence of a toxic metal as previously de-
scribed [26]. Bacterial growth was followed by measuring the increase
of cell numbers in an Olympus BX50 optical microscope and a Petroff-
Hausser counting chamber. To obtain cultures successively adapted to
cadmium, the process was started with a cell inoculum in which mi-
croorganisms had never been in contact with cadmium before. After
that, sequential subcultures were done with increasing CdSO4 con-
centrations. Once the desired metal concentration was reached, 3 suc-
cessive subcultures were performed at the chosen final concentrations
(100 and 200 mM of CdSO4). On the other hand, for cultures subjected
to a cadmium shock, cells never exposed to CdSO4 were first grown
until their early exponential phase. At this point, CdSO4 was added to
the cultures to attain 75 mM CdSO4 concentration. After one hour under
metal exposure, cells were harvested by centrifugation.
E. coli BL21 (DE3) (NEB) and TOP10 cells (Invitrogen) were used for
2
Journal of Proteomics xxx (xxxx) xxx–xxx
heterologous expression of some A. ferrooxidans proteins [23]. These E.
coli strains were grown at 37 °C in Luria-Bertani (LB) medium.
2.2. Total protein extracts preparation for iTRAQ analysis
A. ferrooxidans ATCC 53993 cells grown in absence or presence of
CdSO4 were harvested by centrifugation (10,000 ×g for 2 min), washed
three times with dilute sulfuric acid (pH 1.5) followed by three washes
with 50 mM sodium citrate at pH 7.0. Cells were then resuspended in
sonication buffer (50 mM Tris-HCl pH 8, 1 mM EDTA) containing
100 μg/mL PMSF as protease inhibitor and were disrupted by sonic
oscillation during 60 min on ice by using successive 15 s pulses. Finally,
the lysate was centrifuged at 10,000 ×g for 10 min to remove unbroken
cells and cell debris. Protein concentrations in cell-free extracts were
determined as before [35]. Total protein extracts from three biological
replicates (different independent cultures) were pooled using 50 mi-
crograms of each to obtain a representative sample of each experi-
mental condition, with a total of 150 μg of protein in each case. Pooled
samples were lyophilized for 48 h at −40 °C. Lyophilized samples were
stored at −20 °C until their processing and iTRAQ labeling [36].
2.2.1. Protein digestion and tagging with iTRAQ-4-plex® reagent
Total protein concentration was determined using microBCA pro-
tein assay kit (Pierce). For digestion, 50 μg of protein from each con-
dition was precipitated by methanol/chloroform method. Protein pel-
lets were resuspended and denatured in 20 μL 6 M guanidine
hydrochloride/100 mM HEPES, pH 7.5, (SERVA Electrophoresis
GmbH), reduced with 1 μL of 50 mM Tris (2-carboxyethyl) phosphine
(TCEP, AB SCIEX), pH 8.0, at 60 °C for 30 min and followed by 2 μL of
200 mM cysteine-blocking reagent (methyl methanethiosulfonate
(MMTS, Pierce) for 10 min at room temperature. Samples were diluted
up to 120 μL to reduce guanidine concentration with 50 mM TEAB.
Digestions were initiated by adding 2 μg of sequence grade-modified
trypsin (Sigma-Aldrich) to each sample in a ratio 1/25 (w/w), which
were then incubated at 37 °C overnight on a shaker. Sample digestions
were evaporated to dryness.
Each peptide solution was labelled at room temperature for 2 h with
a half unit of iTRAQ Reagent Multi-plex kit (AB SCIEX, Foster City, CA,
USA) previously reconstituted with 80 μL of 70% ethanol/50 mM TEAB.
The iTRAQ labelling was performed according to the following scheme:
iTRAQ 114 reagent = control A. ferrooxidans; iTRAQ 115 reagent: A.
ferrooxidans adapted to 100 mM CdSO4; iTRAQ 116 reagent: A. fer-
rooxidans 60 min shock in 75 mM CdSO4. After labeling, samples were
combined, and labelling reaction stopped by evaporation in a Speed
Vac.
2.2.2. Liquid chromatography and mass spectrometer analysis
A 2 μg aliquot of each sample was subjected to 2D-nano LC ESI-
MSMS analysis using a nano liquid chromatography system (Eksigent
Technologies nanoLC Ultra 1D plus, AB SCIEX, Foster City, CA) coupled
to high speed Triple TOF 5600 mass spectrometer (SCIEX, Foster City,
CA) with a Nanospray III source. Injection volume was 5 μL and three
independent technical replicas were analyzed. The analytical column
used was a silica-based reversed phase column C18 ChromXP
75 μm × 15 cm, 3 μm particle size and 120 Å pore size (Eksigent
Technologies, AB SCIEX, Foster City, CA). The trap column was a C18
ChromXP (Eksigent Technologies, AB SCIEX, Foster City, CA), 3 μm
particle diameter, 120 Å pore size, switched on-line with the analytical
column. The loading pump delivered a solution of 0.1% formic acid in
water at 2 μL/min. The nano-pump provided a flow-rate of 300 nL/min
and was operated under gradient elution conditions, using 0.1% formic
acid in water as mobile phase A, and 0.1% formic acid in acetonitrile as
mobile phase B. Gradient elution was performed according to the fol-
lowing scheme: isocratic conditions of 96% A: 4% B for 5 min, a linear
increase to 40% B in 205 min, then a linear increase to 90% B for 15
additional min, isocratic conditions of 90% B for 10 min and return to
J. Ramos-Zúñiga et al.
initial conditions in 2 min. Total gradient length was 250 min.
Data acquisition was performed with a TripleTOF 5600 System.
Ionization occurred under the following conditions: ionspray voltage
floating (ISVF) 2800 V, curtain gas (CUR) 20, interface heater tem-
perature (IHT) 150, ion source gas 1 (GS1) 20, declustering potential
(DP) 85 V. All data was acquired using information-dependent acqui-
sition (IDA) mode with Analyst TF 1.5 software (AB SCIEX, USA). For
IDA parameters, 0.25 s MS survey scan in the mass range of
350–1250 Da were followed by 25 MS/MS scans of 150 ms in the mass
range of 100–1500 (total cycle time: 4 s). Switching criteria were set to
ions greater than mass to charge ratio (m/z) 350 and smaller than m/z
1250 with charge state of 2–5 and an abundance threshold of > 90
counts (cps). Former target ions were excluded for 20 S. IDA rolling
collision energy (CE) parameters script was used for automatically
controlling the CE.
2.2.3. Data analysis and statistics
MS and MS/MS data obtained from pooled samples were processed
using Analyst TF 1.5.1 Software (SCIEX). Raw data file conversion
tools-generated mgf files were independently searched against a A.
ferrooxidans database (downloaded from UniprotKB, 20,140,604 ver-
sion) containing 2748 protein entries and their corresponding reversed
counterparts, using the Mascot Server v. 2.3.1 (Matrix Science, London,
UK). Search parameters were set as follows: enzyme, trypsin; allowed
missed cleavages, 2; fixed modifications, iTRAQ 4-plex (N-term and K)
and beta-methylthiolation of cysteine; variable modifications, oxidation
of methionine. Peptide mass tolerance was set to ± 25 ppm for pre-
cursors and 0.05 Da for-fragment masses. The confidence interval for
protein identification was set to ≥95% and only peptides with an in-
dividual ion score above the 1% false discovery rate (FDR) threshold
were considered correctly identified. To obtain iTRAQ protein ratios,
the median was calculated over all distinct peptides assigned to a
protein subgroup in each replicate. Then, each iTRAQ channel was
normalized by dividing each protein ratio by the median of ratio in each
channel. This normalized median in each replicate was used to obtain
the final geometric media of the corresponding protein. After calcu-
lating log2 of geometric media, frequency distribution histograms were
obtained from Microsoft Excel 2010. Log2 protein ratios were fitted a
normal distribution using least squares regression. Mean and standard
deviation values derived from the Gaussian fit were used to calculate P
values and FDR (at quantitation level). The FDR for quantitation was
then calculated as the FDR = (E value/protein rank), with E value = (P
value * total number of quantified proteins) and the protein rank the
individual position of the specific protein after ordered it by its P value.
A 5% quantitation FDR threshold was estimated to consider the sig-
nificant differentially expressed proteins.
2.3. Extraction of total RNA from A. ferrooxidans and cDNA synthesis
To determine the effect of cadmium in the expression of some genes
of interest, cells in batch cultures were adapted to grow in presence or
absence of CdSO4 until late exponential growth phase was reached. At
this time, total RNA was extracted from each culture condition by lysing
cells as previously reported and TRIzol (Invitrogen) was used for the
extraction [22,23]. Between three to five biological replicas were used
for each experimental condition. Any remaining DNA was eliminated
from the RNA preparations by the addition of 4 U of TURBO DNA-free
DNase (Ambion) following the manufacturer's instructions. For cDNA
synthesis, 0.8 μg of total RNA was reverse transcribed for 1 h at 42 °C
using ImProm-II (Promega) reverse transcription system, 0.5 μg of
random hexamers (Promega) and 3 mM MgCl2 [22].
2.4. Primer design and real-time RT-PCR
Primers for quantitative real time PCR (qRT-PCR) were designed by
using Primer3 software. After separating PCR products by
3
Journal of Proteomics xxx (xxxx) xxx–xxx
electrophoresis in a 1% agarose gel (0.5 × Tris–acetate–EDTA pH 8.0
buffer), no cross-amplification or non-specific bands were detected.
Cadmium-resistance related gene expression was analyzed by qRT-PCR
with the Corbett Rotor Gene 6000 system as described previously [24].
Efficiency of each primer pair was calculated from the average slope of
a linear regression curve, which resulted from qPCRs using a 10-fold
dilution series (10 pg–10 ng) of A. ferrooxidans DNA as template. Effi-
ciencies between 90 and 110% were used. Quantification cycle (Cq)
values were automatically determined by Real-Time Rotor-gene 6000
PCR software (Corbett Life Sciences, Thermo Scientific/Qiagen, Hilden,
Germany). For transcriptional analysis of the different genes studied, a
relative quantification method was used which is based in the ratio
between the transcripts of a study sample (in presence of cadmium)
versus a control sample (no cadmium) [37]. 16S rRNAAf was selected as
a reference gene since its expression was found to be the most stable
under our experimental conditions [26]. To carry out real-time PCR,
1.0 μL of 1:20 diluted cDNA or 1.0 μL of 1:200 diluted 16S rRNAAf,
0.2 μL of each primer (10 μM) and 10.0 μL of master mix Rotor-Gene
SYBR Green PCR (Qiagen) in a final volume of 20 μL, completed with
RNA-free water were used. The program used was 10 min at 95 °C fol-
lowed by 40 cycles of 5 s at 95 °C and 20 s at 60 °C.
2.5. Cloning and functional assays of A. ferrooxidans genes expressed in E.
coli
Since there is a lack of efficient and reproducible methodologies to
generate knockout mutants in A. ferrooxidans, genes functionality was
tested by using heterologous expression of A. ferrooxidans genes in E.
coli as carried out before [23,26]. A. ferrooxidans genes of interest were
cloned in the commercial vector pGEMT Easy (Promega). A Shine-
Dalgarno sequence was introduced in the forward primer to be used and
the protein of interest was expressed by IPTG induction of the T7
promoter carried in the vector. For ligation reaction 25 ng of vector
DNA and 10 ng of DNA to be cloned were used, and incubation was
overnight at 4 °C. Then 5 μL of this reaction was used to transform E.
coli One Shot TOP10 (Invitrogen). Transformed E. coli strains were
grown at 37 °C in LB solid plates including 1.5% agar, supplemented
with ampicillin (100 μg/mL), IPTG (1 mM) and X-gal (20 mg/mL) for
selection. The clones obtained were analized for the correct orientation
by enzimatic digestion. Finally, E. coli BL21 (DE3) (NEB) was transform
for functional assays.
To estimate the minimum inhibitory concentration (MIC) of E. coli
strains, cells were grown at 37 °C overnight in LB medium supple-
mented with 100 μg/mL of ampicillin, 1 mM IPTG and CdSO4 in a
concentration range between 0 and 2 mM. Finally, the lowest cadmium
concentration inhibiting total growth was considered to be the MIC for
transformed cells.
3. Results and discussion
3.1. A. ferrooxidans growth in presence of Cd
To determine the effect of Cd in A. ferrooxidans ATCC 53993
growth, bacteria were grown in presence of 0, 25, 50, 75, 100 and
200 mM of CdSO4. As seen in Fig. 1A, at 25 mM CdSO4 microorganisms
grew in a similar way to control cells in absence of Cd, reaching the
same number of cells at stationary phase. The same happened at 50 mM
CdSO4, although the stationary phase was reached in a longer time. At
higher concentrations of Cd, A. ferrooxidans was notably affected by the
metal, growing much less at 75, 100 and 200 mM CdSO4 in the same
number of days. On the other hand, cells adapted to cadmium (100 or
200 mM) showed a similar growth although both of them reached a
smaller number of cells when compared to control cells (Fig. 1B). The
effect of Cd was compared in both ATCC 23270 and ATCC 53993 strains
(Fig. 1C). It is clearly seen that strain ATCC 53993 resists higher con-
centrations of cadmium compared with strain ATCC 23270. This
J. Ramos-Zúñiga et al.
Fig. 1. Growth of A. ferrooxidans in presence of CdSO4. A. Growth curves of A.
ferrooxidans ATCC 53993 in presence of the indicated initially added CdSO4
concentrations. B. Growth of A. ferrooxidans ATCC 53993 succesively adapted
to CdSO4. C. Growth comparison between A. ferrooxidans ATCC 23270 and
ATCC 53993 strains at the indicated Cd concentrations. Each point was ob-
tained from three biological replicates. Application of t-Student test were: **
p ≤ 0.01.
behavior has also been observed in presence of copper, since strain
ATCC 53993 resisted higher concentrations of this metal [22]. As
shown previously, A. ferrooxidans ATCC 53993 has a genomic island
coding for both, genes involved in Cu and Cd resistance. This duplica-
tion of resistance determinants most likely provides an advantage to
this strain to survive at elevated concentrations of copper [21,22] and
cadmium as seen here.
3.2. Quantitative proteomics of A. ferrooxidans grown in presence of
cadmium
To have a global view of the response of A. ferrooxidans ATCC 53993
exposed to cadmium, a quantitative iTRAQ proteomic study was carried
out. The results obtained were grouped according to functional cate-
gories (COGs) of the proteins changing their levels in presence of metal,
as seen in Fig. 2A. The analysis identified 1287 proteins for each con-
dition, of which 151 proteins showed quantitative changes in cells
under cadmium shock and 178 proteins changed in adapted cells
compared to control cells grown in cadmium absence. There was a si-
milar overall pattern of changes under both conditions analyzed, with
poorly characterized proteins showing the greatest number of changes
in presence of Cd (39.8% in cadmium shock; 36.5% for adapted cells).
The next categories in which proteins amounts changed were those
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Journal of Proteomics xxx (xxxx) xxx–xxx
including proteins related to metabolism (25.8% for each condition),
information storage and processing (21%) and proteins involved in
cellular processes and signaling (13.2% in cadmium shock and 16.3% in
adapted cells). For a more detailed analysis of each functional category
in the different conditions analyzed, a breakdown of the changes was
made in each of the functional subcategories seen in Fig. 2B.
Possible identities and putative functions of most proteins regulated
differentially are analyzed next, grouped by their functional categories
and putative or possible roles in cadmium tolerance.
3.2.1. Metabolism
3.2.1.1. Energy production and conversion. Most changes in this sub-
category corresponded to proteins decreasing their levels (Table 2),
however, a MerA mercury reductase (Lferr_2107) was upregulated in
cells subjected to cadmium shock (Table 1). This protein is encoded by
a mer operon, responsible of mercury resistance in cells [12]. In
cytoplasm, MerA in presence of NADPH catalizes Hg+2 reduction to
Hg0, a less toxic form [9,38]. Cadmium can not be detoxified by
reduction, due to its redox potential (−824 mV) [9]. However,
cadmium is able to recognize binding sites in MerR regulators [39],
justifying MerA increment in presence of cadmium. In addition, merA
expression was a potential biomarker for cadmium response in
experiments with Nitrosomonas europaea [40].
In cadmium adapted cells, a decrease in levels of most proteins in-
volved in iron oxidation was seen, specifically those that are part of the
rus operon: Cyc2, Cyc1, AcoP and CoxB (Table 2). Under cadmium
shock, only Cyc2 was downregulated. This suggests the microorganism
respiration was affected in presence of cadmium, both in Cd-shock and
Cd-adapted cells. Previously, A. ferrooxidans ATCC 23270 exposed to
40 mM copper also showed decreased levels of Cyc1, Cyc2 and PetC-1,
suggesting that respiration was affected, and iron oxidation was slower
in presence of copper [24]. Furthermore, some studies indicate that
cadmium is capable of inhibiting complexes II and III of the electron
transport chain in mitochondria [41]. On the other hand, DNA micro-
array studies in E. coli exposed to cadmium showed a change from
aerobic to anaerobic metabolism, suggesting an energy conservation
metabolism after metal exposure [42]. Therefore, changes in respira-
tion of A. ferrooxidans could also be due to an energy strategy against
cadmium exposure. Another option would involve the inactivation of
proteins by cadmium binding, since as already mentioned, this metal
can replace Zn and Fe bound to proteins [43,44]. It is interesting that as
previously reported, Rus and AcoP proteins are upregulated in A. fer-
rooxidans ATCC 53993 subjected to copper, suggesting these proteins
may also be copper resistance determinants [26]. On the other hand, in
presence of cadmium Rus was not upregulated and AcoP was down-
regulated, clearly suggesting they would not be Cd resistance de-
terminants in A. ferrooxidans.
3.2.1.2. Carbohydrate transport and metabolism. A. ferrooxidans is an
obligate chemolithoautotrophic microorganism, which fixes CO2 using
energy and reducing power obtained from oxidation of iron or sulfur
[45]. Carbon fixation is carried out by the pentose phosphate cycle.
Several enzymes of this pathway have been identified in A. ferrooxidans
[46]. This microorganism possesses several genes coding for both
subunits of RuBisCO enzyme [47]. In the present study, both subunits
of this enzyme were downregulated in both Cd-adapted and Cd-shock
cells (Table 2). Actually, there is no explanation for this effect of Cd on
RuBisCO expression in A. ferrooxidans.
3.2.1.3. Inorganic ion transport and metabolism. This functional category
mainly includes possible cadmium resistance determinants associated
with metal efflux systems. P-type ATPases play a fundamental role in
the detoxification of cells in presence of metals, since they export them
from cytoplasm to periplasm using hydrolysis of ATP. Under cadmium
shock and adapted conditions, P-type ATPases Lferr_2691, Lferr_1686
and Lferr_0186 increased their levels (Table 1). Lferr_2691 is an Ion
J. Ramos-Zúñiga et al.
Fig. 2. Functional categories, sub-categories and numbers of A. ferrooxidans ATCC 53993 proteins changing their synthesis levels in cells grown in presence of CdSO4.
A. Pie chart protein categories changing their levels under the indicated conditions. B. Number of proteins varying their amounts in each sub-category. (+) Indicates
upregulated and (−) downregulated proteins.
transport 2-domain protein, which has 100% identity with AFE_3093, a
putative cation channel protein characterized as a cadmium ATPase in
A. ferrooxidans ATCC 23270 by Chen et al., [34]. This protein also has
two homologs (CadA1 and CadA2) in A. ferrooxidans ATCC 23270,
which provide cadmium resistance [34]. Lferr_1686 corresponds to
CopB, a copper ATPase already described in Enterococcus hirae [48,49].
The increase in this protein in the presence of cadmium could be
justified due to the promiscuity in metal binding sites in some of these
transporters. As an example, Zn transporters can bind metals such as Co
and Cd due to their similar chemical characteristics [50].
Lferr_0186, annotated as a heavy metal translocating P-type ATPase
at the exclusive GI of A. ferrooxidans ATCC 53993, increased its levels in
presence of cadmium (Table 1). On the other hand, transcriptional le-
vels of Lferr_0186 did not change in presence of copper [22], suggesting
it could be a specific cadmium transporter. In cells adapted to grow at
100 mM Cd, gene Lferr_1147, coding for a cation diffusion facilitator
family transporter increased its levels 1.5-fold (Table 1). This kind of
transporter helps to detoxify cells by exporting the metal to the peri-
plasmic space by using a proton gradient [18].
To obtain additional information on A. ferrooxidans cadmium re-
sistance, A. ferrooxidans ATCC 53993 genome was analyzed to search
for other cadmium transporters. The bioinformatics approach showed
two groups of genes possibly coding for CzcCBA type RND complexes.
Amongst these are czcA genes: Lferr_2060 and Lferr_1617 (Fig. 3),
which are part of two posible operons with the other components of the
system (czcB; Lferr_2061, Lferr_1618; czcC: Lferr_2062, Lferr_1619).
Inner membrane CzcA proteins are involved in the export of divalent
heavy metal cations such as Co, Zn and Cd. These proteins exhibit a
DFGX3DGAX3VEN consensus sequence in their VI transmembrane do-
main, where aspartate and glutamate residues are essential for metal
transport [51]. Both Lferr_2060 and Lferr_1617 have a possible metal
binding domain similar to the one described above, suggesting that
could also bind Cd, but this should have to be experimentally proven.
A second group of genes coding for type P ATPases was identified:
Lferr_0186 and Lferr_0209 and (Fig. 3). The cytosolic domain of this
type of transporters possesses a CXXC conserved motif that would be
the metal binding site. Additionally, a CPC motif located in the trans-
membrane VI domain has been identified [53], which would be part of
the ionic channel inside the membrane. This channel is found only in P-
ATPases of metal ions such as Zn, Cd and Pb [53]. These domains were
5
Journal of Proteomics xxx (xxxx) xxx–xxx
identified in the gene products of Lferr_209 and Lferr_0186. Interest-
ingly, these genes are in the exclusive GI of A. ferrooxidans ATCC 53993
[22], supporting its greater resistance to cadmium.
Four genes coding for possible CDF type transporters were found:
Lferr_0625, Lferr_1147, Lferr_1214, and Lferr_2050 (Fig. 3). Three of
these four genes (Lferr_0625, Lferr_1147, Lferr_2050) would code for
proteins that have high identity with CDF cadmium transporters, such
as CzcD. In turn, two of them (Lferr_1147 and Lferr_2050) have a
contiguous MerR transcriptional regulator, both in a divergent direction
from CDF genes (Fig. 3) as found previously in A. ferrooxidans ATCC
23270 [50]. MerR regulators control transcription of CDF transporters
and through genetic analysis this kind of regulators have been found in
A. ferrooxidans ATCC 23270 (AFE_1431 and AFE_2421) coding for two
CadR putative proteins in response to cadmium [54]. Interestingly,
these regulators would be located in the chromosome forming diver-
gons with CDF transporters (AFE_2420 and AFE_1430). Transcriptional
regulator MerR (AFE_2421) from A. ferrooxidans ATCC 23270 could
regulate CDF (AFE_2420) expression in response to cadmium [33]. This
CDF transporter (AFE_2420) shows 100% identity to Lferr_2050 from A.
ferrooxidans ATCC 53993 analyzed in the present study. All mentioned
CDF transporters contain the highly conserved DAXHMLTD sequence in
domain II of this type of CDF transporters when compared to those
present in Cupriavidus metallidurans CH34. The conserved said domain
would be related to the specificity of these proteins for metals such as
cadmium in bacteria [55].
To evaluate the transcriptional expression changes of genes men-
tioned in Fig. 3, A. ferrooxidans ATCC 53993 was grown in presence or
absence of cadmium and total RNA was extracted and their expression
profiles were evaluated by qRT-PCR. As seen in Fig. 4, at 50 mM Cd,
only Lferr_2050 showed significant changes, increasing its levels about
50-fold. On the other hand, Lferr_0186 and Lferr_1617 decreased their
expression levels. A 30-fold up-regulated expression of Lferr_2050 was
seen at 75 mM CdSO4 shock. Additionally, genes coding for ATPases
Lferr_0186 and Lferr_0209 showed an approximate 15-fold increase in
their expression levels. The CzcA transporter Lferr_2060 also showed a
similar variation. Lferr_0625 and Lferr_1147 genes, coding for CDF
proteins, were up-regulated only 5-fold. Conversely, in cells adapted to
100 mM CdSO4, Lferr_2050 increased its expression about 80-fold and
Lferr_1617 and Lferr_2060 (CzcA), Lferr_0625, Lferr_1214, Lferr_1147
(CDF) increased their levels only 4–5-fold. Finally, the greatest changes
J. Ramos-Zúñiga et al. Journal of Proteomics xxx (xxxx) xxx–xxx

Table 1
Up-regulation of some identified proteins in A. ferrooxidans ATCC 53993 grown in presence of cadmium.
Fold changea

Accesion number ORF Function/similarity Score Coverage Peptide Shock Adapted

(Lferr) number 75 mM 100 mM

Metabolism
Energy production and conversion
B5EM79 2107 Mercuric reductase (MerA) 895.67 37.73 17 1.761 –
Amino acid transport and
metabolism
B5ELU7 0465 5-methyltetrahydropteroyltriglutamate–homocysteine 3630.33 67.43 100 – 1.505
methyltransferase (MetE)
B5ELV3 0471 Serine hydroxymethyltransferase (GlyA) 1463.67 67.13 40 – 1.455
B5ERU4 1382 Microcompartments protein 637.00 90.80 14 – 1.409
B5ERU5 1383 Microcompartments protein 628.33 90.80 13 – 1.402
Nucleotide transport and
metabolism
B5EN79 2265 Ribonucleoside-diphosphate reductase subunit beta 106.00 6.00 2 2.280 1.848
B5EN78 2264 Ribonucleoside-diphosphate reductase 923.67 32.00 22 – 1.405
Coenzyme transport and
metabolism
B5EN11 0690 S-adenosylmethionine synthase (MetK) 666.67 38.53 17 1.755 1.425
Inorganic ion transport and
metabolism
B5EQI7 2691 Ion transport 2 domain protein 84.33 8.70 2 11.924 16.086
B5EJX7 1686 Heavy metal translocating P-type ATPase (CopB) 44.00 2.50 1 1.628 1.562
B5EK85 0186 Heavy metal translocating P-type ATPase 23.00 2.80 1 1.726 1.404
B5EQN5 1147 Cation diffusion facilitator family transporter 24.50 2.20 1 – 1.561
B5EQW3 2722 Adenylylsulfate reductase, thioredoxin dependent (CysN) 296.67 39.60 6 – 1.501

Cellular Processes and Signaling

Cell cycle control, cell division,
chromosome partitioning
B5EPW5 2566 Maf-like protein 94.50 24.60 3 – 1.504
Cell wall/membrane/envelope
biogenesis
B5EJP1 0085 Glycosyl transferase family 2 18.00 3.60 1 – 1.836
B5EMU2 0618 LPS-assembly lipoprotein LptE 90.00 15.10 2 1.868 –
Post-translational modification,
protein turnover, and
chaperones
B5ELM8 1995 Thioredoxin 58.33 9.93 1 – 1.580
B5EN22 0701 Cytochrome c biogenesis protein transmembrane region (DsbD) 55.00 2.10 1 1.610 –
Defense mechanisms
B5EQ94 1097 ABC transporter related (MsbA) 29.50 5.15 1 2.320 1.459
B5EMW5 0642 ABC transporter related 63.00 10.27 1 – 1.438

Information Storage and Processing

Translation, ribosomal structure
and biogenesis
B5EMK9 2144 Peptide chain release factor 3 56.67 6.67 2 13.208 13.186
B5ELZ2 0510 30S ribosomal protein S14 type Z 61.00 19.70 1 3.440 –
B5ELW9 0487 50S ribosomal protein L1 1070.67 61.17 27 1.765 –
B5ELY6 0504 50S ribosomal protein L16 473.33 48.30 12 1.735 –
B5EPC7 2473 50S ribosomal protein L19 387.00 61.40 8 1.735 –
B5EQH8 2681 50S ribosomal protein L13 335.67 48.60 9 1.719 –
B5ELZ5 0513 50S ribosomal protein L18 428.00 53.80 9 1.707 –
B5ELY9 0507 50S ribosomal protein L14 416.67 61.77 12 1.638 –
B5ENM4 0808 50S ribosomal protein L31 56.00 15.67 2 1.637 –
B5EP97 2443 50S ribosomal protein L9 468.33 65.07 14 1.630 –
B5EN54 2240 50S ribosomal protein L20 330.67 41.20 8 1.630 –
B5ELX9 0497 50S ribosomal protein L3 487.67 44.93 15 1.611 –
B5ELX5 0493 30S ribosomal protein S7 636.67 60.27 22 1.607 –
B5EKI9 1803 50S ribosomal protein L25 407.33 51.33 11 1.588 –
B5EL84 0346 Ribosomal RNA small subunit methyltransferase A 38.00 10.40 2 – 1.427
Transcription
B5EJX3 1682 Phage shock protein A, PspA 901.67 74.03 26 – 1.617
B5ELV4 0472 Transcriptional repressor NrdR 118.33 23.50 3 – 1.498
Replication. recombination and
repair
B5EJT9 1648 Exodeoxyribonuclease 7 large subunit 39.00 7.20 2 – 1.787

a
Fold change: Values higher than 1 indicate upregulation; values lower than 1 indicate downregulation.

took place under the shock at 75 mM CdSO4. could play a pivotal role in cadmium resistance of A. ferrooxidans ATCC
Lferr_2050 gene, coding for a CDF protein, was the one with the 53993. This was the only gene increasing its expression at 50 mM
highest transcriptional levels in response to Cd (Fig. 4). This suggests it CdSO4, suggesting that when the bacterium is subjected to that

6
J. Ramos-Zúñiga et al. Journal of Proteomics xxx (xxxx) xxx–xxx

Table 2
Down-regulation of some identified proteins in A. ferrooxidans ATCC 53993 grown in presence of cadmium.
Fold changea

Accesion number ORF Function/similarity Score Coverage Peptide Shock Adapted

(Lferr) number 75 mM 100 mM

Metabolism
Energy production and conversion
B5EQK0 2705 Cytochrome c class I (cycA1) 130.67 10.87 3 – 0.767
B5EQY9 2748 Cytochrome c oxidase subunit II (coxB) 554.00 40.53 21 – 0.737
B5EQZ1 2750 Cytochrome c class I (cyc1) 409.67 39.30 14 – 0.692
B5ERV0 1388 Ribulose-bisphosphate carboxylase (cbbS) 735.00 88.20 30 0.638 0.685
B5EQZ0 2749 Uncharacterized protein (acoP) 341.00 53.73 7 – 0.683
B5ERS1 1359 NADH/Ubiquinone/plastoquinone (Complex I) 67.67 2.60 2 0.655 –
B5EQZ4 2754 Cytochrome c-like protein 133.33 14.00 2 0.632 –
B5EQZ2 2751 Cytochrome c (cyc2) 500.00 30.00 10 0.626 0.737
B5EQ66 1069 Cytochrome d ubiquinol oxidase, subunit II 52.00 3.20 1 0.623 –
B5EQ95 1098 4Fe-4S ferredoxin iron‑sulfur binding domain protein 161.67 26.80 3 0.538 0.602
B5EQF8 2661 Ribulose-bisphosphate carboxylase (cbbS) 284.67 51.97 10 0.505 –
Amino acid transport and metabolism
B5EPE4 2490 Glycine cleavage system H protein 94.33 18.43 2 0.649 –
Carbohydrate transport and metabolism
B5EL13 1878 Glucan 1,4-alpha-glucosidase 18.50 1.70 1 – 0.731
B5EPB6 2462 ROK family protein 169.33 14.37 3 – 0.697
B5EJY1 1690 Glucose-6-phosphate 1-dehydrogenase 394.33 26.83 10 0.669 –
B5ERV1 1389 Ribulose bisphosphate carboxylase large chain (cbbL) 2226.3 70.23 71 0.645 0.640
B5ES38 1479 Fructose-bisphosphate aldolase 824.00 73.53 14 0.646 –
B5ERS6 1364 Transketolase central region 1609.3 46.23 38 0.591 0.608
B5EMF6 0581 Transaldolase 835.00 56.47 18 0.559 –
B5EQF7 2660 Ribulose bisphosphate carboxylase large chain (cbbL) 759.33 42.00 21 0.580 0.746
Inorganic ion transport and metabolism
B5EM36 2057 Uncharacterized protein (CusF2) 92.67 17.50 3 – 0.753
B5EJ99 1551 Polyphosphate kinase 123.67 10.00 4 – 0.744
B5EPX1 2572 Probable Fe(2+)-trafficking protein 220.00 78.13 6 0.673 –
B5EM44 2066 Copper-translocating P-type ATPase (CopA1) 88.00 6.30 2 0.573 –

Cellular Processes and Signaling

Cell wall/membrane/envelope biogenesis
B5ELC1 0383 UDP-N-acetylenolpyruvoylglucosamine reductase 61.67 6.13 1 – 0.765
(murB)
B5EKC7 1740 Glycosyl transferase group 1 61.33 4.00 1 0.679 –
B5EJM8 0072 Peptidoglycan-associated lipoprotein 355.00 31.93 7 – 0.655
B5ELC0 0382 D-alanine–D-alanine ligase (ddl) 72.00 11.70 2 0.591 –
B5ES80 1529 Glycosyl transferase family 2 (pglI) 132.00 14.63 2 0.546 –
Posttranslational modification, protein
turnover, chaperones
B5EM50 2075 Glutaredoxin 82.67 36.30 2 0.669 –
B5EPV7 2558 Peptide methionine sulfoxide reductase MsrB 89.50 19.10 3 0.651 –
B5EMQ1 2187 SirA family protein 542.00 96.97 13 0.648 –
B5EL98 0360 Anhydro-N-acetylmuramic acid kinase (anmK) 52.00 6.03 1 0.606 –
B5ERR0 1347 Heat shock protein Hsp20 379.33 58.37 9 0.541 –
B5EKD0 1743 Heat shock protein Hsp20 915.33 83.63 29 0.425 –
B5EN20 0699 10 kDa chaperonin (groES) 428.67 88.93 16 0.413 0.465
B5EP94 2440 DNA repair protein radA 33.50 7.10 1 – 0.013
B5EQ85 1088 Peptidase S49 domain protein 25.50 3.90 2 – 0.013
Defense mechanisms
B5ENZ4 0835 Restriction modification system DNA specificity 61.00 5.90 2 – 0.762
domain
Information Storage and Processing
Translation, ribosomal structure and
biogenesis
B5ENA7 2295 50S ribosomal protein L28 166.67 38.50 4 – 0.761
B5ELZ7 0515 50S ribosomal protein L30 140.33 43.17 3 – 0.737
B5ELK7 1974 30S ribosomal protein S21 98.67 44.17 3 – 0.734
B5EMD3 0558 30S ribosomal protein S15 231.00 58.03 4 – 0.703
B5EKF7 1771 Aspartyl/glutamyl-tRNA(Asn/Gln) amidotransferase 173.67 69.13 5 0.668 –
subunit C
B5EMS7 2213 Alpha-L-glutamate ligase, RimK family 98.00 12.95 2 0.653 –
B5EJC7 1579 50S ribosomal protein L32 67.00 18.00 2 – 0.631
B5EKY1 1846 30S ribosomal protein S20 71.67 13.20 2 – 0.609
B5ELY3 0501 30S ribosomal protein S19 161.00 51.63 5 – 0.598
B5ENA6 2294 50S ribosomal protein L33 42.00 29.40 2 – 0.553
B5EN55 2241 50S ribosomal protein L35 81.67 39.00 3 – 0.518
B5EJJ2 0036 50S ribosomal protein L34 42.00 20.50 1 – 0.515
B5EM95 0520 50S ribosomal protein L36 21.00 18.90 1 – 0.452
Transcription
B5EMV4 0630 RNA polymerase sigma factor RpoS 123.67 15.90 3 – 0.753
B5EN39 0718 DNA-directed RNA polymerase subunit omega 126.00 39.97 4 – 0.712
(continued on next page)

7
J. Ramos-Zúñiga et al.
Table 2 (continued)
Accesion number ORF Function/similarity
(Lferr)
B5ELN5 2002 Transcriptional regulator, ArsR family
B5ENF8 0742 Cold-shock DNA-binding domain protein
Replication, recombination and repair
B5EMC4 0549 AAA ATPase central domain protein
B5EQ23 1026 Integration host factor subunit beta
a
Fold change: Values higher than 1 indicate upregulation; values lower than 1 indicate downregulation.
concentration of cadmium, the induction of CDF could be sufficient to
resist the presence of the metal. Similar behavior was seen at 100 mM
CdSO4, where this transporter increased 80-fold. However, there was
also a significant increase in the transcription of other transporters
studied. From this, it is inferred that higher cadmium concentrations
require action of additional resistance mechanisms. Regarding the
75 mM shock of CdSO4, an increase of P-ATPases Lferr_0209 and
Lferr_0186 was seen. These last genes are present in A. ferrooxidans
ATCC 53993 and absent in ATCC 23270. All these results suggest that in
presence of high Cd concentrations all mechanisms to eliminate cad-
mium from both cytoplasm and periplasm would be required.
The possible functionality of A. ferrooxidans genes coding for the
two P-ATPases Lferr_0186 and Lferr_0209 and the four CDFs
Lferr_0625, Lferr_1214, Lferr_2050 and Lferr_1147 was tested by
cloning them in vector pGEMT Easy and these constructions were used
to transform E. coli BL21 DE3. The effect of this heterologous expression
of A. ferrooxidans genes in the transformed E. coli was estimated by
determining its MIC values in presence of different cadmium con-
centrations as seen in Fig. 5.
By expressing the P-type ATPases Lferr_0186 and Lferr_0209,
transformed strains were capable to resist high cadmium concentrations
(Fig. 5). Regarding the expression of the CDF genes from A. ferrooxidans
in E. coli, an increased Cd resistance was also seen, however, its effect
was not as high as that seen with the ATPases genes (Fig. 5). These
results indicate cells would activate its metal extrusion capacity to
avoid the toxicity of high cadmium concentrations in their environ-
ment.
3.2.2. Cellular processes and signaling
3.2.2.1. Cell wall/membrane/envelope biogenesis. Lipopolysaccharide
(LPS) is an amphipathic molecule essential for Gram-negative
microorganisms and is the main component of the outer membrane
[56]. The increased levels of proteins related to LPS synthesis in A.
ferrooxidans could be of great importance for cadmium resistance, since
microorganism such as Pseudomonas aeruginosa can accumulate metals
on the cell surface through electrostatic interactions with LPS present in
its membrane [57]. Thus, the polymers could act as a first barrier
against metal exposure. As seen in Table 1, an increase in proteins
involved in LPS synthesis was observed in cells exposed to Cd.
Lferr_0085, a glycosyl transferase family 2 protein, increased its levels
in cells adapted to 100 mM Cd. This protein belongs to the O-antigen
biosynthetic pathway [58–60] and is 100% identical to AFE_0082, a
rhamnosyltransferase from A. ferrooxidans ATCC 23270, also involved
in O-antigen biosynthesis. Moreover, a bioinformatic analysis using
Absynte tool [52] showed AFE_0082 is in the same genomic context
with other glycosyltransferases involved in the synthesis of
polysaccharides. On the other hand, an increased level of LptE
protein (Lferr_0618), associated to LPS transport, was identified
(Table 1). This protein, along with LptA, LptB, LptC, LptD, LptF and
LptG are part of the transport machinery of the mature LPS from the
inner membrane to the outer membrane. LptE is an essential lipoprotein
of the outer membrane. Together with LptD they form a heterodimeric
translocon that receives LPS from LptA and exports it to the surface of
8
Journal of Proteomics xxx (xxxx) xxx–xxx
Fold changea
Score Coverage Peptide Shock Adapted
number 75 mM 100 mM
125.00 29.00 4 0.646 –
301.00 61.17 6 – 0.642
112.00 11.80 3 – 0.714
212.67 64.00 5 0.663 0.765
the membrane [61,62]. Additionally, in the sub-category “Defense
mechanisms” (Table 1), an ABC transporter (Lferr_1097) showed
increased amounts under both Cd studied conditions. This protein
showed a 100% identity with a transporter of the MsbA family of A.
ferrooxidans ATCC 23270. This protein is a crucial homodimer in LPS
transport, since it translocates the core-lipid A complex from the
cytoplasm to the periplasmic face of the inner membrane of the cell
[61]. In vitro studies have determined that the ATPase activity of this
protein is stimulated by hexa-acetylated lipid A, Kdo2-lipid A or by LPS
to transport the complex [56]. Interestingly, mutant strains of MsbA
demonstrated the accumulation of mature LPS in the inner membrane
of cells [63]. This background strongly suggests an increase in LPS
transport in presence of cadmium, which could be justified by the
affinity of these molecules for divalent cations due to the abundance of
anionic functional groups in their structure [64]. This would cause
cadmium to accumulate on the cell surface as a passive response to
metal exposure [65,66]. Related to the role of LPS in metals resistance,
a recent report indicates that genes involved in LPS biosynthesis also
increased their levels in A. ferrooxidans ATCC 23270 subjected to
copper [28].
3.2.2.2. Post-translational modification, protein turnover, and
chaperones. Lferr_0701 (Table 1), a cytochrome C biogenesis protein,
increased its levels in cadmium shock. This protein showed 100%
identity with a disulfide interchange protein (DsbD) from A.
ferrooxidans ATCC 23270. DsbD is part of the Dsb protein family,
involved in disulfide bridge formation and isomerization in the
periplasm [67]. The oxidation of DsbA introduces disulfide bridges to
proteins. On the other hand, DsbB reoxidizes DsbA and keeps it in an
active state. In contrast, DsbD is the only provider of reducing power in
the periplasm, which is necessary for the correct formation of disulfide
bridges [68,69]. In E. coli, DsbD interacts with cytoplasmic
thioredoxins, transferring electrons via disulfide cascades. In this way
it maintains the active sites of DsbC, DsbE and DsbG in a reduced state
[69]. Previously, an up-regulation of DsbG in A. ferrooxidans ATCC
23270 was reported in presence of copper, possibly to repair affected
periplasmic proteins [24]. Some of the cadmium effects in cells
described by Nies, 1999 [9] correspond to thiol binding and proteins
denaturation. It is possible that up-regulation of DsbD (Lferr_0701) in
Table 1, could be involved in protein repair in cells exposed to
cadmium. DsbD is essential for maturation of cytochromes C in E. coli
[69]. The increased amount of this protein in A. ferrooxidans could be
associated with a response to the cytochrome downregulation already
described (Table 2).
Moreover, some studies have proposed that DsbD could have an
unknown chaperone function involved in metal resistance, since it
contains the previously described CXXC metal binding site [70]. Ob-
viously, future studies should be conducted in A. ferrooxidans to support
these speculations.
There was an unexpected decrease in levels of proteins associated
with stress response (Table 2, subcategory “Postranslational modifica-
tion, protein turnover, chaperons”) mainly under cadmium shock. An
increase in the amounts of these proteins was expected in presence of
J. Ramos-Zúñiga et al.
Fig. 3. Genetic contexts of possible genes related to cadmium resistance in A. ferrooxidans ATCC 53993. The genes of interest are shown in bold letters and gray
arrows. Genomic contexts were obtained by using Absynte tool [52].
Cd, since previous studies in P. aeruginosa under copper shock showed
an increase of this kind of proteins compared with those of cells adapted
to Cu [71]. Nevertheless, cadmium is a divalent cation unable to gen-
erate free radicals directly by Fenton and Haber Weis reactions [72].
Despite this, other microorganisms showed changes in the oxidative
response in presence of Cd, inducing SOD, DnaK, ClpB, GroEL/GroES,
Ahp, Hsp, amongst other [42–44]. As described before, Cd could re-
place Zn and Fe in some proteins, inactivating them. This in turn, would
increase the abundance of these displaced oxidizing metals in the cell,
generating additional oxidative stress [43,44]. Table 2 shows a decrease
of glutaredoxin (Lferr_2075), peptide methionine sulfoxide reductase
9
Journal of Proteomics xxx (xxxx) xxx–xxx
MsrB (Lferr_2558), heat shock proteins Hsp20 (Lferr_1347; Lferr_1743),
and GroES (Lferr_0699). In addition, levels of a poorly characterized
alkylhydroperoxidase like protein AhpD (Lferr_1491) (Table S2) were
also diminished. It is unknown why these proteins were downregulated
after a cadmium shock. However, this could be explained by the in-
ability of Cd to directly generate free radicals in a short time.
3.2.3. Information storage and processing
3.2.3.1. Translation, ribosomal structure and biogenesis. A variation in
the levels of ribosomal proteins was seen under both experimental
conditions. In the Cd-shock condition, 13 of these proteins, encoded by
J. Ramos-Zúñiga et al. Journal of Proteomics xxx (xxxx) xxx–xxx

Fig. 4. Transcriptional levels of several genes possibly related to Cd resistance in A. ferrooxidans ATCC 53993. The transcriptional levels of P-ATPases genes:
Lferr_0209 and Lferr_0186; CzcA RND-type genes: Lferr_1617 and Lferr_2060 and CDF genes Lferr_0625, Lferr_1214, Lferr_1147 and Lferr_2050 were determined at
the indicated cadmium conditions as described in Material and Methods section. Error bars indicate standard deviations based on three different experimental values.
Application of t-Student test were: **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01 and * p ≤ 0.05.

rplA, rplP, rplS, rplM, rplR, rpsZ, rplN, rpmE, rplI, rplT, rplC, rpsG and rplY
increased their levels in response to the sudden presence of Cd (see
Table 1, “Informatiom Storage and Processing category”, Translation,
Ribosomal Structure and Biogenesis sub-category). These results
suggest the translation machinery is further required due to a higher
demand of Cd-resistance determinants. This phenomenon has also been
observed in E. coli P4, where Cd presence accelerates the rate of
translation to synthethize proteins in response to stress [44].
Conversely, in cells adapted to 100 mM Cd, 11 ribosomal proteins
decreased their levels. Constant exposure to high metal concentrations

Fig. 5. Minimum inhibitory concentration (MIC) of E. coli BL21 DE3, transformed with pGEMT Easy vector. A. Cells transformed with putative P-type ATPases. B.
Cells transformed with putative CDF transporters. Control cells carried only the empty vector. Transformed cells were grown at 37 °C overnight and plasmid
expression was induced with IPTG. The MIC was the CdSO4 concentration in which growth of a particular clone was absolutely inhibited. Statistical analysis was done
as in Fig. 4.

10
J. Ramos-Zúñiga et al. Journal of Proteomics xxx (xxxx) xxx–xxx

Fig. 6. Putative new cadmium operon in A. ferrooxidans ATCC 53993. A. Transcriptional levels as determined by qRT-PCR of putative cadmium operon genes under
both Cd-shock and Cd-adapted cells. B. Relative positions of genes Lferr_0208, Lferr_0209, Lferr_0210 and Lferr_0211 (open arrows), locations for the primers used
(black arrows) and the intergenic products and sizes expected (black horizontal bars). C. PCR reactions were done using cDNA templates synthesized in the presence
(+RT) or absence (−RT) of reverse transcriptase to detect the possible presence of genomic DNA contamination by using agarose gel electrophoresis. Amplified
bands with their respective expected sizes are indicated by white arrows. A. ferrooxidans total RNA was extracted as described in Material & Methods from a culture
subjected to a 75 mM CdSO4 shock. cDNA was synthesized with a reverse primer hybridizing toward the 3′ end of Lferr_0211. PCR amplifications of the intergenic
regions between Lferr_0208–0209, Lferr_0209–0210 and Lferr_0210–0211 were carried out with the cDNA and corresponding pair of primers. Statistical analysis was
done as in Fig. 4.

could affect the translational machinery of the cell. This finding is in
agreement with the reduction in the overall rate of protein synthesis
previously seen in response to Cd [73].
Ribosomal proteins variation has been studied in response to other
type of stress such as superoxide, sodium salicylate, low temperatures
and H2O2 [74–76]. In all these cases there was a decrease and then an
increase in ribosomal proteins. Also, studies of transcriptional profiles
in E. coli showed a variation in levels of expression in transcription and
translation in presence of cadmium. In these studies, a short-term de-
crease was observed and after 25 min, an increase of the ribosomal
proteins levels was detected [42]. It is therefore possible that a similar
situation is also taking place in A. ferrooxidans in the present studies. It
has been previously suggested that protein biosynthesis and accumu-
lation may be adjusted specifically toward processes that achieve heavy
metal tolerance and adaptation to high concentrations of toxic heavy
metals in the environment [77].
3.2.4. Poorly characterized proteins
The “Poorly characterized” category presented the greatest number
of proteins changing their levels of expression: 39.5% for cadmium
shock and 36.5% for adapted cells (Fig. 2, Table S1, Table S2). This
group is conformed by several proteins with unknown domains. How-
ever, the uncharacterized protein Lferr_0210 (Table S1) is of great in-
terest since it is present in the exclusive GI of strain ATCC 53993. The
gene enconding this protein is in the same genomic context of the
previously described P-type ATPase (Lferr_0209), an ArsR type metal
regulator (Lferr_0208) and a hypothetical protein (Lferr_0211) (Fig. 3).
A bioinformatic analysis of the Lferr_0210 protein yielded interesting
results. When carrying out multiple alignments, proteins with the same
domain, DUF302, of unknown origin, were identified. These proteins
are found in a similar genomic context to that of Lferr_0210. Finally, 3
possible metal binding amino acids were identified in Lferr_0210 by
using MetalDetector: Cys66, Cys81 and Met98. Considering the char-
acteristics of this protein, its genomic context and the response of ad-
jacent genes to Cd stress, it would be very interesting to determine
whether they are part of an exclusive cadmium resistance operon in
strain ATCC 53993. Remarkably, the transcriptional level of these 4
genes increased similarly in both shock and adapted to Cd cells
(Fig. 6A). Additionally, by using RT-PCR it was demonstrated that these
genes were cotranscribed, suggesting they form a transcriptional unit or
possible operon (Fig. 6B, C). Therefore, this unit may code for a novel P-
type ATPase with a possible function in cadmium resistance in A. fer-
rooxidans, an idea that should be proven.
It will be important to further study this and other possible operons
involved in Cd resistance in A. ferrooxidans ATCC 53993. Most likely,
several of the up-regulated proteins in the presence of cadmium might
be Cd resistance determinants and it will be of great interest to continue
these studies in the future. A summary model illustrating some of the
main over- and under-expressed proteins identifieds in A. ferrooxidans
ATCC 53993 in presence of cadmium is seen in Fig. 7.
4. Conclusions
Proteomics, transcriptional and functional results indicate A. fer-
rooxidans ATCC 53993 cells would activate its metal extrusion capacity
to avoid toxicity of high cadmium concentrations in their environment.
Most of the up-regulated proteins in A. ferrooxidans ATCC 53993 sub-
jected to cadmium are related to those previously described in non-

11
J. Ramos-Zúñiga et al.
Fig. 7. A cartoon model of the main changes in A. ferrooxidans proteins in presence of cadmium. Green, proteins upregulated. Red, proteins downregulated in
presence of cadmium. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)
acidophilic bacteria. However, several of the poorly characterized or
unknown proteins that are part of the GI present in strain ATCC 53993,
such as Lferr_0210, which apparently forms part of a possible operon,
could be new possible cadmium-resistance determinants to be further
investigated. The upregulation of putative cadmium ATPases present in
the exclusive GI of strain ATCC 53993, is also important for cadmium
resistance in this acidophile. The presence of these exclusive transpor-
ters in this strain could also explain its greater resistance to cadmium
compared to strain ATCC 23270.
Conflict of interest
The authors declare no conflict of interest.
Acknowledgments
Supported by FONDECYT grant n° 1150791. The Proteomics Unit of
the Spanish National Biotechnology Centre belongs to ProteoRed
(PRB2-ISCIII) and is supported by FIS grant PT13/0001. The mass
spectrometry proteomics data have been deposited to the
ProteomeXchange Consortium via the PRIDE [78] partner repository
with the dataset identifier PXD012041.
Appendix A. Supplementary data
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.jprot.2018.12.013.
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