Bioinformatics and Transcriptional Study of the Nramp Gene in the Extreme Acidophile Acidithiobacillus ferrooxidans Strain DC

: The family of Nramp (natural resistance-associated macrophage protein) metal ion transporter functions in diverse organisms from bacteria to humans. Acidithiobacillus ferrooxidans ( At. ferrooxidans ) is a Gram-negative bacterium that lives at pH 2 in high concentrations of soluble ferrous ion (600 mM). The AFE_2126 protein of At. ferrooxidans of the Dachang Copper Mine (DC) was analyzed by bioinformatics software or online tools, showing that it was highly homologous to the Nramp family, and its subcellular localization was predicted to locate in the cytoplasmic membrane. Transcriptional study revealed that AFE_2126 was expressed by Fe 2 + -limiting conditions in At. ferrooxidans DC. It can be concluded that the AFE_2126 protein may function in ferrous ion transport into the cells. Based on the ∆ pH of the cytoplasmic membrane between the periplasm (pH 3.5) and the cytoplasm (pH 6.5), it can be concluded that Fe 2 + is transported in the direction identical to that of the H + gradient. This study indirectly conﬁrmed that the function of Nramp in At. ferrooxidans DC can transport divalent iron ions.


Introduction
NRAMP comprises a cluster of highly conserved integral membrane proteins and exists widely in bacteria, archaea, fungi, plants, animals, and humans [1][2][3][4][5][6]. The NRAMP family is a novel family of functionally related proteins defined by a conserved hydrophobic core of ten transmembrane domains [1]. Rodents and humans express two paralogous genes related to iron imbalance and immune diseases [7]. Nramp1 is an integral membrane protein, expressed only in cells of the immune system, and plays a significant role in the membrane of the phagosome during phagocytosis [8]. Nramp2 is a multivalent divalent cation transporter such as Fe 2+ , Mn 2+ , and Zn 2+ [9]. It is highly expressed in the intestine and is the major iron uptake system in mammals that do not depend on transferrin. The yeast proteins Smf1p and Smf2p may also transport divalent cations [10]. The natural resistance of mice to infection by intracellular parasites is controlled by the Bcg locus [11], which regulates the cytostatic/cytocidal activity of phagocytes. AtNRAMP1, AtNRAMP3, and AtNRAMP4 encode functional plant metal transporters [12] and are expressed at higher levels when the low iron content in plants is low [13,14]. In contrast, using semiquantitative reverse transcription PCR (semi-Q-RT-PCR), Agranoff et al. [15] suggested that the Nramp mRNA levels in M. tuberculosis will increase with the increases of surrounding Fe 2+ . Other transporter proteins from multiple 0.5 g MgSO 4 ·7H 2 O, and 0.01 g Ca(NO 3 ) 2 ) containing sulfur (9K + S (9K s ): 0.8% (g/L) filter-sterilized sulfur adjusted to pH 2.2 with H 2 SO 4 ). Based on the research purpose, the 9-K s medium was modified as follows: 9K s + Fe 2+ : 1, 10, 100, 300, and 600 mM FeSO 4 ·7H 2 O.

Growth Curves
Media (9K s + Fe 2+ ) was inoculated with fresh mid-exponential-phase cells of At. ferrooxidans DC pregrown in 9K s + Fe 2+ and incubated at 30 • C. Samples were taken every day and counted in a Petroff-Hausser counting chamber under a phase-contrast microscope at 400× magnification. The number of cells per milliliter was plotted against the incubation time. All experiments were conducted at least twice, with two to three repetitions for each treatment.

Cell Collection
Before collecting the cells, At. ferrooxidans DC cultures for nucleic acid were centrifuged at 4500×g to remove iron precipitates. The cell pellet was resuspended in 9-K salt solution for further use. The washed cells were collected by centrifugation at 12,000×g for 10 min. To reach the same number of the cells before RNA was extracted from different samples, the concentration of each sample was calculated according to the standard curve at A 600 . Then, the corresponding volume was taken to reach 5 × 10 8 cells.

General DNA Techniques
Genomic DNA was extracted from At. ferrooxidans DC by the manufacturers of the reagents (Tiangen Biotech, Beijing, China). Oligonucleotide primers (F1: 5'-GATGTCAATGTATGCCACGC-3' and R1: 5'-TATCCTCTATCGCCGCAATT-3') used in this study and PCR was performed using a 25-µL PCR reaction system and conditions comprising a denaturation step of 3 min at 95 • C, followed by 30 amplification cycles (denaturation for 45 s at 95 • C, annealing for 45 s at 60 • C, and elongation for 90 s at 72 • C) and a final elongation step at 72 • C for 10 min. Each PCR reaction contained a 2-ng template DNA, 1-µM required primers, and 0.2-mM each deoxyribonucleotide in a volume of 25 µL of 2 × PCR mix containing 1.5-mM MgCl 2 (Tiangen). The At. ferrooxidans DC Nramp coding region was amplified from chromosomal DNA preparations with a forward primer (F1) and reverse primer (R1). PCR product was purified from agarose gels with a DNA purification kit (Tiangen Biotech, Beijing, China) and sequenced by Sangon Biotech (Shanghai, China).

RNA Extraction and Reverse Transcription Quantitative Real-Time PCR (RT-qPCR)
Total RNA was extracted from At. ferrooxidans DC grown to the mid-exponential phase in 9-K s medium in the presence of ferrous ion (Fe 2+ : 1, 10, 100, 300, and 600 mM) using the RNAprep Pure Cell/Bacteria Kit (Tiangen Biotech, Beijing, China) according to manufacturer instructions. Then, the RNA was immediately inversed-transcribed to the cDNA in a volume of 20 µL by a Quantscript RT Kit (Tiangen Biotech, Beijing, China). One microgram of total RNA was used for each reaction. The RNA integrality was evaluated by 2.0% agarose gel electrophoresis, and its purity and ratio of absorbance at 260 nm to 280 nm (A260/A280 ratio) were measured with the NanoDrop ® ND-1000 Spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA). RNA was temporarily stored at −80 • C, and the resulting cDNA was stored in aliquots at −20 • C until further use.
The integrity of the total cellular RNA was checked by formaldehyde agarose gel electrophoresis and ethidium bromide staining. The definition of the 23S and 16S bands was very high, and the luminance ratio between 23S and 16S was 2/1. The A260/A280, which was quantified with the NanoDrop ® ND-1000 Spectrophotometer (NanoDrop Technologies, Montchanin, DE, USA), was about 1.75-2.0. These results indicated that the purity and integrity of the total cellular RNA was sufficient for this study.
To confirm that the RNA does not contain DNA, only RNA was used as a negative control in qPCR with other samples. The negative control did not produce a fluorescence signal, indicating that the RNA was not contaminated with DNA. The melt curve had only one peak, so there was no nonspecific product or primer dimer in the PCR products.
Primers for RT-qPCR assays [39] and amplicon size are listed in Table 1. The real-time PCR reactions were performed in the iCycler iQ real-time PCR detection system (Bio-Rad) using the RealMasterMix SYBR Green Kit (Tiangen Biotech, Beijing, China). Each 25-µL reaction contained 1 µL of cDNA template, 12.5 µL of real master mix, including the SyberGreen I fluorescent dye, enabling the monitoring of the PCR amplification, and the gene-specific primer pair at a final concentration of 200 nM for each primer. The cycling protocol was as follows: initial denaturation for 3 min at 95 • C, followed by 40 cycles of 15 s at 95 • C, 20 s at 59 • C, and 35 s at 68 • C. Fluorescence was measured after the extension phase at 68 • C. The PCR products were subjected to a melting curve analysis, which was followed by 80 consecutive cycles of 55 • C for 10 s, with each cycle increased by 0.5 • C. Specific amplification was confirmed by a single peak in the melting curve. The reactions for each target gene were performed in triplicate and the same PCR run. The reverse-transcribed product (cDNA) 10-fold dilutions (ranging from 10 −4 to 10 −9 ) were used to generate a 6-point standard curve for every gene by using the quantification cycle (C q , previously known as the threshold cycle (C t )) value versus the logarithm of each dilution factor. Transcript quantities were calculated from the standard curve by the software accompanying the iCycler iQ real-time PCR detection System (Bio-Rad) set with default parameters. Each experiment included no template control, a negative control (RNA template), and a positive control (DNA template). Every sample was performed in triplicate.

Expression Stability of Candidate Reference Genes
According to the methods of Vandesompele and Andersen implemented in the Visual Basic Application geNorm, the expression of the 7 selected reference genes (map, gmk, rplL, gyrA, recA, era, and rpoC) [40] at six different Fe 2+ concentration were analyzed. A value of M results in a rank, where the best genes are those with the lowest M value [41]. TIGRFAMs were used to classify the reference genes by function (Table 2) [42]. Relative gene expression levels were normalized based on multiple reference genes. The ∆∆Ct-based method (GeNorm software) was used to normalize the expression of AFE_2126 by correlating their Ct values of samples grown in the presence of different concentrations of ferrous iron to the Ct values of the reference genes.

Nucleotide Sequence Accession Number
The nucleotide sequence of the AFE_2126 gene and 7 selected reference genes (map, gmk, rplL, gyrA, recA, era, and rpoC) from At. ferrooxidans DC reported in this paper have been deposited in the National Center of Biotechnology Information of America nucleotide sequence database with accession numbers KC677728, KJ854561, KJ854563, KJ854559, KJ854562, KJ854560, KJ854557, and KJ854558.

Identification and Sequencing of AFE_2126 Gene from At. Ferrooxidans DC
At. ferrooxidans DC was confirmed as At. ferrooxidans by the 16S ribosomal RNA gene (gi:209405433) fragment comparison with At. ferrooxidans ATCC23270 (gi:28194033). Based on the sequence of the At. ferrooxidans ATCC23270 chromosome, two primers, F1 and R1, were designed to amplify the putative open reading frame (ORF) encoding AFE_2126 from the genomic DNA of At. ferrooxidans DC. The amplified product was purified and confirmed by DNA sequencing. The results showed that AFE_2126 has a predicted length of 1371 nt ORF, encoding 456 amino acid residues (molecular mass 49.5 ku), and an isoelectric point of 6.55. It contains 24 acidic amino acids, 32 basic, 94 polar, and 306 nonpolar, covering 19 kinds of amino acids but Cys (cysteine). The percentages were calculated as 5.3% acidic residues, 7.0% basic, 20.6% polar, and 67.1% nonpolar.

Prediction of Conserved Sites and Domains of the NRAMP Homolog
Multiple sequence alignment analysis revealed that there were many conserved sites and domains ( Figure 2). As shown in Figure 2a Multiple sequence alignment analysis revealed that there were many conserved sites and domains (Figure 2), which may be provided with specific roles. Some of them were studied and clarified by some authors, and the others were unclear in function. After multiple sequence alignments from bacteria to humans, the results showed that there were many invariant and variant sites, which may be important for the function of iron transport and the apparent transporter affinity for H + .

Selection and Evaluation of Reference Genes
We studied the mRNA expression levels of seven candidate reference genes from At. ferrooxidans DC in six different Fe 2+ concentrations using RT-qPCR and evaluated their expression stabilities. In short, the geNorm method is based on the pairwise comparison method. It depends on the calculation of an M value, which is defined as the average pairwise variation of a particular gene from all other genes [41]. The M values are less than 1.5, which satisfy the requirements for the stability evaluation of the candidate reference genes (Supplementary File S4). According to geNorm, era, gmk, and rpoC showed the least variability in expression in all conditions evaluated (Supplementary File S5). Descending order of the expression stabilities was era, gmk, rpoC, gyrA, recA, map, and rplL. We chose the first three candidate reference genes (era, gmk, and rpoC) as reference genes of this study to ensure the results were more reliable.

Expression of NRAMP in At. Ferrooxidans DC in Different Growth Conditions
RT-qPCR experiment was designed to measure NRAMP mRNA levels. We chose three candidate reference mRNA (era, gmk, and rpoC) as reference genes to normalize the expression of AFE_2126 mRNA, because they showed the least variability in ferrous iron concentrations. Then, we got a different expression level of AFE_2126 mRNA in different ferrous iron concentrations. In order to easily identify the change trends, we set the minimum value as one and others greater than one ( Figure 3). Figure 3 shows that, when the ferrous iron concentration was 600 mM, the expression level of AFE_2126 mRNA was the least. The relative expression of different ferrous ion concentrations: 300 mM, 100 mM, 10 mM, 1 mM, and 0 mM, compared to 600 mM, were 1.1, 2.3, 11.7, 13.6, and 18.5-fold, respectively (Figure 3 and Supplementary File S6). The results showed that AFE_2126 mRNA levels in At. ferrooxidans DC were upregulated in response to decreases in the ambient Fe 2+ . According to an unpaired t-test at 95% confidence, we found that there was no significant change in the relative expressions at growth conditions of 600-mM and 300-mM ferrous ion concentrations. The ferrous ion concentration may be too high, indicating that it was no longer the limiting factor for bacterial growth. Instead, it became a toxic factor, which limited the expression of ferrous ion uptake genes.

Selection and Evaluation of Reference Genes
We studied the mRNA expression levels of seven candidate reference genes from At. ferrooxidans DC in six different Fe 2+ concentrations using RT-qPCR and evaluated their expression stabilities. In short, the geNorm method is based on the pairwise comparison method. It depends on the calculation of an M value, which is defined as the average pairwise variation of a particular gene from all other genes [41]. The M values are less than 1.5, which satisfy the requirements for the stability evaluation of the candidate reference genes (Supplementary File S4). According to geNorm, era, gmk, and rpoC showed the least variability in expression in all conditions evaluated (Supplementary File S5). Descending order of the expression stabilities was era, gmk, rpoC, gyrA, recA, map, and rplL. We chose the first three candidate reference genes (era, gmk, and rpoC) as reference genes of this study to ensure the results were more reliable.

Expression of NRAMP in At. Ferrooxidans DC in Different Growth Conditions
RT-qPCR experiment was designed to measure NRAMP mRNA levels. We chose three candidate reference mRNA (era, gmk, and rpoC) as reference genes to normalize the expression of AFE_2126 mRNA, because they showed the least variability in ferrous iron concentrations. Then, we got a different expression level of AFE_2126 mRNA in different ferrous iron concentrations. In order to easily identify the change trends, we set the minimum value as one and others greater than one (Figure 3). Figure 3 shows that, when the ferrous iron concentration was 600 mM, the expression level of AFE_2126 mRNA was the least. The relative expression of different ferrous ion concentrations: 300 mM, 100 mM, 10 mM, 1 mM, and 0 mM, compared to 600 mM, were 1.1, 2.3, 11.7, 13.6, and 18.5-fold, respectively (Figure 3 and Supplementary File S6). The results showed that AFE_2126 mRNA levels in At. ferrooxidans DC were upregulated in response to decreases in the ambient Fe 2+ . According to an unpaired t-test at 95% confidence, we found that there was no significant change in the relative expressions at growth conditions of 600-mM and 300-mM ferrous ion concentrations. The ferrous ion concentration may be too high, indicating that it was no longer the limiting factor for bacterial growth. Instead, it became a toxic factor, which limited the expression of ferrous ion uptake genes.

The AFE_2126 Protein Belongs to the NRAMP Family of Membrane Transporters
The NRAMP family is a novel family of functionally related proteins that is defined by a common conserved hydrophobic core of ten transmembrane domains and either one or two nonconserved, highly hydrophobic TM domains [43,46]. The AFE_2126 protein sequence indicated the presence of eleven internal helices. AFE_2126 amino acid sequences share identity with eukaryotes in the range of 15-20%. Still, they have many remote homologies based on discrete sequence motifs, which are highly conserved between many members of the Nramp family ( Figure  2). On the basis of these analyses, it is proposed that the AFE_2126 protein belongs to the NRAMP family of membrane transporters.

AFE_2126 Protein has Many Conserved Motifs Associated with Metal Ion Transport
The predicted extracytoplasmic loop 1 (between TM1 and TM2) contains the Nramp conserved motif D(P/A)G(N/G) (Figure 2), which is of importance for the proper function of the transporter [7]. When DMT1 residue Gly 88 was replaced by Ala, it led to abolishing both the Me 2+ uptake ( 55 Fe 2+ , 54 Mn 2+ and 60 Co 2+ ) and metal-state currents [47]. The motif DAGG extracytoplasmic loop 1 existed in At. ferrooxidans DC and most prokaryotes, and motif DPGN existed in most eukaryotes and few prokaryotes (Supplementary File S3). As shown in Figure 2, TMS1 (transmembrane segments) contained a motif GP(G/A), the extracytoplasmic loop 2 contained a motif E(V/F)xG, TMS 6 contained a motif GxxxxP(H/W)xx(F/Y), and intracytoplasmic loop 2 contained a motif Qx(L/M)xxxxGxxTxxx(L/H)xxx(C/I). The functions of these conserved sites were unclear in At. ferrooxidans DC, so the function will be studied through mutagenesis approaches and a toxicity assay [48] in the future.

The AFE_2126 Protein Belongs to the NRAMP Family of Membrane Transporters
The NRAMP family is a novel family of functionally related proteins that is defined by a common conserved hydrophobic core of ten transmembrane domains and either one or two nonconserved, highly hydrophobic TM domains [43,46]. The AFE_2126 protein sequence indicated the presence of eleven internal helices. AFE_2126 amino acid sequences share identity with eukaryotes in the range of 15-20%. Still, they have many remote homologies based on discrete sequence motifs, which are highly conserved between many members of the Nramp family ( Figure 2). On the basis of these analyses, it is proposed that the AFE_2126 protein belongs to the NRAMP family of membrane transporters.

AFE_2126 Protein has Many Conserved Motifs Associated with Metal Ion Transport
The predicted extracytoplasmic loop 1 (between TM1 and TM2) contains the Nramp conserved motif D(P/A)G(N/G) (Figure 2), which is of importance for the proper function of the transporter [7]. When DMT1 residue Gly 88 was replaced by Ala, it led to abolishing both the Me 2+ uptake ( 55 Fe 2+ , 54 Mn 2+ and 60 Co 2+ ) and metal-state currents [47]. The motif DAGG extracytoplasmic loop 1 existed in At. ferrooxidans DC and most prokaryotes, and motif DPGN existed in most eukaryotes and few prokaryotes (Supplementary File S3). As shown in Figure 2, TMS1 (transmembrane segments) contained a motif GP(G/A), the extracytoplasmic loop 2 contained a motif E(V/F)xG, TMS 6 contained a motif GxxxxP(H/W)xx(F/Y), and intracytoplasmic loop 2 contained a motif Qx(L/M)xxxxGxxTxxx(L/H)xxx(C/I). The functions of these conserved sites were unclear in At. ferrooxidans DC, so the function will be studied through mutagenesis approaches and a toxicity assay [48] in the future.

AFE_2126 Protein Mediates the Uptake of Fe 2+
The results showed that AFE_2126 mRNA levels in At. ferrooxidans DC were upregulated in response to decreases in the ambient Fe 2+ . Navarro et al. [49] suggested that the gene coded for the potential efflux system, due to the increase of gene expressions with the rise of the metal ion concentrations. In contrast, the results in Figure 3 suggest that AFE_2126 is more probably related to a Fe 2+ import protein rather than to a Fe 2+ efflux protein, as had been previously proposed through a bioinformatic prediction and in the reports of other organisms [50]. The cytoplasmic membrane expression of mRNA is increased in iron deficiency, which indicates that the microorganisms take up necessary iron to maintain the normal metabolism of life by increasing the expression of specific functional genes to survive. In the future, we will use the markerless mutagenesis strategy [51,52] to further verify the function of AFE_2126 through the mutant responses under various ferrous ion stresses.

AFE_2126 Protein is a Symporter of H + and Iron (Fe 2+ )
As known to all, the growth-medium pH of At. ferrooxidans is between 1.5-2.5 [20,53], while the periplasm pH is around 3.5 [54], and the cytoplasm pH is thought to be 6.5 [20]. The AFE_2126 gene was predicted to lie in the cytoplasmic membrane (between the cytoplasm and the periplasm). From the results of these analysis, it suggests that AFE_2126 functions to transport Fe 2+ from the periplasm to the cytoplasm ( Figure 4). Overall, these results also conclude that Fe 2+ is transported in the direction identical to that of the H + gradient, i.e., symport (Figure 4), as demonstrated for Nramp2 [55], rather than antiport, as demonstrated for Nramp1 in Xenopus laevis [56]. This finding is consistent with the function in the iron transport from the acidified lumen of the endosomes into the cytoplasm [57]. In this case, the energy transported is provided by an electrochemical proton gradient, rather than directly driven by ATP hydrolysis [58,59]. For the Nramp1 function, ATP hydrolysis is maintained by the activity of the vesicular ATPase [60]. In the normally acidic late-endosomal/lysosomal compartment, Nramp1's function is to transport Fe 2+ and other metal ions from a high pH (cytoplasmic) to a low pH (intravesicular) [56]. Unusually, the proton uptake drives cation symports [60]. Gu et al. [55] indicated that the cotransport of H + could lead to a slight decrease of pH on the inner membrane surface, resulting in maintaining the dissolution of iron before its binding to intracellular proteins. The transfer of ferrous iron into cells may be used to synthesize functional materials such as magnetosomes [34,61]. The results showed that AFE_2126 mRNA levels in At. ferrooxidans DC were upregulated in response to decreases in the ambient Fe 2+ . Navarro et al. [49] suggested that the gene coded for the potential efflux system, due to the increase of gene expressions with the rise of the metal ion concentrations. In contrast, the results in Figure 3 suggest that AFE_2126 is more probably related to a Fe 2+ import protein rather than to a Fe 2+ efflux protein, as had been previously proposed through a bioinformatic prediction and in the reports of other organisms [50]. The cytoplasmic membrane expression of mRNA is increased in iron deficiency, which indicates that the microorganisms take up necessary iron to maintain the normal metabolism of life by increasing the expression of specific functional genes to survive. In the future, we will use the markerless mutagenesis strategy [51,52] to further verify the function of AFE_2126 through the mutant responses under various ferrous ion stresses.

AFE_2126 Protein is a Symporter of H + and Iron (Fe 2+ )
As known to all, the growth-medium pH of At. ferrooxidans is between 1.5-2.5 [20,53], while the periplasm pH is around 3.5 [54], and the cytoplasm pH is thought to be 6.5 [20]. The AFE_2126 gene was predicted to lie in the cytoplasmic membrane (between the cytoplasm and the periplasm). From the results of these analysis, it suggests that AFE_2126 functions to transport Fe 2+ from the periplasm to the cytoplasm ( Figure 4). Overall, these results also conclude that Fe 2+ is transported in the direction identical to that of the H + gradient, i.e., symport (Figure 4), as demonstrated for Nramp2 [55], rather than antiport, as demonstrated for Nramp1 in Xenopus laevis [56]. This finding is consistent with the function in the iron transport from the acidified lumen of the endosomes into the cytoplasm [57]. In this case, the energy transported is provided by an electrochemical proton gradient, rather than directly driven by ATP hydrolysis [58,59]. For the Nramp1 function, ATP hydrolysis is maintained by the activity of the vesicular ATPase [60]. In the normally acidic late-endosomal/lysosomal compartment, Nramp1's function is to transport Fe 2+ and other metal ions from a high pH (cytoplasmic) to a low pH (intravesicular) [56]. Unusually, the proton uptake drives cation symports [60]. Gu et al. [55] indicated that the cotransport of H + could lead to a slight decrease of pH on the inner membrane surface, resulting in maintaining the dissolution of iron before its binding to intracellular proteins. The transfer of ferrous iron into cells may be used to synthesize functional materials such as magnetosomes [34,61].

Conclusions
After genome sequencing and sequence-similarity analyses, it is speculated that AFE_2126 of At. ferrooxidans DC belongs to the Nramp family, its N-terminus is cytoplasmic, followed by 11 transmembrane segments, and the C-terminus is periplasmic, and the loops between TMS1/2, 3/4, 5/6, 7/8, and 9/10. The transporter property of AFE_2126 was studied by indirect measurement (RT-qPCR), and the results presented that AFE_2126 mRNA was upregulated in response to an iron deficiency, showing it can mediate the uptake of Fe 2+ . Based on the ∆pH of the cytoplasmic membrane between the cytoplasm and the periplasm, it can be concluded that the AFE_2126 protein is a symporter of H + and iron (Fe 2+ ). Fe 2+ and H + diffuse through the porins of the outer membrane (OM) and are subsequently transported into the cytoplasm by NRAMP anchored in the inner membrane (IM). The establishment of its expression pattern in bacteria and its subcellular localization will help elucidate the function of the NRAMP protein in bacteria.