3.1 Isolation, magnetic screening and identification
The distinct and vivid color change from colorless to red-brown can be easily observed by naked eyes after inoculating the soil samples into 9K medium in a shaking incubator with 120 rpm at 30°C for 2–3 weeks. The serially diluted cultures were separately spread on the surface of 9K solid medium and incubated at 30°C for 10 days. It can be observed that the bacterial colonies with an average diameter of 1.0 ± 0.2 mm appeared smooth, round or oval with the rust-colored raised center surrounded by a yellow zone (Fig. 1a), which was similar to the results reported by Yan et al. [14]. The single colony was scraped and inoculated into 9K liquid medium for incubation and magnetic enrichment. The Pasteur tube appeared a brown liquid after few minutes under the “race-track” liquid magnetic separation (Fig. 1c). It can be found that the bacterial cells from the tip end of a cut-off Pasteur tube showed weak magnetotaxis on the semisolid 9K plates under an artificial magnetic field, while the magnetostatic phenomenon did not occur in the case of no artificial magnetic field (Fig. 1d). After repeating these procedures, the bacterial cells were collected. SEM analysis showed that the morphology of the bacterium is rod-shaped with 0.2–0.4 µm in width and 0.8–1.4 µm in length (Fig. 1b), which was consistent with the previous report [16].
Sequencing results showed that the length of the 16S rDNA fragment of the isolate was 1429 bp. The NCBI BLAST sequence homology analysis showed the 16S rDNA of the isolate shared 99.93% similarity with At. ferrooxidans ATCC 19859, thus the isolate was named At. ferrooxidans BYM (Fig. 1e). It also can be found that the 16S rDNA of At. ferrooxidans BYM exhibited high similarity (> 97.0%) with six strains of Acidithiobacillus genus, whereas only showed 80.80% homology with M. gryphiswaldense MSR-1.
3.2 Magnetosome-producing potential assay
MTB usually mineralize membrane-bound intracellular iron crystals (magnetosomes) which enable the cells to align and swim along magnetic field lines in a preferred direction. The inorganic component of magnetosomes is magnetite (Fe3O4) or greigite (Fe3S4). In order to biomineralize magnetite, MTB require several orders of magnitude more iron than non-MTB, reaching up to 4.0% of their cell dry weight [17]. There are many similar characteristics between MTB and At. ferrooxidans such as morphological types, trophic type, motility, ecological distribution, and growth temperature. It has been reported that At. ferrooxidans can synthesize magnetosomes and contain more than 2.0% iron based on dry weight [11]. Thus, sufficient iron is the crucial substrate for magnetosome-producing bacteria to form magnetosomes. Results showed that At. ferrooxidans BYM can produce magnetosomes within a wide range of ferrous sulfate concentrations (15–40 g/L) (Fig. 2). It appeared that the cell dry weight of At. ferrooxidans BYM increased with the increase of ferrous sulfate concentration, but the yields of magnetosomes were found to increase first and then decrease. The maximum yields of 7.1137 mg/g magnetosomes occurred at 30 g/L ferrous sulfate. However, the magnetosome yields declined to 4.5294 mg/g when the ferrous sulfate concentration increased to 40 g/L. This finding was supported by the previous study in which the high concentration of ferrous iron promoted the growth of bacteria but inhibited the synthesis of magnetosomes [18]. Additionally, the previous works suggested that the intracellular iron content of M. magneticum AMB-1 cells presented a logarithmic increase with increasing external iron concentration from 10 to 100 µM. However, a mutant of M. magneticum AMB-1 lacking MAI which was unable to form magnetosomes can still incorporate a large amount of iron [19]. Therefore, the iron demand for the growth of MTB cell was not completely consistent with that for magnetosomes synthesis, which is similar to our result.
The genes associated with the synthesis of magnetosomes such as mpsA-like, magA-like and mamB-like existed in the chromosome of At. ferrooxidans BYM (Table 2). It has been reported that the highest expression level of these genes appeared at 150–200 mM of ferrous concentration, while the iron-deficient or iron-excessive conditions (Below or above 150–200 mM ferrous concentration) could adversely affect their expression [20]. Additionally, the jarosite precipitation formed by hydroxylation would increase with the increase of the ferrous concentration, which could gradually cover the bacterial surface and hinder the electron transfer from iron as well as proton diffusion from bacterial cells, resulting in the reduction of magnetosome formation [11].
Table 1
Comparison of the genome characteristics of At. ferrooxidans BYM and other Acidithiobacillus spp. available in GenBank database
strains | Level | DNA types | Length (kb) | GC% | Predicted gene numbers | Protein |
ATCC 53993 | complete | chromosome | 2885.04 | 58.90 | 2926 | 2779 |
ATCC 23270 | complete | chromosome | 2982.40 | 58.80 | 3087 | 2909 |
Hel18 | partial | chromosome | 3109.16 | 58.60 | 3179 | 2955 |
BY0502 | partial | chromosome | 2976.67 | 56.80 | 3186 | 2821 |
CCM 4253 | partial | chromosome | 3196.56 | 58.60 | 3278 | 3049 |
IO-2C | partial | chromosome | 2716.89 | 58.70 | 2822 | 2620 |
YQH-1 | partial | chromosome | 3111.22 | 58.60 | 3189 | 2956 |
DLC-5 | partial | chromosome | 4184.22 | 57.60 | - | - |
RVS1 | partial | chromosome | 2826.31 | 58.80 | 2888 | 2705 |
YNTRS-40 | complete | chromosome | 3257.04 | 58.47 | 3339 | 3053 |
plasmid | 47.104 | 56.43 | 70 | 46 |
BY-3 | partial | chromosome | 3832.34 | 57.80 | 4067 | 3777 |
BYM | complete | chromosome | 3208.389 | 58.54 | 3260 | 2184 |
plasmid | 47.11 | 56.44 | 54 | 27 |
Table 2
Comparison of magnetosome numbers between At. ferrooxidans BYM to other magnetosome-producing bacteria.
Strains | Nutritional type | Oxygen demand | Number per cell | Incubation time (h) | Refences |
M. magneticum AMB-1 | chemoorganoheterotrophic | microaerophilic | 6–18 | 130 | [33] |
M. gryphiswaldense MSR-1 | chemoorganoheterotrophic | aerobic and microaerobic | 26–43 | 60 | [37] |
M. magnetotacticum MS-1 | chemoorganoheterotrophic | microaerophilic | 21.8 | 168 | [32] |
D. magneticus RS-1 | chemoorganoheterotrophic | anaerobic | 40 | 168 | [34] |
Magnetospirillum sp. ME-1 | chemoorganoheterotrophic | microaerophilic | 13–21 | 49 | [40] |
Magnetofaba australis IT-1 | chemolithoautotrophic | microaerophilic | 2–10 | ༞168 | [12] |
chemoorganoheterotrophic | 7–13 | ༞168 | [12] |
Gammaproteobacteria SS-5 | chemoorganoheterotrophic | microaerophilic | 13–27 | ༞168 | [35] |
Gammaproteobacteria BW-2 | chemoorganoheterotrophic | microaerophilic | 21–39 | ༞168 | [35] |
Candidatus Magnetobacterium bavaricum LO-1 | Uncultured | microaerophilic | 100–200 | uncultured | [39] |
Rhodospirillum rubrum “magneticum” | photoheterotrophic | anaerobic | 14 | 160 | [36] |
At. ferrooxidans BY-3 | chemolithotrophic | aerobic | 3–5 | 48 | [11] |
At. ferrooxidans 23270 | chemolithotrophic | aerobic | 2–4 | 146 | [38] |
At. ferrooxidans BYM | chemolithotrophic | aerobic | 6–9 | 48 | This study |
One of the most important factors affecting microbial growth and magnetosome synthesis is oxygen. It has been demonstrated that magnetosome formation in MTB occurred only in a narrow range of low oxygen concentration. Dissolved oxygen (DO) was the major factor affecting the magnetosome production of M. gryphiswaldense MSR-1 and the yields in a microaerobic condition with 5–10 ppm oxygen was found to be 1.43 times higher than that in anaerobic condition [21]. The formation of magnetosomes in M. magneticum AMB-1 was favorably carried out at the low level of O2 (≤ 18.7 µM), but was strongly inhibited under high O2 concentrations (50–100 µM) [22]. The effect of oxygen on the magnetosome-producing potential of At. ferrooxidans BYM were investigated under different aeration rates (0.5–1.5 L/min). Results showed that the formation of magnetosomes produced at any given aeration rate and the yields of magnetosomes ranged from 0.5896 to 4.5336 mg/g (Fig. 2). It was observed that the bacterial biomass significantly increased with the increase of ventilatory capacity due to the typical aerobic characteristic of At. ferrooxidans BYM, while the magnetosome yields tended to saturation and decreased gradually with further increase of air supply. The low aeration rate might cause the low level of oxygen dissolved in 9K medium, which could inhibit the growth of At. ferrooxidans BYM, resulting in a lower final cell density and further a lower magnetosome production. This finding indicated that the oxygen demand for cell growth of At. ferrooxidans BYM was higher than that for magnetosome formation.
It appeared that the magnetosome yields began to decrease once the aeration rate exceeded 1.2 L/min (Fig. 2). The enhancement of oxygen mass transfer in the fermentation system possibly caused by the increase of aeration rate could cause high autooxidation of ferrous ion [23], which would compete with biological oxidation mediated by bacteria, leading to an advice effect on the growth of At. ferrooxidans BYM. The reactive oxygen species (ROS) produced by the metabolism of At. ferrooxidans BYM with O2 as the terminal electron acceptor can damage DNA ultimately leading to cell death. It has been reported that the formation of magnetosomes can efficiently scavenge the intracellular ROS in MTB [24]. Therefore, the magnetosome formation in At. ferrooxidans might play critical role in removing ROS to avoid damage to cells. These indicated that the suitable aeration rate can not only satisfy the oxygen requirement for bacterial oxidation of ferrous ions, but also maintain the balance between ROS scavenging and magnetosome formation.
Nitrogen source is also an important impact factor for the growth of MTB and magnetite magnetosome biomineralization. It can be seen from Fig. 2 that the ammonium sulfate in the concentration range of 0.24 to 1.80 g/L was well documented to favor magnetosome formation. The magnetosome yields increased from 2.0099 to 13.1291 mg/g with ammonium sulfate concentration (Fig. 2). It has been reported that there is a substantial demand for nitrogen to form proteins for the assembly of magnetosome vesicles [25]. Additionally, the magnetosome-associated membrane proteins play a significant role in magnetosome crystals growth inside MTB [26]. Therefore, in the case of a sufficient supply of nitrogen sources, At. ferrooxidans BYM can efficiently form magnetosomes. Generally, the formation of magnetosomes would be adversely affected once the concentration of ammonium sulfate exceeds a certain value. It can be observed that the magnetosome yields decreased to 7.6584 mg/g when the ammonium sulfate concentration exceeded 1.2 g/L. In such cases, the production of massive amounts of jarosite layer on the cell surface at high ammonia ion concentration could prevent the metabolism of bacteria [23], resulting in the reduction of magnetosome yield. Therefore, the nitrogen source was not only necessary for the cell growth of magnetosome-producing bacteria but was also essential for magnetosome formation. Our finding was consistent with the previous studies on MTB [21, 27]. It has been confirmed that NaNO3 was more likely to favor the magnetosome formation of M. gryphiswaldense MSR-1 than NH4Cl and (NH4)2SO4, since NaNO3 was used as terminal electron acceptor to alternate oxygen for the survival of microaerobic MTB. Although the cell growth rate of M. gryphiswaldense MSR-1 increased by 27% with the increase of NaNO3 concentrations from 20 to 60 mM, the magnetosome yields decreased [21]. Low nitrate concentration (0.004 M) was found to promote the magnetosome formation of M. gryphiswaldense MSR-1 significantly. In contrast, the magnetosome yield decreased but the cell growth was not affected in nitrate concentrations ranging from 0.01 to 0.02 M [27].
Although organic compounds usually appear adverse effects on the growth of At. ferrooxidans, our results suggested that the gluconic acid in concentrations ranging from 5 to 15 mM can promote the yields of magnetosomes from 2.6009 to 6.8854 mg/g (Fig. 2). The gluconic acid might act as an iron-chelating agent to chelate ferric ions and enhance the bioavailability of iron by forming ferric–ligand complexes. This phenomenon was in agreement with the results of Edouard Alphandéry et al., who noted the growth of M. magneticum AMB-1 and the production of magnetosomes were improved by the chelating agents including ethylenediaminetetraacetic acid (EDTA), ascorbic acid, citric acid, alendronic acid, and neridronic acid [28]. The magnetosome formation depended on siderophores with a high and specific affinity for the ferric ion to incorporate iron, which has been described in diverse groups of MTB [29]. It has been reported that At. ferrooxidans can synthesize several potential siderophore outer membrane receptors (OMRs) involved in transporting ferric ions to the cytoplasm [30]. Therefore, the ferric-chelating agents can save energy consumption for synthesizing siderophores and OMRs, thus providing more energy for cell growth. Additionally, gluconic acid could alleviate the passivation formation and remove the precipitate layer on the surface of bacteria, since it can chelate with ferric iron of jarosite which is the main component for passivation and precipitate layer [28]. Thus, the adverse effect of ferrous iron metabolic by-products on nutrient mass transfer and energy metabolism of At. ferrooxidans BYM could be relieved to a certain extent, resulting in a favorable conidiation for bacterial growth and magnetosome formation. Moreover, the redox potential of the cultivation system of At. ferrooxidans is dominated by the ratio of dissolved ferric to ferrous ions. Adding gluconic acid might affect the redox potential and pH because jarosite formation can be eliminated or partially alleviated in high redox potential and low pH [31]. It was observed that the magnetosome yields began to decrease with the increase of the gluconic acid concentration ranged from 15 to 50 mM (Fig. 2). Two possible explanations for this phenomenon can be considered. One is that high concentration of organic chelating agent might cause toxicity to autotrophic At. ferrooxidans BYM and affect its growth. The other reason is that the reactive oxygen species generated as the result of the alcohol functional unbinding from iron could damage the out membrane of bacterial cells.
3.3 TEM, STEM and HRTEM observation
It has been documented that the magnetosomes exist as one, two, or multiple chains inside living MTB [3]. Due to the presence of these magnetosomes, MTB have the ability to move along the applied external magnetic field. It can be observed that At. ferrooxidans BYM was capable of synthesizing intracellular magnetosomes which were not arranged in chain but distributed irregularly in cells (Fig. 3a). The magnetotaxis and magnetism of At. ferrooxidans BYM are very weak (Fig. 1d), which might be attributed to the random distribution of magnetosomes, which is a way of storing iron sources in the cell [23].
TEM observation indicated that the number of magnetosomes per cell was 6–9 in At. ferrooxidans BYM (Fig. 3a, white arrows). A comparison of the magnetosome number between At. ferrooxidans BYM to other magnetosome-producing bacteria was shown in Table 2 [11, 12, 32–40]. Although the magnetosome number per cell of At. ferrooxidans BYM is similar to or lower than most MTB, At. ferrooxidans BYM is still promising for magnetosome production due to its mild culture condition, simple nutritional requirement, and short incubation time. The particle size of the magnetosomes in At. ferrooxidans BYM varied from 20 to 80 nm (Fig. 3a, b and i), which was similar to the finding in At. ferrooxidans BY-3 [15]. It has been addressed that the size of the mature magnetosomes in most MTB fall within a narrow range of 35–120 nm, which were in the magnetic-single- domain range [3]. Therefore, the magnetosomes formed in At. ferrooxidans BYM appeared a single-domain (SD) size, possibly resulting in the permanent magnetism of the magnetosomes and thus the weak magnetotaxis behavior of cells.
We performed STEM-EDXS elemental mapping and HRTEM investigations on the magnetosomes to localize and characterize the intracellular iron mineral phases. It appeared that At. ferrooxidans BYM accumulated iron and intracellularly formed amorphous granules with an elemental composition dominated by Fe, C, N and O (Fig. 5b-h). This indicated that the inorganic crystal was magnetite and composed of organic membrane. The finding was supported by the previous studies, in which the magnetosomes of MTB and ferrous oxidizers from freshwater were consisted of magnetite crystal and phospholipid bilayer membrane [4]. It can be seen from Fig. 5b (white arrows) and Fig. 5i that the crystals mainly exhibited elongated prismatic shape which usually existed in the MTB affiliated with the alpha, eta and gamma subclasses of Proteobacteria [5]. The presence of the irregular shaped crystals in Fig. 5b (black arrows) might be due to immaturity in magnetosome biomineralization. Additionally, no crystal defects such as twinning, stacking fault and cation vacancies were detected in the isolated magnetosomes (Fig. 5b and i), indicating that no obvious oxidization occurred within magnetite crystals during magnetosome extraction. The Fast Fourier Transform (FFT) analyses indicated that the magnetosome crystals expressed {110} crystal faces (Fig. 5j), and the lattice spacing of nanoparticles was about 2.8 Å, which was consistent with the distance of the {111} crystal face. These results implied that the magnetite crystals elongated along the [111] direction. The similar finding was also reported in the previous studies. The lattice spacings of nanoparticles in A. ferrooxidans ATCC 23270 were found to vary from 2.43 to 2.95 in the previous study [38]. It has been reported that the magnetosomes in M. magnetotacticum MS-1 were elongated-prismatic and grew parallel to [111] direction [41].
3.4 Whole-genome sequencing and magnetosome-producing candidate gene prediction
The rapid development of genome sequencing has obtained the clear insight on the magnetosome-producing mechanism of MTB. Deciphering the whole genome sequence is necessary for understanding the genes involved in the formation process of magnetosomes. It has been reported that ten strains among more than 500 At. ferrooxidans sensu stricto isolates have been sequenced so far, but only three strains (ATCC 23270, ATCC 53993, YNTRS-40) were sequenced to obtain the complete genome sequences. Illumina sequencing showed that the genome of At. ferrooxidans BYM consisted of a circular chromosome of about 3208.389 kb with 58.54% GC content, ORF number of 3260, 3 CRISPRs, and a plasmid of 47.11 kb with a GC content of 56.44%, ORF number of 54. The genes categorized in “biological process”, “cellular component” and “molecular function” were determined in the genome of At. ferrooxidans BYM. The presence of numerous signal transduction components suggested that the At. ferrooxidans could greatly regulate the cellular function to adapt to varying environmental conditions. GO functional annotation indicated that a total of 314 genes and 8 genes closely were related to iron metabolism on chromosome and plasmid, respectively (Fig. 4). Among them, 94 genes were associated with iron-sulfur cluster binding, followed by 49 genes related to iron ion binding. The genes concerned with ferric iron transport, ferrous iron transport, iron assimilation and iron chelate transport participated in the multiple metabolic pathways of At. ferrooxidans BYM.
Blast analysis indicated that several genes possibly related to magnetosome formation existed in the genome of At. ferrooxidans BYM, including the major category mam genes, followed by feoAB, magA, mpsA and mms6 (Table 3) [35, 42–47]. The result indicated that the mamA-like gene might exist in At. ferrooxidans BYM because the gene fragment in the genome of BYM shared 87.88% sequence identity with mamA from Alphaproteobacterium LM-1. It has been proved that the magnetosome-associated protein MamA encoded by mamA gene (651bp) covers the outside of magnetosomes and plays a critical role in protein sorting through protein-protein interactions [48, 49]. The function was found to be based on its special structure because MamA folds as a sequential tetratricopeptide repeat (TPR) protein with three protein–protein interaction sites including a concave site, a convex site, and a putative TPR repeat in M. magneticum AMB-1 and M. gryphiswaldense MSR-1. MamA self-assembles through its putative TPR motif and its concave site create a large homooligomeric scaffold that can interact with other magnetosome-associated proteins via the convex site [49]. It has been demonstrated that the TPR motifs existed in a wide variety of proteins from prokaryotes are important for cells to execute the desired functions such as protein transport, protein folding, transcription and splicing, and cell cycle control [48]. Additionally, another function based on studies of a mamA deletion mutant has been proposed that MamA appears to activate or prime the preformed magnetosomes to biomineralization. Deletion of mamA has no effect on magnetosome membrane invagination, but fewer crystals were formed in M. magneticum AMB-1 [50].
Table 3
A list of predicted genes related to magnetosome formation in At. ferrooxidans BYM.
Predicted genes | Referenced genes | Locus | Source | Similarity (%) | E-value | Predicted function | References |
mamA-like | mamA | JN406508 | Alpha proteobacterium LM-1 | 87.88 | 0.003 | activate the magnetosome vesicles, stabilize the magnetosome chain | [44] |
mamB-like | mamB | KF787127 | Magnetospirillum moscoviense BB-1 | 83.78 | 0.001 | iron transport, magnetosome vesicle formation | [45] |
mamE-like | mamE | JX628767 | Gamma proteobacterium SS-5 | 70.19 | 2e-05 | redox control, protein sorting | [35] |
mamM-like | mamM | JX628771 | Gamma proteobacterium SS-5 | 87.88 | 0.005 | iron transport, magnetite nucleation and crystal growth | [35] |
mamO-like | mamO | KF787139 | Magnetospirillum marisnigri SP-1 | 92.59 | 0.003 | magnetite nucleation, activation of MamE | [45] |
mamQ-like | mamQ | JF429807 | Alpha proteobacterium SS-4 | 80.39 | 0.001 | membrane formation | [44] |
feoAB-like | feoAB | EF120624 | Magnetospirillum gryphiswaldense MSR-1 | 68.41 | 2e-28 | iron transport | [43] |
magA-like | magA | AB001699 | Magnetospirillum magnetotacticum MS-1 | 79.55 | 0.007 | iron uptake, iron transport | [46] |
mpsA-like | mpsA | D87827 | Magnetospirillum magneticum AMB-1 | 64.82 | 4e-12 | membrane formation | [42] |
mms6-like | mms6 | AB096081 | Magnetospirillum magneticum AMB-1 | 89.66 | 0.002 | morphological regulation | [47] |
The genes of mamB in M. moscoviense BB-1and mamM in Gammaproteobacterium SS-5 separately shared 83.78% and 87.88% similarity with the sequences in At. ferrooxidans BYM (Table 2). It has been reported that MamB and MamM are conserved and abundant integral membrane magnetosome-associated proteins with a molecular weight of 31.9 and 34.4 kDa, respectively [51]. They were thought to be involved in iron transport during magnetosome formation because of their high similarity to the members of the cation diffusion facilitator family (CDF), which generally transport metal cations such as ferrous ions from the cytoplasm into intracellular compartments or into the extracellular space using the proton motive force [52]. These might be the reason that could account for the increase of magnetosome yields of At. ferrooxidans BYM with increasing ferrous sulfate concentration. It has been confirmed that MamB not only serves as iron transporter in magnetite biomineralization, but also acts as a landmark protein to initiating the formation of magnetosome vesicle in a transport independent process [53]. The previous studies suggested that the stable expression of MamB depended on the presence of MamM which could protect MamB from proteolytic degradation due to complex formation [52]. Additionally, the stabilization of MamB and MamM was found to directly involve in the regulation of magnetite crystal growth in magnetosome formation [54]. Therefore, the potential proteins coded by mamB-like and mamM-like genes in At. ferrooxidans BYM might execute iron transport and crystal size control.
It has been reported that mamE and mamO genes might play critical roles in the early stage of magnetite magnetosome formation in M. magneticum AMB-1. MamE, a HtrA protease consisted of 655 amino acids in M. gryphiswaldense MSR-1, which is responsible for the regulation of the Fe(II)/Fe(III) ratio to promote magnetite nucleation, and also appears to play an important role in protein sorting to the magnetosome membrane [51, 54, 55]. Deletion of mamE can cause the empty magnetosome vesicles and the mislocalization of several proteins within the cell [55]. It was found that mamE gene in Gammaproteobacteria SS-5 shared 70.19% identity with the gene fragment (mamE-like) in At. ferrooxidans BYM (Table 2). The previous study showed that Gammaproteobacteria SS-5 isolated from the southeastern shore of the Salton Sea could mineralize octahedral magnetite crystal chains [35]. Similar to MamE, MamO is a second HtrA/DegP family protease containing 632 amino acids [56]. Additionally, MamO contains a degenerate active site, rendering it incapable of exerting protease activity. It has been reported that MamO can promote magnetosome formation through two genetically distinct, noncatalytic activities including the activation of MamE-dependent proteolysis of biomineralization factors and direct binding to transition metal ions [57]. The GO results indicated that 49 genes related to iron ion binding existed in At. ferrooxidans BYM. The mamO in Magnetospirillum marisnigri SP-1 displayed a strong homology (92.59%) with the sequence in At. ferrooxidans BYM, suggesting that the potential mamO-like gene in At. ferrooxidans BYM might specifically promote magnetite nucleation. It has been proved that MamK and MamJ play key roles in the formation of magnetosome chain in MTB [51]. However, we did not find the genes with high similarity to mamK and mamJ in genome of At. ferrooxidans BYM, which was supported by the TEM observation that the magnetosomes appeared to arrange dispersively.
Table 2 showed that the mamQ gene in Alphaproteobacterium SS-4 shared 80.39% sequence identity with the possible gene in At. ferrooxidans BYM. It has been suggested that magnetosome membrane formation was genetically induced via the mamQ gene by acting as a hub for the early organization of magnetosome proteins prior to membrane invagination. The deletion of mamQ in M. magneticum AMB-1 cells resulted in the complete loss of magnetosome formation [56]. It has been reported that an integral membrane protein MamQ with 273 amino acids was found to be homologous to the LemA protein family which bears a potential resemblance to BAR domain proteins and involves bending membranes in eukaryotic cells [51, 58].
It can be found that the magA-like gene might exist in At. ferrooxidans BYM due to it shared high similarity (79.55%) with magA sequence in M. magnetotacticum MS-1. MagA consisted of 434 amino acids was found to have high homology with the cation efflux proteins including the potassium ion-translocating protein KefC in Escherichia coli, and the putative Na+/H+-antiporter NapA from Enterococcus hirae. It has been reported that MagA was an iron transporter in E. coli which was confirmed by direct iron uptake measurements in membrane vesicles [59]. More importantly, the gene fragment with high similarity with magA gene was also found to exist in At. ferrooxidans 23270 and its maximum expression level occurred at 150–200 mmol/L ferrous [20]. In the present study, the highest magnetosome yield was observed when the concentration of ferrous sulfate was 30 g/L (Fig. 2). These results indicated that the potential protein coded by magA-like gene in At. ferrooxidans BYM could participate in the iron transport during the process of magnetosome formation.
It has been reported that mpsA with a length of 954 bp plays a key role in the formation of magnetosome membrane invaginated from cytoplasmic membrane. The MpsA protein consisted of 317 amino acids has been identified from M. magneticum AMB-1 [42]. It is homologous to acyl-CoA carboxylase of E. coli, which was classified under the category of lipid metabolism, providing evidence that the invagination of the cytoplasmic membrane to form magnetosome membrane were mediated by acylation [60]. The mpsA gene in M. magneticum AMB-1 was found to share 64.82% identity with the sequence in At. ferrooxidans BYM. These indicated that mpsA-like gene might play a critical role in the process of magnetosome formation in At. ferrooxidans BYM, which served a similar function in MTB. The previous report suggested that the mpsA gene in At. ferrooxidans reached the highest expression level at 150–200 mmol/L of ferrous and its expression increased with the decrease of oxygen concentration [61]. These findings indicated that mpsA-like gene was significantly affected by ferrous concentration and oxygen concentration and tightly associated with the magnetosome formation in At. ferrooxidans.
Mms6, a small acidic protein consisted of 136 amino acids in M. gryphiswaldense MSR-1 strain, was predicted to control the size of the magnetite crystal [62]. It was also isolated from M. magneticum AMB-1 along with Mms5, Mms7 and Mms13. The Mms6 protein has a low complexity hydrophobic N-terminal region with a hydrophobic glycine-leucine sequence, and a hydrophilic C-terminal region rich in acidic amino acids (aspartic-glutamic) with iron-binding activity [63]. It has been reported that the mms6 gene deletion mutant of M. magneticum AMB-1 was found to synthesize the smaller magnetite crystals with uncommon crystal faces, while the wild-type and complementation strains synthesized highly ordered cubo-octahedral crystals [62]. The sequence of mms6 gene in M. magneticum AMB-1 shared 89.66% similarity with the mms6-like sequence in At. ferrooxidans BYM, indicating Mms6 protein might regulate crystal morphology during magnetite biomineralization in At. ferrooxidans BYM.
3.5 Hypothetical mechanism of magnetosome formation in At. ferrooxidans
So far, the understanding of magnetosome formation and iron biomineralization process in the magnetosome-producing bacteria mainly comes from two model MTB strains M. gryphiswaldense MSR-1 and M. magneticum AMB-1 [5]. It has been demonstrated that the synthesis of bacterial magnetite magnetosomes is not a simple transformation from ionic iron to nano-magnetite particles, but includes a series of processes such as invagination of the cytoplasmic membrane, formation of magnetosome vesicles, iron uptake and transport, formation of iron mineral precursors and crystal nucleus growth and maturation [64]. The magnetosome gene island (MAI), a set of about 40 genes located in five operons including mamGFDC, mms6, mamAB, mamXYZ, and feoAB, was proved to involve in the step-wise magnetosome genesis mentioned above.
Therefore, based on the prediction of the magnetosome-producing candidate genes in At. ferrooxidans BYM and the information of magnetosome formation mechanism in MTB, we proposed a possible mechanism for the formation of magnetosomes in At. ferrooxidans. The hypothetical model of magnetosome formation in At. ferrooxidans is present in Fig. 5. The formation of magnetosomes in At. ferrooxidans is hypothesized to comprise three independent and constitutionally coupled processes. (1) The protein encoded by mamA-like gene could initiate invagination of the cytoplasmic membrane to form magnetosome membrane once the ferric uptake regulator (Fur) is able to sense the external ferrous iron ions. (2) The Fe(II) then transport from the outer membrane into the periplasm via the iron transporter FeoAB and/or Mnth. Meanwhile, a portion of Fe(II) is oxidized by Cyc1 and/or CycA1 to Fe(III) [9]. Then MamB-like, MamM-like and MagA-like proteins transport Fe(II) and Fe(III) into the magnetosome vesicle to generate hydrated ferric oxide (Fe2O3·nH2O), which triggers the growth of magnetosome vesicle involving the proteins of MamB-like, MamQ-like and MpsA-like. (3) The dehydration and redox of hydrated ferric oxide simultaneously occurred to form the magnetite precursor with the involvement of MamE-like, MamM-like, MamO-like and Mms6-like [3]. A stoichiometric ratio of Fe(III)/Fe(II) = 2:1 and the low redox potential inside magnetosome vesicle were regarded as necessary for crystal formation. Thus, the occurrence of the crystal nucleation mediated by MamM-like and MamO-like might result in the maturation of magnetosome from magnetite precursor.