Mechanism of manganese oxidization of Bacillus safensis strain ST7 isolated from the soil of mineral area


 Background: The manganese pollution is very serious surrounding the mine area, which could be enriched and harmful to animal, plant and human. Manganese oxidation bacteria (MOB) can completely remove the toxicity of Mn(II) with diverse mechanisms.

Results: To seek a resource and disclose the oxidation mechanism of MOB, we isolated the Bacillus safensis strain ST7 from the soil of Songtao manganese mine in Guizhou province, China. Strain ST7 could survive in media containing 2200 mg/L Mn(II) with the Mn(II) removal efficiency of 82% after seven days cultivation. The rate was 7.75 μmol/L of Mn(II) each day detected by LBB method. The manganese oxides appeared after stationary growth phase and lots of irregular precipitates were observed on the surface of bacteria by scanning electron microscopy (SEM). We further constructed eight cDNA libraries at two growth stages of strain ST7, at which the first stage is the mid-exponential growth phase (stage1) and the second one at the onset of stationary phase (stage2). The gene expression patterns were analyzed across the entire transcriptome under 250 mg/L Mn(II) stress by using Illumina Hiseq platform. After mapping to the reference B. safensis genome, we detected 3574 expressed genes from the eight libraries. At the first stage, 1040 differently expressed genes (DEGs) were determined with 502 genes up-regulated in Mn(II) dealt group. For the second stage, 760 genes were increased and 702 genes down-regulated under Mn(II) stress. Of those, the expressed trend of seventeen random selective genes were confirmed by RT-qPCR method. Only nine high expressed DEGs were screened out and all of them were up-regulated in the manganese dealt group at stage1. The great changes at stage 1 were focused on the genes related with siderophore synthesis to help Mn(II) uptake and oxidation and gene cheA to elevate the chemotaxis and the motility of bacteria. It was observed that the motility of strain ST7 was much active in the media with Mn(II) supply. And the expression level of gene601, coded for a multicopper oxidase (MCO) enzyme-like protein, raised about 3.66 times than its control group at stage 1. By using homologous recombination technology, it was demonstrated that the Mn(II) oxidase ability decreased obviously when the gene601 of B. safensis strain ST7 was knocked out. For stage 2 of strain ST7 dealt with Mn(II), there were nineteen genes related with sporulation and most of flagellum genes were inhibited. However, lots of transporters genes were augmented to function as pumps to extrude manganese outside of the bacterium cell.

Conclusions: In a brief, the isolated B. sanfensis took two strategies against Mn stress including manganese oxidation at exponential growth stage and transformation of Mn(II) at stationary phase. The strain could be used to treat the environmental manganese pollution to minimize the use of chemical oxidants as a cost-effective technology.

report that the Mn concentrations in ground water is exceeding the permitted limits, including China, Austria, the United States etc [10].
There is no doubt that the water or river near mining processing plant have high level of Mn(II). The dominant contamination originated from mining and metal smelting industries. In China, the abundant manganese reserves and the largest manganese industries gathered in the border area of three provinces, known as the "manganese triangle", which located in the northeast of Guizhou, northwest of China. The manganese pollution has impacted on the growth and development of some children surrounding those mine areas. For a high concentration of manganese, chemical methods, such as filter of manganese sand, are often used to remove manganese in waste water from mine factories. In natural world, not all of Mn(II) could be oxidized into Mn(III) or Mn(IV) by oxygen and it is impossible for these manganese metal is eliminated completely by the way of sedimentation and aeration. And manganese pollution in a low concentration need microorganism to remove thoroughly. Many kinds of bacteria can transform thoroughly the Mn(II) to its oxides [11]. It is reported that kinds of manganese-oxidizing bacteria (MOB) harbour multicopper oxidase (MCO)-type enzyme or heme peroxidase [12,13] with the capacity of oxidization the soluble Mn(II) to insoluble Mn(IV). MOB can elevate the Mn(II) removal efficiency by about five folds [14,15]. Nevertheless, it appear that the details of the mechanisms why bacteria are able to catalyze the oxidation of manganese might be diverse and remain largely unknown [16,17]. In the present work, we isolated a strain of MOB from the soil of manganese mine and investigated its transcriptome profiles under high level of manganese stress.

Isolation of MOB and detection of manganese oxidation capacity
To separate MOB strains with high ability, soil was sampled from an abandoned manganese mine located in Guizhou, China. A total of seven bacteria strains were isolated. After training, the strain ST7 appeared the highest Mn(II) oxidation capacity, which could grow on media containing 2200 mg/L Mn(II) with the Mn removal efficiency of 82%. Based on the characteristics of dye LBB specifically oxidized by Mn(III) and Mn(IV) [18,19], the oxide formation of Mn(III/IV) was further observed that a blue colour appeared on the colonies of strain ST7 grown on solid media supplied with 250 mg/L MnCl2 while the blue colour was much deeper on the plate contained 2200 mg/l MnCl2 (Fig. 1). Qualitative test for Mn(III, IV) oxide in liquid cultures gave the similar results (Fig. 2). The capacity of strain ST7 was 7.75 μmol/(L.d). Moreover, lots of irregular precipitates covered on the surface of bacteria could be visualized by SEM after incubated for seven days in 2200 mg/L MnCl2 liquid media (Fig. 3B).

The motility of strain ST7
Base on the puncture experiment, the strain ST7 present motility capacity under 250 mg/L Mn(II) stress (Fig. 4B). The colonies spread about two times in diameter when the strain ST7 was placed on soft-agar plate supplied with 250 mg/L Mn(II) (Fig. 4D). It indicated that the motility of strain ST7 could be stimulated by manganese.

Identification for strain ST7
Strain ST7 was positive in Gram-staining (Fig. S1), and able to produce ornithine decarboxylase, catalase, and H2S, but not to produce lysine decarboxylase, β-galactosidase. The citrate test was positive but both of Voges-Proskauer test and methyl red (MR) test were negative. The bacteria ST7 could hydrolyze glucose, gelatin and malonate. Acid from ethanol, dulcitpl, sorbitol and starch is negative.
The evaluated characteristics coincided with the records of Bacillus sp. in the Bergey's Manual of Systematic Bacteriology.
The 16S rRNA gene fragments were amplified using the genome extracted from strain ST7. Then, the PCR products with 1421 bp in length were obtained and sequenced. Based on BLAST searching with the related sequences deposited in the nucleotide database of NCBI, the phylogenetic tree were constructed using MEGA7 [20]. Based on nucleotide similarity of 16S rRNA gene sequence, the isolated strain ST7 was clustered with Bacillus safensis and Bacillus pumilus in the phylogenetic tree (Fig. S2A).
It might be the reason that both species are too much close to distinguish from each other just based on the 16 S rRNA gene similarity [21][22][23]. Another evolutionary analyse was further built based on gene6 sequence encoded DNA gyrase subunit A (gryA). The nucleotide sequence (2505 nt) of gene6 from strain ST7 could be clustered with the known sequences from all of Bacillus safensis with identity of 97.41-99.72% while it was less than 92.53% with Bacillus pumilus and the other Bacillus species (Fig.   S2B). Taken together, the strain ST7 was identified as Bacillus safensis according to the phenotypic and biochemical characteristics and sequence similarities of both 16S rRNA and gryA genes.
Expression profile of transcriptome of B. safensis strain ST7

Concentration of Mn(II) stress
The strain ST7 grew fast in the PYCM medium without manganese (Fig. 5). It reached the mid-exponential phase cultured for 8 hs and the onset of stationary phase needed 12 hs. When the strain was cultured in media supplied with 2200 mg/L MnCl2, the growth was inhibited obviously reaching to the onset of stationary phase after 44 hs. Compared with the control group, the growth rate decreased about 50% by the stress of 250 mg/L MnCl2, in which the mid-exponential phase appeared in 16 hs and the stationary phase started at 24 hs. To balance a higher response on the manganese stress and the growth velocity of bacteria, the ED50 of Mn(II) of 250 mg/L was taken as the concentration used for transcriptome RNA-Seq.

Analysis of transcriptome data
To investigate the response on the manganese stress of strain ST7, we constructed eight cDNA libraries at stage1 and stage2. The levels of gene expression were screened for the whole transcriptome under manganese stress by using Illumina Hiseq X-ten platform to sequence the libraries. All of the cDNA libraries generated 106.7 million of clean reads with each read in PE150 base-pair (bp) after quality control and filtering. All samples showed similar matching percentages, with 80.67% on average of reads mapping onto the reference genome of B. safensis and the Q30 above 89.34% (Table 1). The results showed that eight libraries presented high quality, and obtained high coverage of the B. safensis reference genome. It allowed us to compare the expression patterns from strain ST7 between groups with and without manganese stress treatment.  (Table S1) were selected as expressed genes with high quality, while it was 1462 genes from total 3152 genes at stage 2.
We used two softwares of EdgeR and DESeq2 to analyze the differently expressed genes (DEGs).
At stage1, 1040 genes differently expressed between two groups after join results from both softwares with the threshold of |logFC|≥1, in which, 502 genes were increased and 538 genes were decreased in the Mn(II) dealt group (Fig. 6A). The range of log2FC values of DEGs was varied from -3.79 to 6.03. For stage 2, all of the 1462 quality expressed genes were transcribed differently with log2FC from -13.77 to 8.49, in which 760 genes were up-regulated and 702 genes were down-regulated in the Mn(II) dealt group compared with its control (Fig. 6B). And 575 DEGs were shared between two stages with 624 genes specific at stage 1 while 887 genes expressed only at stage2 (Fig. 7).  The high-quality expressed genes between two stages.

Candidates of response genes under manganese stress
To find out the response genes under manganese stress, the high expressed DEGs were screened out according to the criteria of CPM larger than 10000 and Log2FC larger than 4. Only nine genes were qualified and all of them were up-regulated in manganese dealt group at stage1 (Table S2). Of those, three genes matched KO terms, in which gene1073 was in the lysine degradation pathway, gene3829 was in the porphyrin and chlorophyll metabolism pathway, and gene1591 occupied two pathways, which were the two-component system pathway and bacterial chemotaxis pathway (  Fig. 8). Total of 140 genes were great varied between two stages. The expressed level of 51 genes in stage2 increased ten times than that at stage1. Of these, total of twenty-six genes enriched in carbohydrate, energy, lipid, amino acid, cofactors and vitamins metabolism pathways. Some of them took part in more than one pathways including nine genes, gene89, gene363, gene2844, gene2371, gene2070, gene2036, gene2035, gene201 and gene1923. And eleven genes gathered in transporter pathways were up-regulated including gene425. There found 89 genes decreased about ten times in the stationary phase, including two genes, gene3709 and gene3346, specific expressed in the mid-exponential phase. Total of 24 genes enriched in the cell growth pathways, and seven genes were down-regulated in three pathways or brite hierachy, which were ribosome, peptidases and inhibitors, oxidative phosphorylation (Table S5). For the whole 140 variety genes between two stages, more than 37% of non redundant genes (52/140) were classified into metabolism category (A09100) ( Table S5). Compared with stage1 of strain ST7, most of genes related with carbohydrate metabolism (B09107 and B09101) together with amino acid metabolism were increased at stage 2, but genes participated nucleotide metabolism (B09104) and secondary metabolites (B09109 and B09110) were decreased. In the process of genetic information processing, six genes coded for 50S ribosomal protein L6, L10, L14, L16, L18 together with 30S ribosomal protein S5 and S8 in the ribosome assembly were down-regulated. In the cellular processes (A09140), biofilm formation related two genes (B09145), gene3036 and gene3564 coded for S-ribosylhomocysteine lyase and glycosyltransferase, were increased while four flagellar genes related with cell motility (B09142) and Bacterial chemotaxis (C02030) decreased. Interestingly, we found 33 genes related with flagellum which were up-regulated in the first stage, while only eleven of them expressed at stage 2 with 8 of 11 down-regulated ( Table 3). The pathway for these genes enriched in both of flagellar assembly and chemotaxis, and Brite hierarchies of transporters and bacteria motility.

Confirmation of DEGs
We selected randomly seventeen expressed genes to confirm by using RT-qPCR method. The expression profile of seventeen genes were consistent with the trend detected by transcriptomic abundance changes based on RNA-seq data ( Fig. 9). Fig. 9 The pattern of expressed genes confirmed by RT-qPCR method cultured in media supplied with 250 mg/L Mn(II).

Function demonstration of gene601
By homologous recombination technology, the partial fragment of gene601 without stop codon with 696 bp in length was generated as described previously from the genomic DNA of strain ST7 [24]. The partial fragment of gene601 was fused after the complete kanamycin gene by PCR method and then transformed into the wild strain ST7 using electroporation. According to the resistance against kanamycin, the knockout colony mutant Δgene601 was selected out. The in-frame deletion of gene601 were verified by PCR amplification and sequencing. Although the blue colour between two bacteria colonies was not obvious cultivated for 16 hours, but the colour was much weak of the mutant Δgene601 than that of wild strain ST7 after incubated for seven days under Mn(II) stress (Fig. 10). It demonstrated that the ability of mutant Δgene601 was decreased after knockout of gene601.

Discussion
In the present paper, a strain ST7, was isolated from the surface soil of the Songtao manganese mine, Guizhou province. The isolated strain ST7 could survive on 2200 mg/L MnCl2 media, although the growth velocity was very slow (Fig. 5). The manganese oxidation activity of strain ST7 was further demonstrated to oxide Mn(II) into Mn(III) or Mn(IV) oxides by LBB quantification with remove capacity of 82%. The ability of strain ST7 was near to Arthrobacter sp. strain HW-16 with oxidation ration of 66.28% in 3000 mg/L Mn(II) media [25] and much higher than that of B. cereus strain P1 [26] and Streptomyces spinoverrucosus strain NB-7 [27].
Based on the physiological and biochemical properties together with the similarity of 16S rRNA gene sequence, it could not distinguish two close species between Bacillus safensis and Bacillus pumilus because the sequences similarity of 16S rRNA gene is more than 98% (Fig. S2). Previous reports showed that B. safensis and B. pumilus, along with other Bacillus, are hard to distinguish from one another if only classify them on the differences of colony morphology, physiological and biochemical detection and 16S rRNA gene similarity [21]. These closely related Bacillus species are sorted to be B.
pumilus group. Multiple genome comparison is performed among species of B. pumilus group which gives a hint that the gyrA gene phylogenetic distances is much similar with that by using whole genome phylogenetic analysis methodology on 65 whole genome sequences of Bacillus [22]. Herein, the NJ tree was constructed using the gyrA gene sequences, in which the distance of strain ST7 gyrA gene was much close to that of B. safensis than the other Bacillus sp. The strain ST7 was then identified to be B.
It is reported that the time point to precipitate Mn(III, IV) from Mn(II) on the cell surface is upon reaching stationary phase in both of two strains, Pseudomonas putida MnB1 and GB-1. The time to produce a kind of Mn(II) oxidizing protein is from late logarithmic to early stationary stage [28]. But in Pseudomonas aeruginosa strain PAO1, cultured liquid at logarithmic stage could produce manganese oxidizes [29]. Therefore, we designed to detect the gene expression profiles in both the mid-exponential growth phase and the onset of stationary phase.

The adaptation of strain ST7 under Mn(II) stress at exponential growth phase
In the mid-exponential growth phase of strain ST7, there found nine high expressed DEGs under Mn(II) stress. Of those, gene1073, encoded lysine 6-monooxygenase, also named as lysine N6-hydroxylase (iucD) [EC:1.14.13.59]. The enzyme from Nocardia farcinica catalyzes the hydroxylation of l-Lys in the biosynthetic pathway of the nocobactin, a kind of siderophore [30]. Siderophore is low molecular weight iron chelator that is secreted by bacteria growing under low iron condition [31]. Recently, lots of previous works demonstrated that manganese could inhibit the uptake of iron and the chelator of iron can combine with Mn(III) as well [32]. The binding of chelator and Mn(III) further accelerate the oxidation of Mn(II) into Mn(III) [33,34]. Moreover, the complexes of Mn(III) and the siderophore function as superoxide dismutase and help the oxidation of Mn(II) [35,36]. It was interested that both of gene1071 and 1074, coded for IucA/IucC family siderophore biosynthesis protein were high expressed with 3.72 and 4.47 times higher than the control group. And iucA, iucD and iucC genes coded for three enzymes to participate the biosynthesis of aerobactin, a kind of siderophore, in Escherichia coli [37]. Herein, the Mn(II) oxidation process in stage1 may rely on siderophore of IucA/IucC family in strain ST7.
The gene3829 coded for iron ABC transporter ATP-binding protein, clustered in heme oxygenase, took part in porphyrin and chlorophyll metabolism. It is reported that some kinds of ABC transporter, such as PsaA protein from Streptococcus pneumoniae, could uptake Mn(II) and possibly Zn(II) as well [38,39]. In the patient epithelial cells, the high-affinity manganese and zinc transporters of Salmonella are upregulated in the niche with high-degree limitation of metal ions [40]. It might present a similar function for the protein coded by gene3829 when the strain ST7 was cultured in iron deficiency PYCM media.
The gene1591 coded for chemotaxis protein CheA and gathered in the bacterial chemotaxis related with cell motility process (Table 2). In alphaproteobacteria Ruegeria sp. TM1040, chemotaxis proteins are upregulated to stimuli motility and escape the high Mn(II) concentration, in which both of CheW and CheB together with flagellar proteins, FlgL, FlgK, FlgB and FlgG, increased more than five times in 200 uM Mn(II) solution [41]. Furthermore, cheA could excise regulator as a central member of two-component system to sense and transmit the signal of high Mn(II) environment, which then coordinate multiple cellular functions including metabolism, growth and survival of bacteria [42]. Both of chemotaxis and phototaxis are disappear when the central core of cheA gene is deleted in Halobacterium salinarium. And the CheA, similar to E. coli, is thought to be the transmitter protein that relays signals from both chemoreceptors and photosensory transducers to the flagellar motor switch [43].
In both of Eubacteria and Archaea, two-component signaling system is much conserve manner to execute response to chemical or light signals. The mechanism of chemotaxis and phototaxis is that a chemo-or phototactic signal causes CheW-mediated selfphosphorylation activity of CheA by ATP, then the phosphorylated CheA provides phosphate to CheY, the phosphorylated CheY stimulates the flagellar motor switch and change the motility and direction of bacterium. In strain ST7, all of thirty-three genes coded for flagella assembly proteins were up-regulated in the group dealt with 250 mg/L Mn(II) at the mid-exponential growth phase (Table 3). It was corresponding to the increase motility ability of strain ST7 at Mn(II) stress (Fig. 4).
It was worthy to notify the gene601 of strain ST7, which encoded a copper oxidase, and the product of gene601 is classified as spore coat protein A (cotA), manganese oxidase  [29,[45][46][47][48]. However, the four signature domain regions for copper binding could be found out from the cotA of strain ST7 when compared with other reported proteins with manganese oxidation ability by using Mega and muscle program (Fig. S3). The multicopper oxidase (MCO) family is composed of distantly related domains to bind with copper ions that are involved in electron transfer during the oxidation of various substrates, like Fe(II) or Mn(II) [49]. It includes three members of MCO family: laccase, ascorbate oxidase, and ferroxidase. All of them contained the four domain enriched in histidine residues as the regions from domain 1 to 4 in Figure S3.
The putative copper-binding motifs in the cotA of strain ST7 were assigned to range from residues 102 to 110, 149 to 154, 419 to 426, and 491 to 502, with highly conserved of ten histidine occupied 42.9% of 35 residues in Fig. S3. It indicated that the gene601 of strain ST7 might involve in producing a protein to oxidize the soluble manganese compound just like the other members of multicopper oxidase family. In the group with Mn(II), the expression level of gene601 is 3.66 times higher than that in control group at stage1 (Table S1). It indicated that the protein cotA of strain ST7 could oxidize Mn(II) at the first stage, and the knockout of gene601 did reduce the Mn oxidation capacity (Fig. 10).
In brief, the great change at stage 1 might focus on uptake of Mn(II) by increasing of siderophores, and cheA protein to elevate the chemotaxis and motility of bacteria, and the main enzyme to oxidize Mn(II) was relied on cotA coded by gene601.

The response of strain ST7 at stationary phase under Mn(II) stress
Of those high expressed genes at stationary phase (Table S3, Table S5), thirty-two genes were up-regulated. Most of them were in the process of catabolism of large molecules such as glycolysis, citrate cycle, many kinds of amino acids degradation, peptidases etc. However, a cluster of fifteen gene functioned as transporters were increased in the group dealt with Mn(II). It was noted that two clusters of genes were high expressed. The first cluster was gene3340, gene3341, gene3342, gene3343, gene3344. The second one included gene3830, gene3831, gene3833, gene3834, gene3835. They might express in common as operons. The fifteen genes were annotated to be nine kinds of transporter proteins, which were ABC transporter substrate-binding protein, ABC transporter ATP-binding protein, iron-uptake system-binding protein, , iron ABC transporter permease, iron(3+)-hydroxamate-binding protein fhuD, heme ABC transporter substrate-binding protein IsdE, PTS galactitol transporter subunit IIC, amino acid permease, and copper-binding protein. The ABC transporters are widely distributed membrane proteins and import/export molecules across the membrane from bacteria to human cell [50].
Type I transporters import sugars and amino acids metabolites, and the second type of transporters uptake complexes of organic compound and metal, including vitamin B12, iron-siderophores, and heme.
All ABC transporters are basically consisted of two domains, the first one is two NBDs (intracellular nucleotide-binding domain), and the second one is two TMDs (trans-membrane domain). ATP binds with the NBD domain and provides energy by ATP, and the TMDs domains provide a path for the cargo to go through the cell membrane. Additionally, ABC transporter needs a SBP, substrate-binding protein, to carry the substrate outside the membrane reaching to the TMD domain of ABC transporters [51]. Of the nine annotated proteins, two of them classified to be ABC transporters in cell membrane, included ABC transporter ATP-binding protein and iron ABC transporter permease. And four of them were SBPs to help ABC transporter to uptake iron and other metal ions, which were ABC transporter substrate-binding protein, iron(3+)-hydroxamate-binding protein fhuD, iron-uptake system-binding protein, heme ABC transporter substrate-binding protein IsdE. The mechanism of PTS transporter is based on group translocation, much different from both of ABC-type and NRAMP transporters. The phosphoenolpyruvate-dependent carbohydrate transport system (PTS) performs the translocation together with concomitant phosphorylation of sugars, galactitol and hexitols [52]. The amino acid permease mainly uptake amino acids into the cell and some of them are ABC-type transports [53]. The copper-binding protein, coded by gene425, is the carrier to bind with Cu(I). It is demonstrated that the copper-binding protein, CopL, is lipoprotein on cell surface, which holds four Cu(I) on the outer surface of the cell and contributes to the Cu effluxer effects of proteins CopA or CopB [54]. It has been reported that one of copper-binding protein, prion protein (PrP), could bind with both of copper and manganese [55]. It suggested that these transporters including copper-binding protein coded by gene425 might contribute to extrude Mn(II) from bacterium.
In Bacillus subtilis, there reported two specific transform systems of Mn(II), mntABCD and mntH.
The mntABCD operon encodes a four ABC transporters of Mn binding lipoprotein, ATP-binding protein and two permeases. MntH codes for the proton-coupled manganese transporter. solution [56]. However, the annotation of reference genome in Bacillus safensis (Assembly no. GCF_001895885.1) is imperfect, in which there does not contain the annotation of mntABCD, mntH and mntP except for mntR coded by gene2410. We herein realigned the clean data from eight libraries taking the Bacillus subtilis as reference with assembly no. of GCF_000009045.1. Then, four genes, the mntB, mntD, mntH and mntP, were annotated to be gene3359, gene3360, gene474, gene3661 in the strain ST7, the other mnt genes were not found out. However, all of four genes decreased obviously except for gene2410 coded for mntR, which was increased with log2FC of 2.17 times in stage2. It suggest that both of mntABCD and mntH might not functioned in the transport of Mn in B. safensis strain ST7.
A total of nineteen genes were decreased and clustered into the cell growth path with sporulation.
Of those, four genes were hypothetical proteins, and eleven genes coded for protein of spore assembly in the sporogenesis process. The other four genes coded four enzymes related with sporogenesis. Both of gene1627 and 1628 coded for dipicolinic acid synthetase subunits A and B take part in synthesis of dipicolinic acid (DPA) during sporulation period in the mother cell [57,58]. The gene2117 coded for glycosyltransferase family 2 protein, which catalyzes the transference and modification of monosaccharide in the crust during late stage of sporulation [59]. And gene3170 coded for KapD, which inhibit the spore formation in Bacillus thuringiensis [60]. Nearly half numbers of down-regulated genes blocked the spore growth of bacteria.

The great changeable genes between two stages
The greatest variant 140 genes between two stages related with types of biological process were sorted out, such as metabolism, cellular process and genetic information processes etc (Table S5) operon [63]. It was coincided with the increased motility of strain ST7 at Mn(II) stress (Fig. 4).
Compared with stage1, the main variation in stage2 was focused on those decreased genes for spore formation and flagella assembly together with the increased genes for transporters to extrude manganese outside of the bacteria.

Conclusions
The gene coded for siderophores were strong transcribed to uptake Mn(II) as cofactor to enzyme and the gene601 coded for cotA to oxide the excessive Mn(II) as the bacteria grew very fast at the mid-exponential growth phase (stage1). When entering into the stationary phase (stage2), genes related with spore formation and flagella assembly were great down-regulated while the transporter genes were increased to extrude too much Mn(II) outside of bacteria to avoid the harmness of Mn(II) as the slowing down of bacteria growth.

Isolation and training of MOB strain
Samples were collected from 0 to 20 cm depth of the subsoil located in Songtao manganese mine

Detection of Mn removal efficiency
The bacteria suspensions of strain ST7 at exponential growth phase were transformed into PYCM with 250 mg/L MnCl2 on a scale of 1:20, and cultured at oscillation of 180 rpm at 28℃. Aliquots supernatant were collect after seven days and detected the rest Mn(II) content in medium by using atomic absorption spectrophotometry in triplicates.
The Mn removal efficiency (RE) was calculated by equation: where CS is the concentration of rest MnCl2 in the supernatant after a certain period of cultivation and C0 is the concentration of MnCl2 at the beginning [64].

Determination of the motility of strain ST7 under Mn(II) stress
Strain ST7 was punctured into soft-agar medium tube of PYCM containing 0.3% agar to observe the bacteria motility capacity. The equivalent amount of strain ST7 from exponential growth phase was further placed on PYCM soft-agar plates supplied with or without 250 mg/L Mn(II) and incubated for seven days. The diameter of spread colony was measured with ruler.

Identification of isolated strains
The biochemical characterization and Gram staining were carried out by regular process. Total genomic DNA was extracted by TIANamp Bacteria DNA Kit (TIANGEN) based on the kit protocol. Fragments of gene (16S rRNA and gyrA) were amplified by PCR method using the bacteria genomic DNA as templates [65]. The products of amplification after purification were sequenced directly from two ends of strand by Sanger sequencing method. The consensus phylogenetic tree were constructed by UPGMA method using MEGA7 program [20]. The phylogenetic trees were constructed by bootstrap method after 1000 repetitions.

Mn(II) oxidation activity assays
Quantitative and qualitative analysis methods were used to test the activity of manganese oxidation of strain ST7 as previous reports [15,61]. In brief, taking Staphyloccocus aureus Rosenbach as control, bacteria ST7 cultures were spread and cultivated for seven days on agar solid PYCM plates. The colonies on plates or liquid cultures were monitored by the colorimetric dye solution of leucoberbelin blue (LBB) (0.04% w/v) to detect the Mn oxides products including Mn(III) and Mn(IV) [18,19]. The manganese oxidation activity of strain ST7 was determined quantitatively by using LBB taking KMnO4 solution for standard curve as previously described [15,66].

Visualization of Mn(II) oxides by scanning electron microscopy
The bacteria pellets were prepared for observation by using scan electron microscopy (SEM) as previous report [67]. The strain ST7 pellets cultured for seven days with 2200 mg/L MnCl2 or not were collected by centrifugation and were washed in PBS (pH7.4) solution. The pellets surface morphology were observed in a Hitachi S-3400N scan electron microscope with 20,000 V accelerating voltage.

Determination of the proper concentration of Mn(II) stress
Strain ST7 was cultured in the PYCM liquid medium with sterile MnCl2 at concentration of 0, 250, 500,

Analysis of transcripts expression profile
The strain ST7 was incubated in PYCM liquid medium supplemented with 250 mg/L MnCl2 as Mn dealt group, and taking equivalent aliquot of cultures as control group in medium without Mn(II). Two samples were taken out from each group at the mid-exponential growth phase (stage1) or onset of stationary phase (stage2), respectively. Samples L01 and L02 were prepared from the control group without Mn(II) at stage1 cultivated for 8 hs (cfu/mL = 3.183 × 10 8 ), and L03 and L04 from the Mn dealt group at stage1 for 16 hs (cfu/mL = 3.182 × 10 8 ). For stage 2, both of L05 and L06 were sampled from control group in 12 hs (cfu/mL = 4.635 × 10 8 ) while L07 and L08 were from the dealt group in 24 hs (cfu/mL = 4.573 × 10 8) .
Pellets from eight samples were washed two times by PBS solution (pH7.0) at centrifugation of 10,000 g × 5 min at 4°C. Based on the protocol of Genedenovo Biotechnology Co., Ltd (Guangzhou, China), cDNA library was constructed. In brief, total RNA of bacteria was prepared by using TRIzol method (Life Technologies, CA, USA) followed by chloroform extraction. The total RNA was digested by RQ1 DNase (Promega, Madison, WI) to clear genomic DNA contamination, and purified by phenol and chloroform extraction and anhydrous alcohol precipitation. The RNA quality was detected by NanoPhotometer® spectrophotometer (IMPLEN, CA, USA) and the RNA integrity (RIN) was measured to be from 8.1 to 9.9 using Agilent Bioanalyzer 2100 system (Agilent, Santa Clara, CA). Based on method for lncRNA library,

Analysis for different expression genes
Raw data were filtered to remove low-quality reads if it contained larger than 10 % unidentified nucleotides (N), larger than 50 % bases containing the phred quality score less than twenty or only barcode adapter using NGSQC Toolkit (http://www.nipgr.ac.in/ngsqctoolkit.html). The reads were then aligned with the reference genome of Bacillus safensis strain KCTC 12796BP (Assembly no. GCF_001895885.1, containing 4088 genes) using STAR program (version 2.7) (https://github.com/alexdobin/STAR) allowing no mismatches, reads mapped to rRNA were removed.
The expression patterns of genes was calculated according to the value of counts per million (CPM) to standardize the gene expression. CPM = 10,000,000*(A/mapped reads) , A is the read counts of one gene.
Both of DESeq2 and edgeR packages on R platform (http://www.r-project.org/) were used to identify deferentially expressed genes (DEGs) across groups. The high quality expressed gene was defined according to the threshold that the CPM value was larger than 200 together with the FDR value ≤ 0.1.
The up-or down-regulated genes were recognized if the value of |log2 (fold change)| was more than or equal to one, and was further displayed in the volcano plot taking FDR value and log2FC as coordinate axes by Sangerbox program online (http://sangerbox.com/Tool).

Gene enrichment analysis by KEGG
The amino acid sequence of DEGs were generated from the assembled transcripts by trinity v2.85 [68].
The amino acid sequences of DEGs were input to analyze the KEGG Orthology by the KEGG Automatic Annotation Server online (KAAS) (https://www.genome.jp/tools/kaas/) taking gene lists from 30 species of Bacillus as references containing 148,323 complete genome sequences.

Validation of DEGs
The same aliquot total RNA for RNA-seq was used to validate DEG by RT-qPCR method. Specific primers for genes were designed by primer5.0 software (Table S6) taking 16S rRNA as internal reference gene. The qPCR reaction was performed according to previous research [69,70]. The corresponding RT-qPCR efficiency (E) was in the range of 90.1-101.9% (Fig. S4). All assays were performed in triplicates.

Knockout of gene601
The partial region of gene601 (copper oxidase), were generated as described previously based on homologous recombination technology [24]. In brief, The fragment F1 without stop codon of the target gene was amplified using primers cotA-F/cotA-R to get the 5`-terminus fragment about 700 bp (Table S6) from the genomic DNA of strain ST7. The complete kanamycin gene (fragment F2) was obtained from plasmid pPIC9K (Invitrogen life technologies) by PCR method guided by primers km-F/km-R (Table S6).               Table 3 The expressed gene related with flagellum at two stages of strain ST7.

Supplement information
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