An operon consisting of a P-type ATPase gene and a transcriptional regular gene given the different cadmium resistances in Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25

Cadmium (Cd) is a severely toxic heavy metal to most microorganisms. Many bacteria have developed Cd2+ resistance. In this study, we isolated two different Cd2+ resistance Bacillus sp. strains, Bacillus vietamensis 151-6 and Bacillus marisflavi 151-25, which could be grown in the presence of Cd2+ at concentration up to 0.3 mM and 0.8 mM, respectively. According to the genomic sequencing, transcriptome under cadmium stress and related biological experiments, a gene cluster in plasmid p25 containing orf4802 and orf4803, which encode an ATPase transporter and a transcriptional regulator protein, respectively, was a major contributor to Cd2+ resistance in B. marisflavi 151-25. Although 151-6 has much lower Cd2+ resistance than 151-25, they contain similar gene cluster, but in different locations. A gene cluster on the chromosome containing orf4111, orf4112 and orf4113, which encode an ATPase transporter, a cadmium efflux system accessory protein and a cadmium resistance protein, respectively, was found to be a major role on the Cd2+ resistance for B. vietamensis 151-6. Based on homologies to the cad system (CadA-CadC) in Staphylococcus aureus, the mechanisms of cadmium resistance in B. vietamensis 151-6 and B. marisflavi 151-25 were as same as the cad system. Our study on the cadmium mechanism for B. vietamensis 151-6 and B. marisflavi 151-25 proved preliminarily that the cad system is widespread in Bacillus sp. bacteria.


Introduction
Soil contamination with heavy metals is becoming an increasingly urgent problem worldwide. Among all heavy metals, one of the most hazardous is cadmium (Cd) (Abdu et al. 2016;Liu et al. 2016). Cd is a non-essential heavy metal element that is toxic, teratogenic and carcinogenic to humans. When critical Cd levels in soil are reached, biodiversity, agricultural productivity, food safety and human heath can be threatened, as 3 Cd can be accumulated in the food chain (Feng et al. 2018;Lehembre et al. 2013;Op De Beeck et al. 2015;Rehman et al. 2018;Satarug et al. 2003). Consequently, solutions to remediate heavy metal-contaminated soil, in particular Cd-contaminated soil, are urgently needed. Many remediation techniques, such as chemical immobilization, electrokinetic extraction, phytoremediation, and bioremediation, have been proposed for soils contaminated with heavy metals ). Among these technologies, bioremediation is considered an innovative and promising remediation method based on its cost effectiveness and low environmental impact (Emenike et al. 2018;Jacob et al. 2018;Peng et al. 2018). To use microorganisms to remediate heavy metal contamination, in particular Cd-contaminated soil, it is first necessary to isolate bacteria that are resistant to Cd.
Many organisms have developed strategies to withstand the presence of Cd in the environment, such as exclusion, compartmentalization, deployment of inorganic polyphosphates, cell wall binding, and expression of metal binding proteins (Abbas et al. 2018;Gillan 2016;Kulakovskaya 2018;Reddy et al. 2016;Yu et al. 2018). The most prominent mechanism of resistance to Cd 2+ is the use of efflux pumps (Bruins et al. 2000).
One of the most thoroughly characterized Cd 2+ resistance efflux systems is the czc (Cd 2+ , Zn 2+ , and Co 2+ resistance) system in the gram-negative bacterium Alcaligenes eutrophus (Diels et al. 1995;Nies et al. 1989). The CzcA, CzcB and CzcC proteins comprise an active efflux pump complex driven by a cation-proton antiporter (Nies et al. 1989). Briefly, CzcA, located in the inner membrane, is essential for cation transport and can remove heavy metals (Cd 2+ , Zn 2+ , and Co 2+ ) from the cytoplasm using an H + ion gradient (Diels et al. 1995;Nies 1992). CzcB belongs to a family of bacterial membrane fusion proteins. It may create a pathway for the removal of cations from CzcA (Diels et al. 1995;Rensing et al. 1997b). CzcC relies on CzcB to function and may act as a substrate (Cd 2+ , Zn 2+ , and Co 2+ ) switch for the efflux pump (Bruins et al. 2000).
Another well-characterized Cd 2+ resistance system is the cad system( cadA-cadC) in the gram-positive bacterium S. aureus (Nucifora et al. 1989;. The Cdefflux ATPase is encoded by the cadA gene, which contains six predicted membranespanning regions. The fourth membrane span is thought to be involved in the cation translocation pathway, and includes a conserved Cys-Pro-Cys tripeptide (Silver and Phung 1996). CadC is a regulatory protein encoded immediately downstream of cadA, and is also required for Cd 2+ resistance in S. aureus . CadC is a member of the ArsR/SmtB family (Busenlehner et al. 2003;Saha et al. 2017), which can bind to the promoter-operator area of the cadA-cadC system and acts as a transcriptional repressor in vitro (Endo and Silver 1995 (10 µg/mL), or tetracycline(5 µg/mL) was added as necessary.

Isolation of Cd-resistant bacteria
Cd-contaminated soil was collected from a former industrial site in Hunan Province (27°46′N, 112°52′E) and analyzed for Cd content through acid digestion followed by the use of a 7,700 × inductively coupled plasma mass spectrometer (Agilent Technologies, Tokyo, Japan). To isolate Cd-resistant and safe bacteria, aerobic Bacillus sp. were isolated from the soil by plating on LB agar plates containing progressively higher concentrations of cadmium chloride (0, 0.5, 1 and 2 mM) after bacterial enrichment cultures were heatshocked at 80℃ for 20 min. Bacterial genomic DNA was isolated using the TIANamp Bacteria DNA kit (TIANGEN Biotech). The 16S rRNA gene was amplified from the extracted DNA using the universal primers 16S rRNA-F/16S rRNA-R (Table S2) and the amplification products were cloned in the pGM-T (TIANGEN Biotech) vector using competent E. coli TOP10 cells (TIANGEN Biotech). Sequencing was carried out using T7 and SP6 primers and 6 compared to the GenBank database using the NCBI BLAST program.

Evaluation of cadmium resistance and growth curve
To evaluate growth in a liquid medium, the minimum inhibitory concentration (MIC) of Cd 2+ (MIC-Cd) was determined. LB medium (800 μL) with different concentrations of Cd 2+ was dispensed into 96-well (12 × 8) microtiter plates (96 × 2-mL wells) with a multichannel micropipette. Single colonies of the test strains were inoculated into 3 mL of LB medium and cultured overnight. The test culture (15 μL) was then inoculated into each well of the prepared 96-well plate. After 24 h at 37℃ and 750 rpm in an incubator (Heidolph, Viertrieb, Germany), 200 μL of the cell suspension was transferred to a 96-well plate and the turbidity at OD 600 was measured.
To determine the tolerance to Cd 2+ of bacteria, growth curves at different concentrations of Cd 2+ were analyzed. For the growth assay, single colonies of the test strains were cultured overnight and then diluted 1:100 into 100-well plates containing 200 μL of LB and various concentrations of Cd 2+ in quintuplicate. The growth curve was measured at 1-h intervals using a Bioscreen C automatic growth curve analyzer (Bioscreen, Helsinki, Finland).

Genome sequencing and analysis
Bacterial genomic DNA was extracted using the sodium dodecyl sulfate (SDS) method (Lee et al. 2003). A total of 5 μg DNA was used to generate each library, and this DNA was sheared using Covaris g-Tubes to generate sheared fragments >10 kb in length. The sheared DNA fragments were then prepared using the SMRT bell template preparation kit (Pacific Biosciences, Menlo Park, CA, USA) according to the manufacturer's instructions.
Then, the filtered reads were transferred for the next step of gap closing. Transfer RNA (tRNA) genes were predicted with tRNAscan-SE (Lowe and Eddy 1997). Ribosomal RNA (rRNA) genes were analyzed using rRNAmmer (Lagesen et al. 2007). Coding genes were identified with the GeneMarkS program (Besemer et al. 2001). The predicted coding genes were annotated based on the non-redundant protein database (NR) of the National Center for Biotechnology Information (Li et al. 2002) and Gene Ontology (GO) (Ashburner et al. 2000).

RNA sequencing and transcriptome analysis
An overnight culture was diluted 1:100 in LB medium in the presence (0.1 mM) and absence of Cd 2+ , and these cultures were grown at 37℃ and 200 rpm to the exponential phase. Total RNA from both groups (each group has three replicates) were extracted using a TRIzol kit (TIANGEN Biotech) according to the manufacturer's instructions. A total of 1 μg RNA per sample was used as input material for RNA sample preparation. Sequence
Meanwhile, the primers for quantitative reverse transcription-polymerase chain reaction (qRT-PCR) (Table S2) located on plasmid p25 were also used to detect the plasmid (  (Table S2). The results were aligned with the genome sequence of B. vietamensis 151-6 to confirm the sequence of the inserted fragments.
To compare the level of Cd 2+ resistance of two selected strains with other Bacillus sp.
bacteria, we examined their MIC-Cd, as well as those of three other Bacillus sp. strains, BS,BA and BL, in liquid medium supplemented with various Cd 2+ concentrations. 151-25 and 151-6 were grown in the presence of Cd 2+ at concentrations up to 0.8 mM and 0.3 mM, respectively (Fig. 1a), while the growth of the other three Bacillus sp. strains was inhibited in the presence of 0.3 mM Cd 2+ (Fig. 1a). The growth curves at 0 mM Cd 2+ demonstrated that all four Bacillus sp. strains exhibited similar growth trends in liquid LB medium (Fig. S1a). However, under culture conditions with 0.1 mM or 0.3 mM Cd 2+ , 151-6 and 151-25 exhibited greater growth potential than the other three Bacillus sp. strains ( Fig. S1b and Fig. 1c). And only 151-25 could grow at 0.5 mM Cd 2+ (Fig. S1d). These data indicated that compared to other Bacillus sp. strains, 151-6 and 151-25 exhibited stronger tolerance to Cd 2+ . And the Cd 2+ resistance of 151-25 was significantly higher than 151-6.

Genomic analysis of 151-6 and 151-25
To compare the mechanism of Cd 2+ resistance for 151-6 and 151-25, their genome were sequenced. The results showed that 151-6 contains a single chromosome (4,556,861bp and 4,952 predicted genes) and one plasmid, designated p6 (40,946bp and 66 predicted genes) (Fig. S2). And 151-25 also contains a single chromosome (4,411,234 bp and 4,734 predicted genes) and one plasmid, designated p25 (138,020 bp and 156 predicted genes) (Fig. S3). Simultaneously, the results of non-redundant protein database (NR) annotation of the predicted coding genes presented that strain 151-6 canbe identified as B. To determine whether plasmid p6 and p25 were responsible for Cd 2+ resistance in B.

A gene cluster consisting of orf4802 and orf4803 confers high Cd 2 + resistance in B. marisflavi 151-25
To determine the genes related to the Cd 2+ resistance of B. marisflavi 151-25, its transcriptome under Cd 2+ induction was analyzed by RNA sequencing. Fragments per kilobase of exon model per million mapped reads values were used to measure transcript abundance. As shown in Table S4 and Fig. S5, a total of 65 differentially expressed genes were identified in the presence of Cd 2+ (47 up-regulated genes and 18 down-regulated genes). Among the 47 up-regulated genes, 9 were located on plasmid p25, and the top three up-regulated genes in terms of fold change were also from plasmid p25 (Tables 1   and S4). Briefly, transcripts of a transcriptional regulator gene (orf4803) and a coppertranslocating P-type ATPase gene (orf4802) were increased 72.53-and 63.83-fold, respectively. These results were confirmed through qRT-PCR (Fig. 4b), and it further indicated that plasmid p25 is related to Cd 2+ resistance in B. marisflavi 151-25. Based on these results, we hypothesized that nine up-regulated genes located on plasmid p25, orf4774, 4775, 4776, 4777, 4779, 4781, 4782, 4802 and 4803 may be involved in Cd 2+ resistance of B. marisflavi 151-25. To confirm this hypothesis, we overexpressed these genes using their own promoter with five gene clusters (Fig. 4a) in E. coli and B. subtilis, to examine their contributions to Cd 2+ resistance. As shown in Fig. 4c and 4d were identified (585 up-regulated genes and 683 down-regulated genes), significantly higher than that of B. marisflavi 151-25 ( Fig. S4 and Table S5). Moreover, 585 upregulated genes in the presence in Cd 2+ were located on the chromosome, and among the 683 down-regulated genes, only 16 were located on plasmid p6. These data verified that plasmid p6 does not influence the Cd 2+ resistance of B. vietamensis 151-6. To further identify the key Cd 2+ resistance genes from those differentially expressed genes, a fosmid library of B. vietamensis 151-6 genomic DNA was constructed in E. coli. And the clones were screened by the Cd 2+ resistance to determine the genes that contribute to Cd 2+ resistance. Among the total of 1204 clones, there were 25 and 3 clones could be grown on LB agar plates with 1.2 mM and 1.5 mM Cd 2+ , respectively. In liquid LB medium with 1.2 mM Cd 2+ , there were 27 clones could be grown. Only 2 clones, B2 and C2, could be grown in liquid LB medium with 1.5 mM Cd 2+ . The MIC-Cd of B2 and C2 were determined. As shown in Fig. S8, B2 and C2 exhibit higher Cd 2+ resistance than negative control strain EPI300-T1 R . The fosmid DNAs isolated from B2 and C2 were sequenced, respectively.
Sequence analyses of B2 revealed that it contained 32,263bp insert fragment, which consisted 31 annotated genes (Table 2) (And the fosmid DNA isolated from C2 was failed by sequencing many times, so the clone was not analyzed). Moreover, the analysis of the transcriptome showed that insert fragment of B2 contained 8 up-regulated genes (orf4108, orf4109, orf4088,orf4087, orf4090, orf4093, orf4106, orf4107) and 3 down-regulated genes (orf4086, orf4101, orf4120). These results were confirmed through qRT-PCR and the transcription levels of other 16 genes in the insert fragment of B2 were also evaluated by qRT-PCR. The results presented that the transcripts of the orf4108, orf4109, orf4088, orf4087, orf4104, orf4106 and orf4107 were increased 156. 64, 130.84, 102.58, 87.26, 14.19, 12.68 and 9.07-fold, respectively (Table 2 and Fig. 5b). Based on these results, four gene clusters (4108-4109, 4087-4088, 4104, 4106-4107) containing their own promoter were overexpressed in E. coli and B. subtilis to examine their contributions to the Cd 2+ resistance. Due to the fact that ATPase gene and oxidoreductase gene have been reported to involved in Cd 2+ resistance (Wang and Crowley 2005), three related gene clusters (4093-4094-4095, 4102-4103 and 4111-4112-4113) containing their own promoter were also overexpressed in E. coli and B. subtilis (And the construction of the recombinant vectors containing 4104 and 4106-4107 were failed by many times, so the results were not showed). As shown in Fig. 5c and Fig. 5d, the gene cluster containingorf4111 (coppertranslocating P-type ATPase gene), orf4112 (cadmium efflux system accessory protein gene, cadC) and orf4113 (cadmium resistance protein gene, cadD) allowed recombinant E.
coli and B. subtilis to exhibit higher Cd 2+ resistance than the negative control strain. and orf4113 confers Cd 2+ resistance for B. vietamensis 151-6.

Discussion
The present study showed that the gene cluster4802-4803 located on plasmid p25 and the gene cluster 4111-4112-4113 located on chromosome were involved in Cd 2+ resistance for strain B. marisflavi 151-25 and B. vietamensis 151-6, respectively. Many microorganisms have been reported to use heavy-metal-transporting ATPases, such as the proteins CadA and ZntA, to overcome Cd 2+ toxicity (Nucifora et al. 1989;Rensing, et al. 1997a).
Specifically, CadA from S. aureus has a length of 727 amino acids (Nucifora et al. 1989), and its amino acid sequence is highly similar to 4111 (63.31%) and 4802 (65.21%) (Fig.   S9). Alignment with CadA from S. aureus showed that the protein sequences of orf4111 from B. vietamensis 151-6 and orf4802 from B. marisflavi 151-25 all included a conserved (Cys-Pro-Cys) tripeptide for Cd 2+ , Pb 2+ or Zn 2+ binding . CadC from S. aureus including 122 amino acids was related to the divalent cation ATPase (Silver and Phung 1996). Expression of CadA is regulated by CadC, which is a homodimeric repressor that dissociates from the cad operator/promoter upon binding (Nucifora et al. 1989). CadC is a member of the ArsR/SmtB family of metalloregulatory proteins (Busenlehner et al. 2003;Saha, et al. 2017). Its crystal structure was resolved in 2005, and showed that two regulatory metal-binding sites for the inducer Cd 2+ are formed by Cys-7 and Cys-11 from the N terminus of one monomer and Cys-58 and Cys-60 of the other monomer . Alignment with CadC from S. aureus showed that the protein sequence of orf4112 from B. vietamensis 151-6 and of orf4803 from B. marisflavi 151-25 contained these four Cys residues (Fig. S10). Moreover, the similarity between CadC and Orf4803 or Orf4112 are 86.89% and 82.64%, respectively. These analysis indicated that the Cd 2+ resistance mechanisms of B. vietamensis 151-6 and B. marisflavi 151-25 were all the cad system, and a gene clusterconsisting a P-type ATPase gene and a transcriptional regular gene given the different cadmium resistances in B. vietamensis 151-6 (4111-4112 located on chromosome) and B. marisflavi 151-25 (4802-4803 located on plasmid). The hypothetical cad model was showed in Fig. S11.
19 up-regulated genes located on the chromosome that exhibited a more than 4-fold change according to transcriptome data were analyzed using qRT-PCR. Compared to wildtype B. marisflavi 151-25, the transcript levels of seven genes showed marked fold increases in 151-25△3 (Fig. S12a). These genes were orf666 (TetR family transcriptional regulator gene), orf667 (cysteine ABC transporter substrate-binding protein gene), orf668 (ABC transporter permease gene), orf1240 (ArsR family transcriptional regulator gene), orf1241 (copper-translocating P-type ATPase gene), orf3892 (hypothetical protein gene) and orf3894 (cation transporter gene). To confirm whether these genes related toCd 2+ resistance for B. marisflavi 151-25, three gene fragments also with 4802-4803 were overexpressed in B. subtilis and the Cd-MIC values for those recombinant strains were determined. As shown in Fig. 12b, the operon containingorf4802 and orf4803 allowed recombinant B. subtilis to exhibit greater Cd 2+ resistance, while the other three fragments would not increase Cd 2+ resistance of the recombinant B. subtilis. Our results suggested that the operon of 4802-4803 plays a leading role for Cd 2+ resistance of B. marisflavi 151-25, and that when plasmid p25 (containing the main Cd-efflux pump genes, orf4802 and orf4803) was deleted, the transcript level of other efflux pump genes would be enhanced.

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The interaction between these genes remains to be further study.
Overall, we identified that the Cd 2+ resistance mechanism of B. vietamensis 151-6 and B. marisflavi 151-25 were all cad system. The cad system was also reported in B. firmus and B. subtilis (Ivey et al. 1992;Solovieva and Entian 2002), but not in B. vietamensis and B.
marisflavi. Our results further confirm that the cad system is widespread in Bacillus sp. bacteria.

Declarations
Identification of cadmium-binding proteins from rice (Oryza sativa L.