Physiological and Genetic Response Characteristics of Stenotrophomonas Rhizophila JC1 Under Heavy Metal Stress

In this study, the Cu 2+ (120 mg/L) and Cr 6+ (80 mg/L) removal rate of S. rhizophila JC1 reached at 79.9% and 89.3%, respectively. Scanning electron microscopy showed that Pb 2+ and Zn 2+ had no obvious effect on cell structure, but the cells became smaller and brighter under Cu 2+ stress, and many nanoparticles formed on the cell surface under Cr 6+ stress. The physiological response analyses demonstrated that moderate change of membrane permeability was necessary for adsorption. FI-IR and EDS analyses showed that exopolysaccharides (EPS) and the replacement of basic elements (ie., C, O) might be the main means of heavy metals adsorption by strain. In addition, 323 transport proteins were predicted in the genome of S. rhizophila JC1. Among them, two, six and ve proteins of the cation diffusion facilitator, resistance-nodulation-division eux and P-type ATPase families were respectively predicted. The expression of genes showed that the synergistic action of transport proteins played an important role in the process of adsorption. The comparative genomics analysis revealed that S. rhizophila JC1 has long-distance evolutionary relationships with other strains, but the eux system of S. rhizophila JC1 contained the same types of metal transport proteins as other metal-resistant bacteria.


Introduction
Precipitating, chelating, or altering the oxidation state of various heavy metals by using microorganisms has become a hotspot of research and a topic of industrial application in the eld of environmental remediation (Kang and So. 2016). To ensure normal growth and metabolism under heavy-metal stress, microorganisms have developed homeostatic systems by controlling the processes of intake, transport, and e ux of heavy metals (Ma et al.2009; Valencia et al. 2013).
E ux is a main approach applied by bacteria to command metal ions transport to reduce intracellular metal concentrations. There are three recognized e ux systems that play crucial roles in the heavy metal detoxi cation by bacteria. The cation diffusion facilitator (CDF) family is ubiquitous in bacteria, archaea and eukaryotes (Paulsen and Saier 1997), and it transports heavy metals, including Co 2+ , Cd 2+ , Zn 2+ and possibly Ni 2+ , Cu 2+ and Hg 2+ (Kambe 2012). Lots of members of the CDF family consist of 300-550 amino acids, which include six transmembrane domains, the regulatory cytoplasmic C-terminal domain and a histidine-rich region that may be a potential metal-binding site (Barber et al. 2016). The P-type ATPase, located on the plasma membrane, promotes the metal e ux system through hydrolysis (Arguello et al. 2011). It transfers substrates from the outside of the cell or the peripheral cytoplasm to the cytoplasm, as well as from the cytoplasm to the extracellular or peripheral cytoplasm through P-type ATPases (Hiraizumi et al. 2019). Simultaneously, owing to the stimulating effects of sulfhydryl compounds on the metal e ux activity, CPx-type ATPases may also use glutathione to expel metals from the cytoplasm (Meng et al. 2015). The transporter-dependent resistance-nodulation-division (RND) e ux system has been considered to play a crucial role in heavy-metal resistance of bacteria (Saier et al. 1994). In most cases, the gene adjacent to the encoded protein is the member of membrane fusion protein (MFP) family (Nagakubo et al. 2002). Along with the outer membrane factors (OMFs) (Maseda et al. 2002), three of these protein families form an excretion protein complex, which can excrete the matrix from the cytoplasm, cell membrane or periplasmic space through the outer membrane (Kim et al. 2011;Yang et al. 2014 ).
In this paper, the adsorption ability of S. rhizophila JC1 (CPO50062) for Cr 6+ , Cu 2+ , Pb 2+ and Zn 2+ of different concentration was validated. Furthermore, the morphological feature of strain JC1 stressed by different heavy metals was observed through scanning electron microscopy (SEM), the element composition The strain JC1 stressed at 80mg/L of Pb 2+ , Cr 6+ , Cu 2+ and Zn 2+ were also collected for functional groups and element composition analysis. Cell sediments were collected by centrifugation at 10 000 g for 10 minutes and dried at 45 ℃. After that, the infrared absorption spectra in the range of 4000-400 cm -1 was measured by Fourier Transform Infrared Spectrometry (FT-IR) and screened by 325 meshes for Energy Dispersive X-ray spectroscopy (EDS) examination  .
The physiological response of S. rhizophila JC1 under different metal ions stress To reveal the physiological response mechanism of strain JC1 after heavy metal ions stress, membrane permeability and intracellular macromolecular (ie., protein, phospholipid, alkaline phosphatase) substances were analyzed. Each treatment was repeated three times.
The physiological response of membrane permeability The cell outer membrane (OM) permeability of S. rhizophila JC1 under different metal ions stress was analyzed by the method described by Loh (Loh et al. 1984). The strain JC1 was respectively stressed with 0, 40, 80, 120mg/L concentration of Pb 2+ , Cr 6+ , Cu 2+ and Zn 2+ for 24h. After that, N-phenyl-1naphthylamine (NPN) solution with the nal concentration was 10 μmol/L was added, then slowly oscillate at 37 ℃ about 3 minutes. Finally, the uorescence value was measured at excitation wavelengths was 350nm and emission wavelengths was 440 nm with uorescence spectrophotometer. Three repeated experiments were performed.
The cell inner membrane (IM) permeability of S. rhizophila JC1 stressed by different metal ions was analyzed by measuring the β-galactosidasea activity (Liu et al. 2004). The logarithmic phase cells that cultured in LB medium containing 2% lactose were collected and washed twice with 0.85% NaCl solution, then respectively stressed with 0, 40, 80, 120mg/L concentration of Pb 2+ , Cr 6+ , Cu 2+ and Zn 2+ for 24h. Thereafter, O-Nitrophenyl-β-D-Galactopyranoside (ONPG) solution with the nal concentration was 1.5 mM was added, then slowly oscillate at 37 ℃about 3 minutes. Finally, the absorption value was measured at 415 nm with Spectrophotometer. Three repeated experiments were performed.

The physiological response of intracellular macromolecular
The strain JC1 treated with 0, 40, 80, 120mg/L concentration of Pb 2+ , Cr 6+ , Cu 2+ and Zn 2+ was cultured to the logarithmic growth stage, then the cell culture medium was collected. The protein content was determined by the Bradford method, and measured with BSA as standard (Panja et al. 2008, Wang et al. 2021).
The chromogenic substrate p-nitrophenylphosphate (pNPP) was used to investigate alkaline phosphatase enzyme activity (Schlesinger 1989). The strain JC1 was initially cultured in tris-glucose medium to the logarithmic growth stage. Then washed three times with phosphorus-free tris-glucose before cultured in phosphorus-free tris-glucose solution at 37 ºC for 40 minutes. After that, the cell sediments were re-suspended in deionized water after washed, then treated with treated with 0, 40, 80, 120mg/L concentration of Pb 2+ , Cr 6+ , Cu 2+ and Zn 2+ . Finally, the mixture involving 1.0 mL metal-laden bacteria and 2.0 mL pNPP of 200 mM were incubated at 37°C about 15 minutes. The absorbance at 420 nm was measured.
Three repeated experiments were performed of above experiments.

Genomes
The genomic protein sequences and 16S rRNA of S. rhizophila JC1 were obtained from whole-genome sequencing results. Corresponding sequences of other strains were obtained from NCBI (https://www.ncbi.nlm.nih.gov/).

Transport protein classi cation and comparation
Diamond software was used to compare the amino acid sequence of the target species by TCDB database and match genes of the target species with their corresponding functional annotation information. SWISSPROT and TrEMBL databases were used for function analyses. The phylogenetic relationship of 15 comparative strains was obtained by 16S rRNA sequences analysis (Rozycki and Nies 2009).

Functional validation of predicted genes by qPCR
To identify the function of transport proteins of S. rhizophila JC1 under four heavy metal ions stress, four genes (i e., czcD, czcB, ZntA, Cu 2+ -exporting ATPase) were examined by quantitative real-time PCR (qPCR). The reverse-transcribed was carried out according to the instructions of Super RT Kit (TaKaRa). Each qPCR procedure was carried out according to the description by Novinscak and Filion (Novinscak and Filion 2011). The 16S rRNA and β-actin gene were used as an internal standard. Three rounds of independent qPCR reactions were used to verify the expression of each gene and the data were showed as arithmetic means ± the standard deviation. The P-value less than 0.05 was considered to be statistically signi cant ).

Results And Discussion
Metal ions removal e ciency The toleration and adsorption to different metal ions of strain JC1 showed great difference. For instance, during S. rhizophila JC1 was respectively cultured in LB+Cu 2+ , LB+Cr 6+ , LB+Zn 2+ that the concentration of metal ions was 40~120 mg/L , 40~200 mg/L, 40 mg/L for 24 hours, there were no signi cant difference in OD 600 value. However, during the concentration of Zn 2+ and Pb 2+ were increased to 160 mg/L, the strain JC1 hardly grew. The adsorption e ciency was 79.8% when the concentration of Cu 2+ was 120 mg/L -1 , while it reduced to 26.1% when the concentration reached to 160 mg/L. More interesting was that with the concentration was 40~200 mg·L -1 of Cr 6+ , there had no signi cant suppression on the growth of bacteria, but only showed maximum adsorption e ciency of 89.3% when the concentration was 80 mg/L. In terms of Pb 2+ and Zn 2+ , the strain JC1 hardly showed adsorption ability (Fig. 1). Hence, this phenomenon indicated that the adsorption of metal ions by bacteria was selective, which was also suggested by Ye (Ye et al.2014) Analysis of cells structure under metal stress by SEM In this study, the morphological feature of strain JC1 comparison between metal ions stressed-cells and unstressed-cells was performed with SEM analysis. In different metal ions stress condition, different cell morphology was observed (Fig. 2). For unstressed-cells, the cell structure was intact, showed rod-shaped ( Fig. 2a). However, the rod-shaped of JC1 after Cr 6+ stress was almost impossible observed, there were crowds of particles gathered on the surface and the cells were wrapped in the membrane (Fig. 2b). In the case of Cu 2+ , cells showed shorter and brighter (Fig. 2c). The change caused by Pb 2+ and Zn 2+ was similar. In brief, the surface of the JC1 became rougher, but the difference was that the cells condensed into clusters after Zn 2+ stress (Fig. 4d&e).
The change of cell morphology was the result of bacteria response to heavy metal stress. The decrease in cell size may be due to dehydration of the cells under Cu 2+ stressed and also may be explained as a negative response of bacteria against further uptake of metal by decreasing the area of contact with the In this study, the functional groups of strain JC1 for adsorption with four heavy metals was analyzed by Fourier transform infrared spectroscopy (FI-IR).
Obviously, among four metal-laden samples, signi cant shifts of C-O-C and C=O were observed after the treatment of Cr 6+ , signi cant shifts of O-H was observed after the treatment of Cu 2+ and Zn 2+ , signi cant shifts of C-O was observed after the treatment of Pb 2+ (Fig. 2f). According to the FI-IR analysis, we suggested that the binding by EPS was one of the important way for heavy metal detoxi cation. In addition, the detoxi cation that depends on the functional groups of the bacteria and the valence state of metal ions is selective (Ye et al. 2014, Zhang et al. 2017).

Analysis of element composition under metal stress by EDS
Generally, non-metallic elements such O, C, Si and non-toxic metal elements such as Ca 2+ , Fe 2+ /Fe 3+ were the basic elements to maintain normal growth and metabolism of microorganism. However, some toxic metals such as Cr 6+ or Pb 2+ will exist in the cells through sites replacement due to atomic radius, cell structure. The result of element composition analysis after metal ions stress was shown in Fig. 3. Obviously, the corresponding metal elements were increased in the bacteria when they were stressed by different metals. Moreover, the metal content is consistent with the adsorption e ciency. Further analysis we found that, Cr 6+ and Cu 2+ mainly replaced the O element, while the Pb 2+ and Zn 2+ mainly replaced the C element, this may be related to the radius of the metal.
Analysis of physiological response of S. rhizophila JC1 under different metal ions stress Determination of cell membrane permeability The change of OM permeability under different heavy metal stress was shown in Fig. 4. Distinctly, the OM permeability of strain JC1 was signi cantly affected by the type and concentration of metal ions. What is more, its variation trend was positively correlated with the adsorption capacity of Cr 6+ and Cu 2+ (Fig. 1 4).
However, it was interesting to note that the change induced by Pb 2+ and Zn 2+ were higher than Cr 6+ and Cu 2+ , but the cell survival rate and adsorption capacity were contrary to this. Secondly, the higher the concentration of Pb 2+ and Zn 2+ , the higher the change of OM permeability, but the cell survival rate and adsorption capacity were also contrary to this (Fig. 1 4). We speculated that: 1): detoxi cation factors for Cr 6+ and Cu 2+ were more abundant compared with Pb 2+ and Zn 2+ in cells of strain JC1, 2): cells will die when the membrane permeability changes exceeds its self-repair ability.
Similar to the OM permeability, the change of IM permeability were also affected by the type and concentration of metal ions. But it did not mean that the higher of the concentration of metal ions, the greater the change of membrane permeability, nor the stronger of the adsorption capacity. In summary, the change of membrane permeability was the key factor for bacteria to adsorb heavy metals, but when degree of change exceeded its self-repair capacity, it became non-resistant or low-resistant.

Analysis of changes in bioactive macromolecules
The excessive release of macromolecules such as proteins (Pr), phospholipid (PL), alkaline phosphatase (ALP) will de nitely affect the normal function of cells. Without a doubt, with the change of membrane permeability, macromolecules will release to the outside of the cells. In terms of proteins and phospholipid, the variation trend was consistent with the change of membrane permeability (Fig. 4 5), which was also the reason that why cells hardly grow under the stress of Pb 2+ and Zn 2+ . On the contrary, the release trend of alkaline phosphatase was consistent with the heavy metal adsorption capacity of strain JC1.We speculated that alkaline phosphatase participates in the hydrolysis reaction of microorganisms and generates phosphate ions (-PO 4 3-) and free hydroxyl groups(-OH), then the -PO 4 3and -OH complexed with the positive metal ions such as Cr 6+ and Cu 2+ , also with Pb 2+ and Zn 2+ .
Transport proteome of S. rhizophila JC1 In total, 323 transport proteins belonging to nine major transport protein classes were predicted in the genome of S. rhizophila JC1 (4.28 Mb) ( Table S1). Most of them were primary active transporters (TC#3), electrochemical potential-driven transporters (TC#2) and channels/pores (TC#1). They represented only 9% of the total number of predicted proteins. In addition, only 33% 50% of superior heavy metal-resistant bacteria which genome larger than 4 Mb (Rozycki and Nies 2009). The distribution of transport proteins was similar to these bacteria. Thus, the adsorption and resistance of S. rhizophila JC1 to heavy metals may be independent of the number of transporters, the types of transporter may be the key factor.

Heavy-metal transport proteins
Because cells do not contain the NADPH level required for reductase activities and there is a lack of methylation or other covalent modi cation mechanisms, some divalent metal ions cannot be discharged from cells independently. However, the three e ux systems (CDF, RND and P-type ATPases), containing almost all of the metal transport proteins, actively mediate the intracellular to extracellular excretion of divalent metal ions (Rozycki and Nies 2009). Therefore, the numbers and functions of transport proteins involved in the three e ux systems were analyzed in S. rhizophila JC1.
The cation diffusion facilitator (CDF) system The CDF system members are chemiosmotic ion/proton exchangers that are involved in the e ux of divalent metal cations (Nies 2003) and the transport of metals from the cytoplasm across the cytoplasmic membrane into the periplasmic space (Higuchi et al. 2009) As shown in Table 1, two predicted proteins constituted the CDF system in S. rhizophila JC1. The czcD/zitB (JC1_GM001116) was predicted as Co 2+ /Zn 2+/ Cd2+ e ux system protein that included cation e ux, cation transporter ATPase C terminal as well as zinc transporter dimerization domains. Furthermore, its homolog was con rmed to bind Cu 2+ and Ni 2+ in Ralstonia metallidurans (Anton et al. 2004). The other CDF system member (TC # 2.A.4.4.7) (JC1_GM001883) was predicted to be an integral membrane protein, which was con rmed as a lead (Pb 2+ ) e ux transporter (PbtF) in Achromobacter xylosoxidans (Hložková et al. 2013), and it may provide sites for the targeted utilization of proteins. The sequences of these two proteins were provided in Additional le 1.

The resistance-nodulation-division (RND) e ux system
On the basis of transport substrate by bacteria, the RND was subdivided into six sub-groups: RND1 (Zn 2+ , Co 2+ and Cd 2+ ), RND2 (Co 2+ and Ni 2+ ), RND3a Six heavy-metal e ux proteins were predicted in S. rhizophila JC1 (Table 1). Among them, two types of czcCBA operons (TC#2.A.6.1.15 and TC#2.A.6.1.16) form a complete cobalt-zinc-cadmium resistance system homologous to that in Escherichia coli (Tam et al. 2019). czcA is the RND protein, having at least one transmembrane domain and a membrane-spanning helical or beta-stranded domain that embeds in the membrane. czcC is a member of the OMP family that forms a trimeric channel and a long helical barrel that allows the export of a variety of substrates in Gram-negative bacteria. In S. rhizophila JC1, the czcCBA operon may transport Cd 2+ and Zn 2+ , as well as Co 2+ , because there is a gene downstream of czcA that encodes a Cd 2+ /Zn 2+ -exporting ATPase (Fig.  6).
No regulatory gene was predicted near the two types of czcCBA operon, which was in contrast to the cnr in genome of R. metallidurans CH34 (cnrYXHCBA), czc in genome of R. metallidurans and Alcaligenes eutrophus (czcCBADRS/czcNICBADRS) and ncc in genome of Achromobacter xilosoxidans 31A The low similarity level of these proteins suggested that they have bifurcated considerably in the process of evolution, they may have obtained unique way in protecting strain JC1 from being damaged by heavy metals (Valencia et al. 2013). The Ni 2+ /Co 2+ -uptake system was not predicted in the genome of strain JC1, but fortunately the Mg 2+ /Co 2+ -uptake system was predicted. It may be that S. rhizophila JC1 decreased its Ni 2+ /Co 2+ -detoxi cation function, but to maintaining homeostasis through controlling the e ux of outer membrane (Rozycki and Nies 2009). This nding meant the stepwise evolutionary from the ancestor of S. rhizophila had already able to deal with heavy metals (Nies 2003, Rozycki andNies 2009).
P-type ATPase S.rhizophila JC1 contains a relatively low number (5) of predicted P-type ATPases (Table 1). After concerned, copA involved a polypeptide domain of approximately 50 amino acid residues with two cysteines. Owing to the presence of this domain that the copA may be related to the detoxi cation of Cu + /Ag + , but ineffective to divalent ions that in contrast to copB. However, in Bradyrhizobium liaoningense by Liang (2016), copA is responsible for resistance to Cu + , Zn 2+ and Cd 2+ . Moreover, copA induced gold (Au 2+ ) detoxi cation due to a cytoplasmic metal-binding protein (Pontel et al. 2007).
The ZntA(JC1_GM001891) predicted in S. rhizophila JC1 was the P5-type ATPase, having plasma membrane C-terminal and N-terminal auto inhibitory domains. As described in Cupriavidus metallidurans, ZntA was the downstream gene of czcCBA operon that encoded the Cd 2+ /Zn 2+ -exporting ATPase, and its side chains, containing Met254, Cys476 and His807 contributed to Cd 2+ , Co 2+ and Zn 2+ binding and transporting, respectively (Smith et al. 2017).
In summary, the e ux system of S. rhizophila JC1 contained the same types of metal transport proteins as many other metal-resistant bacteria. Its low number of transport proteins may be the result of evolution.
Comparative genomics analysis

Molecular evolutionary analysis
A molecular evolutionary analysis is an valuable measure to evaluate similarities or differences, and to match functional gene information, between model organisms and uncharacterized newly sequenced genomes(Gabaldón and Koonin 2013).
The evolutionary relationships of the 15 investigated bacteria were shown in Fig. S1. S.rhizophila JC1 had long-distance evolutionary relationships with other strains, which indicated that there may be great differences in gene expression levels. In addition, the lower number of transporters in genome of S. rhizophila JC1 compared with the other strains was the result of evolution.

Numbers of paralogs and transporters in the compared bacteria
For more information about the origin of metal-resistance gene of S. rhizophila JC1, the paralogs of predictive gene products of 15 investigated bacteria were analyzed by BLAST comparisons. On the whole, the frequency level of paralogs among compared bacteria was approximately 8% ( Table 2). The relatively closest relationship with Salmonella enterica P-stx-12, has 30% paralogs, indicating that most of its increased genome size may be due to gene replication events during the bacterial species formation process.
In terms of most investigated strains, the frequency of paralogs of transporters was close to the 7% (Table 2). High percentages of paralogous proteinencoding genes were located on plasmids in S. rhizophila JC1 (28%), unlike the plasmid in Enterococcus hirae NCTC12204 (5%). When only considered the plasmid encoding paralogs of transporters, 29% of the S. rhizophila JC1 proteins represented paralogs. Therefore, duplicating genes encoding transport proteins, relocating them to plasmids and altering the substrate range and their products' expression patterns may have been important evolutionary processes in the evolution of S. rhizophila JC1.

Comparison of transition-metal transporters
A higher number of heavy metal-speci c transport proteins were predicted of strain JC1 through comparing with 15 investigated strains, especially proteins belonging to three e ux system (Table 3). For instance, the number of P-Type ATPases that contribute to Cd 2+ /Zn 2+ even to Cu + /Cu 2+ was nearly twice of other compared strains. Regrettably, the number of zinc transporters and mercuric transporters were relatively lower. The Mg 2+ /Co 2+ -transport protein (TC # 9.A.40.1.2, JC1_GM001315) that belong to the HlyC/CorC family was predicted in S. rhizophila JC1. We speculated that it may have great detoxi cation ability to divalent cations due to its approximately 440 amino acids with an N-terminal four TMS domain. Here, we further speculated that the metal resistance gene of S. rhizophila JC1appeared to have evolved by horizontal acquisition or duplication (Rozycki and Nies 2009). The detoxi cation ability to heavy metal was perfect, even though the number of detoxi cation system had decreased.

Validation of predicted genes by qPCR
The expression level of 4 genes that respectively represents three e ux systems were identi ed by qPCR. Each gene to be validated was successfully ampli ed and produced a single band. CzcD, czcB and ZntA were up regulated after treated with Cr 6+ , Pb 2+ and Zn 2+ as compared to the CK. Cu 2+ -exporting ATPase and ZntA were up regulated in after treated with Cu 2+ as compared to the CK. Among them, CzcD, czcB and ZntA exhibited higher expression levels in S. rhizophila JC1 treated with Cr 6+ compared to samples treated with Pb 2+ and Zn 2+ . The expression level of each gene used for identi cation showed signi cantly difference by P-value evaluation. The expression trend of 4 selected genes was consistent with the metal ions-removal e ciency and predicted results, which further con rmed that transport proteins play important roles in process of heavy metal adsorption.

Conclusion
The S. rhizophila JC1 showed great adsorption capacity for heavy metals, especially for Cu 2+ and Cr 6+ . The moderate change of membrane permeability and replacement of essential element sites may be the key factors for the adsorption of heavy metals by bacteria, and the exopolysaccharides (EPS) is the main Tables   Table 1 Heavy   The toleration and adsorption to different metal ions of strain JC1 showed great difference. For instance, during S. rhizophila JC1 was respectively cultured in LB+Cu2+, LB+Cr6+, LB+Zn2+ that the concentration of metal ions was 40~120 mg/L , 40~200 mg/L, 40 mg/L for 24 hours, there were no signi cant difference in OD600 value. However, during the concentration of Zn2+ and Pb2+ were increased to 160 mg/L, the strain JC1 hardly grew. The adsorption e ciency was 79.8% when the concentration of Cu2+ was 120 mg/L-1, while it reduced to 26.1% when the concentration reached to 160 mg/L. More interesting was that with the concentration was 40~200 mg·L-1 of Cr6+ , there had no signi cant suppression on the growth of bacteria, but only showed maximum adsorption e ciency of 89.3% when the concentration was 80 mg/L. In terms of Pb2+ and Zn2+, the strain JC1 hardly showed adsorption ability The morphological feature of strain JC1 comparison between metal ions stressed-cells and unstressed-cells was performed with SEM analysis. In different metal ions stress condition, different cell morphology was observed Figure 3 Generally, non-metallic elements such O, C, Si and non-toxic metal elements such as Ca2+, Fe2+/Fe3+ were the basic elements to maintain normal growth and metabolism of microorganism. However, some toxic metals such as Cr6+ or Pb2+ will exist in the cells through sites replacement due to atomic radius, cell structure. The result of element composition analysis after metal ions stress was shown in Fig. 3.

Figure 5
The excessive release of macromolecules such as proteins (Pr), phospholipid (PL), alkaline phosphatase (ALP) will de nitely affect the normal function of cells. Without a doubt, with the change of membrane permeability, macromolecules will release to the outside of the cells. In terms of proteins and phospholipid, the variation trend was consistent with the change of membrane permeability Figure 6 Six heavy-metal e ux proteins were predicted in S. rhizophila JC1 (Table 1). Among them, two types of czcCBA operons (TC#2.A.6.1.15 and TC#2.A.6.1.16) form a complete cobalt-zinc-cadmium resistance system homologous to that in Escherichia coli (Tam et al. 2019). czcA is the RND protein, having at least one transmembrane domain and a membrane-spanning helical or beta-stranded domain that embeds in the membrane. czcC is a member of the OMP family