Identi ﬁ cation of cadmium resistance and adsorption gene from Escherichia coli BL21 (DE3) †

Cadmium is recognized as one of the most toxic heavy metals, and chronic cadmium exposure threatens plant, animal and human health. Because certain bacteria can play an important role in remedying heavy metal pollution, it is essential to further our understanding and clone cadmium adsorption and resistance genes from microorganisms. In this study, the cadmium resistance of di ﬀ erent Escherichia coli strains including BL21 (DE3), JM109, DH5 a , Top10 and Mach was determined. Among those strains, the E. coli BL21 (DE3) exhibited the highest cadmium resistance. Sequence analyses of four cadmium resistant clones screened from the BL21 (DE3) fosmid library showed that the insertion of each fosmid plasmid contained a fragment located at the lambda phage DE3 region. Next, we overexpressed several genes of this fragment in BL21 (DE3) and identi ﬁ ed that cadmium resistance of E. coli is acquired through the capB gene. Indeed, overexpression of the capB gene increased the adsorption rate of cadmium. Taken together, these results indicated that the capB gene confers E. coli BL21 (DE3) with increased cadmium resistance by adsorption of cadmium. This study provided new insight into the mechanism of microbial cadmium resistance and expanded our understanding of cadmium resistance, adsorption and bioremediation by microorganisms.


Introduction
Cadmium is a nonessential nutrient element for organisms and is regarded as one of most toxic heavy metals with an unknown biological function. 1,24][5][6] Chronic cadmium exposure can not only affect the growth, yield and quality of the crops, 7 but cadmium can also enter the body through the food chain and consequently endanger human health; Itai-Itai disease, respiratory and kidney disease. 8,9ccording to previous studies, bacteria evolved several cadmium resistance mechanisms: 1,2,10-12 metal exclusion by a cellular permeability barrier such as the cell membrane or envelope; intracellular and extracellular sequestration through exopolysaccharide coating; 1-3 reduced metal sensitivity of cellular targets and transport efflux pumps. 13Particularly the intracellular and extracellular binding of metal is of interest to researchers in the eld of bioremediation as this method could be used to immobilize and adsorb target metals.
5][16][17][18][19][20][21] Currently, most studies focus on the overexpression of cadmium resistant genes from Gram-positive bacteria to reverse cadmium pollution or to isolate strains in heavy metal contaminated areas: 2 cadA from S. aureus, 22 czc system from Alcaligenes eutrophus, 23 and MTS 16,23,24 and phsABC 25 from Salmonella typhimurium.In fact, E. coli is intrinsically tolerant to high levels of cadmium up to 0.9-1.0mM. 16,25,26However the exact mechanism of cadmium resistance in E. coli is not fully understood.8][19] Interestingly, most cadmium resistance mechanisms currently known in microbiology are cadmium exclusion.Thus, the identication of cadmium resistant genes, especially cadmium adsorption genes, in E. coli would be of great importance for applications in remedying cadmium contamination.
In this study, we determined the cadmium resistance of different strains of E. coli including BL21 (DE3), JM109, DH5a, Top10, and Mach.We discovered that BL21 (DE3) had the highest cadmium resistance, up to 1.0 mM.Consequently, we constructed a BL21 (DE3) fosmid library to look for genes conferring the high resistance of E. coli to cadmium.Four clones with higher cadmium resistance than the wild type were identied, and the insertion fragments of the four strains were overlapped.Through overexpression of different genes in this section, we were eventually able to identify the gene related to cadmium resistance and absorption.Based on our knowledge, this is the rst study to report the key gene related to cadmium absorption of BL21 (DE3).This work has paved the way for bioremediation and increasing cadmium resistance.

Bacterial strains and growth conditions
The plasmids and bacterial strains used in this study are listed in Table 1; EPI300-T1R came with the Fosmid Library Construct Kit.All the strains were grown in Luria Bertani (LB) medium (10 g L À1 trypoton, 5 g L À1 yeast extract, 10 g L À1 NaCl) overnight at 37 C with shaking at 200 rpm.All reagents used in this study were purchased from Sigma-Aldrich (St. Louis, Missouri, USA).

Determination of minimum inhibitory concentration (MIC) of cadmium
The MIC values 27 were assessed by measuring the growth rate of different strains estimated from the optical density at 600 nm (OD 600 ).For the assay, the overnight culture of the test strains was transferred into fresh LB medium and incubated at 37 C until an OD 600 of 0.6-0.8 was reached.Next, the different strains (1%) were inoculated into 96-well plates containing different concentrations of CdCl 2 (0-1.4 mM) in triplicate.Aer incubation at 37 C for 20-24 hours, the OD 600 was measured with a uorescence absorbance cuvette.

Construction and screening of BL21 (DE3) genomic fosmid library
The BL21 (DE3) total genomic DNA was extracted with the Bacterial Genome Rapid Extraction Kit (BioTeKe, China), according to the manufacturer's protocol.DNA concentration and quality were checked by absorbance cuvette (A 260 /A 280 $ 1.8) and gel electrophoresis (1%).Approximately 0.5 mg mL À1 DNA extract was used to construct a fosmid library with the Copy-Control HTP Fosmid Library Production Kit and pCC1FOS Vector (Epicentre, Madison, WI, USA), according to the manufacturer's instructions.Twelve fosmid clones were randomly selected for evaluating the insert size by BamHI digestion and agarose gel electrophoresis analysis (Fig. S1 †).A total of 1443 fosmid clones (insert size range 25-40 kb) were obtained and stored at À70 C in 40% glycerol.
To screen for clones with cadmium resistance, all fosmid clones were replicated in 96-well plates containing LB broth supplemented with chloramphenicol (12.5 mg mL À1 ) and cultivated at 37 C for 8-12 h.The cultures were then transferred onto solid LB medium supplemented with chloramphenicol (12.5 mg mL À1 ) containing 1.2 mM CdCl 2 and incubated at 37 C for 36 h.
Fosmid DNA was isolated from the positive fosmid clones with the High Purity Plasmid Extraction Kit (TIANGEN, China) and sequenced from both ends with an ABI 3730 DNA analyzer and the pCC1FOS vector universal primers fosmid-T7 and fosmid-RP (Table.S1 †).The sequences were analyzed by BlastN (NCBI) to determine the location of the inserted fragments in the entire genome.

Gene overexpression in E. coli
Genes containing their promoters predicted using Web Promoter Scan Services (https://www-bimas.cit.nih.gov/molbio/proscan/) were amplied by PCR from the positive plasmids with the primers listed in Table S1, † and digested with KpnI and XbaI.These fragments were inserted into the KpnI/XbaI double digest of pUC19-GFP, containing a reporter green uorescent protein (GFP) gene gfp, to create plasmids pUC19-capA-GFP, pUC19-capB-GFP, pUC19-capJ-GFP, pUC19-regI-GFP, pUC19-regII-GFP and pUC19-regIII-GFP (Table.S1 †).Each plasmid was veried by DNA sequencing and transformed into E. coli BL21 (DE3) to express the GFP-tagged proteins CapA, CapB, CapJ, CapC, CapD, CapE, CapFi, CapFii, CapV, CapQ and CapT, respectively.All the host cells harboring the recombinant vectors were grown in LB at 37 C for 20 h.To conrm gene expression, uorescence of the cultures was measured with a uorescence absorbance cuvette.Fluorescence in the wells was measured at an excitation/emission wavelength of 484/507 nm, using a microplate reader SpectraMax M2 (Molecular Devices, USA).BL21 (DE3) cells harboring the empty pUC19-GFP expression vector were used as the negative controls.

Cd 2+ removal by protein CapB
The capB overexpression strains and control strains (BL21 (DE3)) with empty vector pUC19-GFP were cultivated in LB broth supplemented with ampicillin (100 mg mL À1 ) for 12 h in a shaking incubator (200 rpm), before transferring them into 3 mL LB supplemented with 0.1 mM Cd 2+ or 0.05 mM Cd 2+ for 24 h.Then the cultures were transferred into 1.5 mL tubes and centrifuged at 10 000g for 10 min.The supernatant was separated and diluted 100 folds using 0.6 M HCl and the Cd 2+ concentration was determined with an atomic absorption spectrophotometer (AAS). 28,29Based on the mean absorbance, concentration of Cd 2+ was determined by comparing with mean absorbance of the standard Cd 2+ .The experiments were performed in triplicate.

Resistance of E. coli stains to Cd 2+
To determine the degree of Cd 2+ resistance of E. coli, we examined the growth of different E. coli stains including BL21 (DE3), JM109, DH5a, Top10 and Mach in LB liquid medium with various Cd 2+ concentrations.As depicted in Fig. 1a, the strain BL21 (DE3) resisted Cd 2+ concentrations of up to 1.0 mM.Strain Top10 resisted up to 0.8 mM Cd 2+ and Mach, JM109 and DH5a up to 0.6 mM Cd 2+ .Clearly, from all the E. coli stains used in this study, BL21 (DE3) had the highest cadmium resistance (1.0 mM AE 0.02).The evident difference between BL21 (DE3) and the other four genetically engineered E. coli stains is that BL21 (DE3) carries a lysogenic lambda phage DE3 that expresses T7 RNA polymerase under the control of a lacUV5 promoter. 30o we speculated that this specic DE3 sequence, located at 748 396-791 320 in the BL21 (DE3) genome, might confer the higher resistance to cadmium.
The fosmid DNA isolated from these four clones was sequenced and by aligning the sequences with the BL21 (DE3) genome (accession number: NC_012892), we found that the inserted fragment from the four plasmids all contained a DNA fragment located between 768 481 and 801 476 (Table 2).Indeed, this region overlapped with the aforementioned DE3 sequence.The result conrmed that the DE3 sequence is important for the cadmium resistance of BL21 (DE3).

Overexpression of protein CapB can improve the cadmium resistance of BL21 (DE3)
To identify the cadmium resistant gene(s) in the DE3 sequence, we further analyzed the genetic composition and gene function of the fragment locating between 768 481 and 791 320.It contained 24 genes coding for DNA packing proteins, capsid components and tail components (Fig. 2).In order to identify the target genes conferring the cadmium resistance, some of these 24 genes were overexpressed in BL21 (DE3) to see whether they could increase the cadmium resistance of BL21 (DE3).The selected genes were fusion expressed with GFP to conveniently detect the expression of target genes by measuring uorescence.
As shown in Fig. 3a, the genes capA, capB and capJ were successfully overexpressed.However, only overexpression of capB could increase the MIC of Cd 2+ for BL21 (DE3) from 1.0 mM to 1.2 mM (Fig. 3b).In order to conrm that the CapB protein is expressed under its own promoter, we also performed SDS-PAGE (Fig. S2 †).In addition, overexpression of capB in DH5a was also shown to improve the Cd 2+ resistance of DH5a (Fig. 3c).
CapB encodes a capsid component of the lambda phage DE3 and the isoelectric point (pI) of this protein was 5.31.In the neutral pH environment in the cell, CapB has a negative charge that could bind to the positive ion of cadmium.Similarly, also the pIs of the proteins CapC, CapD, CapE, CapFi, CapFii, CapV, CapG and CapT were low (Table S2 †).We therefore also selected these to be overexpressed in BL21 (DE3) in the hope to identify more Cd 2+ resistance related genes.Based on the promoter and position of these genes, we expressed three fragments separately: region I (containing genes capC and capD), region II (containing genes capE, capFi and capFii) and region III (containing genes capV, capG and capT) (Fig. 2).Although the proteins were successfully expressed in BL21 (DE3) (Fig. 3a), none of the fragments changed its Cd 2+ resistance (Fig. 3c).Taken together, the results suggested that the CapB protein encoded by capB contributes to the Cd 2+ resistance of E. coli.

Absorption of cadmium ions by the protein CapB
As CapB is not a membrane protein, we speculated that it may be expressed in cells to increase Cd 2+ resistance by absorbing Cd 2+ .To verify this, the nal concentration of Cd 2+ was measured in culture media supernatant aer cultivation of capB overexpressing strains.We found that the Cd 2+ removal rate of capB overexpressing strains was signicantly higher than the wild type BL21 (DE3) strain (Fig. 4).At 0.05 mM Cd 2+ , the Cd 2+ removal rate of capB overexpressing strains was about two-fold greater than that of the wild type BL21 (DE3).At 0.1 mM Cd 2+ , the Cd 2+ removal rate of capB overexpressing strains was also higher than that of the wild type (Fig. 4).It was speculated that such a high concentration of metal ions was toxic to cells and the gradual saturation of adsorption sites by cadmium ions would have limited the capacity of Cd 2+ removal. 31The above results suggested that the CapB protein indeed increased cadmium resistance by absorbing more Cd 2+ .Unlike the earlier discovered metallothionein, 10,16,32,33 CapB does not contain a lot of cysteine short peptides.The pI of CapB (5.31) was lower than the neutral pH, implying that the protein surface will have many negative charges that can bind the positive cadmium ions in the cell.Perhaps it is just those functional negative groups that chelate with cadmium ions. 34However, a low pI is just one of the characteristics of CapB since not all proteins with a low pI could adsorb cadmium.Therefore, in addition to the low pI, the special sequence or structure is also fundamental to conferring cadmium resistance.Our future studies will thus focus on the binding characteristics of the CapB protein.
Our results reiterated that bacterial proteins could improve heavy metal resistance or adsorption.With advances in bioinformatics and the newly acquired knowledge on CapB, we may be able to improve upon the design of such proteins to remedy heavy metal pollution in the future.

Fig. 4
Fig.4Determination of cadmium adsorption capacity of recombinant strains with pUC19 as the expression vector.CapB overexpressing strains were cultivated at 0.05 mM and 0.1 mM Cd 2+ , indicated as 0.05 mM and 0.1 mM respectively.pUC19-GFP was used as the control.**, p < 0.01.Ordinate represents the cadmium removal efficiency.

Table 1
The plasmids and bacterial strains used in this study