Project Introduction

1. Successful Modification of Plasmids

We begin by examining the widely used pYYDT plasmid as a heterologous expression vector for Acidithiobacillus ferrooxidans, which serves as a versatile vector complying with the RFC10 standard. However, the Escherichia coli strain employed in our study, E. coli sm10, inherently possesses kanamycin resistance. This characteristic prevents us from using kanamycin as a selection marker for subsequent strain selection. Given that our chassis organism, A. ferrooxidans ATCC 23270, thrives in acidic environments, we decided to substitute kanamycin resistance with streptomycin resistance, which remains effective under acidic conditions. Details of this modification process are outlined in the engineering section.

We introduced both the unmodified pYYDT plasmid and the modified pYDT plasmid into E. coli DH5α, cultivating them in LB media supplemented with kanamycin and streptomycin, respectively, in order to evaluate their growth characteristics. The same experimental procedures were conducted with the E. coli sm10 strain. Initially, we confirmed that E. coli sm10 WT exhibits kanamycin resistance but is devoid of streptomycin resistance (Fig. 1A). As demonstrated in Fig. 1B, E. coli DH5α/pYYDT, which harbors the unmodified pYYDT plasmid, grows normally in kanamycin-supplemented media, while it fails to grow in streptomycin-supplemented media. In contrast, E. coli DH5α/pYDT, which contains the modified pYDT plasmid, flourishes in streptomycin-supplemented media but cannot grow in kanamycin-supplemented media (Fig. 1C).

These results confirm that we have successfully modified the vector plasmid pYYDT into the pYDT plasmid, making it suitable for our chassis cells. This foundational work paves the way for the subsequent successful conjugative transfer of the target gene into the chassis cells.

Fig 1 The growth curves of E. coli DH5α and E. coli sm10 containing different plasmids. (A) The growth curve of E. coli sm10 WT under kanamycin and streptomycin resistance. (B) The growth curve of E. coli DH5α/pYYDT containing the expression vector pYYDT under kanamycin and streptomycin resistance. (C) The growth curve of E. coli DH5α/pYDT containing the expression vector pYDT under kanamycin and streptomycin resistance. Experiments were conducted in triplicate and the error bar represent SD.

2. A dynamic synthesis module for cyclic di-GMP has been constructed, successfully elucidating a highly efficient synthesis module for c-di-GMP

To facilitate the efficient formation of biofilms by engineered strains and to enhance the effectiveness of bioleaching, we have opted to overexpress the second messenger cyclic di-GMP synthase in our target strain. During the engineering phase, we constructed a recombinant vector, pYDT_yedQ, which utilizes the IPTG-inducible promoter P _tac to regulate the expression of the GDC gene yedQ, sourced from E. coli BL21. Additionally, we developed recombinant plasmids pYDT_1373 and pYDT_1379, designed to express the GDC genes AFE_1373 and AFE_1379, respectively, obtained from A. ferrooxidans.

To evaluate whether these genes could enhance the intracellular concentration of c-di-GMP and consequently improve the biofilm formation capability of the chassis cells, we initially utilized E. coli BL21 as the model organism. We began by investigating suitable cultivation conditions, assessing the growth curves in M9 media supplemented with 5%, 10%, and 20% LB. As shown in Fig. 2, the growth of E. coli BL21 with the plasmid was minimally affected at an LB concentration of 10%. Therefore, we proceeded to choose the addition of 10% LB to the M9 medium for the subsequent biofilm experimentation with the engineered strains.

Fig 2 The growth curve of E. coli BL21 in LB medium at different concentrations. (A) The growth curve of E. coli BL21 is depicted at a concentration of 5% LB. (B) The growth curve of E. coli BL21 is depicted at a concentration of 10% LB. (C) The growth curve of E. coli BL21 is depicted at a concentration of 20% LB. Experiments were conducted in triplicate and the error bar represent SD.

Research has shown that biofilm formation significantly enhances leaching efficiency ^[1]. Consequently, it is crucial to promote substantial biofilm growth by bacteria on tailings to optimize leaching performance. In our study, we evaluated the biofilm content of E. coli BL21/pYDT_yedQ, E. coli BL21/pYDT_1373, and E. coli BL21/pYDT_1379 in response to the induction by 1 mM IPTG at various time intervals. As depicted in Fig. 3, the addition of IPTG in E. coli BL21 led to a significant increase in biofilm formation for E. coli BL21/pYDT_1373, E. coli BL21/pYDT_1379, and E. coli BL21/pYDT_yedQ when compared to the control group that did not receive IPTG, In contrast, no significant IPTG-induced biofilm increase was observed in the control strain E. coli BL21/pYDT.Notably, E. coli BL21/pYDT_1373 demonstrated the most robust biofilm formation. Through the overexpression of DGE genes, we effectively enhanced the biofilm-forming capabilities of the engineered strains.

Fig 3 (A) Bar chart of biofilm formation of E. coli BL21. (B) Biofilm formation of E. coli BL21 at 12 hours. Biofilm formation of E. coli BL21 at 24 hours. (C) Biofilm formation of E. coli BL21 at 48 hours. (D) E. coli BL21 at 24 hours. Biofilm formation of E. coli BL21 at 72 hours. Experiments were conducted in triplicate and the error bar represent SD.Two-sided Student’s t-test was used to analyze the statistical significance (*0.01< P < 0.05; **P < 0.01, NS: No significance).

Direct quantification of the normaliezd intracellular c-di-GMP showed that the c-di-GMP concentration in E. coli BL21/pYDT_1373, E. coli BL21/pYDT_1379, E. coli BL21/pYDT_yedQ, and E. coli BL21/pYDT of 24-h cultures with 1 mM IPTG was significantly higher than the cells without IPTG. The presence of 1 mM IPTG successfully increased the c-di-GMP level. The c-di-GMP content was determined based on the standard curve presented in Fig. 4B. As illustrated in Fig. 4, the c-di-GMP levels in E. coli BL21/pAFE_1373, E. coli BL21/pAFE_1379, and E. coli BL21/pYedQ displayed a significant increase following the addition of the inducer IPTG compared to the control group. In contrast, the intracellular concentration of c-di-GMP in the empty vector strain did not show any significant changes.

Fig 4 The schematic diagram of c-di-GMP content in E. coli BL21/pYDT, E. coli BL21/pYDT_1373, E. coli BL21/pYDT_1379, and E. coli BL21/pYDT_yedQ. (A) The c-di-GMP content of E. coli BL21/pYDT, E. coli BL21/pYDT_1373, E. coli BL21/pYDT_1379, and E. coli BL21/pYDT_yedQ. (B) The standard curve for c-di-GMP. (C) The total protein concentrations of E. coli BL21/pYDT, E. coli BL21/pYDT_1373, E. coli BL21/pYDT_1379, and E. coli BL21/pYDT_yedQ. (D) The standard curve for protein concentration is depicted. Experiments were conducted in triplicate and the error bar represent SD.Two-sided Student’s t-test was used to analyze the statistical significance (*0.01< P < 0.05; **P < 0.01, NS: No significance).

A. ferrooxidans is a chemolithoautotrophic bacterium that oxidizes sulfide minerals to release metal ions while producing sulfuric acid, thereby facilitating further metal dissolution. Due to the difficulty in culturing this strain, we employed ferrous iron and sulfur as energy sources to more intuitively and effectively study the growth characteristics of A. ferrooxidans ^[2]. Our observations indicate that when ferrous iron was used as the energy source, the engineered strains A. ferrooxidans/pYDT_1379, A. ferrooxidans/pYDT_0053, and A. ferrooxidans/pYDT_1373 displayed a lag phase during the mid-growth period. We hypothesize that the enhanced biofilm formation capability may impact the growth rate of the bacteria in a planktonic state (Fig. 5A). In contrast, when cultured on sulfur, the lag phase exhibited by the three engineered strains A. ferrooxidans/pYDT_1379, A. ferrooxidans/pYDT_0053, and A. ferrooxidans/pYDT_1373 was significantly reduced (Fig. 5B). This reduction is likely attributed to the solid powdered form of sulfur, which may mitigate the lag phenomenon associated with enhanced biofilm formation.

Fig 5 The growth curve of A. ferrooxidans.(A) The growth characteristic curve of A. ferrooxidans when using ferrous iron as the energy source. (B) The growth characteristic curve of A. ferrooxidans using sulfur as an energy source. Experiments were conducted in triplicate and the error bar represent SD.

After assessing the growth characteristics of A. ferrooxidans, we proceeded to measure the biofilm content of the strains in the presence of the inducer IPTG at various time intervals. Due to the interference caused by ferrous ions with the spectrophotometry used for detecting biofilm content, we chose to culture the strains using sulfur as the energy source. At the peak of biofilm formation at 36 hours, A. ferrooxidans/pYDT_0053 displayed the thickest biofilm, which was significantly different from the control group. Furthermore, A. ferrooxidans/pYDT_1379 and A. ferrooxidans/pYDT_1373 also contributed to biofilm formation, demonstrating significant differences (P＜0.05) that indicate a substantial enhancement in biofilm development (Fig. 6).

Fig 6. Bar chart of biofilm formation of A. ferrooxidans. Experiments were conducted in triplicate and the error bar represent SD.Two-sided Student’s t-test was used to analyze the statistical significance (*0.01< P < 0.05; **P < 0.01, NS: No significance).

3. Successfully constructing a gold-specific detection module

To recover gold from tailings, we designed a specific detection module for gold in the engineering section. We added the gfp gene following the golS gene to create the expression vector pYDT-golS-P_gol-gfp, which enables the production of a detectable green fluorescent protein during gene expression. To verify the successful expression of the gold-specific module, we introduced the target vector into the E. coli BL21 strain and added various metal salt solutions to the culture system, specifically HAuCl₄, NiCl₂, CdCl₂, CuSO₄, and ZnCl₂, with all metal ion concentrations (Au[III], Ni[II], Cd[II], Cu[II], Zn[II]) set at 20 μM. By examining the growth characteristics of the engineered bacteria and the normalized fluorescence intensity in cultures with different ions, As the Fig. 7 shows, we observed a significant increase in fluorescence intensity upon the addition of 20 μM Au[III], while no notable changes were detected for Ni[II], Cd[II], Cu[II], and Zn[II]. These results demonstrate that our constructed module can specifically detect gold and effectively drive downstream gene expression.

Fig 7 Schematic diagram illustrating the growth characteristics and expression features of E. coli BL21 in response to Au[III] specificity. (A) The growth curve of E. coli BL21. (B) The normalized fluorescence intensity of E. coli BL21 in different metal ions at the same concentration is illustrated. (C) The normalized fluorescence intensity of E. coli BL21 cultured for 12 hours in different metal ions at the same concentration. Experiments were conducted in triplicate and the error bar represent SD.

Futher, we evaluated the sensitivity of the gold detection module by establishing a gradient of Au[III] concentrations. The results indicated that the transformed E. coli BL21 strain containing the pYDT-golS-P_gol-gfp construct was capable of responding to a minimum concentration of 0.25 μM Au[III] (Fig. 8B). Furthermore, as the concentration of Au[III] increased, the fluorescence intensity of the E. coli BL21 strain to Au[III] increased in response (Fig. 8C).

Fig 8 Schematic diagram illustrating the growth characteristics and expression features of E. coli BL21 in response to the sensitivity of Au[III]. (A) Growth curve of E. coli BL21 in different concentrations of Au[III]. (B) Schematic diagram of the normalized fluorescence intensity of E. coli BL21 at different concentrations of Au[III]. (C) The normalized fluorescence intensity of E. coli BL21 cultivated for 12 hours in different concentrations of Au[III]. Experiments were conducted in triplicate and the error bar represent SD. Two-sided Student’s t-test was used to analyze the statistical significance (*0.01< P < 0.05; **P < 0.01, NS: No significance).

4. Successful incorporation of a kill switch in engineered bacteria

To prevent the leakage of GMOs into the environment, we have developed a gold ion-dependent kill switch. To verify the successful construction of this kill switch, we introduced Au[III], IPTG, and their combination during the culture of the engineered strains, and subsequently assessed their growth characteristics. When both the inducer IPTG and Au[III] were present, the bacteria displayed normal growth. However, in the absence of either IPTG or Au[III], or when both were absent, the expression of the toxic protein gene mazF was induced, resulting in bacterial death (Fig. 9). This outcome confirms the effective operation of the kill switch module.

Fig 9 The growth curves of E. coli BL21 were obtained under three different conditions: in the presence of both the inducer IPTG and Au[III], in the presence of only IPTG or Au[III], and in the absence of both IPTG and Au[III]. Experiments were conducted in triplicate and the error bar represent SD.

References:
[1] Rohwerder, T., Gehrke, T., Kinzler, K. et al. Bioleaching review part A:. Appl Microbiol Biotechnol 63, 239–248 (2003).
[2] Inaba Y, West AC, Banta S. Glutathione Synthetase Overexpression in Acidithiobacillus ferrooxidans Improves Halotolerance of Iron Oxidation. Appl Environ Microbiol. 2021 Sep 28;87(20):e0151821.