Competition of Cd(II) and Pb(II) on the bacterial cells: a new insight from bioaccumulation based on NanoSIMS imaging

Abstract

Polymetallic exposure causes complex toxicity to microorganisms. In this study, we investigated the responses of Escherichia coli under co-existence of cadmium (Cd) and lead (Pb), primarily based on biochemical analysis and RNA sequencing. Cd completely inhibited bacterial growth at a concentration of 2.41 mmol/L, with its removal rate as low as <10%. In contrast, the Pb removal rate was >95% under equimolar sole Pb stress. In addition, the Raman analysis confirmed the loss of proteins for the bacterial cells. Under the co-existence of Cd and Pb, the Cd toxicity to E. coli was alleviated. Meanwhile, the biosorption of Pb cations was more intense during the competitive sorption with Cd. Transmission electron microscopy images showed that a few cells were elongated during incubation, i.e., the average cellular length increased from 1.535 ± 0.407 to 1.845 ± 0.620 µm. Moreover, NanoSIMS imaging showed that the intracellular distribution of Cd and Pb was coupled with sulfur. Genes regulating sulfate transporter were also upregulated to promote sulfate assimilation. Then, the subsequent production of biogenic sulfide and sulfur-containing amino acids was enhanced. Although this strategy based on S enrichment could resist the polymetallic stress, not all related genes were induced to upregulate under sole Cd stress. Therefore, the S metabolism might remodel the microbial resistance to variable occurrence of heavy metals. Furthermore, the competitive sorption (in contrast to sole Cd stress) could prevent microbial cells from strong Cd toxicity.IMPORTANCEMicrobial tolerance and resistance to heavy metals have been widely studied under stress of single metals. However, the polymetallic exposure seems to prevail in the environment. Though microbial resistance can alleviate the effects of exogenous stress, the taxonomic or functional response to polymetallic exposure is still not fully understood. We determined the strong cytotoxicity of cadmium (Cd) on growth, and cell elongation would be driven by Cd stress. The addition of appropriate lead (Pb) showed a stimulating effect on microbial bioactivity. Meanwhile, the biosorption of Pb was more intense during co-existence of Pb and Cd. Our work also revealed the spatial coupling of intracellular S and Cd/Pb. In particular, the S assimilation was promoted by Pb stress. This work elucidated the microbial responses to polymetallic exposure and may provide new insights into the antagonistic function during metal stresses.

Keywords: Cd; Escherichia coli; NanoSIMS; Pb; transcriptomic profiling.

Fig 1

Physiological properties of E. coli cultures. Growth curves of E. coli cell density measured by OD₆₀₀. (A) Growth curve of E. coli in the CK treatments. (B) Growth curve of E. coli in the CdT treatments. (C) Growth curve of E. coli and Pb removal rate in the PbT treatments. (D) Growth curve of E. coli and Pb removal rate in the PbCd treatments.

Fig 2

TEM images of E. coli cells incubated for (A) 48 h, (B) 96 h, and (C) 144 h in the PbCd treatments.

Fig 3

TEM images with EDS analysis on the sections of the E. coli cells in the PbCd treatment. (A–C) The typical EDS spots on the membrane and within the cytoplasm. (D) TEM image of a typical lysed cell (EDS spots on P1 and P2).

Fig 4

Raman spectra and imaging on the same E. coli cells. (A) Raman spectra on the bacterial cells collected from CK and PbT treatments. Each spectrum was averaged from five spectra. (B) The E. coli cells from CK treatment under SEM (left) and LM (right) modes. (C) The E. coli cells from the PbT treatment under SEM (left) and LM (right) modes. (D) Peak fitting of the Raman spectra on the E. coli cells from CK treatment. (E) Peak fitting of the Raman spectra on the E. coli cells from PbT treatment.

Fig 5

NanoSIMS imaging on the E. coli cells incubated for 96 h in the PbCd treatment. Secondary ions of (A, E, and I) ¹²C¹⁴N, (B, F, and J) ³²S, (C, G, and K) ¹¹⁴Cd¹⁶O, and (D, H, and L) ²⁰⁸Pb¹⁶O were analyzed.

Fig 6

Key genes of sulfur metabolism that responds to exogenous stress in E. coli. (A) The pathway of assimilatory sulfate reduction. The metabolic pathways, related enzymes, and genes are indicated. The pathway was adapted from the KEGG pathway 00920 (sulfur metabolism). APS, adenylyl sulfate; PAPS, 3′-phosphoadenylyl sulfate. (B) The heatmap illustrates the fold change of gene expression (Sbp, CysP, CysU, CysW, CysA, CysN, CyD, CyC, CyH, CyJ, and CyI) in all samples exposed to HMs based on RNA-Seq.