The Effect of Heavy Metals on Conjugation Efficiency of an F-Plasmid in Escherichia coli

Abstract

Conjugation, the process by which conjugative plasmids are transferred between bacteria, is regarded as a major contributor to the spread of antibiotic resistance, in both environmental and clinical settings. Heavy metals are known to co-select for antibiotic resistance, but the impact of the presence of these metals on conjugation itself is not clear. Here, we systematically investigate the impact that five heavy metals (arsenic, cadmium, copper, manganese, and zinc) have on the transfer of an IncF conjugative plasmid in Escherichia coli. Our results show that two of the metals, cadmium and manganese, have no significant impact, while arsenic and zinc both reduce conjugation efficiency by approximately 2-fold. Copper showed the largest impact, with an almost 100-fold decrease in conjugation efficiency. This was not mediated by any change in transcription from the major Py promoter responsible for transcription of the conjugation machinery genes. Further, we show that in order to have this severe impact on the transfer of the plasmid, copper sulfate needs to be present during the mating process, and we suggest explanations for this.

Keywords: Escherichia coli; antibiotic resistance; bacterial conjugation; heavy metals; horizontal gene transfer; plasmids.

Figure 1

Effect of heavy metals on conjugation efficiency of an F-plasmid in E. coli. Donor and recipient strains were grown to log phase and then allowed to mate in the presence of heavy metals at the indicated concentrations. Conjugation efficiency is expressed as the ratio of transconjugants formed per donor. Bars represent the mean conjugation efficiency relative to the control (no heavy metal added), and the error bars are the standard error of the mean (SEM) of 2–6 biological replicates with 2 technical replicates each. p-values were computed using a one-sample unpaired t-test comparing the sample mean to a hypothetical value of 1 (the mean relative conjugation efficiency of the control). The level of significance is indicated as follows: * p < 0.05, ** p < 0.01, *** p < 0.001.

Figure 2

CuSO₄ lowers conjugation efficiency proportional to the concentration added. Cultures of the donor (HA14) and recipient strain (HA4) were grown to log phase and then mixed and allowed to mate in the presence of various concentrations of CuSO₄ for 30 min. Conjugation efficiency was calculated as in Figure 1 and as described in Materials and Methods. Symbols indicate the mean and error bars and represent the standard error of the mean (SEM) of 2 biological replicates with 2 technical replicates each.

Figure 3

CuSO₄ does not alter expression from the P_Y promoter. A P_Y-lacZ strain (PAS3) was grown to log phase, after which the culture was split and 3 mM CuSO₄ added (open symbols) or not added (closed symbols). OD600 is shown as gray squares and β-galactosidase activity as black circles. Symbols represent the mean, and the error bars are the standard error of the mean (SEM) of 2 biological replicates.

Figure 4

CuSO₄ lowers conjugation efficiency when present during mating. ‘Donor’/’Recipient’: the donor (HA14) or recipient strain (HA4) was grown to log phase while exposed to 3 mM CuSO₄, after which the culture was pelleted and resuspended in fresh LB, mixed with untreated donor or recipient, and allowed to mate in the absence of CuSO₄. ‘Mating’: the donor and recipient strains were grown to log phase in the absence of CuSO₄, and then allowed to mate in the presence of 3 mM CuSO₄. The bars represent the mean conjugation efficiency relative to the control (0 mM), and the error bars represent the standard error of the mean (SEM) of 2 biological replicates with 2 technical replicates each.