Sublethal concentrations of the aminoglycoside amikacin interfere with cell division without affecting chromosome dynamics

Abstract

Aminoglycosides bind to the 16S rRNA at the tRNA acceptor site (A site) and disturb protein synthesis by inducing codon misreading. We investigated Escherichia coli cell elongation and division, as well as the dynamics of chromosome replication and segregation, in the presence of sublethal concentrations of amikacin (AMK). The fates of the chromosome ori and ter loci were monitored by visualization by using derivatives of LacI and TetR fused to fluorescent proteins in E. coli strains that carry operator arrays at the appropriate locations. The results showed that cultures containing sublethal concentrations of AMK contained abnormally elongated cells. The chromosomes in these cells were properly located, suggesting that the dynamics of replication and segregation were normal. FtsZ, an essential protein in the process of cell division, was studied by using an ectopic FtsZ-cyan fluorescent protein fusion. Consistent with a defect in cell division, we revealed that the Z ring failed to properly assemble in these elongated cells.

FIG. 1.

Analysis of E. coli IL05(pWX6) cells growing in the presence or the absence of 4 μg/ml AMK. (A) Growth curves; (B) cells visualized after 14 h in the presence of gelatin, as described in Materials and Methods. Under these conditions the nucleoids appear as light masses inside the cells. Arrows indicate inclusion body-like formations inside the cells (black arrow) or in the medium (white arrow).

FIG. 2.

Effects of sublethal concentrations of AMK on chromosome dynamics and Z-ring assembly. The cartoons show circular E. coli chromosomes indicating the target regions and the plasmids coding for fusion proteins. For simplicity, regardless of the nature of the fluorescent protein, the foci corresponding to the ori regions have been colored green. Fluorescent signals indicating the ter region or the FtsZ protein have been colored red. (A) E. coli IL05(pWX6) cells cultured in the presence of 4 μg/ml AMK examined under phase contrast only (left) or the overlay of phase contrast and CFP and YFP fluorescence signals. (B) Representative E. coli IL05(pWX6) cells of the five types found under different growth conditions (with 0, 4, 8, 16, or 32 μg/ml AMK). Although the distribution of cell types is described in Table 1, this figure also shows the values for 0 and 4 μg/ml AMK. The proposed progression of the cell cycle in the absence and the presence of AMK is shown by the black and the red arrows, respectively. Dashed arrows indicate the stages at which cell division may occur. (C) E. coli IL06(pWX6) cultured in the presence of 4 μg/ml AMK (see text). (D) E. coli IL06(pCP12) cultured in the absence or the presence of 4 μg/ml AMK.