Organization of ribosomes and nucleoids in Escherichia coli cells during growth and in quiescence

Abstract

We have examined the distribution of ribosomes and nucleoids in live Escherichia coli cells under conditions of growth, division, and in quiescence. In exponentially growing cells translating ribosomes are interspersed among and around the nucleoid lobes, appearing as alternative bands under a fluorescence microscope. In contrast, inactive ribosomes either in stationary phase or after treatment with translation inhibitors such as chloramphenicol, tetracycline, and streptomycin gather predominantly at the cell poles and boundaries with concomitant compaction of the nucleoid. However, under all conditions, spatial segregation of the ribosomes and the nucleoids is well maintained. In dividing cells, ribosomes accumulate on both sides of the FtsZ ring at the mid cell. However, the distribution of the ribosomes among the new daughter cells is often unequal. Both the shape of the nucleoid and the pattern of ribosome distribution are also modified when the cells are exposed to rifampicin (transcription inhibitor), nalidixic acid (gyrase inhibitor), or A22 (MreB-cytoskeleton disruptor). Thus we conclude that the intracellular organization of the ribosomes and the nucleoids in bacteria are dynamic and critically dependent on cellular growth processes (replication, transcription, and translation) as well as on the integrity of the MreB cytoskeleton.

Keywords: Antibiotics; Cell Division; Cytoskeleton; DNA Replication; FtsZ Ring; Nucleoid; Ribosomes; Subcellular Organelles; Transcription; Translation.

FIGURE 1.

Construction and characterization of E. coli strain QC101 (L9-mCherry), QC901 (S6-TurboGFP), QC702 (EF-Tu-mCherry), and QC801 (EF-Tu-mCherry and L9-TurboGFP). A, scheme showing the strategy for fusion of red fluorescent protein mCherry with ribosomal protein L9. λ-Red recombineering was used to insert a linear DNA containing tandemly arranged genes for mCherry and Kan replacing the stop codon of rplI gene on MG1655 chromosome. The resulting recombinant QC101 produced an in-frame fusion at the 3′-end of rplI gene with the gene for mCherry (see “Experimental Procedures”). B, colony PCR screening for mCherry-Kan (1.5 kb) fusion to rplI (0.5 kb) using primers flanking rplI gene. Lane 1, DNA ladder; lane 2, PCR from colonies of MG1655; lane 3, recombinant QC101 with pSIM5; lane 4, QC101. C, Western blot analysis of cellular fractions from QC101 to trace the L9-mCherry fusion protein. The cell lysate, ribosomal pellet (r-pellet), supernatant (collected after pelleting ribosomes by ultracentrifugation), purified ribosomal particles 70 S, 50 S, and 30 S from QC101, and purified mCherry protein (31.5 kDa) were subjected to Western blot using α-mCherry antibody. Antibodies to the ribosomal proteins S1 and L12 (gift from J. P. Ballesta, CBMSO, Spain) were used to specify 30 S and 50 S subunits. The blot corresponding to L9-mCherry fusion (42.5 kDa) was visible in r-pellet and 70 S and 50 S subunits but not in the supernatant and 30 S subunits. D, fluorescence spectra of 70 S, 50 S, and 30 S ribosomal particles from QC101. Characteristic mCherry fluorescence (excitation at 555 nm and emission λ_max 610 nm) was seen in 70 S ribosomes and 50 S subunits but not in the ribosome-free supernatant. Small fluorescence seen in the 30 S subunits could be from minor contamination of 50 S particles. E, Western blot analysis of the cell lysate, ribosomal pellet, ribosome-free supernatant, and the ribosomal particles 70 S, 50 S, and 30 S from QC901 cells separated on a 14% SDS-PAGE using antibodies α-turboGFP, α-S1 (specific to 30 S), and α-L12 (specific to 50 S). Purified TurboGFP (30.5 kDa) was used as a control. Immunoblotting detected S6-turboGFP fusion (40.7 kDa) in the ribosomal pellet and 30 S subunits but neither in the supernatant nor in the 50 S subunits. F, Western blot analysis of cell lysate, ribosomal pellet, and ribosome-free supernatant from QC702 and QC801 cells with α-turboGFP, α-mCherry, and α-L12 antibodies. Purified TurboGFP (∼36 kDa) and mCherry (31.5 kDa) were loaded as positive controls and size markers. In both QC702 and QC801 EF-Tu-mCherry fusion (∼70 kDa) was detected in the lysate and in the supernatant fraction, whereas L9-TurboGFP (∼41 kDa) was present exclusively in the ribosomes of QC801.

FIGURE 2.

Distribution of ribosomes and nucleoids in exponential and stationary phase. Fluorescence images of the ribosomes alone, the nucleoids alone, and both superimposed and digitally colored are as labeled on the top of the figures. Color codes are red for mCherry tagged ribosomes and green for DAPI stained nucleoids. The bar indicates 5 μm. A, images of QC101 cells growing in the exponential phase. The first three panels show a field view with multiple cells; images of mCherry-ribosome alone (1) and (DAPI-stained) nucleoid alone (2), and both superimposed and digitally colored in red and green, respectively (3), as labeled on the top of the images. Panels 4 and 5 show examples of a typical long and a short cell, respectively, following the same color code as in panel 3 together with the intensity scans of the ribosome (red) and the DNA (green) along the longitudinal axis of the cells. a.u., arbitrary units. B, Z-scan images of the exponential phase QC101 cells showing distribution of ribosomes and nucleoids along the longitudinal sections as indicated by the symbols and the labels. C, the distribution of the ribosomes and the nucleoids in a filamentous QC101 cells treated with 10 μg/ml cephalexin, an inhibitor of cell division. D, a field view of exponentially growing QC901 cells; ribosome alone or together with the DNA as indicated in the label. E, a filed view of stationary phase QC101 cells showing ribosomes alone (1), nucleoids alone (2), and both superimposed and colored in red and green, respectively (3). Examples of three enlarged stationary phase cells together with the intensity scans of the ribosomes (red) the DNA (green) are added in the panels 4–6. The bar indicates 5 μm for all panels.

FIGURE 3.

Following QC101 cells through growth and division. A, time-lapse images of ribosome distribution in QC101 (L9-mCherry) cells (#1, #2, and #3) and corresponding ribosomal intensity scans of the cell #1 (along the long axis) for each time point (indicated by the number in the images). The fluorescence intensity is integrated over the whole cell-width and normalized to the same level for comparison. The x axis in the intensity scan diagrams indicates the distance relative to cell center (marked with 0) for 0–80-min plots and distance from the new cell pole for 100 and 110-min plots. The gray bars in the 100- and 110-min plots indicate the plane for cell division. B, the change in cell-length (left y axis, the length of a full-grown cell is normalized to 1) and the number of ribosome and nucleoid peaks (right y axis) relative to the generation time. The values are based on the time-lapse images in A, the average from 10 independent cells. The error bars indicate S.D. C, a field view showing mCherry-ribosome (red) distribution in relation to FtsZ ring (yellow) in BS433 (L9-mCherry + FtsZ-GFP) cells. D, zoomed images of three typical fully grown QC101 cells just before dividing or freshly divided showing ribosomes (red) and DNA (green) together with intensity scans of the ribosome along the longitudinal axis of the cells. The black circles highlight the zone for cell division. The gray bars in the intensity scans signify the plane for cell division. The bar in all panels corresponds to 5 μm. a.u., arbitrary units.

FIGURE 4.

Distribution of EF-Tu in E. coli. A, distribution of EF-Tu-mCherry during exponential and stationary phase of growth. The images are taken using QC702 (EF-Tu-mCherry) cells. B, the distribution of Turbo-GFP tagged ribosomes, EF-Tu-mCherry, DAPI-stained DNA alone or in superposition of all three. The images were taken using QC801 (L9-TurboGFP + EF-Tu-mCherry) cells growing in the exponential phase. The bar indicates 5 μm.

FIGURE 5.

The effect of various antibiotics on ribosome and nucleoid distribution in E. coli. Subcellular distribution of the ribosomes and DNA alone or superimposed and digitally colored in red and green, respectively, in QC101 (L9-mCherry) cells after exposure to three independent translation-inhibiting antibiotics (200 μg/ml chloramphenicol (Chl), 50 μg/ml tetracycline Tet, and 100 μg/ml streptomycin (Strep)) for 10 min (A), transcription inhibitor rifampicin (200 μg/ml) for 5, 30, and 120 min (B), gyrase inhibitor nalidixic acid (30 μg/ml) for 5, 60, and 120 min (arrows highlight the vacuole-like empty spaces (C), both nalidixic acid (30 μg/ml) and chloramphenicol (200 μg/ml) for 120 min (D), and MreB inhibitor drug A22 (10 μg/ml) for 60 min (E). The bar indicates 5 μm for all panels. F, total protein synthesis in QC101 cells were measured by incorporation of [³H]valine without (●) or with drugs chloramphenicol (200 μg/ml) (▴) and A22 (10 μg/ml) (□).