Fig 1.
Growth of S. aureus clpX mutants is stimulated by β-lactams.
(A) SA564 wild-type and SA564clpX were plated at 37°C and tested for susceptibility to oxacillin in a disc diffusion assay; arrow points to zone of improved growth. Disks contain 1 μg oxacillin. (B) The S. aureus wild-type strains, SA564 (MSSA) and JE2 (MRSA) and the corresponding clpX deletion mutants were grown exponentially in TSB at 37°C. At OD600 = 0.5, the cultures were diluted 101, 102, 103 and 104-fold, and 10 μl of each dilution was spotted on TSA plates +/- oxacillin and the plates were subsequently incubated at 30°C for 24 h. Note that fast growing suppressor colonies appear with high frequency when the SA564 clpX cells are plated at 30°C as described in [20]. (C) Final yield (OD600 or cfu ml-1) reached by SA564 wild type and SA564clpX cells when grown in microtiter plates for 24 h at 30°C in the absence or presence of increasing concentrations of oxacillin as indicated. (D) Growth rates (h-1) for S. aureus SA564 wild-type and SA564clpX when grown at 30°C in the absence or presence of increasing concentrations of oxacillin were determined in a Bioscreen C instrument. The average growth rate (h-1) and standard deviations from three biological replicates were plotted. Numbers above bars indicate average doubling time in minutes. Asterisks indicate significantly improved growth rate (P < 0.05). The P values were obtained by comparing the growth rates at each concentration to the growth rate without antibiotics and were calculated using Student’s t-test.
Fig 2.
Single cell analysis reveals that oxacillin prevents premature growth arrest and spontaneous lysis of S. aureus clpX mutants.
Still images from time-lapse microscopy (phase contrast) of SA564 wild-type and clpX cells growing on a semisolid surface at 30°C, without (A) or supplemented with 0.01 μg ml-1 oxacillin (B). In the upper and middle panel (T = -90) cells were exposed to 0.01 μg ml-1 oxacillin 90 min prior to imaging; in the lower panel (T = 0), clpX cells were grown in the absence of oxacillin prior to imaging. The still images are taken from movies (see S1 Movie) showing the typical growth of one micro-colony among at least 20 imaged micro colonies, except that the clpX cells depicted in (A) belong to the minority of clpX cells that were capable of initiating growth and forming a micro colony. N, corresponds to the number of living cells at the endpoint (T = 360). Scale bar, 5 μm.
Fig 3.
S. aureus clpX cells grown at 30°C display aberrant septum ingrowth and initiate daughter cell separation prior to septum closure.
TEM (left panels) and SEM (right panel) images of SA564 wild-type (A) or clpX cells (B-F) grown in TSB to mid-exponential phase at 30°C. Images show characteristic morphologies of SA564 wild-type or clpX cells at 30°C as determined from at least three biological replicates. clpX cells displaying the normal coccoid morphology (B); the typical appearance of lysed clpX cells (C); clpX cells with non-divided cytoplasma displaying premature splitting of daughter cells (D); clpX cells displaying asymmetrical septum ingrowth, (E); clpX cells displaying mesosome-like structrues at the septal site (F). White arrows point to the intact peripheral cell wall at the site of septum that is typical for wild-type cells with unclosed septa, while black arrows point to signs of daughter cell splitting initiating from the peripheral wall in cells with closed division septa. Asterisk mark clpX cells that despite displaying an incomplete septum show signs of daughter cell splitting initiating from the peripheral wall. The displayed morphological changes are typical for clpX cells also in other S. aureus strain backgrounds tested (8325–4, and JE2). Scale bar, 0.2 μm.
Fig 4.
Oxacillin restores progression of the cell cycle in S. aureus clpX cells grown at 30°C.
SA564 wild-type and clpX cells were grown at 37°C or 30°C as indicated in the absence or presence of 0.05 μg ml-1 oxacillin (A and B); cells were then stained with the membrane dye Nile Red (red) and the cell wall dye Van-FL (green) before imaging by SR-SIM. To examine if ClpX alters progression of the growth cycle, 300 cells (from each of two biological replicates) were scored according to the stage of septum ingrowth: no septum (phase 1), incomplete septum (phase 2), or non-separated cells with complete septum (phase 3), according to the examples images shown in the (A) top panel. To estimate the fraction of dead cells, the DNA dye Hoechst 3334 was used to identify anucleated and lysed cells–see two examples depicted to the right in the top panel. Scale bar, 0.5 μm. (B) Phase 2 cells (200 phase 2 cells for each biological replicate) were additionally scored according to the state of septum ingrowth (cells with less than 15% septum ingrowth were scored as “early” (E), while cells with more than 15% septum ingrowth were scored as “late” (L)), and whether the ingrowth was asymmetrical, as shown in the example images in the top panel. The proportion of cells presenting premature split was estimated based on the Van-FL staining. Scale bar, 0.5 μm.
Fig 5.
Aberrant progression of septal PG synthesis in S. aureus clpX cells is rescued by oxacillin.
S. aureus wild-type (SA564) and clpX cells were grown at 30°C in the absence (A and C) or presence of 0.05 μg ml-1 oxacillin (B and C), and PG synthesis was followed by sequentially labeling with NADA for 10 min, followed by washing and labeling with HADA for additional 10 min before SR-SIM imaging. In order to improve contrast, the NADA signal is displayed in magenta, while the HADA signal is displayed in cyan. In the absence of oxacillin (A) septal PG synthesis progressed predictably in wild-type cells and in some clpX cells (= non-overlapping septal NADA and HADA signals, marked with green arrows). In contrast, some clpX cells that initiated septum formation during the first period of labeling showed co-localization of NADA and HADA signals in an early septum ingrowth + HADA signal in the peripheral wall (examples are marked with white arrows), and in some of these cells splitting of the premature septum was observed (examples marked with red arrows). Enlarged examples are depicted in the lower panel. (B) In the presence of sub-lethal concentrations of oxacillin, some wild-type cells displayed overlapping NADA and HADA septal signals (examples displayed in middle panel), a phenotype that was not observed for clpX cells grown in the presence of oxacillin. (C) To examine progression of septal PG synthesis in clpX cells displaying premature septal split, 50 cells from each of three biological replicates (grown +/- oxacillin) that initiated septum formation during incubation with NADA, and displayed premature splitting were randomly selected. PG synthesis was followed by assessing HADA incorporation. (i-iii) show examples and distribution of the three phenotypes observed. (i) show the number of cells where septum synthesis was continued; (ii) shows the number of cells that did not continue septum synthesis and instead displayed a HADA signal in the peripheral wall; (iii) shows the number of cells where no HADA signal was detected. Numbers are given as the mean and SD of the three biological replicates. Scale bars, 0.5 μm. *** P < 0.001; statistical analysis was performed using the chi square test for independence.
Fig 6.
FtsZ localization and Z-ring dynamics appear similar in S. aureus wild-type and clpX mutant cells.
FtsZ localization and dynamics were analyzed in S. aureus wild-type and clpX mutant expressing an eYFP-tagged derivative of FtsZ from an IPTG-inducible promoter. (A) Still images from time-lapse fluorescence microscopy showing FtsZ-eYFP dynamics in S. aureus wild type and clpX cells growing on a semi-solid matrix in the presence of 100 uM IPTG at 30°C; (S2 Movie). Images are shown as overlay of phase contrast and the YFP signal, scale bar 1 μm. (B) The FtsZ-eYFP signal localizes ahead of the site of active PG synthesis: localization of FtsZ relative to PG synthesis was analyzed by sequentially labeling S. aureus wild-type and clpX cells growing in TSB + 50 uM IPTG at 30°C with TADA (red, but displayed in magenta) for 10 minutes followed by washing and labeling with HADA (blue, but displayed in cyan) for 10 min prior to SR-SIM imaging. (i-iv) Examples of cells that started septum synthesis in the first period of labeling: wild-type cell (i); clpX cell with normal progression of PG synthesis (ii); clpX cell with stalled septum synthesis (HADA-signal localizes at the peripheral cell wall) (iii); clpX cell displaying premature split (iv). Overview images can be found in S6 Fig. Scale bars 0.5 μm.
Fig 7.
β-lactam antibiotics targeting PBP1or BPB3, and WTA (TarO) inhibitors specifically promote growth of the clpX mutant.
(A) The S. aureus wild-type strains, SA564 (MSSA) and USA300 JE2 (MRSA) and the clpX deletion strains derived here from were grown exponentially in TSB at 37°C. At OD600 = 0.5, cultures were diluted 101, 102, 103 and 104-fold, and 10 μl of each dilution was spotted on TSA plates in the presence or absence of subinhibitory concentrations (1/5 MIC of wild-type) of β-lactams with different PBP specificities as indicated; the TarO inhibitors tunicamycin (0.5 ug/ml), and tarocin A1 (0.5 ug/ml), or the TarG inhibitor, targocil (0.2 ug/ml), and incubated at 30°C for 24 h. (B) S. aureus SA564 wild type and the clpX deletion strains were grown overnight at 37°C, diluted 1:200 and grown at 37°C until mid-exponential phase. These cultures were then diluted into TSB alone (control) or containing increasing concentrations of tunicamycin, tarocin A or targocil in a 96-well format, and the plates were incubated for 24 h at 30°C. The average final OD and standard deviations from three biological replicates were plotted. (C) The S. aureus SA564 wild-type strain, and the indicated mutant strains were grown exponentially in TSB at 37°C. At OD600 = 0.5, cultures were diluted 101, 102, 103 and 104-fold, and 10 μl of each dilution was spotted on TSA plates and incubated at 30°C for 24 h.
Fig 8.
Model of temperature dependent lysis of S. aureus clpX mutant.
At 37°C (upper panel), progression from an early (A) to a late septal ingrowth (B) occurs in the absence of ClpX activity. Upon septum closure, release of cross-linking substrates from the TP domain of PBPs in combination with teichoic acid biosynthesis serve as signals to activate autolytic splitting of daughter cells (C). At 30°C (lower panel), progression from an early to a late septal ingrowth becomes more dependent on assistance from the ClpX chaperone, and in the absence of ClpX, septum synthesis occasionally stalls in an early septal ingrowth that put the cells at risk for activation of Sle1- mediated autolytic splitting from the peripheral wall (D). Cells with premature splitting will be prone to lysis due to the risk of turgor pressure forces breaking the thin and mechanically weak cell wall at the tip of the ingrowing septum (E).