Fast Mechanically Driven Daughter Cell Separation Is Widespread in Actinobacteria

ABSTRACT Dividing cells of the coccoid Gram-positive bacterium Staphylococcus aureus undergo extremely rapid (millisecond) daughter cell separation (DCS) driven by mechanical crack propagation, a strategy that is very distinct from the gradual, enzymatically driven cell wall remodeling process that has been well described in several rod-shaped model bacteria. To determine if other bacteria, especially those in the same phylum (Firmicutes) or with similar coccoid shapes as S. aureus, might use a similar mechanically driven strategy for DCS, we used high-resolution video microscopy to examine cytokinesis in a phylogenetically wide range of species with various cell shapes and sizes. We found that fast mechanically driven DCS is rather rare in the Firmicutes (low G+C Gram positives), observed only in Staphylococcus and its closest coccoid relatives in the Macrococcus genus, and we did not observe this division strategy among the Gram-negative Proteobacteria. In contrast, several members of the high-G+C Gram-positive phylum Actinobacteria (Micrococcus luteus, Brachybacterium faecium, Corynebacterium glutamicum, and Mycobacterium smegmatis) with diverse shapes ranging from coccoid to rod all undergo fast mechanical DCS during cell division. Most intriguingly, similar fast mechanical DCS was also observed during the sporulation of the actinobacterium Streptomyces venezuelae.

(DCS), is typically a slow process requiring several minutes. In many well-characterized bacteria, including Escherichia coli and Caulobacter crescentus, DCS is achieved by gradual symmetric constriction coupled with construction of new hemispherical poles at the junction between the presumptive daughters (1,2), while other bacteria such as Bacillus subtilis initially build a flat septum that then undergoes gradual resolution around the periphery to allow symmetric DCS (3). In contrast, the Grampositive coccus Staphylococcus aureus undergoes rapid (millisecond time scale) DCS (4,5), and the resulting daughters remain connected asymmetrically by a hinge, hallmarks of separation driven by mechanical rupture rather than by gradual enzymatic remodeling of the peripheral cell wall (4).
In order to determine whether this mechanism of fast mechanical DCS is unique to S. aureus or also found among other bacterial species, we surveyed representative species across three major bacterial phyla, including the Firmicutes (low GϩC Gram positives), Actinobacteria (high GϩC Gram positives), and Proteobacteria (Gram negatives), with particular attention to include diverse species that share the coccoid (near-spherical) shape of S. aureus (6) ( Fig. 1; see Table S1 in the supplemental material). For all species, we directly examined their cytokinesis and DCS processes using time-lapse microscopy, observing both changes in overall cell shape with phase-contrast imaging and reorganization of cell membrane using the intercalating dye FM 4-64 (Fig. 1). Where initial time-lapse characterization using 5-min imaging intervals indicated the possibility of fast mechanical DCS, we further examined cell division using high-speed phase-contrast imaging at 10-ms intervals (see Fig. S1 and Movie S1 in the supplemental material) and scanning electron microscopy (SEM) to characterize the shapes and surface characteristics of cells immediately before and after DCS (Fig. 2).
We first set out to determine whether close relatives of S. aureus in the Staphylococcaceae family employ fast mechanical DCS. Indeed, Macrococcus caseolyticus, which has a similar cell shape but slightly larger size (7), divided like S. aureus, such that the round cell gradually formed a septum generating two "hemispherical" OBSERVATION crossmark daughters which then separated rapidly (within 10 ms) accompanied by a drastic shape conversion (  Table S1 in the supplemental material). Surprisingly, two other coccoid members of the Staphylococcaceae, Salinicoccus roseus (a halophile that grows optimally with 10% salt [8]  (a member of a genus originally isolated from the Korean fish sauce jeotgal [9]), showed no evidence of fast mechanical DCS and instead separated by gradual and symmetrical resolution of the septum ( Fig. 1; see Fig. S2A and B in the supplemental material). Notably, S. roseus formed regular, symmetrical cuboidal clusters ( Fig. 1; see Fig. S2A) rather than the irregular "grape-like" clusters characteristic of S. aureus, consistent with the idea that irregular clusters are likely to be a consequence of the randomly positioned asymmetric hinge attachment generated by fast mechanically driven DCS (4) while cuboidal clusters of coccoid bacteria may reflect slow and symmetric DCS events.
To compare the behavior of these coccoid Staphylococcaceae to related species in the same order, Bacillales, we next examined Sporosarcina ureae, a large coccoid soil bacterium, and Listeria monocytogenes, a rod-shaped pathogen. Both species separated by gradual resolution of the septum (Fig. 1). Additionally, S. ureae formed cuboidal clusters similar to Salinicoccus roseus ( Fig. 1; see Fig. S2C), consistent with symmetric DCS. In addition to the Bacillales, we inspected Streptococcus mutans and Lactococcus lactis, two members of a related order, Lactobacillales, both of which have ovoid shapes that divide in a single plane to form chains. Cytokinesis in S. mutans and L. lactis appeared very similar, where a septum was formed and resolved gradually to form the new poles ( Fig. 1), similar to B. subtilis. Thus, the closely related genera Staphylococcus and Macrococcus are the only examples we found of fast mechanically driven DCS among the Firmicutes, and this particular behavior was not even observed among all Staphylococcaceae.
To explore beyond Firmicutes, we next examined two coccoid Gram-negative species among the Proteobacteria with different cell sizes: the betaproteobacterium Neisseria sicca and the gammaproteobacterium Moraxella catarrhalis. Both N. sicca and M. catarrhalis constricted gradually at the division site to form the new poles while separating the daughters (Fig. 1). This is consistent with the cytokinesis process well documented in rod-shaped Gram-negative bacteria, where DCS coincides with septation to coordinate outer membrane synthesis (1).
Next we turned to the other major Gram-positive phylum besides the Firmicutes, the high-GϩC Actinobacteria. Again we began with a well-characterized coccoid species, Micrococcus luteus, the type strain of the genus Micrococcus within the Actinomycetales (10) known for the discovery of lysozyme (11). Similar to S. aureus, daughter cells of M. luteus separated rapidly (slower than S. aureus, but still within a few tens of milliseconds) (see Fig. S1C in the supplemental material), leaving behind clearly hinged sister pairs (Fig. 2D) and irregular clusters as a result. Similar fast DCS was also observed in Brachybacterium faecium, another member in the Micrococcineae suborder with a slightly elongated cell shape (12) (Fig. 2E; see Fig. S1D).
One well-known suborder in Actinobacteria is the mycolateproducing Corynebacterineae, which contains the genera Corynebacterium and Mycobacterium, both polar-growing rods that have been reported to undergo drastic "V-snapping" at the final step of cell division (13)(14)(15). Indeed, we observed that C. glutamicum and M. smegmatis snapped rapidly following septation, with a characteristic DCS time of~10 ms ( Fig. S1E and F), very similar to the mechanically driven DCS described above for the various coccoid species. Because these organisms are rod shaped, the newly separated daughters connected by a hinge point had an overall V shape as previously described (13)(14)(15) (Fig. 2F and G).
For M. smegmatis, besides the characteristic V-snapping, we observed another more subtle form of separation where the two daughters remained aligned and symmetric postseparation (labeled "straight" in Fig. S3A to D in the supplemental material), resembling the straight cell form previously reported for Mycobacterium cultures (14). However, unlike the gradual symmetric DCS observed in the Firmicutes such as Listeria, the straight mode of DCS in M. smegmatis occurred rapidly with a time scale comparable to that of the V-snapping (within 20 ms; see Fig. S3B), suggesting a similar mechanical mechanism. Given the thin rod shape of M. smegmatis (lowest pole size/cell length ratio among all of the species undergoing fast DCS), we wondered whether the fast straight DCS could rise from a scenario in which the torque generated during the asymmetric fracture of the peripheral ring is not strong enough to overcome the resistance for the daughters to rotate around the hinge. Indeed, factors that increase the rotation resistance, such as physical confinements (see Fig. S3E) and adhesions between daughters at the septum presumably due to the mycomembrane (see Fig. S3F), did raise the likelihood of straight DCS.
Finally, we looked at Streptomyces, the largest genus in Actinobacteria with a complex life cycle, including a vegetative growth stage that yields multigenomic hyphae (substrate mycelia) and a later sporulation stage in which the aerial hyphae septate into spores, typically in response to unfavorable conditions (16). We imaged the sporulation of Streptomyces venezuelae hyphae (17) by exposing them to the spent media of a sporulated culture either in microfluidic chambers (see Movie S2 and Fig. S4 in the supplemental material) or on agarose pads (Fig. 1) and observed that separation of the spores is fast and hinged, similar to the "Vsnapping" observed in other Actinobacteria. Interestingly, we often observed a "chain reaction" process where several parts on the same hypha would snap simultaneously or in rapid succession, possibly due to the buildup of tension in the hypha as a result of adjacent cells snapping (see Movie S2 and Fig. S4). Asymmetric hinge point connections between neighboring spores in a single hyphal chain were readily observable by SEM (Fig. 2H). Thus, so far, all five species representing five distinct families in Actinobacteria that we examined undergo fast DCS.
Taken together, our results indicate that cell shape (coccoid, rod, or hyphal) is not a determining factor for whether a particular bacterial species can undergo fast mechanical DCS, while a thick layer of peptidoglycan (Gram positive) together with the formation of a flat septum may be prerequisites. The species we identified here as sharing this feature represent a substantial phylogenetic diversity, yet the mechanisms they use are likely very similar to that of S. aureus, with the key factor being the septum structure, where the two daughter cells are predominantly only connected by the peripheral ring postseptation (see Fig. S5 in the supplemental material). Transmission electron microscopy (TEM) images of several Actinobacteria species confirmed this septum geometry (15,(18)(19)(20)(21)(22). It is intriguing that fast mechanically driven DCS is narrowly distributed in Firmicutes, observed in only Staphylococcus and Macrococcus, while widely adopted in the distantly related Actinobacteria. Overall, our findings revealed that the mechanical rupture of the peripheral cell wall is a common strategy implemented by diverse Gram-positive bacteria to accomplish DCS.
Methods. (i) Bacterial strains and growth conditions. The strains and corresponding growth conditions are summarized in Table S1 in the supplemental material. For all experiments, over- Zhou et al. night cultures were diluted 1:100 into fresh medium and grown until the mid-exponential phase. Live cell imaging was performed on 1% agarose pads prepared with fresh media or in CellASIC B04A plates (EMD Millipore, Inc.). One microgram/ml FM 4-64 (Life Technologies) was added to cultures or agarose pads when needed to stain the cell membrane for time-lapse microscopy.
(ii) Light microscopy. Two-dimensional (2D) time-lapse imaging was performed on a Nikon Eclipse Ti inverted fluorescence microscope with a 100ϫ (NA 1.40) oil-immersion objective (Nikon Instruments) and MicroManager v1.4. Cells grown on agarose pads were maintained at the targeted temperature during imaging with an active-control environmental chamber (Haison Technology). An iXon3 888 electron-multiplying charge-coupled device (EMCCD) camera (Andor) was used for fluorescent timelapse microscopy experiments, and a Zyla 5.5 sCMOS camera (Andor) was used for millisecond phase-contrast imaging of cell separation.
(iii) Scanning electron microscopy. Bacterial cells (mid-log phase) were pelleted and resuspended in cold phosphate-buffered saline (PBS) and were fixed with 2% glutaraldehyde and 4% paraformaldehyde in 0.1 M sodium cacodylate buffer (pH 7.3) at 4°C overnight. Fixed cells were settled onto poly-L-lysine (Sigma-Aldrich)-treated coverslips for 2 min on ice and washed with 0.1 M sodium cacodylate buffer three times, postfixed with 1% OsO 4 at 4°C for 1 h, dehydrated in a series of increasing concentrations of ethanol (50, 70, 95, and 100%), and inserted into an Autosamdri-815 series A critical point dryer (Tousimis) to remove residual ethanol with carbon dioxide. The dehydrated samples were then sputter coated with gold-palladium to an~60 Å thickness and visualized with a Sigma series field emission scanning electron microscope (Zeiss).