The genetic determinants of septal Z-ring assembly and cell wall synthesis processes in mycobacteria are not completely known. In other rod-shaped bacteria, actin- and tubulin-like cytoskeletal elements direct the cell wall synthesis and cell division processes, respectively (reviewed in references 35
). For example, it is reported that the MreB protein coordinates the cell wall elongation steps, whereas the tubulin-like FtsZ protein initiates the cell division process by promoting the assembly of septasomal components and helps direct septum formation (2
). Interactions among proteins involved in the two pathways are believed to be essential for coordinating growth, cell division, cell wall synthesis, and cell shape maintenance (35
). M. tuberculosis
and other mycobacterial members lack MreB-like proteins but contain FtsZ and its interacting partners, some of which are novel proteins not present in other bacteria (7
). Thus, it is an enigma how the cell wall synthesis process occurs and is coordinated with the Z-ring assembly. In the present study, we provide evidence that CwsA is an important protein involved in cell wall synthesis and cell shape maintenance in mycobacteria. The observed membrane, polar, and septal localization patterns of CwsA (), reduction in the rates of 14
C-labeled GlcNAc incorporation (), increased autolysis rates (), susceptibility of the isolated cell walls to the lysozyme (), and importantly, defects in cell shape with bulged cell morphology of the ΔcwsA
mutant are consistent with the notion that CwsA is required for cell wall synthesis and cell shape maintenance (). Earlier studies reported that CrgA and Wag31 proteins contribute to some steps of cell wall synthesis and cell shape (19
). Thus, CwsA joins the list of the components comprising the mycobacterial cell wall synthesis machinery. It is also likely that CwsA, owing to its interactions with CrgA, is important for coordination of the cell wall synthesis process with the Z-ring assembly (see below).
Our results also showed physical and possibly functional interactions of CwsA with Wag31 and CrgA proteins. One functional consequence of the CwsA interaction with CrgA is to promote Wag31 localization because the Wag31 localization pattern was compromised in the mutants with single mutations of cwsA
(), although the latter gene product does not interact with Wag31 (see Fig. S8 in the supplemental material). CrgA interacts with FtsI, a Wag31-interacting protein; and FtsI localization is shown to be severely compromised in the ΔcrgA
). CrgA also interacts with FtsZ, FtsQ, and possibly other unidentified cell division proteins (29
). Thus, the decreased localization of Wag31 in the crgA
background could be due to a compromised FtsI-CrgA interaction, which then influences FtsI-Wag31 interactions and, in turn, cell wall synthesis. Therefore, CwsA directly promotes and CrgA indirectly promotes Wag31 localization. We propose that CrgA and CwsA proteins together contribute to the FtsI- and Wag31-mediated cell wall synthesis steps and that a coordinated action of these four proteins is required for optimal cell wall synthesis (). Disruption of CrgA and CwsA interactions could compromise the stability of the PG synthesis network and thereby impact cell wall synthesis.
Fig 9 Protein-protein interactions among cell division and cell wall synthesis proteins in mycobacteria. Interactions of CrgA and CwsA with each other and with FtsI and Wag31, respectively, contribute to cell division and cell wall synthesis (). Green (more ...)
Characterization of the DKO strain in this study, however, provided clues as to how interactions between CwsA and CrgA lead to a regulated cell wall synthesis and possibly cell shape maintenance in mycobacteria. The DKO strain showed significant defects in growth, viability, PG synthesis, and cell shape morphology, as well as increased autolysis and cell wall turnover compared with either of the mutants with a single cwsA
mutation ( and ). Furthermore, the localization patterns of Wag31 and FtsI were severely compromised in the DKO background (). These results support a model involving two interdependent but closely connected routes for PG synthesis and cell shape maintenance (). The presence of two routes for PG synthesis ensures continual PG synthesis even when the functions of CwsA or CrgA proteins are compromised. For example, in the absence of CwsA function, cell wall synthesis and cell division proceed, albeit defectively, via a route involving CrgA, FtsI, and Wag31, whereas in the absence of CrgA, this route could involve the actions of the CwsA, Wag31, and FtsI proteins. We propose that interactions between CwsA and CrgA are critical for tethering the complex together and promoting optimal cell wall synthesis. Finally, it is known that FtsZ and FtsI proteins interact with each other via FtsW (9
). Thus, it is also possible that cell wall synthesis in the cwsA crgA
DKO strain proceeds in a compromised manner involving the actions of FtsZ, FtsW, FtsI, and Wag31 ().
Close inspection of autolysis and cell wall turnover data revealed that the CrgA and CwsA proteins differentially modulate these processes. For example, while both proteins are required for cell wall synthesis, the absence of crgA only modestly reduced turnover but did not affect autolysis, whereas the absence of cwsA increased autolysis but did not affect turnover. On the other hand, the absence of both proteins increased autolysis and turnover. These results suggest that the underlying mechanisms regulating autolysis and turnover involving CwsA and CrgA and, possibly, their interactions are rather complex and require more detailed investigations. It should be noted that the CrgA localization was modestly enhanced in the cwsA background (see Fig. S5 in the supplemental material). The possibility that elevated levels of CrgA somehow modulate autolysis rates in the cwsA background remains open.
One interesting aspect of our data is that the FtsZ-GFP localization was severely compromised in the cwsA crgA
DKO strain (). It was reported earlier that although CrgA interacts with FtsZ, the absence of CrgA does not affect FtsZ localization (29
). It seems likely that CrgA and CwsA function as a complex and help coordinate FtsZ-ring assembly at the septa with downstream cell wall synthesis events. Defective FtsZ septal assembly either due to compromised cell wall synthesis or due to defective growth of DKO might then affect the loading of other divisome components at the septa and therefore productive cell division. This is an not unreasonable assumption, as perturbations to cell wall integrity due to overproduction or loss of the cell wall hydrolase ChiZ have been shown to affect FtsZ-ring stability in M. smegmatis
and M. tuberculosis
). Further studies are required to address how cell wall synthesis coordination with Z-ring assembly takes place in mycobacteria. In summary, the results presented in this study indicate that CwsA and CrgA together with Wag31 and FtsI are members of a multiprotein complex engaged in PG synthesis in mycobacteria. Large multiprotein complexes made up of proteins spanning different cellular compartments, viz.
, the cytoplasm, inner membrane, and periplasm, are proposed to be involved in various stages of PG synthesis in E. coli
, Bacillus subtilis
, and other bacteria (reviewed in reference 38
). Further studies on the roles of CrgA and CwsA will likely aid our understanding of cell division and cell wall synthesis processes in mycobacteria.