The FtsQBL is an essential molecular complex sitting midway through bacterial divisome assembly. To visualize and understand its structure, and the consequences of its membrane anchorage, we produced a model of the E. coli complex using the deep-learning prediction utility, AlphaFold 2. The heterotrimeric model was inserted into a 3-lipid model membrane and subjected to a 500-ns atomistic molecular dynamics simulation. The model is superb in quality and captures most experimentally derived structural features, at both the secondary structure and the side-chain levels. The model consists of a uniquely interlocking module contributed by the C-terminal regions of all three proteins. The functionally important constriction control domain residues of FtsB and FtsL are located at a fixed vertical position of ∼43-49 Å from the membrane surface. While the periplasmic domains of all three proteins are well-defined and rigid, the single transmembrane helices of each are flexible and their collective twisting and bending contribute to most structural variations, according to principal component analysis. Considering FtsQ only, the protein is more flexible in its free state relative to its complexed state-with the biggest structural changes located at the elbow between the transmembrane helix and the α-domain. The disordered N-terminal domains of FtsQ and FtsL associate with the cytoplasmic surface of the inner membrane instead of freely venturing into the solvent. Contact network analysis highlighted the formation of the interlocking trimeric module in FtsQBL as playing a central role in mediating the overall structure of the complex.

The filamentous, multicellular cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a prokaryotic model for the study of cell differentiation and cell-cell interactions. Upon combined-nitrogen deprivation, Anabaena forms a particular cell type, heterocyst, for aerobic nitrogen fixation. Heterocysts are semiregularly spaced among vegetative cells. Heterocyst differentiation is coupled to cell division, but the underlying mechanism remains unclear. This mechanism could be mediated by the putative protease HetF, which is a divisome component and is necessary for heterocyst differentiation. In this study, by suppressor screening, we identified PatU3, as a negative regulator acting downstream of HetF for cell division and heterocyst development. The inactivation of patU3 restored the capacity of cell division and heterocyst differentiation in the ΔhetF mutant, and overexpression of patU3 inhibited both processes in the wild-type background. We demonstrated that PatU3 was a specific substrate of the protease activity of HetF. Consequently, PatU3 accumulated in the hetF-deficient mutant, which was responsible for the resultant mutant phenotype. The cleavage site of PatU3 by HetF was mapped after the Arg117 residue, whose mutation made PatU3 resistant to HetF processing, and mimicked the effect of hetF deletion. Our results provided evidence that HetF regulated cell division and heterocyst differentiation by controlling the inhibitory effects of PatU3. This proteolytic pathway constituted a mechanism for the coordination between cell division and differentiation in a prokaryotic model used for studies on developmental biology and multicellularity.

FtsQBL is a transmembrane protein complex in the divisome of Escherichia coli that plays a critical role in regulating cell division. Although extensive efforts have been made to investigate the interactions between the three involved proteins, FtsQ, FtsB, and FtsL, the detailed interaction mechanism is still poorly understood. In this study, we used hydrogen-deuterium exchange mass spectrometry to investigate these full-length proteins and their complexes. We also dissected the structural dynamic changes and the related binding interfaces within the complexes. Our data revealed that FtsB and FtsL interact at both the periplasmic and transmembrane regions to form a stable complex. Furthermore, the periplasmic region of FtsB underwent significant conformational changes. With the help of computational modeling, our results suggest that FtsBL complexation may bring the respective constriction control domains (CCDs) in close proximity. We show that when FtsBL adopts a coiled-coil structure, the CCDs are fixed at a vertical position relative to the membrane surface; thus, this conformational change may be essential for FtsBL\'s interaction with other divisome proteins. In the FtsQBL complex, intriguingly, we show only FtsB interacts with FtsQ at its C-terminal region, which stiffens a large area of the β-domain of FtsQ. Consistent with this, we found the connection between the α- and β-domains in FtsQ is also strengthened in the complex. Overall, the present study provides important experimental evidence detailing the local interactions between the full-length FtsB, FtsL, and FtsQ protein, as well as valuable insights into the roles of FtsQBL complexation in regulating divisome activity.

Bacterial cell shape is determined by the peptidoglycan (PG) layer. The cyanobacterium Anabaena sp. PCC 7120 (Anabaena) is a filamentous strain with ovoid-shaped cells connected together with incomplete cell constriction. When deprived of combined nitrogen in the growth medium, about 5-10% of the cells differentiate into heterocysts, cells devoted to nitrogen fixation. It has been shown that PG synthesis is modulated during heterocyst development and some penicillin-binding proteins (PBPs) participating in PG synthesis are required for heterocyst morphogenesis or functioning. Anabaena has multiple PBPs with functional redundancy. In this study, in order to examine the function of PG synthesis and its relationship with heterocyst development, we created a conditional mutant of mraY, a gene necessary for the synthesis of the PG precursor, lipid I. We show that mraY is required for cell and filament integrity. Furthermore, when mraY expression was being limited, persistent septal PG synthetic activity was observed, resulting in increase in cell width. Under non-permissive conditions, filaments and cells were rapidly lysed, and no sign of heterocyst development within the time window allowed was detected after nitrogen starvation. When mraY expression was being limited, a high percentage of heterocyst doublets were found. These doublets are formed likely as a consequence of delayed cell division and persistent septal PG synthesis. MraY interacts with components of both the elongasome and the divisome, in particular those directly involved in PG synthesis, including HetF, which is required for both cell division and heterocyst formation.

Bacterial cell division is a highly controlled process regulated accurately by a diverse array of proteins spatially and temporally working together. Among these proteins, FtsZ is recognized as a cytoskeleton protein because it can assemble into a ring-like structure called Z-ring at midcell. Z-ring recruits downstream proteins, thus forming a multiprotein complex termed the divisome. When the Z-ring scaffold is established and the divisome matures, peptidoglycan (PG) biosynthesis and chromosome segregation are triggered. In this review, we focus on multiple interactions between FtsZ and its accessory proteins in bacterial cell cytokinesis, including FtsZ localization, Z-ring formation and stabilization, PG biosynthesis, and chromosome segregation. Understanding the interactions among these proteins may help discover superior targets on treating bacterial infectious diseases.