Single-molecule imaging reveals that Z-ring condensation is essential for cell division in Bacillus subtilis

Abstract

How proteins in the bacterial cell division complex (the divisome) coordinate to divide bacteria remains unknown. To explore how these proteins collectively function, we conducted a complete dynamic characterization of the proteins involved, and then examined the function of FtsZ binding proteins (ZBPs) and their role in cytokinesis. We find that the divisome consists of two dynamically distinct subcomplexes: stationary ZBPs that transiently bind to treadmilling FtsZ filaments, and a directionally-moving complex that includes cell wall synthases. FtsZ filaments treadmill at steady state and the ZBPs have no effect on filament dynamics. Rather, ZBPs bundle FtsZ filaments, condensing them into Z rings. Z ring condensation increases the recruitment of cell wall synthesis enzymes to the division site, and this condensation is necessary for cytokinesis. Main Text:The mechanism by which bacteria divide remains poorly understood. In most bacteria, division begins when filaments of FtsZ, a tubulin homolog, form a "Z ring" at midcell (1). The Z ring then recruits other cell division proteins, collectively called the divisome (Fig 1A). The first group of these proteins (early proteins) arrives concurrently with FtsZ and includes the actin homolog FtsA and several other FtsZ binding proteins (ZBPs): EzrA, SepF, and ZapA. The second group of integral membrane proteins (late proteins) is then recruited, including DivIB, DivIC, and FtsL, and the cell wall synthesis enzymes Pbp2B and FtsW (2, 3). During cytokinesis, the Z ring constricts while the associated cell wall synthesis enzymes build a septum that divides the cell in half (4). Recent work has shown that FtsZ filaments treadmill around the division plane, moving at the same rate as the transpeptidase Pbp2B. (5, 6) (Movie S1). FtsZ treadmilling dynamics are critical for cell division: In Bacillus subtilis, the rate of treadmilling limits Pbp2B motion, the rate of septal cell wall synthesis, and the overall rate of septation (5).To understand how these proteins work to divide cells, we sought to build a dynamic characterization of how this multi-component machine functions in B. subtilis. We first worked to identify groups of divisome proteins that move together, then investigated how the FtsZ-.

A Novel Z-Ring Associated Protein ZapA-Like Protein (PA5407) From Pseudomonas aeruginosa Promotes FtsZ to Form Double Filaments

“…It is different from the tight bundles caused by divalent ions, which will greatly reduce the activity of FtsZ GTPase and slow down their dynamics ( Yu and Margolin, 1997 ; Mukherjee and Lutkenhaus, 1999 ; Chen and Erickson, 2009 ). Recent research discovered that ZapA does not affect the FtsZ treadmilling rate in vivo and in vitro and suggested the dynamics of FtsZ polymers in vivo may be intrinsic to the polymer itself ( Caldas et al, 2019 ; Walker et al, 2020 ; Squyres et al, 2021 ). The double filament formed by FtsZ-ZapAL also has a loose structure; the gap between two FtsZ filaments is about 3 nm.…”

“…In B. subtilis , previous studies showed that FtsZ binding proteins contain ZapA, SepF, and EzrA, and knockout of zapA or sepF gene alone did not alter cell morphology, but when ezrA is knocked out together, the cell displays severe division impairment ( Gueiros-Filho and Losick, 2002 ). Squyres et al (2021) suggested that FtsZ binding proteins bundle FtsZ filaments into a condensed Z-ring, which is important to recruit downstream proteins, including cell wall synthesis enzymes to the division site. Without FtsZ-binding proteins, ZapA and EzrA, Z-ring condensation disappears.…”

“…Without FtsZ-binding proteins, ZapA and EzrA, Z-ring condensation disappears. Furthermore, FtsZ suppressor mutant K86E that enhances its lateral interactions partially restores Z-ring condensation ( Squyres et al, 2021 ). A variety of specific FtsZ bundling proteins were recently reported in different bacterial species ( Bhattacharya et al, 2017 ; Eswara et al, 2018 ; Ramos-Leon et al, 2021 ; Tan et al, 2021 ), these also indicate the importance of FtsZ stabilizers for bacterial division.…”

“…Different from FtsZ tight bundling induced by divalent cations, which significantly inhibit FtsZ GTPase activity and dynamic ( Yu and Margolin, 1997 ; Mukherjee and Lutkenhaus, 1999 ; Chen and Erickson, 2009 ), the loose bundles or sheets promoted by ZapA has only mild reduction of FtsZ GTPase activity ( Gueiros-Filho and Losick, 2002 ; Mohammadi et al, 2009 ; Rahman et al, 2020 ). Further studies by super-resolution fluorescence microscopy revealed that ZapA did not alter FtsZ treadmilling rates in vivo ( Walker et al, 2020 ; Squyres et al, 2021 ) and in vitro ( Caldas et al, 2019 ).…”

“…FtsZ bundling may be an important process for bacterial division. Recent research suggested that the FtsZ stabilizers increase the Z-ring condensation, beneficial for recruiting downstream proteins to the division site ( Squyres et al, 2021 ). Several different FtsZ stabilizers were also identified in different bacterial species, including GpsB in Staphylococcus aureus ( Eswara et al, 2018 ), SepH in actinobacteria ( Ramos-Leon et al, 2021 ), WhmD in Mycobacterium smegmatis ( Bhattacharya et al, 2017 ), and MsmK in Streptococcus suis ( Tan et al, 2021 ).…”