FtsZ-Ring Regulation and Cell Division Are Mediated by Essential EzrA and Accessory Proteins ZapA and ZapJ in Streptococcus pneumoniae.

Amilcar J Perez,Madeline L Danforth,Orietta Massidda,Malcolm E Winkler,Jesus Bazan Villicana,Ho-Ching T Tsui,Mattia Benedet

doi:10.3389/fmicb.2021.780864

Amilcar J Perez, Madeline L Danforth + Show 5 more

Open Access

https://doi.org/10.3389/fmicb.2021.780864

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Abstract

The bacterial FtsZ-ring initiates division by recruiting a large repertoire of proteins (the divisome; Z-ring) needed for septation and separation of cells. Although FtsZ is essential and its role as the main orchestrator of cell division is conserved in most eubacteria, the regulators of Z-ring presence and positioning are not universal. This study characterizes factors that regulate divisome presence and placement in the ovoid-shaped pathogen, Streptococcus pneumoniae (Spn), focusing on FtsZ, EzrA, SepF, ZapA, and ZapJ, which is reported here as a partner of ZapA. Epi-fluorescence microscopy (EFm) and high-resolution microscopy experiments showed that FtsZ and EzrA co-localize during the entire Spn cell cycle, whereas ZapA and ZapJ are late-arriving divisome proteins. Depletion and conditional mutants demonstrate that EzrA is essential in Spn and required for normal cell growth, size, shape homeostasis, and chromosome segregation. Moreover, EzrA(Spn) is required for midcell placement of FtsZ-rings and PG synthesis. Notably, overexpression of EzrA leads to the appearance of extra Z-rings in Spn. Together, these observations support a role for EzrA as a positive regulator of FtsZ-ring formation in Spn. Conversely, FtsZ is required for EzrA recruitment to equatorial rings and for the organization of PG synthesis. In contrast to EzrA depletion, which causes a bacteriostatic phenotype in Spn, depletion of FtsZ results in enlarged spherical cells that are subject to LytA-dependent autolysis. Co-immunoprecipitation and bacterial two-hybrid assays show that EzrA(Spn) is in complexes with FtsZ, Z-ring regulators (FtsA, SepF, ZapA, MapZ), division proteins (FtsK, StkP), and proteins that mediate peptidoglycan synthesis (GpsB, aPBP1a), consistent with a role for EzrA at the interface of cell division and PG synthesis. In contrast to the essentiality of FtsZ and EzrA, ZapA and SepF have accessory roles in regulating pneumococcal physiology. We further show that ZapA interacts with a non-ZapB homolog, named here as ZapJ, which is conserved in Streptococcus species. The absence of the accessory proteins, ZapA, ZapJ, and SepF, exacerbates growth defects when EzrA is depleted or MapZ is deleted. Taken together, these results provide new information about the spatially and temporally distinct proteins that regulate FtsZ-ring organization and cell division in Spn.

Highlights

Bacterial cell division initiates by polymerization of the highly conserved and essential tubulin-like protein FtsZ into a dynamic Z-ring composed of FtsZ treadmilling filaments (Bi and Lutkenhaus, 1991; Lutkenhaus et al, 2012; Haeusser and Margolin, 2016; Bisson-Filho et al, 2017; Yang et al, 2017; Perez et al, 2019)
We used the B2H assay to test for interactions between EzrA(Spn) and several proteins not tested in coIP assays. These assays indicate possible direct interactions between EzrA and MacP (Fenton et al, 2018), RodA (SEDS GTase in Peripheral PG (pPG) synthesis) (Meeske et al, 2016), MreD and RodZ (PG elongasome/pPG regulators) (Massidda et al, 2013; Briggs et al, 2021), MpgA (Taguchi et al, 2021), and FtsQ/L (Noirclerc-Savoye et al, 2005; Briggs et al, 2021). These results show that EzrA(Spn) forms complexes and interacts with many key proteins that mediate Z-ring regulation, cell division, and PG synthesis (Figure 9B and Supplementary Figure 21), consistent with its extended spectrin-like repeated structure (Supplementary Figure 1D) and the diverse interactions reported for EzrA in other bacteria
We show that EzrA and FtsZ are both essential for the growth, division, ovoid shape, and normal size of pneumococcal cells (Figures 2, 7)

Summary

Introduction

Bacterial cell division initiates by polymerization of the highly conserved and essential tubulin-like protein FtsZ into a dynamic Z-ring composed of FtsZ treadmilling filaments (Bi and Lutkenhaus, 1991; Lutkenhaus et al, 2012; Haeusser and Margolin, 2016; Bisson-Filho et al, 2017; Yang et al, 2017; Perez et al, 2019). Many studies of FtsZ-ring regulation and cell division have been performed on model rod-shaped bacteria, including Escherichia coli (Eco; Gram-negative), Bacillus subtilis (Bsu; Gram-positive), and Caulobacter crescentus (Ccr; Gramnegative) (Thanbichler and Shapiro, 2006; Coltharp et al, 2016, Yu et al, 2021). In these bacteria, the position of the Z-ring is dictated largely by negative regulatory systems (Min and nucleoid occlusion in Bsu and Eco and MipZ in Ccr); yet, a many other bacteria with different cell shapes do not follow the paradigms from these model rod-shaped bacteria (reviewed by Monahan et al, 2014). The mechanisms leading to midcell Z-ring placement and regulation in cell division and septal PG synthesis have only recently begun to be understood in ovoid-shaped bacteria like Spn (Briggs et al, 2021), despite their potential to reveal vulnerabilities for the discovery of new antibiotics and vaccines

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