Assembly properties of bacterial tubulin homolog FtsZ regulated by the positive regulator protein ZipA and ZapA from Pseudomonas aeruginosa

Abstract

Bacterial tubulin homolog FtsZ self-assembles into dynamic protofilaments, which forms the scaffold for the contractile ring (Z-ring) to achieve bacterial cell division. Here, we study the biochemical properties of FtsZ from Pseudomonas aeruginosa (PaFtsZ) and the effects of its two positive regulator proteins, ZipA and ZapA. Similar to Escherichia coli FtsZ, PaFtsZ had a strong GTPase activity, ~ 7.8 GTP min-1 FtsZ-1 at pH 7.5, and assembled into mainly short single filaments in vitro. However, PaFtsZ protofilaments were mixtures of straight and “intermediate-curved” (100–300 nm diameter) in pH 7.5 solution and formed some bundles in pH 6.5 solution. The effects of ZipA on PaFtsZ assembly varied with pH. In pH 6.5 buffer ZipA induced PaFtsZ to form large bundles. In pH 7.5 buffer PaFtsZ-ZipA protofilaments were not bundled, but ZipA enhanced PaFtsZ assembly and promoted more curved filaments. Comparable to ZapA from other bacterial species, ZapA from P. aeruginosa induced PaFtsZ protofilaments to associate into long straight loose bundles and/or sheets at both pH 6.5 and pH 7.5, which had little effect on the GTPase activity of PaFtsZ. These results provide us further information that ZipA functions as an enhancer of FtsZ curved filaments, while ZapA works as a stabilizer of FtsZ straight filaments.

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

“…Negative stain electron microscopy (EM) was used to visualize FtsZ filaments, as described previously ( Rahman et al, 2020 ). Briefly, samples were incubated with GTP to polymerize for 1–3 min at room temperature.…”

“…GTPase activity was determined by a continuous assay coupled with a GTP regeneration system, as described previously ( Rahman et al, 2020 ). The solution included 1 mM Phosphoenolpyruvic acid monopotassium, 0.9 mM NADH, 10 units/ml pyruvate kinase and lactate dehydrogenase (Sigma-Aldrich), and 0.5 mM GTP, in HMK buffer (50 mM HEPES, pH 7.5, 5 mM MgAc, and 100 mM KAc), and a 3 mm path cuvette was used for measurement.…”

“…Even though ZapA is functionally redundant under normal conditions, and ZapA knockout strains show minor morphological changes, previous results suggested that zapA deletion strain in B. subitilis had serious defects in bacterial division function if it is knocked out together with the ezrA gene, or the level of FtsZ is reduced (Gueiros-Filho and Losick, 2002). 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).…”

“…ZipA was previously considered a Z-ring stabilizer since it could promote FtsZ into bundles ( Raychaudhuri, 1999 ; Hale et al, 2000 ). However, several in vitro studies showed that ZipA only induces FtsZ bundling when pH is below 7 ( Mateos-Gil et al, 2012 ), and when the pH is larger than 7, ZipA has the function of enhancing and stabilizing the FtsZ curved conformation, not bundling ( Chen et al, 2017a ; Rahman et al, 2020 ). ZapA, ZapC, and ZapD are all FtsZ stabilizers, which are located at midcell and promote FtsZ to form bundles, but they do not share any primary sequence identity ( Gueiros-Filho and Losick, 2002 ; Hale et al, 2011 ; Durand-Heredia et al, 2012 ; Huang et al, 2013 ).…”

“…Both ZapC and ZapD mainly exist in γ–proteobacteria, and ZapA is broadly conserved in almost all bacteria. ZapA is a small cytoplasmic protein; previous results reported that ZapA dimers or tetramers cross-link adjacent FtsZ protofilaments to associate into long straight loose bundles and/or sheets in vitro and thus increase the stability of the Z-ring in vivo ( Gueiros-Filho and Losick, 2002 ; Low et al, 2004 ; Mohammadi et al, 2009 ; Pacheco-Gomez et al, 2013 ; Roseboom et al, 2018 ; Rahman et al, 2020 ). Even though ZapA is functionally redundant under normal conditions, and ZapA knockout strains show minor morphological changes, previous results suggested that zapA deletion strain in B. subitilis had serious defects in bacterial division function if it is knocked out together with the ezrA gene, or the level of FtsZ is reduced ( Gueiros-Filho and Losick, 2002 ).…”