Bacterial strains were grown in LB or on LB agar at 37°C supplemented with the following antibiotics when necessary: 100 μg/ml Carbenicillin and 50 μg/ml Tetracycline. Liquid cultures were grown under aeration at 37°C at 250 rpm.

Plasmids and oligonucleotides used to generate or to verify strains and plasmids are listed in Tables 1 and 2, respectively.

Expression vectors for FtsZ, ZapAL, and ZapA from P. aeruginosa were constructed in the plasmid pET15b at the NdeI/BamHI sites. After the plasmid transformed into an E. coli strain BL21(DE3), protein expression was induced at 16°C overnight by the addition of 0.5 mM isopropyl β-D-1-thiogalactopyranoside (IPTG). Following sonication and centrifugation, the soluble His6-tag proteins were purified by affinity chromatography on a Talon column (Clontech Lab, Inc.). Proteins were firstly washed with 0–30 mM imidazole, and then eluted with the elution buffer containing 50 mM Tris pH 7.7, 300 mM KCl, and 300 mM imidazole. After dialysis with HMK buffer (50 mM HEPES, pH 7.5, 5 mM MgAc, and 100 mM KAc), ZapAL and ZapA proteins with His6 tag were stored at −80°C.

Since FtsZ with His-tag may affect its activity, the purified His6-PaFtsZ was incubated with 2 units/ml of thrombin for 2 h at room temperature to remove the His6-tag. A further purification followed by chromatography on a source Q 10/10 column (GE healthcare) with a linear gradient of 50–500 mM KCl in 50 mM Tris, pH 7.9, 1 mM EDTA, and 10% glycerol. The purified proteins were dialyzed into HMK buffer and stored at −80°C.

A SacB-based strategy was applied to generate the P. aeruginosa gene knockout mutants, modified as previously described (Hoang et al., 1998; Peng et al., 2020). Briefly, upstream and downstream fragments of the genes were amplified by PCR using the corresponding primers (Table 1). Each fragment contained about 2,000 bp. After digested with appropriate restriction enzymes, both upstream and downstream fragments were ligated and inserted into pEX18Amp plasmid. To avoid errors introduced by PCR, the DNA inserts were verified by DNA sequencing. The resultant plasmids were electroporated into PAO1 and subjected to selection for carbenicillin-resistance. Colonies were then selected on Pseudomonas isolation agar plates with 10% sucrose, which indicates a double-crossover event and therefore gene replacement occurring. The resultant mutants were verified by PCR on the region containing the target gene.

The assembly kinetics of FtsZ filaments and bundles with or without ZapA and ZapAL were measured by the light scattering method at room temperature. After FtsZ and the ZapAL or ZapA mixture is quickly mixed with GTP, the changes of the light scattering signal are tracked to obtain their assembly kinetics. Both excitation and emission were set to 340 nm with a Shimadzu RF-5301 PC spectrofluorometer. Each measurement was repeated two or three times.

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. Then, 10 μl samples were added to a carbon-coated copper grid. After standing for about 5 s, the solution was quickly absorbed with filter papers. Grids were immediately stained with 3–5 drops of 2% uranyl acetate, and the excess droplets were dried with filter papers. Images were obtained on a Philips EM420 equipped with a CCD camera.

To determine the composition of the FtsZ-ZapAL polymers, a sedimentation assay was applied, modified as previously described (Huang et al., 2018). The mixture of 15 μM FtsZ and 15 or 30 μM ZapAL were incubated with 1 mM GTP or without GTP at room temperature for 10 min and centrifuged at 80,000 rpm for 30 min at 25°C in a Beckman TLA100 rotor. The supernatant was carefully removed, and the pellet was resuspended in the same volume of buffer. The protein in the pellet and supernatant was assayed by SDS-PAGE. The ratio of supernatant and pellet was analyzed using ImageJ software (Schneider et al., 2012), and the protein concentrations of FtsZ or ZapAL in the pellet were calculated from the percentage of total protein concentration. The measurement was repeated three times.

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. In this assay, when a molecule of GTP is hydrolyzed to GDP, a molecule of NADH in the solution will be consumed in the subsequent reaction, and at the same time, GDP is regenerated to GTP. The GTP concentration in the solution remains constant, and it can avoid the effect of GDP accumulation on the GTPase activity in the solution. The GTP hydrolysis rate is equal to the NADH consumption rate, which is measured at room temperature using the extinction coefficient 0.00567 μM⁻¹ cm⁻¹ at 350 nm with an L5 UV-vis spectrophotometer (INESA analytical Instrument Co.). The hydrolysis rate was plotted as a function of FtsZ concentration, and the slope of the line above the critical concentration (Cc) represents the GTPase activity per unit FtsZ concentration. Each measurement was repeated two or three times.

Pseudomonas aeruginosa PAO1 carrying pME6032-zapAL and pME6032-zapAL-gfp were cultured in LB with 50 μg/ml Tetracycline until OD600 value reached ~0.4, and 0.5 mM IPTG was added and induced at 37°C for 1.5 h. PAO1 and the deletion mutants were cultured in LB for 4 h at 37°C until OD600 reached ~0.5.

The cells in the agarose pad were visualized with phase contrast and fluorescence microscopy using a Leica DMI3000B fluorescence microscopy. PAO1 cell lengths were measured for each mutant, and the results were reported as the mean ± SD.

ZapA is a broadly conserved Z-ring associate protein in most bacteria (Gueiros-Filho and Losick, 2002). When we searched for the ZapA protein sequences in the database (Winsor et al., 2016), we found there were two small proteins from P. aeruginosa, PA5227 and PA5407; both were marked as ZapA in some sequencing data. We used Clustal Omega (Sievers et al., 2011) to align these two proteins and found that they share only 20% sequence identity and around 40% similarity (Figure 1). PA5227 contains 104 amino acids, which is ZapA. Another protein PA5407 contains 96 amino acids and is mainly found in Pseudomonas species. We name this protein ZapAL.

To characterize ZapAL, we first investigated whether or not ZapAL interacts with FtsZ and affects FtsZ assembly. Using negative-stain EM, we found that ZapAL induced FtsZ to form long, straight double filaments (Figure 2). FtsZ from P. aeruginosa alone assembles into mostly the mixture of single straight and arc-shaped filaments in the presence of GTP (Figure 2A). After ZapAL was added, FtsZ-ZapAL mostly assembles into long, double straight filaments in the presence of GTP (Figures 2B,C). It is different from ZapA which promotes FtsZ to form large bundles/sheets composed of multiple FtsZ filaments (Figure 2D, also previous publication; Rahman et al., 2020). From EM images of FtsZ-ZapAL, we could observe the striations across the FtsZ-ZapAL double filaments, indicating ZapAL cross-links (Figures 2B,C, arrows). The distance between ZapAL molecules is around 3.7 ± 0.4 nm, which suggests that ZapAL binds to each FtsZ molecule. Similar to FtsZ-ZapA, the FtsZ-ZapAL double filaments are also loose structures. The average distance from the outer edge of one FtsZ protofilament to another is about 9.2 ± 1.1 nm. Considering the thickness of FtsZ filaments in our measurement is around 3 nm, the interval distance between filaments is approximately 3.2 nm.

We next used sedimentation and SDS-PAGE to analyze the stoichiometry of the FtsZ-ZapAL copolymers (Figure 2E). Following centrifugation at 80,000 rpm, most copolymers were in pellets. Without GTP, little protein could be seen in the pellet. In the presence of GTP, FtsZ and ZapAL were pre-mixed at the ratio of 1:1 or 1:2, and the stoichiometry of the copolymer in the pellet was estimated by SDS-PAGE analysis from the percentage of total protein amounts. In the mixture of 15 μM FtsZ and 15 μM ZapAL, FtsZ concentration is around 11.5 ± 1.2 μM, and ZapAL is around 10.7 ± 1.8 μM in the pellet. In the mixture of 15 μM FtsZ and 30 μM ZapAL, FtsZ concentration is around 10.4 ± 1.5 μM, and ZapAL is around 12.6 ± 1.0 μM in the pellet. The molar ratio of FtsZ and ZapAL in the polymer is almost 1:1. We proposed a model to show the FtsZ-ZapAL double filaments (Figure 2F). Acting as a bridge, ZapAL connects two FtsZ protofilaments to form long straight double filaments.

Since FtsZ-ZapAL assembles into large, straight double filaments, a light-scattering assay will be a useful tool to measure its assembly dynamics. In our experiments, we used the light-scattering assay to study how ZapAL affects the FtsZ assembly kinetics. Figure 3 shows the comparison of FtsZ assembly kinetics with ZapAL and ZapA. FtsZ alone assembles into single filaments and its light-scattering signal is very weak. ZapAL induced FtsZ to form long double filaments, corresponding to a strong light-scattering signal. Figure 3A shows the assembly of a 1:1 mixture of 5 and 10 μM FtsZ-ZapAL in the presence of 1 mM GTP. After a short fast rising, the light-scattering signal increased slowly and reached a plateau at around 300 s. However, the light-scattering signal of FtsZ-ZapAL was completely stable, and no decrease was observed in our measurements, up to 1,200 s. We repeated the measurement of 10 μM FtsZ-ZapAL with 1 mM GTP and 0.1 mM GTP, and almost similar results were obtained (Figure 3B). It is different from the assembly of FtsZ-ZapA (Figure 3C). The assembly of FtsZ-ZapA is faster; it took around 100 s to reach the plateau. Comparing the polymerization kinetics of 10 μM FtsZ-ZapA at different concentrations of GTP, we observed that the light scattering signal with 0.1 mM GTP began to decrease rapidly at about 100 s, and then decreased to near the baseline at about 250 s (Figure 3C). This shows that when GTP is used up, FtsZ-ZapA will completely depolymerize, consistent with the decrease of light scattering signal. However, it seems that the FtsZ-ZapAL copolymers are stable, and no depolymerization could be observed even with 0.1 mM GTP in our measurements (Figure 3B). The similarity between ZapAL and ZapA makes it possible to form heterodimers between them. The Figure 3D compares the effects of ZapA, ZapAL, and a mixture of ZapA and ZapAL in a ratio of 1:1 on the FtsZ polymerization kinetics in the presence of 0.1 mM GTP. We found that the light scattering intensity of the mixture of FtsZ and ZapAL-ZapA was weaker and faster, reaching the maximum in less than 20 s. Then, it began to decline, showing that it would depolymerize after GTP was hydrolyzed, similar to FtsZ-ZapA. This result implies that ZapA and ZapAL are very likely to form a heterodimer and promote FtsZ to form a small bundle, which maintain the dynamic characteristics of the FtsZ filaments.

To obtain more information, we next investigated how ZapAL affects the GTPase activity of FtsZ. Figure 3E shows the GTPase activity of FtsZ at different concentrations with or without ZapAL at room temperature. The GTPase activity of FtsZ is around 4.57 ± 0.34 GTP/FtsZ/min, and it drops to 4.14 ± 0.33 GTP/FtsZ/min with 5 μM ZapAL and to 3.39 ± 0.12 GTP/FtsZ/min with 10 μM ZapAL, only a 26% reduction. We concluded that ZapAL has a mild effect on the GTPase activity of FtsZ, similar to ZapA. If there is only 0.1 mM GTP in the solution, 10 μM FtsZ-ZapAL mixtures will hydrolyze all GTP in about 3 min; however, no depolymerization of FtsZ-ZapAL copolymers was observed in our measurements of light-scattering signals (Figure 3B), meanwhile, the filaments formed by FtsZ-ZapA and FtsZ-ZapA-ZapAL can be depolymerized after GTP is hydrolyzed.

Since ZapAL interacts with FtsZ in vitro, we wanted to know its localization and its possible physiological function in vivo. Fused with GFP at C-terminus, we expressed ZapAL-GFP in wildtype PAO1 strain. Fluorescence microscopy of a GFP-ZapAL producing strain revealed narrow bands or spots in the middle of cells (Figure 4), a localization pattern characteristic of division proteins, similar to ZapA. It suggests that ZapAL is also one of the components of the division machinery.

To investigate the physiological function of ZapAL, we knocked out the ZapAL gene from P. aeruginosa PAO1 strain. However, we found that knockout ZapAL exhibited no recognizable defects in bacterial cell division and growth. Similar results were obtained after we knocked out the zapA gene. Since both protein ZapAL and ZapA show a function to stabilize FtsZ protofilaments, we knocked out both of the zapA and ZapAL genes, and there were still no obvious effects on bacterial morphology (Figure 5A). These results suggest that both ZapAL and ZapA are not essential for cell division under normal laboratory conditions.

In an attempt to uncover a role for ZapAL in bacterial division and growth, we also investigated the effects of the overexpressed ZapAL protein on bacterial cell division. Induced by 0.5 mM IPTG, the ZapAL protein was expressed in P. aeruginosa PAO1 containing pME6032-zapAL plasmid. However, few changes were observed when ZapAL was overexpressed (Figure 5B). But when ZapAL was overexpressed in the zapA knockout strain, we observed an obvious increase in bacterial length (Figure 5B). After overexpression of ZapAL, the bacterial length is around 2.5 ± 0.4 μm (n = 329) in wildtype PAO1 strain, 5.1 ± 2.2 μm (n = 300) in zapA deletion strain, and 5.5 ± 2.1 μm (n = 333) in both zapA and zapAL deletion strain (Figure 5C). It suggests that overexpression of ZapAL would mildly impair bacterial cell division without ZapA. Meanwhile, overexpression of ZapA in PAO1 strain and zapAL deletion has no obvious effect on bacterial division and growth under normal conditions (data not shown).