In vitroassembly of the bacterial actin protein MamK from ‘CandidatusMagnetobacterium casensis’ in the phylumNitrospirae

Magnetotactic bacteria (MTB), a group of phylogenetically diverse organisms that use their unique intracellular magnetosome organelles to swim along the Earth’s magnetic field, play important roles in the biogeochemical cycles of iron and sulfur. Previous studies have revealed that the bacterial actin protein MamK plays essential roles in the linear arrangement of magnetosomes in MTB cells belonging to the Proteobacteria phylum. However, the molecular mechanisms of multiple-magnetosome-chain arrangements in MTB remain largely unknown. Here, we report that the MamK filaments from the uncultivated ‘Candidatus Magnetobacterium casensis’ (Mcas) within the phylum Nitrospirae polymerized in the presence of ATP alone and were stable without obvious ATP hydrolysis-mediated disassembly. MamK in Mcas can convert NTP to NDP and NDP to NMP, showing the highest preference to ATP. Unlike its Magnetospirillum counterparts, which form a single magnetosome chain, or other bacterial actins such as MreB and ParM, the polymerized MamK from Mcas is independent of metal ions and nucleotides except for ATP, and is assembled into well-ordered filamentous bundles consisted of multiple filaments. Our results suggest a dynamically stable assembly of MamK from the uncultivated Nitrospirae MTB that synthesizes multiple magnetosome chains per cell. These findings further improve the current knowledge of biomineralization and organelle biogenesis in prokaryotic systems. Electronic supplementary material The online version of this article (doi:10.1007/s13238-016-0253-x) contains supplementary material, which is available to authorized users.


Supplemental Methods
Cloning, expression and purification. The mamK gene was amplified from Mcas genomic DNA using the primers WB643 (AAGGCATATGACAAAAACAAAGATACTTAACA) and WB567 (TTCAAGCTTTTATCGCTTGCTTACTTCGTCCCA) and then digested with NdeI and HindIII. The digested products were purified and ligated with the pET28a vector (Invitrogen) and digested with the same restriction enzymes to create the plasmid pWYE369 for expressing N-terminal his-tagged MamK. To construct C-terminal his-tagged MamK expressing plasmid pWYE403, the mamK gene was amplified using primers WB643 and WB860 (GTGAAGCTTTCGCTTGCTTACTTCG) and then cloned into the pET22b vector (Invitrogen) at restriction sites NdeI/HindIII.
The plasmids pWYE369 and pWYE403 were transformed into Escherichia coli C43 (DE3) cells and verified by DNA sequencing. The cells were grown in LB medium at 37°C and induced with 0.5 mM IPTG after reaching an OD 600 of 0.8. After 4 hours of induction, the cells were harvested and disrupted by sonication in buffer A containing 150 mM NaCl, 10% glycerol and 20 mM Tris-HCl (pH 8.0). After centrifugation at 15,000 ×g for 30 min, the his-tagged MamK protein in the supernatant was purified by Ni 2+ affinity chromatography (GE healthcare, USA) and gel filtration (Superdex 75 10/300 GL high-performance column, GE healthcare, USA). To obtain protein without his tag, purified N-terminal his-tagged MamK was subsequently incubated with immobilizing bovine thrombin (Sigma, USA) at 4°C to 3 remove the poly-histidine tags. The resulting MamK protein only with three residues at the N-terminus was eluted from the thrombin-agarose, and passed through a Ni 2+ affinity column again to remove the remove uncleaved protein and the cleaved His tags. The purified protein was centrifuged at 500,000 ×g for 30 min at 4°C to remove the aggregates and stored in aliquots at -80°C for further study. The protein concentration was determined using the BCA protein assay kit (Pierce, Rockford) (1) where n is the dilution factor and 25,120 cm -1 M -1 is the molar extinction coefficient of the MamK protein at 280 nm (estimated using the ProtParam tool, http://web.expasy.org/protparam/). The degree of labeling (D, moles dye per mole protein) was calculated as equation 2: where n is the dilution factor and 71,000 cm -1 M -1 is the molar extinction coefficient of the Alexa Fluor 488 dye at 494 nm.
Characterization of MamK ATPase features. Enzyme activity was determined using a modified malachite-green assay as described above, with slight further modifications. Purified protein at a final concentration of 2.0 μM in the nucleotide hydrolysis reaction was used to determine ATPase activity under various conditions. The optimum temperature was determined after incubating the reaction mixtures at different temperatures (30-70°C). The effect of pH on enzyme activity was measured at different pH values (5-11) at 37°C. The relative enzyme activity was calculated as a percentage of the maximal activity. For the thermal stability assay, MamK was incubated at various temperatures (30-70°C) for 30 min, and the residual enzyme activity was subsequently determined. The residual enzyme activity was calculated as a percentage of the starting activity.
The effects of various metal ions on the enzyme activity were determined after incubating the reaction mixtures with MgCl 2 , CaCl 2 , FeSO 4 , CuCl 2 and NiSO 4 at concentrations ranging from 0 to 2.0 mM. To avoid the oxidation of iron (II), FeSO 4 was dissolved in the reaction buffer containing a reducing agent (2 mM ascorbic acid), which had been determined to have no effects on the MamK ATPase activity.
The enzyme activity assayed under the same condition, but in the absence of metal ions, was set at 100%.

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To evaluate the effect of salt, the enzyme activity was measured in the presence of 0-500 mM KCl and NaCl. The enzyme activity assayed under the same conditions, but in the absence of salts, was set at 100%.
The kinetic parameters were determined through Lineweaver-Burk curves using varying concentrations of ATP from 0.01 to 0.50 mM. Substrates at different concentrations were incubated with MamK using a standard ATPase activity assay as described above. The velocity (μM min -1 Pi /μM protein, y axis) was defined as the change in catalytic rate of the concentration of released phosphate per minute and per μM protein, which was plotted against the concentration of the substrate (μM, x axis). Subsequently, the curves were fitted using the Lineweaver-Burk curve method, from which the Michaelis constants (Vmax and Km) for each enzymatic reaction were derived. The initial velocities were used to determine Vmax and Km.
The catalytic constant (Kcat) was determined after dividing the Vmax by the concentration of the enzyme. The catalytic efficiency was defined as Kcat/Km (min -1 μM -1 ). Approximately 4.5 μM of MamK was doped with 15% Alexa 488-labelled monomer in polymerization buffer and loaded onto a cover slip, followed by polymerization using saturating amounts of ATP at 37°C. The fluorescently labelled filaments were observed at 0 to 25 μm from the bottom of the slide using a Leica SP8 confocal microscopy.

Determination of the physicochemical conditions for polymerization. Various
Determination of the polymerization by pelleting assay. Because the ATPase 7 activity and assembly of MamK from Mcas was inhibited at 4°C (Fig. 3a), polymerization was assessed using a pelleting assay. To simultaneously determinate the ATP hydrolysis and the polymerization of MamK protein, the polymerizing sample at the same time point was separated in two parts. One was mixed with 4 volumes of chromogenic reagent and incubated at 37°C for 30 min, while another was centrifuged at 500,000 ×g at 4°C for 30 min. Subsequently, they were subjected to determine the amount of Pi (630 nm) and protein monomers concentration (595 nm) by spectrophotometer and the pellet by SDS-PAGE.
Statistical analyses. Significant differences were analyzed using SPSS software (version 13.0; http://www-01.ibm.com/software/analytics/spss/). The data conducted under various conditions were compared by one-way analysis of variance (ANOVA) and Duncan's multiple range tests. All measurements were performed at least in triplicate, and each value represented the mean ± standard deviation (SD). P-values less than 0.05 were considered statistically significant.