Figure 1.N‐atp operon and c‐subunit sequence of Burkholderia pseudomallei aligned with other known c‐subunits
Gene order of the F‐type atp operon (top) compared to the N‐type atp operon (bottom). In the N‐atp operon, the atpD and atpC precede atpB and are followed by atpQ and atpR, which are absent in the F‐atp operon. Arrows indicate coding sequences. The theoretical molecular masses of the F‐ATP and the N‐ATPase gene products are given in kDa (brackets) above each gene. In B. pseudomallei, the F‐type operon is located on the genomic chromosome 1 and the N‐type operon is located on genomic chromosome 2, flanked by virulence‐specific genes .
Amino acid alignment of selected H+‐ and Na+‐specific F‐type c‐subunits with the N‐type c‐subunit of B. pseudomallei. The conserved loop region is bold and conserved residues involved in ion binding are colored. The conserved glycine motif as discussed in the text is highlighted in gray. Species are as follows: Burkholderia pseudomallei (numbering), Geobacter sulfurreducens, Ilyobacter tartaricus, Fusobacterium nucleatum subsp. nucleatum, Spirulina platensis, Saccharomyces cerevisiae, Bacillus pseudofirmus OF4, Mycobacterium phlei, and Escherichia coli.
Figure 3.Effect of Na+ and high pH on the kinetics of the NCD‐4 modification of the Burkholderia pseudomallei N‐type ATPase rotor ring
100 μM NCD‐4 was added (blue arrows) to the purified c‐ring in 1.0% DDM and 2‐(N‐morpholino)ethanesulfonic acid (MES) buffer, pH 6.0. Continuous increase of fluorescence upon NCD‐4 binding to Glu61 of the c‐ring.
Addition of 15 and 150 mM NaCl (red arrows) decelerated the reaction due to unspecific salt effects.
Increasing the pH to 9.0 by adding 2.5 M Tris base stock solution (red arrow) abolished NCD‐4 binding to Glu61. Results of typical experiments are shown. The experiments were repeated three times each.
Figure EV2.Effect of Li+ and Cs+ on the kinetics of the NCD‐4 modification of the Burkholderia pseudomallei N‐type ATPase c‐ring
100 μM NCD‐4 was added to the purified c‐ring in 1.0% DDM and MES buffer, pH 6.0. The trace shows the continuous increase of fluorescence upon NCD‐4 binding to Glu61 of the c‐ring.
Addition of 15 or 150 mM LiCl resulted in a drop in labeling efficiency to 55 or 17%.
Addition of 15 or 150 mM CsCl reduced the labeling efficiency to 44 or 24%, illustrating the effect of salt in the buffer  and the overall dilution of the reaction volume. Results of typical experiments are shown. Each experiment was repeated three times.
Figure 4.Electron cryo‐microscopy of the Burkholderia pseudomallei N‐type ATPase rotor ring
A–D Representative 2D class averages of the rotor ring showing the ring from the side and top as well as slices through 3D maps after refinement for (A) LDAO, (B) DDM, (C) C12E8, and (D) amphipol A8‐35.
E. Top view of the rotor ring in LDAO with 17 identical hairpin‐like c‐subunits fitted.
F. Side view of the c‐ring in LDAO with the refined model fitted. The helices in the outer ring are slightly shorter than the inner helices. Two adjacent c‐subunits form a functional ion‐binding unit. The positions of residues involved in ion binding or defining the membrane boundary are shown in different colors.
Figure EV4.Subunits adjacent to the c‐ring of N‐type ATPase
Alignment of F‐type and N‐type atpG, atpC, and atpB, comparing sequences of the predicted γ‐, ε‐, and a‐subunits of Burkholderia pseudomallei (BC), Spirulina platensis (SP), Bacillus pseudofirmus OF4 (OF), Ilyobacter tartaricus (IT), and Escherichia coli (EC) having a c17, c15, c13, c11, and c10 ring. Differences were observed in the γ‐subunit of B. pseudomallei.
Figure EV5.Cartoon model of the Escherichia coli γ‐subunit
The structure from Cingolani & Duncan  was used. The positions of different residues marked in magenta in Fig EV4 are highlighted in the E. coli structure. Glu208 and Tyr207, which are exchanged to Val and His in Burkholderia pseudomallei interact with the c‐ring .
Figure 5.Proposed role of the Burkholderia pseudomallei N‐type ATPase in H+ homeostasis and phagosome escape during the early state of infection
Burkholderia pseudomallei enters macrophage phagosomes that are acidified by a host cell‐specific V‐type ATPases (1). Due to the high pH gradient, protons leak through the cell membrane of B. pseudomallei and acidify the B. pseudomallei cytosol (2). The proposed role of the N‐type ATPase is to redress the internal pH by pumping protons out of the bacterial cell and to maintain protein homeostasis (3). B. pseudomallei expresses secretion apparatus (bsa) system, which is essential for phagosome escape (4). Drawing adapted from Wiersinga et al .