Roles of the Sodium-Translocating NADH:Quinone Oxidoreductase (Na⁺-NQR) on Vibrio cholerae Metabolism, Motility and Osmotic Stress Resistance

The Na⁺ translocating NADH:quinone oxidoreductase (Na⁺-NQR) is a unique respiratory enzyme catalyzing the electron transfer from NADH to quinone coupled with the translocation of sodium ions across the membrane. Typically, Vibrio spp., including Vibrio cholerae, have this enzyme but lack the proton-pumping NADH:ubiquinone oxidoreductase (Complex I). Thus, Na⁺-NQR should significantly contribute to multiple aspects of V. cholerae physiology; however, no detailed characterization of this aspect has been reported so far. In this study, we broadly investigated the effects of loss of Na⁺-NQR on V. cholerae physiology by using Phenotype Microarray (Biolog), transcriptome and metabolomics analyses. We found that the V. cholerae ΔnqrA-F mutant showed multiple defects in metabolism detected by Phenotype Microarray. Transcriptome analysis revealed that the V. cholerae ΔnqrA-F mutant up-regulates 31 genes and down-regulates 55 genes in both early and mid-growth phases. The most up-regulated genes included the cadA and cadB genes, encoding a lysine decarboxylase and a lysine/cadaverine antiporter, respectively. Increased CadAB activity was further suggested by the metabolomics analysis. The down-regulated genes include sialic acid catabolism genes. Metabolomic analysis also suggested increased reductive pathway of TCA cycle and decreased purine metabolism in the V. cholerae ΔnqrA-F mutant. Lack of Na⁺-NQR did not affect any of the Na+ pumping-related phenotypes of V. cholerae suggesting that other secondary Na⁺ pump(s) can compensate for Na⁺ pumping activity of Na⁺-NQR. Overall, our study provides important insights into the contribution of Na⁺-NQR to V. cholerae physiology

Plasmids used in this study.

<p>Plasmids used in this study.</p

Transcription level of plasmid-encoded <i>versus</i> chromosomally encoded <i>pomB</i> in <i>V</i>. <i>cholerae</i> determined by qRT-PCR.

<p>A: analysis of amplification products obtained from qRT-PCR reactions with template (cDNA) amounts of 100 ng, 10 ng, 1 ng and 0.1 ng by agarose gel electrophoresis. The efficiency of the primer pair “pomB fwd” / “pomB rev” was 115% with R² = 0.998, as calculated using the CFX Manager software (Bio-Rad). A 100 bp DNA ladder (GeneRuler, Thermo Scientific) served as molecular size marker. B: The correct size (134 bp) of the products from the qRT-PCR reactions (technical triplicates, I–III) was confirmed by agarose gel electrophoresis. C: Relative transcription level of <i>pomB</i> in the <i>V</i>. <i>cholerae</i> reference strain and in <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i> pAB induced with 10 mM L-arabinose. For both strains, qRT-PCR reactions were performed in technical triplicates. Transcription levels of <i>pomB</i> were calculated using the CFX Manager software (Bio-Rad, version 2.1.1022.0523), and the transcription level of the chromosomally encoded <i>pomB</i> (reference strain) was set to 1.</p

Distribution of swimming speeds of the <i>V</i>. <i>cholerae</i> reference strain and of <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i> expressing <i>pomA</i> and <i>pomB</i> in trans.

<p>Tracks were recorded in LB-Na⁺ adjusted with Tris to pH 7.0 (light grey bars), pH 8.0 (white bars), or pH 9.0 (dark grey bars). Tracking results of individual cells were assigned to three main classes of velocities (“slow”, “medium” and “fast”). The numbers in the bars indicate the number of tracks used to calculate the mean values and standard deviations. Minimum, median and maximum values are given in the supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123518#pone.0123518.s001" target="_blank">S1 Table</a>).</p

Na⁺ flux through the flagellar PomA₄PomB₂ complex by a channel-like mechanism.

<p>Na⁺ enters the PomA₄PomB₂ stator complex in its hydrated state from the periplasm via a water-filled access channel. The conserved amino acid residues S26 and D23 within the membrane-bound region of PomB interact with water molecules surrounding the central Na⁺, hereby perturbing its hydration shell. The sodium ion passes the narrow constriction site, or selectivity filter, in its dehydrated state. After passage through the filter, Na⁺ is hydrated and released towards the cytoplasmic side of the stator. The downhill flux of Na⁺ from the periplasm to the cytoplasm is driven by the electrochemical Na⁺ gradient across the inner membrane.</p

Expression levels of plasmid-encoded PomB variants in <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i>.

<p>Wild type PomB-Strep, PomB-S26A-Strep and PomB-S26T-Strep were detected in extracts from <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i> cells transformed with variants of plasmid pAB on a Western blot. PomB-Strep with an apparent molecular weight around 46 kDa was expressed in the presence (+) but not in the absence (‒) of 10 mM L-arabinose, and it was also absent in extracts from <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i> transformed with the empty vector (pISC-H). Cells were grown for 4 h at 30°C, harvested and resuspended in SDS loading buffer to a final OD₆₀₀ of 5.0. Per lane, an aliquot of 18 μg total cell protein was loaded. The bands (*) above the 58 kDa marker result from detection of an endogenous, biotinylated protein of <i>V</i>. <i>cholerae</i> by the Strep-Tactin-horseradish peroxidase conjugate [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123518#pone.0123518.ref052" target="_blank">52</a>].</p

Influence of inducer concentration on motility of <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i> overproducing His₆-PomA together with PomB-Strep, PomB-S26A-Strep, or PomB-S26T-Strep.

<p>The average and standard deviations were calculated of eight experiments (the lowest and highest values from a total of ten experiments were omitted). Black bars: <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i> pAB; open bars: <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i> pAB-S26A; hatched bars: <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i> pAB-S26T.</p

Influence of pH and salt condition on the swimming speeds of <i>V</i>. <i>cholerae</i> Δ<i>pomAB</i> cells overproducing His₆-PomA together with PomB-Strep, PomB-S26A-Strep, or PomB-S26T-Strep.

<p>Tracks were recorded in different LB media adjusted with Tris to pH 7.0 (light grey bars), pH 8.0 (white bars), or pH 9.0 (dark grey bars). A: LB-Na⁺; B: no salt added, with residual concentrations of 11 mM Na⁺ and 12 mM K⁺; C: LB-K⁺. Tracking results of individual cells were assigned to three main classes of velocities (“slow”, “medium” and “fast”). The numbers in the bars represent the number of tracks recorded under the specified condition. The mean values and standard deviations of velocities are presented. Minimum, median and maximum values are given in the supporting information (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123518#pone.0123518.s002" target="_blank">S2</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123518#pone.0123518.s004" target="_blank">S4</a> Tables).</p

Rotation of the single polar flagellum from <i>V</i>. <i>cholerae</i> is driven by a Na⁺ flux through the PomA₄PomB₂ stator complex.

<p>A: The flagellum of <i>V</i>. <i>cholerae</i> consists of rotating and static parts. The filament, the hook, which connects the filament to the motor complex, the membrane embedded L and P rings in the outer membrane (OM), and the C ring in the inner membrane (IM) rotate against several stator complexes, each composed of four PomA and two PomB proteins. The H ring functions as a bushing to enable rotation against the peptidoglycan layer (PG). The T ring connects the stator complexes with the peptidoglycan layer. Proton-driven flagella, such as found in <i>E</i>. <i>coli</i> and <i>Salmonella</i>, do not have H and T rings. Export of protein components of the flagellar filament is catalyzed by a type III secretion system located at the base of the flagellum. B: Four PomA and two PomB subunits form the flagellar stator complex. PomA possesses four transmembrane helices (TM I–IV), PomB only one. The translucent, purple boxes in the front each represent one PomA subunit. The flux of Na⁺ through the stator complex (red arrow) along the chemical gradient drives the rotation of the C ring against the stator elements. It is assumed that Na⁺ ions pass through a channel built by helices III and IV of PomA and the single transmembrane helix of PomB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123518#pone.0123518.ref017" target="_blank">17</a>,<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123518#pone.0123518.ref031" target="_blank">31</a>]. The peptidoglycan binding domain (PBD) in the C-terminal part of PomB stabilizes the stator complexes within the peptidoglycan layer. The conserved amino acid residues D42, S26 and D23 of PomB promote the transport of Na⁺ through the stator [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0123518#pone.0123518.ref052" target="_blank">52</a>].</p

Serine 26 in the PomB Subunit of the Flagellar Motor Is Essential for Hypermotility of <i>Vibrio cholerae</i>

<div><p><i>Vibrio cholerae</i> is motile by means of its single polar flagellum which is driven by the sodium-motive force. In the motor driving rotation of the flagellar filament, a stator complex consisting of subunits PomA and PomB converts the electrochemical sodium ion gradient into torque. Charged or polar residues within the membrane part of PomB could act as ligands for Na⁺, or stabilize a hydrogen bond network by interacting with water within the putative channel between PomA and PomB. By analyzing a large data set of individual tracks of swimming cells, we show that S26 located within the transmembrane helix of PomB is required to promote very fast swimming of <i>V</i>. <i>cholerae</i>. Loss of hypermotility was observed with the S26T variant of PomB at pH 7.0, but fast swimming was restored by decreasing the H⁺ concentration of the external medium. Our study identifies S26 as a second important residue besides D23 in the PomB channel. It is proposed that S26, together with D23 located in close proximity, is important to perturb the hydration shell of Na⁺ before its passage through a constriction within the stator channel.</p></div