The effect of KCl and NaCl on swimming speed of <i>B. subtilis</i> and <i>E.coli</i> strains.

<p>The effect of KCl and NaCl on swimming speed of <i>B. subtilis</i> strains <i>(A)</i> and <i>(B)</i>, The velocity was measured at several different pH values in phosphate buffer that contained 200 mM Na⁺, 150 mM Na⁺ plus 50 mM K⁺, 100 mM Na⁺ plus 100 mM K⁺, 50 mM Na⁺ plus 150 mM K⁺, or 200 mM K⁺. The blue line and blue filled circles, the green line and green filled circles, and the red line and red filled circles show the data at pH 7.0, 7.5, and 8.0, respectively. The effect of KCl and NaCl on swimming speed of <i>E. coli</i> strain <i>(C)</i> The velocity was measured at pH 7.0 in phosphate buffer that contained 200 mM Na⁺, 150 mM Na⁺ plus 50 mM K⁺, 100 mM Na⁺ plus 100 mM K⁺, 50 mM Na⁺ plus 150 mM K⁺, or 200 mM K⁺. The blue line and blue filled circles and the red line and red filled circles show the data for EC-BAPS and EC-BAPS-MotS_M33L, respectively. The swimming speed was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046248#s2" target="_blank">Materials and Methods</a> section. The results that are shown represent the averages of three independent experiments in each of which the swimming speeds of 20 independent cells as calculated in each experiment. The error bars indicate the standard deviations of the values.</p

A <em>Bacillus</em> Flagellar Motor That Can Use Both Na⁺ and K⁺ as a Coupling Ion Is Converted by a Single Mutation to Use Only Na⁺

<div><p>In bacteria, the sodium ion (Na⁺) cycle plays a critical role in negotiating the challenges of an extremely alkaline and sodium-rich environment. Alkaliphilic bacteria that grow optimally at high pH values use Na⁺ for solute uptake and flagellar rotation because the proton (H⁺) motive force is insufficient for use at extremely alkaline pH. Only three types of electrically driven rotary motors exist in nature: the F-type ATPase, the V-type ATPase, and the bacterial flagellar motor. Until now, only H⁺ and Na⁺ have been reported as coupling ions for these motors. Here, we report that the alkaliphilic bacterium <em>Bacillus alcalophilus</em> Vedder 1934 can grow not only under a Na⁺-rich and potassium ion (K⁺)-poor condition but also under the opposite condition in an extremely alkaline environment. In this organism, swimming performance depends on concentrations of Na⁺, K⁺ or Rb⁺. In the absence of Na⁺, swimming behavior is clearly K⁺- dependent. This pattern was confirmed in swimming assays of stator-less <em>Bacillus subtilis</em> and <em>Escherichia coli</em> mutants expressing MotPS from <em>B. alcalophilus</em> (BA-MotPS). Furthermore, a single mutation in BA-MotS was identified that converted the naturally bi-functional BA-MotPS to stators that cannot use K⁺ or Rb⁺. This is the first report that describes a flagellar motor that can use K⁺ and Rb⁺ as coupling ions. The finding will affect the understanding of the operating principles of flagellar motors and the molecular mechanisms of ion selectivity, the field of the evolution of environmental changes and stresses, and areas of nanotechnology.</p> </div

Oligonucleotides used in this study.

<p>Extra nucleotides that were added to introduce restriction sites are <u>underlined</u>.</p><p>Substituted nucleotides that were added to introduce point mutation sites are shown by a small letter.</p

Effect of KCl on the growth and intracellular ion content of various <i>E. coli</i> TK2420 transformants.

<p>The growth of <i>E. coli</i> strain DH5αMCR transformed with control plasmid pBAD24 (filled blue circles) and <i>E. coli</i> strain TK2420 transformed with pBAD24 (open blue circles), pBAPS (filled red circles) and pBAPS-MotS_M33L (open red circles). Cells were shaken in the TK2420 minimal medium adding 10 mM <i>(A)</i>, 25 mM <i>(B)</i> or 50 mM <i>(C)</i> KCl at 37°C under aerobic conditions. Cell growth was monitored at 600 nm. Intracellular [K⁺] and [Na⁺] levels in <i>E. coli</i> DH5αMCR transformed with control plasmid pBAD24 (filled light blue bar) and <i>E. coli</i> strain TK2420 transformed with pBAD24 (open light blue filled bar), pBAPS (filled red bar) and pBAPS-MotS_M33L (open red bar). Cells were shaken in the TK2420 minimal medium adding 25 mM <i>(D)</i>, or 50 mM <i>(E)</i> KCl at 37°C under aerobic conditions. The results are the averages of three independent duplicate experiments, with error bars representing the standard deviations.</p

Motility of <i>B. pseudofirmus</i> and <i>B. alcalophilus</i> in liquid medium.

<p><i>B. pseudofirmus</i> OF4 and <i>B. alcalophilus</i> cells in the logarithmic growth phase that were grown at 30°C in MYE medium (pH 10.5) and KMYE medium (pH 10.5), respectively, were harvested and resuspended in 30 mM Tris-HCl (pH 9.0) that contained 5 mM glucose and the indicated sodium <i>(A)</i>, potassium <i>(B)</i> or rubidium <i>(C)</i> concentrations as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046248#s2" target="_blank">Materials and Methods</a> section. The red line and red open circles show the data for the <i>B. pseudofirmus</i> OF4 strain, and the blue line and blue filled circles show the data for the <i>B. alcalophilus</i> strain. The swimming speed was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046248#s2" target="_blank">Materials and Methods</a> section. The results that are shown represent the averages of three independent experiments in each of which the swimming speeds of 20 independent cells as calculated in each experiment. The error bars indicate the standard deviations of the values.</p

The swimming speed of two alkaliphiles dependent upon pH and concentrations of NaCl and KCl.

<p>The relationship between the swimming speed and several different pH values at 200 mM Na⁺<i>(A)</i> or K⁺<i>(B)</i> is illustrated. The relationship between swimming speed in 30 mM Tris-HCl containing 5 mM glucose (pH 9.0) and the various indicated concentrations of KCl and NaCl is shown in <i>(C)</i>. The red line and red open circles show the data for the <i>B. pseudofirmus</i> OF4 strain, and the blue line and blue filled circles show the data for the <i>B. alcalophilus</i> strain. The swimming speed was determined as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046248#s2" target="_blank">Materials and Methods</a> section. The results that are shown represent the averages of three independent experiments in each of which the swimming speeds of 20 independent cells as calculated in each experiment. The error bars indicate the standard deviations of the values.</p

Stained flagellar of <i>B. alcalophilus</i> and alignment with flagella motor sequences from other bacteria.

<p>Alignments of the region containing the single transmembrane segment of <i>E. coli</i> MotB (EC_MotB), <i>B. subtilis</i> MotB (BS_MotB) and MotS (BS_MotS), <i>B. licheniformis</i> MotB (BL_MotB) and MotS (BL_MotS), <i>Geobacillus kaustophilus</i> MotB (GK_MotB), <i>Oceanobacillus iheyensis</i> MotB (OI_MotB) and MotS (OI_MotS), <i>B. clausii</i> MotB (BCl_MotB), <i>B. alcalophilus</i> MotS (BA_MotS), <i>B. pseudofirmus</i> MotS (BP_MotB), <i>B. halodurans</i> MotS (BH_MotB), <i>B. megaterium</i> MotB (BM_MotS), <i>V. alginolyticus</i> MotB (VA_MotB) and PomB (VA_PomB), <i>V. parahaemolyticus</i> MotB (VP_MotB) and PomB (VP_PomB), <i>V. mimicus</i> MotB (VM_MotB), <i>V. splendidus</i> PomB (VS_PomB), and <i>V. fisheri</i> PomB (VF_PomB). The position of D32 in EC_MotB is known to be critical for rotation and is highlighted in green. The MotAB of <i>B. clausii</i> can use Na⁺ instead of H⁺ to promote flagellar rotation at high pH values. The V37L mutation was critical for sodium selectivity and a combination of the V37L mutation and either the A40S or the G42S mutation was required for production of the BCl-MotB (the ninth line) form that exhibits sodium-coupling at low pH <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0046248#pone.0046248-Terahara1" target="_blank">[14]</a>. The position of V43 in EC_MotB (the first line) is conserved among all of the MotB-H⁺-type proteins and is highlighted in light blue. The position of L32 in BP_MotS (the eleventh line from the top) is conserved among all of the MotS-Na⁺-type proteins with the exception of BA_MotS and is highlighted in yellow. The same position in <i>B. alcalophilus</i> MotS encodes methionine instead of the conserved leucine residue, and it is highlighted with violet.</p

Comparison of gene contents and numbers with reference strain KCTC11537.

<p>As assessed by RAST functional categories grouped among insertion/deletion genes, between the KRS-02083 and KRS-02109 <i>S. parauberis</i> genomes.</p

Genetic structure of the chromosomal region where the CRISPR/Cas system is integrated in KRS-02109.

<p>Genetic structure of the chromosomal region where the CRISPR/Cas system is integrated in KRS-02109.</p

Circular maps from NGS comparing the genomes of <i>S. parauberis</i> strains KRS-02083 and KRS-02109 with that of reference strain KCTC11537.

<p>Beginning with the outermost ring, ring 1 shows the CRISPR/Cas and Tn916 regions encoded in the KRS-20083 genome. Rings 2 and 3 show large contigs from 454 GS-FLX and pair-end reads from the Illumina GA alignment of KRS-02083. Rings 4 and 5 show large contigs from 454 GS-FLX and pair-end reads from Illumina GA alignment in KRS-02109. Ring 6 shows four phage-associated regions in the KRS-02083 genome. Rings 7 and 8 show the GC content and GC skew, respectively, with respect to reference strain KCTC11537. The inner circle shows the scale (bp).</p