Image_1_The Surface Layer Homology Domain-Containing Proteins of Alkaliphilic Bacillus pseudofirmus OF4 Play an Important Role in Alkaline Adaptation via Peptidoglycan Synthesis.PDF

<p>It is well known that the Na⁺ cycle and the cell wall are essential for alkaline adaptation of Na⁺-dependent alkaliphilic Bacillus species. In Bacillus pseudofirmus OF4, surface layer protein A (SlpA), the most abundant protein in the surface layer (S-layer) of the cell wall, is involved in alkaline adaptation, especially under low Na⁺ concentrations. The presence of a large number of genes that encode S-layer homology (SLH) domain-containing proteins has been suggested from the genome sequence of B. pseudofirmus OF4. However, other than SlpA, the functions of SLH domain-containing proteins are not well known. Therefore, a deletion mutant of the csaB gene, required for the retention of SLH domain-containing proteins on the cell wall, was constructed to investigate its physiological properties. The csaB mutant strain of B. pseudofirmus OF4 had a chained morphology and alkaline sensitivity even under a 230 mM Na⁺ concentration at which there is no growth difference between the parental strain and the slpA mutant strain. Ultra-thin section transmission electron microscopy showed that a csaB mutant strain lacked an S-layer part, and its peptidoglycan (PG) layer was disturbed. The slpA mutant strain also lacked an S-layer part, although its PG layer was not disturbed. These results suggested that the surface layer homology domain-containing proteins of B. pseudofirmus OF4 play an important role in alkaline adaptation via peptidoglycan synthesis.</p

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

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

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

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

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

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

Data_Sheet_1_Isolation and Cs+ resistance mechanism of Escherichia coli strain ZX-1.PDF

This research aims to elucidate the physiological mechanisms behind the accidental acquisition of high-concentration cesium ions (Cs+) tolerance of Escherichia coli and apply this understanding to develop bioremediation technologies. Bacterial Cs+ resistance has attracted attention, but its physiological mechanism remains largely unknown and poorly understood. In a prior study, we identified the Cs+/H+ antiporter TS_CshA in Microbacterium sp. TS-1, resistant to high Cs+ concentrations, exhibits a low Cs+ affinity with a Km value of 370 mM at pH 8.5. To enhance bioremediation efficacy, we conducted random mutagenesis of TS_cshA using Error-Prone PCR, aiming for higher-affinity mutants. The mutations were inserted downstream of the PBAD promoter in the pBAD24 vector, creating a mutant library. This was then transformed into E. coli-competent cells. As a result, we obtained a Cs+-resistant strain, ZX-1, capable of thriving in 400 mM CsCl—a concentration too high for ordinary E. coli. Unlike the parent strain Mach1™, which struggled in 300 mM CsCl, ZX-1 showed robust growth even in 700 mM CsCl. After 700 mM CsCl treatment, the 70S ribosome of Mach1™ collapsed, whereas ZX-1 and its derivative ΔZX-1/pBR322ΔAp remained stable. This means that the ribosomes of ZX-1 are more stable to high Cs+. The inverted membrane vesicles from strain ZX-1 showed an apparent Km value of 28.7 mM (pH 8.5) for Cs+/H+ antiport activity, indicating an approximately 12.9-fold increase in Cs+ affinity. Remarkably, the entire plasmid isolated from ZX-1, including the TS_cshA region, was mutation-free. Subsequent whole-genome analysis of ZX-1 identified multiple SNPs on the chromosome that differed from those in the parent strain. No mutations in transporter-related genes were identified in ZX-1. However, three mutations emerged as significant: genes encoding the ribosomal bS6 modification enzyme RimK, the phage lysis regulatory protein LysB, and the flagellar base component protein FlgG. These mutations are hypothesized to affect post-translational modifications, influencing the Km value of TS_CshA and accessory protein expression. This study unveils a novel Cs+ resistance mechanism in ZX-1, enhancing our understanding of Cs+ resistance and paving the way for developing technology to recover radioactive Cs+ from water using TS_CshA-expressing inverted membrane vesicles.</p

The Effect of DNA Adsorption on Optical Transitions in Single Walled Carbon Nanotubes

Photoluminescence (PL) from a single-walled carbon nanotube (SWNT) is sensitively influenced by molecular adsorption on the surface of SWNT, and is thus useful for analysis of molecular adsorption. We investigated PL spectra from DNA and SWNT hybrids and successfully obtained PL spectra from isolated DNA–SWNTs on a substrate under the dry condition. PL peak energies from single-stranded DNA–SWNT hybrids depended on the DNA base type, namely thymine, adenine, cytosine, and guanine. The base type dependence was attributed to the polarizability of the electronic charge distribution between the DNA base molecules and SWNT, which correlates to the adsorption energy of the DNA bases on the SWNT surface. The present results demonstrate that PL provides information on the DNA and SWNT binding