Strains and plasmids used in this study.

R<p>resistant; ^Ssensitive;</p>*<p>Bacillus Genetic Stock Center.</p

Cadmium and arsenate resistance assays.

<p>Cadmium resistance assay (A) was performed in LB medium and arsenate resistance assay (B) was performed in low phosphate medium containing increasing concentrations of Cd²⁺ (A) or As⁵⁺ (B). Bacterial growth was measured by optical density at 595 nm after 20 h of growth at 30°C. Each data point represents the mean value of triplicate cultures. The error bars indicate the standard deviation values. OD₅₉₅, optical density at 595 nm.</p

Physical and genetic map of plasmid pGIAK1.

<p>The map was visualized in CGView <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072461#pone.0072461-Stothard1" target="_blank">[44]</a>. The outer circles indicate the predicted CDS. The blue and light green block arrows show the predicted genes with different transcription directions. The numbers beside the arrows indicate the corresponding genes. The black circle represents the GC content and the green/purple circles represent the GC-skew. The predicted genes involved in replication, cadmium resistance, arsenic resistance and conjugation are indicated. The single cloning site <i>Bam</i>HI is also indicated.</p

Comparison of pGIAK1 and the heavy metal resistant plasmid pWCFS103 from <i>L. plantarum</i>.

<p>The displayed bar represents a length of 5,000 basepairs. The conserved regions are blue-shaded, the color intensity indicating the identity levels (from 35 to 100%). Genes that displayed homologies are indicated by the colored boxes shown below. The alignment was made by the Easyfig 2.1 program <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0072461#pone.0072461-Sullivan1" target="_blank">[49]</a><b>.</b></p

Cells and plasmids of strain JMAK1.

<p>(A) Bacterial cells were observed under a BX50 phase contrast microscope (Olympus, Japan). V, vegetative cell; S, spore. The bar is 5 µm. (B) Plasmid profile of strain JMAK1. The plasmid preparation was run in a 0.5% agarose gel and stained with Ethidium Bromide. The chromosomal DNA referred to the degraded linear chromosomal material. Lane 1, <i>B. thuringiensis</i> serovar <i>israelensis</i> AND508; Lane 2, JMAK1.</p

Cloning and Analysis of a Large Plasmid pBMB165 from <i>Bacillus thuringiensis</i> Revealed a Novel Plasmid Organization

<div><p>In this study, we report a rapid cloning strategy for large native plasmids via a contig linkage map by BAC libraries. Using this method, we cloned a large plasmid pBMB165 from <i>Bacillus thuringiensis</i> serovar <i>tenebrionis</i> strain YBT-1765. Complete sequencing showed that pBMB165 is 77,627 bp long with a GC-content of 35.36%, and contains 103 open reading frames (ORFs). Sequence analysis and comparison reveals that pBMB165 represents a novel plasmid organization: it mainly consists of a pXO2-like replicon and mobile genetic elements (an inducible prophage BMBTP3 and a set of transposable elements). This is the first description of this plasmid organization pattern, which may result from recombination events among the plasmid replicon, prophage and transposable elements. This plasmid organization reveals that the prophage BMBTP3 may use the plasmid replicon to maintain its genetic stability. Our results provide a new approach to understanding co-evolution between bacterial plasmids and bacteriophage.</p> </div

Comparison of pBMB165 and homologous plasmids and phages by Easyfig alignment.

<p>Coding Sequences (CDSs) are represented by colored arrows. Predicted functions/homologies are indicated by the color key featured below. The pXO2-like replicon is highlighted with a green frame. Color coding for the genes is as follows: olive green, plasmid replication; deep green, prophage replication; deep yellow, plasmid stabilization system; orange, regulatory; red, a predicted camelysin; blue, mobile DNA; purple, phage related; grey, hypothetical protein; midnight blue, conjugation-related proteins; wine, capsule synthesis related proteins; and brown, other determinants. Highly conserved segments of the plasmids and phages are paired by shaded regions, with the darker shading reflecting a greater amino acid identity, from 66% (A) or 63% (B) to 100%. The regions outside the shaded regions lack homology between plasmids and phages. The outer scale is marked in kilobases.</p

Identifying BMBTP3 among the total induced phage DNA from B.

<p><b><i>thuringiensis</i> strain YBT-1765</b>. <b>A</b>. Southern hybridization with a replication-associated protein gene specific probe (probe-rep). Lane 1, the total plasmid DNA extracted from YBT-1765; Lane 2, digested total plasmid DNA by <i>Hin</i>dIII; Lane 3, digested total induced phage DNA by <i>Hin</i>dIII; Lane 4, digested total plasmid DNA by <i>Hin</i>cII; Lane 5, digested total induced phage DNA by <i>Hin</i>cII; Lane 6, digested total plasmid DNA by <i>Hpa</i>I; Lane 7, digested total induced phage DNA by <i>Hpa</i>I. <b>B</b>. Southern hybridization with a phage terminase large subunit gene specific probe (probe-term). Lane 1, the total plasmid DNA extracted from YBT-1765; Lane 2, digested total plasmid DNA by <i>Hin</i>dIII; Lane 3, digested total induced phage DNA by <i>Hin</i>dIII; Lane 4, digested total plasmid DNA by <i>Eco</i>RV; Lane 5, digested total induced phage DNA by <i>Eco</i>RV. The sizes of the signal bands are labeled with arrows. In each lane for total plasmids and digested products we loaded 0.7 μg plasmid DNA (lanes 1, 2, 4, 6 in Figures 3A and 1, 2, 4 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081746#pone-0081746-g003" target="_blank">Figure 3B</a>), and for the purified phage DNA and digested products, we loaded 1.3 μg in each lane (lanes 3, 5, 7 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081746#pone-0081746-g003" target="_blank">Fig. 3A</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081746#pone-0081746-g003" target="_blank">3</a>, 5 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0081746#pone-0081746-g003" target="_blank">Fig. 3B</a>). <b>C</b>. The schematic drawing shows the structure of the restriction fragments with the ORF015 (probe-rep), ORF120 (probe-term) and the predicted cos site. The dashed line denotes the DNA of pBMB165, and the sizes of fragment digested by the restriction enzymes and the predicted cos site.</p

Circular representation of plasmid pBMB165 and graphical representation of the annotation and the structure of the prophage BMBTP3.

<p>The inner circle represents the GC bias [(G - C)/(G + C)], with positive and negative values in reddish brown and cobalt blue, respectively; the second circle represents the GC-content, with positive and negative values in grey and black, respectively; and the outer circle represents the predicted genes on the reverse and forward arrows. The pXO2-like replicon is highlighted with a green arching frame. Regions of transposon and prophage BMBTP3 are annotated beside the corresponding arrows and separated by straight lines. The main functional genes of BMBTP3 are annotated above the extended corresponding arrows at the bottom. Different structural and functional regions are annotated and separated by vertical lines. Color coding for the genes is as follows: olive green, plasmid replication; deep green, prophage replication; deep yellow, plasmid stabilization system; orange, regulatory; red, a predicted camelysin; blue, mobile DNA; purple, phage related; grey, hypothetical protein. The outer scale is marked in kilobases. </p

Effects of Solution Flow on the Growth of Colloidal Crystals

For the versatile potential applications of colloidal crystals, precisely controlling their growth is required to achieve properties such as high crystallinity and large-area crystals. Because colloidal crystallization is a self-assembly process of dispersed particles in a solution, solution flow directly and markedly changes the behavior of particles. Thus, the effects of solution flow on the growth of colloidal crystals were investigated in the present study. We found three different effects of solution flow on the growth of colloidal crystals: enlarging the first layer, facilitating the growth of superlattice structures, and forming a new circular packing structure. Specifically, in the single-component system, because the flow speed is lower closer to the bottom of the cell, the second and further layers dissolve owing to the large flow speed, whereas the first layer remains undissolved at the appropriate flow speed. The dissolved particles (particles that are detached from the crystals and returned back into the aqueous medium) are transported near the first layer, where they facilitate the growth of the first layer. In a binary system, when colloidal crystals with different particles are neighboring each other, the flow dissolves the surface of each crystal, which forms a dense, melt-like phase between crystals, from which a superlattice structure such as AB2 grows. The flow often moves the second layer more than the first layer because the flow speed varies with the distance from the bottom. This causes the second layer to slide above the first layer of the neighboring crystals composed of different particle sizes, which transform from the initial face-centered cubic structure of the first layer into a circular pattern with strain. These findings contribute to establishing a sophisticated control method for growing colloidal crystals