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

The pathway of nematicidal crystal proteins entering <i>M. hapla</i> J2.

<p>The confocal laser scanning microscope image showed the ingestion manner and process of Cry55Aa (A), Cry6Aa (B), or Cry5Ba (C) by <i>M. hapla</i> J2 in the presence of resorcinol (Res) or tomato root exudates (TRE). <i>M. hapla</i> J2 were incubated in rhodamine-labeled crystal toxins for three different times then imaged using a merged image. The rhodamine 6G (D) was treated as the same and used as a control. Toxin was detected inside the treated <i>M. hapla</i> J2, but not in the control (CK). The anterior of <i>M. hapla</i> is positioned within the upper region. Abbreviation: s = stylet; el = esophageal lumen; h = head of <i>M. hapla</i> J2. The scale bar of all the images is 40.43 µm.</p

The information of Cry55Aa, Cry6Aa, and Cry5Ba used in this study.

<p>The information of Cry55Aa, Cry6Aa, and Cry5Ba used in this study.</p

Detection of size changes of Cry55Aa, Cry6Aa, and Cry5Ba in <i>M. hapla</i> J2 by Western blot analysis.

<p><i>M. hapla</i> J2 were incubated with Cry55Aa protein (Panel A) and Cry6Aa protein (Panel B), and then detected by Western blot at 0, 12, 36, and 72 hpi, using an anti-crystal antibody; <i>M. hapla</i> J2 were incubated with Cry5Ba protein (Panel D, F) and then detected by Western blot at 0, 12, 22, 50, and 96 hpi, using Cry5Ba protein antibody. Line CK: Controls of crystal protein without being incubated by <i>M. hapla</i> J2. Panel C: SDS-PAGE showed the molecular mass of purified Cry5Ba. Panel E: the Cry5Ba was incubated by <i>M. hapla</i> J2 for 12 h, centrifuged at 12000 rpm for 10 min to remove <i>M. hapla</i> J2, and then detected by Western blot using Cry5Ba antibody.</p

Additional file 2 of Three novel leaderless bacteriocins have antimicrobial activity against gram-positive bacteria to serve as promising food biopreservative

Additional file 2: Fig. S2. Proposed primary structure of thucin A3 and LC–MS/MS analysis of fraction A3. Fragment ions are indicated. “*” indicates that the N-terminal amino acid, methionine, was formylated

Additional file 1 of Three novel leaderless bacteriocins have antimicrobial activity against gram-positive bacteria to serve as promising food biopreservative

Additional file 1: Fig. S1. Proposed primary structure of thucin A2 and LC–MS/MS analysis of fraction A2. Fragment ions are indicated. “*” indicates that the N-terminal amino acid, methionine, was formylated

Activities of rhodamine labeled Cry6Aa, Cry55Aa, and Cry5Ba against <i>M. hapla</i> J2.

<p><i>M. hapla</i> J2 were exposed to three doses of non-labeled crystal protein or rhodamine labeled Cry6Aa (A), Cry55Aa (B), Cry5Ba (C), and rhodamine-6G (D) in the presence of resorcinol. <i>M. hapla</i> J2 were exposed to three doses of crystal protein. Data shown represent the percentage of animals that were intoxicated when fed crystal proteins or rhodamine labeled crystal protein. Each data point represents the average size of 60 animals. Error bars represent the S.D. from the mean of averages over three independent experiments.</p