Novel Plasmids and Resistance Phenotypes in Yersinia pestis: Unique Plasmid Inventory of Strain Java 9 Mediates High Levels of Arsenic Resistance

Growing evidence suggests that the plasmid repertoire of Yersinia pestis is not restricted to the three classical virulence plasmids. The Java 9 strain of Y. pestis is a biovar Orientalis isolate obtained from a rat in Indonesia. Although it lacks the Y. pestis-specific plasmid pMT, which encodes the F1 capsule, it retains virulence in mouse and non-human primate animal models. While comparing diverse Y. pestis strains using subtractive hybridization, we identified sequences in Java 9 that were homologous to a Y. enterocolitica strain carrying the transposon Tn2502, which is known to encode arsenic resistance. Here we demonstrate that Java 9 exhibits high levels of arsenic and arsenite resistance mediated by a novel promiscuous class II transposon, named Tn2503. Arsenic resistance was self-transmissible from Java 9 to other Y. pestis strains via conjugation. Genomic analysis of the atypical plasmid inventory of Java 9 identified pCD and pPCP plasmids of atypical size and two previously uncharacterized cryptic plasmids. Unlike the Tn2502-mediated arsenic resistance encoded on the Y. enterocolitica virulence plasmid; the resistance loci in Java 9 are found on all four indigenous plasmids, including the two novel cryptic plasmids. This unique mobilome introduces more than 105 genes into the species gene pool. The majority of these are encoded by the two entirely novel self-transmissible plasmids, which show partial homology and synteny to other enterics. In contrast to the reductive evolution in Y. pestis, this study underlines the major impact of a dynamic mobilome and lateral acquisition in the genome evolution of the plague bacterium

Poly-γ-Glutamate Capsule-Degrading Enzyme Treatment Enhances Phagocytosis and Killing of Encapsulated Bacillus anthracis

The poly-γ-d-glutamic acid capsule confers antiphagocytic properties on Bacillus anthracis and is essential for virulence. In this study, we showed that CapD, a γ-polyglutamic acid depolymerase encoded on the B. anthracis capsule plasmid, degraded purified capsule and removed the capsule from the surface of anthrax bacilli. Treatment with CapD induced macrophage phagocytosis of encapsulated B. anthracis and enabled human neutrophils to kill encapsulated organisms. A second glutamylase, PghP, a γ-polyglutamic acid hydrolase encoded by Bacillus subtilis bacteriophage ΦNIT1, had minimal activity in degrading B. anthracis capsule, no effect on macrophage phagocytosis, and only minimal enhancement of neutrophil killing. Thus, the levels of both phagocytosis and killing corresponded to the degree of enzyme-mediated capsule degradation. The use of enzymes to degrade the capsule and enable phagocytic killing of B. anthracis offers a new approach to the therapy of anthrax

Genomic architecture of the conjugal transfer system.

<p>The conjugal transfer system on plasmids pJARS36 (<b>A</b>) and pJARS36 (<b>B</b>) share high homology and synteny with corresponding plasmid-borne loci in the insect inhabitant <i>A. culicicola</i> pAC3249A (<b>C</b>) and <i>E. coli</i> R6K (<b>D</b>). The scale in base-pairs indicates the respective genomic location of the plasmid–borne type IV systems. Genes shared between these loci are highlighted with similar colors.</p

Conjugal transfer plasmids.

<p>Circles from outer to inner circles for (<b>A</b>) pJARS35 and (<b>B</b>) pJARS36: (circles 1 and 2) predicted coding sequences on the plus (1) and minus strands (2), colored according to the respective MANATEE role IDs. (circle 3) GC-skew. (circle 4) Plasmid features. <i>Tn2503</i> insertion (red). (circles 5 to 9) Comparative plasmid analysis to <i>Y. enterocolitica</i> pYVe227-<i>Tn2502</i> (circle 5), pJARS36 (circle 6), <i>A. culicicola</i> pAC3249-TypeIV (circle 7) and the <i>E. coli</i> plasmids R721 (circle 8) and R6K (circle 9). Chi-square (circle 10).</p

Transposon architecture and coding capabilities.

<p>The <i>Y. enterocolitica</i> transposon pYVe227-<i>Tn2502</i> (<b>A</b>) shows homology to <i>Tn2503</i> carried on all four indigenous Java 9 plasmids (<b>B</b>). Comparison of the <i>Yersinia</i>-derived arsenic resistance transposons reveals a defect mobility loci in <i>Tn2502</i>. SNP discovery (<b>C</b>) identified the <i>arsH</i> gene as mutational hot spot. Genes shared between these loci are highlighted with similar colors., arsenic resistance and mobility loci are marked in blue and red. IIR, flanking imperfect inverted repeats (IIR). res, resolution site.</p

Target sites of transposon <i>Tn2503</i>.

<p>After excision of <i>Tn2503</i> at the target sites (black wedge), the plasmid states prior transposition for all four indigenous Java 9 plasmids were reconstructed to delineate the effects on neighboring genes. The transposon is found intergenic on pPCP disrupting the <i>IS100</i> sequence (<b>A</b>) and the conjugal transfer plasmids pJARS35 (<b>C</b>) and pJARS36 (<b>D</b>), while on pCD (<b>B</b>) insertion leads to truncated fertility inhibition protein FinO. The scale in base pairs indicates the respective genomic location of the <i>Tn2503</i> insertion loci. Genes shared between these loci are highlighted with similar colors.</p