Results: 4

1.
Figure 4

Figure 4. Schematic Representation of V. cholerae Serogroup Conversion by Chitin-Induced Transformation and Phage-Mediated Selection. From: Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs.

V. cholerae serogroups O1 (I) and O139 (II) are shown as green and red bacteria, respectively. They differ in the size and composition of the O-antigen moiety of LPS and by the presence of a polysaccharide capsule on the surface of the O139 serogroup. These differences are encoded by the O-antigen–specific gene cluster depicted below each bacterium. Black arrows (I, II, and VIII) denote the location of homologous genes present in both serogroups (for detail, see Figure 1). V. cholerae O1, grown on a chitin surface, becomes competent for transformation (III). Free DNA from the O139 donor strain (IV) is taken up (V) and integrated into the chromosome by homologous recombination (VI). Exchange of the O1 gene cluster for the O139 cluster leads to serogroup conversion. The transformed recipient produces the O139 O antigen and CPS (VIII) and becomes resistant to O1-specific bacteriophages (VII).

Melanie Blokesch, et al. PLoS Pathog. 2007 June;3(6):e81.
2.
Figure 1

Figure 1. Comparison of the O-Antigen–Encoding DNA Region (wb*) of Strain A1552 (O1) and MO10 (O139). From: Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs.

The genetic organization of the O1-specific and O139-specific DNA clusters on chromosome I of V. cholerae are shown (adapted from Heidelberg et al. [31], Stoeher et al. [28], Yamasaki et al. [33], Chatterjee and Chaudhuri [30], and the whole genome shotgun sequence of V. cholerae MO10). Genes and their orientation are denoted by arrows. Gene color code: green, O1-specific; red, O139-specific; black, homologous genes. The blue arrow depicts the KanR cassette (aph gene) integrated between wbfA and wbfB in strain VCO139-Kan. The bars below show the minimum size of the exchanged DNA fragments. The asterisks mark the 12 O139-specific genes present on the microarrays. PCR primers for the O139 region were designed to obtain the indicated O139-specific PCR fragments 1 through 8 shown at the bottom. ORFs: 0848 = VchoM_01000848 (homolog to VC0239), 0822 = VchoM_01000822 (homolog to VC0265), 0821 = VchoM_01000821 (homolog to VC0267), 0820 = VchoM_01000820 (homolog to VC0268), 0819 = VchoM_01000819 (homolog to VC0271).

Melanie Blokesch, et al. PLoS Pathog. 2007 June;3(6):e81.
3.
Figure 2

Figure 2. PCR and CGH Studies of Donor and Transformant gDNA Show Exchange of O-Antigen–Encoding Gene Clusters. From: Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs.

(A) gDNA from acceptor strain A1552 (O1), donor strain VCO139-Kan (O139 with KanR marker), and a representative transformant were used as template in a PCR experiment. Lanes: PCR fragments 1 to 8 spanning the whole O139-antigen–encoding region according to Figure 1.
(B) CGH data. CGH was used to compare genes present in the V. cholerae O1 A1552 acceptor strain (labeled with Cy3) with genes absent/present in the donor strain VCO139-Kan (lane 1; labeled with Cy5) or two representative transformants of the acceptor strain (lanes 2 and 3; labeled with Cy5). Oligonucleotides corresponding to genes of the large chromosome (Chr I) and small chromosome (Chr II) of the sequenced strain N16961 [31] were spotted on the microarray (depicted as N16961) in addition to oligonucleotides derived from 12 O139-specific genes and from 82 genes corresponding to the SXT element (denoted as non-N16961; for details see Figure S5).
(C and D) Close-up of the O139-antigen–specific genes (C) and of the O1-antigen–specific gene cluster (D).

Melanie Blokesch, et al. PLoS Pathog. 2007 June;3(6):e81.
4.
Figure 3

Figure 3. O1-to-O139 Transformants Produce O139 LPS and Antigenic Determinant and Are Encapsulated. From: Serogroup Conversion of Vibrio cholerae in Aquatic Reservoirs.

The acceptor strain A1552 (lane 1), the donor strain VCO139-Kan (lane 2), and a representative transformant (lane 3) were analyzed for LPS structure and for the presence of CPS and O-antigenic determinants.
(A) LPS analysis. Purified LPS was subjected to SDS-PAGE (15% gel) and stained with silver. Specific signals, by arrows, depict the lipid A-core (I), the lipidA-core-O139 semi-rough LPS (II), and the lipidA-core-O1 LPS (III). The O139 parental strain MO10 [21] is shown in lane C.
(B) O139-specific immunoblot analysis. Cell-lysates were separated in a 15% SDS gel, transferred onto a polyvinylidene fluoride membrane, and reacted with α-O139 polyclonal antibodies. Capsule-specific bands (SM, slowly migrating; MM, medium migrating) and O-antigen–specific bands (RM, rapidly migrating [14]) are indicated by arrows.
(C) Detection of the CPS by electron microscopy. Cells were treated with polycationic ferritin and the CPS then visualized by transmission electron microscopy. The location of the capsule is indicated by arrows. Scale bar: 0.1 μm.

Melanie Blokesch, et al. PLoS Pathog. 2007 June;3(6):e81.

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