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. 2014 Aug;196(16):2921–2933. doi: 10.1128/JB.01747-14

FIG 1.

FIG 1

Effects of large deletions within pXO1 on plasmid stability. (A) The pXO1 structure with the minireplicon (MR), type III plasmid partition system (type III PPS), and pathogenicity island (PAI) indicated. The MR structure is adopted from reference 1 and shown with the Ω-km cassette inserted into pXO1 GBAA_pXO1_0022 to produce pXO1K. The type III PPS structure is adopted from reference 15 and includes the newly described tubY gene, important for functionality of the type III PPS. The PAI structure with indication of IS1627 insertion sequences and B. anthracis toxin components was prepared according to the complete sequence of Ames Ancestor plasmid pXO1 (NCBI reference sequence NC_007322.2). The numbers located below and on both sides of pXO1 represent nucleotide numbers in the complete sequence. The GBAA_pXO1_ symbols along the top of pXO1 and along the bottom line are locus tag numbers for the Ames Ancestor plasmid pXO1. The pXO1 regions deleted in each construct are shown in parentheses following the Greek letter delta (the numbers are for the ORFs of the area deleted). Orientations of loxP sites that replaced the deleted region are indicated by arrows. The numbers located below the deletions (dashed lines) are the first and last base pairs included in the deletions. (B) PCR verification of the deletions. Primers used to verify retention of specific segments are indicated at the top of the gel. Lanes: 1, Ames 35K Δ(29–190); 2, Ames 35K Δ(29–133); 3, Ames 35K Δ(137–17); 4, Ames 35K Δ(89–17); 5, Ames 35K Δ(85–19); and 6, Ames 35K Δ(79–17). Mr is a GeneRuler DNA ladder mix for size determination (e.g., 1,500 bp). (C) Percentage of kanamycin-resistant (i.e., plasmid-containing) bacteria in cultures of B. anthracis Ames 35 containing either pXO1K or pXO1K with deletions produced in pXO1. Bacteria were subcultured every 12 h on LB agar without kanamycin and grown at 37°C. Bacteria from each subculture were diluted and plated on LB agar with and without kanamycin (20 μg/ml) to determine the fraction retaining the plasmids. Maximum root mean square deviations in the percentage of kanamycin-resistant colonies did not exceed 10%. (D) Percentage of kanamycin-resistant (i.e., plasmid-containing) bacteria in cultures of B. anthracis Ames 35 containing pXO1K with deletions extending clockwise around the circular plasmid from bp 181677 (and ORF 217) past bp 1. Maximum root mean square deviations in the percentage of kanamycin-resistant colonies did not exceed 10%.