(A) Agarose gel electrophoresis of alkaline lysis preparation from H:z66+ and H:z66− strains. Lanes 1 and 9, E. coli 39R861; lane 2, S. Typhi 404Ty; lane 3, S. Typhi In20; lane 4, S. Typhi SGB32 (pBSSB2); lane 5, S. Typhi Ty2; lane 6, S. Typhi CT18; lane 7, E. coli SGB33 (pBSSB2), and lane 8, E. coli TOP10.
(B) Southern blot of transferred DNA from (A). The transferred DNA was probed with a labelled PCR amplicon of the fljB^z66 gene and amplified using primers z66flag_F and z66flag_R (Table S2). Lanes are as shown in (A) and fragment sizes are estimated in comparison to (A).

(A) Plot of percentage GC content of pBSSB1, ranging from 24% to 49%, with an average of 36.6%.
(B) Plot of GC skew ((G−C)/(G+C)), ranging from −0.08 to 0.14 and averaging 0.03.
(C) CDS map of pBSSB1, consisting of the forward (upper) and the reverse (lower) DNA strand. The CDSs are identified with sequential numbers. The previously sequenced genes of Huang et al. [11] (030, fljB^z66, and fljA) are labelled yellow. The tirs are labelled in blue, CDSs with similarity to previously described sequences are coloured red, and CDSs with no similarity to previous sequences are coloured green. *, the change in coding bias associated with a possible origin of replication. †, the location of the kanamycin resistance gene insertion.
(D) Size scale in 2-kbp fragments of pBSSB1 (27,037 bp). Endonuclease cut sites are labelled for XbaI (2,708 bp), PmeI (7,704 and 21,995 bp), SacI (18,611 and 23,778 bp), and SpeI (11,137 bp); additionally, the cut sites located nearest the tirs are labelled for EcoRV (1,279 and 25,255 bp) and SspI (1,715 and 25,799 bp).

(A) Southern blot of transferred digested genomic DNA from S. Typhi 404Ty, probed with purified and labelled pBSSB1 plasmid DNA. Sizes are estimated with respect to Hyperladder I, as measured prior to transfer. Predicted digestion sites from the DNA sequence are highlighted in Figure 2.
(B) Southern blot of the tir region of pBSSB1 using labelled DNA prepared from a PCR amplicon from S. Typhi 404Ty DNA (primers tir_F and tir_G, Table S2) as a probe against digested genomic DNA from S. Typhi 404Ty.

PFGE of agarose plugs prepared with E. coli SGB33 or pBSSB2 DNA. Lane 1, Hyperladder VI; lane 2, E. coli TOP10; lane 3, untreated E. coli SGB33; lane 4, pBSSB2 DNA treated with S1 nuclease; lane 5, pBSSB2 DNA treated with exonuclease III; lane 6, pBSSB2 DNA treated with lambda exonuclease.