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Fig. 4. Identification of orthologous ORF pairs on linear plasmid lp28-2. NUCMER (MUMMER 2.12 package; ref. 1) was used to identify the N40 scaffold (A) that uniquely matches a B31 scaffold (B). Such matches were considered orthologous scaffolds. We subsequently identified long (>250 nt) ORFs (forward in red, reverse in green) by using GLIMMER 2.13 (2) on each of the genome scaffolds. ORFs in syntenic positions between the two scaffolds are identified as orthologous pairs. The B31 finished annotation (C) is drawn at the bottom for reference. Such a comparison also reveals ORF fusions or splits, such as N40 ORF21 and ORF22, which are present as one larger ORF (BBG32) in B31.

1. Delcher, A. L., Phillippy, A., Carlton, J. & Salzberg, S. L. (2002) Nucleic Acids Res. 30, 2478–2483.

2. Delcher, A. L., Harmon, D., Kasif, S., White, O. & Salzberg, S. L. (1999) Nucleic Acids Res. 27, 4636–4641.

Fig. 5. Distributions of nucleotide (A) and amino acid (B) sequence differences between orthologous ORF pairs from B31 and JD1. The distributions (gray bars) were tested against Poisson expectations (black dots) based on the assumption of neutral nucleotide and amino acid substitutions. We selected high-density nucleotide polymorphisms [HDNPs, ORFs having significantly (P < 0.001) more nucleotide sequence differences than expected] as markers for multilocus sequence typing.

Fig. 6. Examples of recombination identified on the main chromosome. (A) An unrooted phylogeny of B31, JD1 and N40. A nucleotide substitution (red bars) on each lineage (branches) generates a SNP belonging to one of the three phylogenetic partitions (n, number of SNPs in each partition). Significant clustering of variable sites (thin vertical black lines) of the same partition (red boxes in panels B, C, and D) is considered evidence for recombination (1). (B) Nucleotide polymorphisms in BB0032. The 5' and 3' sections contain significant clusters of same-partition substitutions, showing distinctly different evolutionary origins of the two parts. (C) Nucleotide polymorphisms in BB0081-BB0084. The region contains significant clustering of all three types of substitutions. The BB0082 and the 5' section of BB0084 are introduced into N40 by recombination, as verified by the subsequent MLST studies. (D) Nucleotide polymorphisms in BB0833. All substitutions are derived on one lineage and the region contains significant runs of unvaried sites (Stephen’s test looks for significant runs of single-partition polymorphisms as well as runs of unvaried sites, both evidence for recombination).

1. Stephens, J. C. (1985) Mol. Biol. Evol. 2, 539–556.

Fig. 7. Two unrooted HDNP gene trees. Trees and branch support are inferred by using MrBayes (1). (A) BBC02 on cp9. Three major-group alleles (B31 lineage in red, N40 lineage in blue, and JD1 lineage in green) were identified based on deep branches on the tree. (B) BBJ19 on lp38. There are also three deep-branching alleles based on this tree. Isolates grouped as B31 (red) lineage at BBC02 split into two BBJ19 lineages, as do the isolates grouped as a single N40 (blue) lineage at BBC02. Such incongruent gene genealogies are indicative of recombination between the two loci, likely by way plasmid exchange. More gene trees are available from the authors.

1. Huelsenbeck, J. P. & Ronquist, F. (2001) Bioinformatics 17, 754–755.

Fig. 8. Nucleotide polymorphisms at BB0082. Isolates sharing an ospC type are represented as horizontal lines shaded with the same color. For each isolate, polymorphisms relative to the B31 sequence are plotted as tick marks: A, green; T, red; C, blue; G, black. The SNP at site 17 occurs in multiple clonal complexes, most likely caused by recombination. The SNP at site 823 is unique and likely a recent mutation within the ospC G clonal complex. A recombination to mutation ratio of 3:1 were observed at this locus using the methods of Guttman and Dykhuizen (1) and Feil et al. (2).

1. Guttman, D. S. & Dykhuizen, D. E. (1994) Science 266, 1380–1383.

2. Feil, E. J., Smith, J. M., Enright, M. C. & Spratt, B. G. (2000) Genetics 154, 1439–1450.