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Fig. 5. PCR protocols for the detection of multiple inversions in the SusC cluster 1 on the BF strain YCH46 chromosome (region 17 in Table 6). As shown (Top), this region contains three IRs: IR-1 (black arrowheads), IR-2 (red arrowheads), and IR-3 (blue arrowheads). Three segments of 2.4, 7.8, and 14 kb flanked by each IR can be inverted independently. By the combination of the three inversions, seven kinds of genome organizations can be generated in this locus. To analyze the genome organization of the locus, we prepared eight PCR primers (17-1F, -2F, -3F, -4F, -1R, -2R, -3R, and -4R) as indicated by arrows. When no inversion occurs (i.e., the genome organization is the same as the final sequence assembly), primer pairs of 17-1F + 17-4R, 17-2F + 17-3R, 17-3F + 17-2R, and 17-4F + 17-1R are predicted to produce 1,030-, 690-, 620-, and 960-bp fragments, respectively. Note that primers 17-1F and 17-1R anneal to noninvertible regions. Using these primers, all possible combinations of inversion events presented can be detected. For example, 17-3F and 17-4F are oriented to the same direction before inversion, but to the face-to-face orientation after inversion 1 to produce a 737-bp fragment. Primer pairs that yield PCR products specific to each inversion event are indicated by red letters. Expected sizes of these PCR products are also indicated.

Fig. 6. Gene duplication in BF, BT, and PG. Gene repertoires and the patterns of duplication in the genomes of BF strain YCH46, BT strain VPI-5482, and PG strain W83 were compared. Total numbers of nonduplicated or duplicated genes in each species are indicated above the bars. Numbers of paralogous gene families identified in each genome are indicated in parentheses.

Fig. 7. Functional distribution of nonduplicated and duplicated genes in BF strain YCH46, BT strain VPI-5482, and PG strain W83. Gene functions were classified according to the COG classification (1): C, energy production and conversion; D, cell division and chromosome partitioning; E, amino acid transport and metabolism; F, nucleotide transport and metabolism; G, carbohydrate transport and metabolism; H, coenzyme metabolism; I, lipid metabolism; J, translation, ribosomal structure, and biogenesis; K, transcription; L, DNA replication, recombination, and repair; M, cell-envelope biogenesis, outer membrane; N, cell motility and secretion; O, posttranslational modification, protein turnover, chaperones; P, inorganic-ion transport and metabolism; Q, biosynthesis, transport, and catabolism of secondary metabolites; R, general function prediction only; S, function unknown; and T, signal transduction mechanisms.

1. Tatusov, R. L., Natale, D. A., Garkavtsev, I. V., Tatusova, T. A., Shankavaram, U. T., Rao, B. S., Kiryutin, B., Galperin, M. Y., Fedorova, N. D. & Koonin, E. V. (2001) Nucleic Acids Res. 29, 22-28.

Fig. 8. Genomewide distribution of invertible regions on the Bacteroides genomes. Invertible regions were classified into six classes according to the consensus motif within IRs and plotted on the genome maps. Invertible regions colocalized with site-specific recombinases are indicated by open circles. Details are available in Tables 6 and 7.

Fig. 9. Demonstration of DNA inversions at invertible regions (other than the seven PS loci) identified on the BF strain YCH46 genome. (A) The annealing positions of the PCR primers for detecting DNA inversion. PCR protocol F amplifies a genomic DNA segment of the same orientation as the final sequence assembly. PCR protocol R amplifies a segment generated by DNA inversion via the IRs (indicated by blue arrowheads). (B) Agarose gel electrophoresis analyses of the PCR products yielded from each invertible region by two different PCR protocols (indicated by F or R above each lane). Region numbers correspond to those described in Table 6. Information for the primer sequences and the predicted sizes of each PCR product are available in Tables 1 and 2, respectively. M, 100-bp DNA ladder markers. Data for PCR analyses of three shufflon-like invertible regions (regions 17, 30, and 31) are shown in Figs. 3, 10, and 11, respectively.

Fig. 10. Multiple DNA inversions in the SusC/D cluster 2 (region 30 in Table 6). (A) The gene organization of the SusC/D cluster 2 and locations of the PCR primers used to detect DNA inversions. The function of each gene is distinguished by color: blue, SusC homologs; green, a SusD homolog; and pink, a tyrosine type site-specific recombinase. There are three repeat sequences of 26 bp (indicated by orange arrowheads) constituting two pairs of IRs in this locus. Each repeat sequence contains a class V internal consensus motif that forms a stem-loop structure (a 9-bp stem and a 1-bp loop). In the genome sequence obtained as a final assembly, the Bacteroides consensus promoter sequence (1) (indicated by an open triangle) was found only in the upstream region of BF0666. In addition, among the three SusC homologs existing in this locus, only BF0666 has an N-terminal signal sequence (indicated by a hatched box). Two types of DNA inversions via the two IRs, however, deliver the promoter and the coding region for N-terminal signal sequence to other SusC homologs (BF0668 or BF0670). A tyrosine type site-specific recombinase (BF0667) probably mediates the DNA inversions in this locus. (B) Detection of DNA inversions by PCR analyses. PCR products generated by various primer combinations were analyzed by agarose gel electrophoresis. Primer pair J2-K1 yielded a 368-bp fragment that was presumably derived from the excised and circularized segment 1. This excision event, which occurs probably by the homologous recombination between the internal consensus sequences of the IR, results in the all-off phenotype for SusC homologs in this locus. However, the amplification of a 422-bp chromosome segment that should have been generated by the excision of segment 1 was not detected by the primer pair, J1-K2, indicating that this event occurs at a very low frequency. Nucleotide sequences of each PCR primer and predicted sizes of each PCR product are available in Tables 1 and 2, respectively.

1. Bayley, D. P., Rocha, E. R. & Smith, C. J. (2000) FEMS Microbiol. Lett. 193, 149-154.

Fig. 11. Multiple inversions in the SusC/D cluster 3 (region 31 in Table 6). (A) The gene organization of the SusC/D cluster 3 and locations of the PCR primers used to detect DNA inversions. The function of each gene is distinguished by color: blue, SusC homologs; green, SusD homologs; pink, a -L-fucosidase; and light blue, hypothetical or conserved hypothetical proteins. Open triangles indicate the locations and orientations of Bacteroides consensus promoter sequences (1), and the pink arrowheads indicate those of IRs. Four types of DNA inversions occur in this locus via these IRs. (B) Detection of DNA inversions by PCR analyses. PCR products generated by various primer combinations were analyzed by agarose gel electrophoresis. Nucleotide sequences of each PCR primer are available in Table 1. Expected sizes of each PCR product indicated by asterisks and possible mechanisms generating these products are described in Table 3. M, lambda HindIII-digest markers.

1. Bayley, D. P., Rocha, E. R. & Smith, C. J. (2000) FEMS Microbiol. Lett. 193, 149-154.