(A) Schematic representation of the Flag-2 gene cluster in E. coli 042. The 48.8-kb region between nucleotide positions 281000 and 329800 from the finished genome of E. coli 042 is indicated by a solid black line. GLIMMER-predicted CDSs are indicated by arrows approximately to scale and colored according to operon and function. Flag-2 genes are designated according to the scheme proposed in this paper. Nt, nucleotides. (B) Schematic representation of the Flag-2 gene clusters of other bacteria. The solid black lines indicate the genome fragments in E. coli K-12 strain MG1655 (11.4 kb), S. enterica serovar Typhi Ty2 (4.9 kb), V. parahaemolyticus (11.7-kb region 1 and 23.2-kb region 2 from chromosome 2), Y. pestis CO92 (44.4 kb), and Y. pestis KIM (28.5 kb) that are equivalent to the lateral flagellar cluster of E. coli 042. E. coli K-12 and S. enterica serovar Typhi Ty2 are representative of other sequenced E. coli strains and S. enterica strains, respectively (data not shown). The dotted lines indicate deletions or missing genes relative to the E. coli 042 Flag-2 gene cluster. CDSs are indicated by arrows colored according to the E. coli 042 scheme shown in panel A. CDSs that are insertions (IS) relative to the E. coli 042 genome are indicated by arrows below the backbone. (C) Tiling path PCR used to map the Flag-2 gene cluster of E. coli strains. Primers and their orientations are indicated by small triangles. The red triangles indicate the lfhA-lafU primers used to confirm the absence of the lateral flagellar cluster in E. coli strains. The orange and blue triangles indicate the lfhAB and lafTU primers, respectively, that were used to confirm the presence of genes between lfhA and lafU. E. coli strains suspected of harboring a Flag-2 gene cluster were further analyzed with primer sets 1 to 8 (lines with triangles at each end) by using long-range PCR to determine the tiling path. ECOR strains that were found to harbor the Flag-2 gene cluster could be divided into four types, type 1 (ECOR4, -49, and -50), type 2 (ECOR-1, -3, -5, -12, -17, -24, -64, -65, -67), type 3 (ECOR-35 and -36), and type 4 (ECOR48), according to the tiling path. Successful long-range PCRs of fragments 1 to 8 (approximately 5 kb each) are indicated by solid black lines. Unsuccessful PCRs are indicated by dotted lines. The red lines indicate cases in which long-range PCR with complementary primers from adjacent successful amplifications was used to span regions that could not be amplified.