Figure 5

Identification of the functional replication region within the three plasmids. (A) Schematic diagram of the L. interrogans–E. coli shuttle vectors derived from pGKBLe24. KmR, kanamycin resistance cassette. Unique restriction sites used for shuttle vector construction are indicated. pGK-lcpL and pGK-lcpS are shuttle vectors containing the predicted parA-parB-rep and rep gene alone, respectively. (B) PCR-verified transformants. PCR amplification was performed using genomic DNA isolated from transformants that were picked from plates and enriched in liquid EMJH. (C) Lung lesion from a hamster infected with pathogenic L. interrogans transformants harboring the shuttle vectors. (D) PCR verification of pathogenic L. interrogans transformants recovered from liver. Primers are listed in Additional file 1: Table S3. Patoc I-lcp3L/S represents the confirmation of vectors lcp3L/S transformed into Patoc I strains. The same as for Patoc I-lcp2L/S, Patoc I- pGKBLe24, 56610-lcp1L, 56610-lcp3S, 56606-lcp3L, 56601-lcp1L, 56601-lcp3L. P-lcp3L is the PCR product from plasmid primers amplifying the fragment including the inserts. The same as for P-lcp3S, P-lcp2L, P-lcp2S, P-orf5, P-lcp1L. ilvA, primers for the specific gene LEPBI_I1590 in L. biflexa serovar Patoc strain Patoc 1; heb, primers for the O-antigen-specific gene heb from the L. interrogans serogroup Hebdomadis strain 56610; aut, primers for the O-antigen-specific gene aut from the L. interrogans serogroup Autumnalis strain 56606; ict, primers for the O-antigen-specific gene ict from the L. interrogans serogroup Icterohaemorrhagiae strain 56601; lipL32, primers for the L. interrogans lipoprotein gene lipL32 [56] of strain 56610, 56606 and 56601.