Figure 4.

Targeted allelic exchange mutagenesis to restore Ech_0379 gene. Panel A is similar to Fig. 2 except that the illustration depicting the genomic segment at the top portion of the panel represents the genome from Ech_0379 mutant. (B) Ech_0379 gene restoration mutant culture expressing mCherry. The restored mutant organisms cultured in ISE6 cells were assessed for the mCherry expression by confocal microscopy using 40x magnification lens. Panels C, D, and E are similar to Fig. 2 panels B, C and D, respectively, except that the inserted sequence in the gene disruption mutant is identified in yellow arrowhead line (panel D) and restriction enzyme and probe used for the Southern blot experiment (panel E) were Cla I and a DNA segment representing Ech_0379 gene, respectively. In panel E, the lanes M₁ and M₂ represent data from genomic DNA recovered from the E. chaffeensis Ech_0379 mutant culture recovered from DH82 and ISE6 culture, respectively. Similarly, R₁ and R₂ represent data from genomic DNA recovered from the E. chaffeensis Ech_0379 reverted mutant culture recovered from DH82 and ISE6 culture, respectively. (Full-length gels and blots were included in the Supplementary Figure file, as parts of the Figure had cropped images).