C, cytoplasm; P, periplasm; a, strain N169-dtatABC; b, strain N16961; c, strain N169-dtatABC (pBAD24); d, strain N169-dtatABC-cp. Growth and morphology of the tatABC mutant The E. coli Tat system is required for the translocation of amidases, and tat mutants display impaired cell division and chain-forming phenotypes . We found that both the wild type strain and the tatABC mutant N169-dtatABC exhibited normal vibrioid
morphology (Fig. 4A and 4B), except that some of mutant cells showed the curved or contorted form. The chains of bacterial cells of the mutant were not observed. Therefore, the Tat protein translocation system did not seem to obviously affect the cell morphology of N16961. Under both aerobic and anaerobic conditions at 37°C, the mutant strain N169-dtatABC did not show any obvious growth deficiencies (data not shown); hence, the Tat protein translocation system did not seem to affect its growth and division. Figure 4 Phenotypes Liver X Receptor agonist of the tatABC mutant N169-dtatABC. A, Electron selleck chemicals llc micrograph of the wild type strain N16961 (×2400); B, Electron micrograph of the mutant N169-dtatABC (×2800); C, the motility of N169-dtatABC in 0.25% LBA, 37°C, 12 h; D, the motility of N16961 in 0.25% LBA, 37°C, 12 h; E and F, Smooth colonies of the wild type strain (E) and rugose colonies of the mutant N169-dtatABC
(F) in LBA after 16 days in room temperature. The magnified inset images show the single colonies. Like the wild type Morin Hydrate strain, the tatABC mutant colonies were smooth and moist in fresh LBA medium for the first 7 days at room temperature. Interestingly, in contrast to the wild type strain, some of N169-dtatABC colonies started to shift to the rugose (wrinkled) phenotype 7 days after inoculation at room temperature, and all the colonies of the mutant shifted to the rugose phenotype 16 days after inoculation, while colonies of the wild type strain were still smooth (Fig. 4E and 4F). Therefore, in contrast to the wild type strain, the tatABC mutant was easier to shift to the rugose phenotype at room temperature. Outer membrane
integrity assay To test the integrities of the outer membrane of V. cholerae tat mutants, we quantified the sensitivity of the mutants with respect to the hydrophobic drug Get and the detergent SDS, based on the concentration of Get or SDS that is required to kill 50% of the cells in liquid culture (LD50). LB without SDS or Get was used as the negative control. We compared the OD600 of the wild type strain and the mutant strains cultured in LB with different dilutions of SDS or Get, and did not find any changes of OD600 and LD50 when compared the wild type strain N16961 with the different tat gene mutants, therefore we did not find any integrity defect in the Tat mutants, including N169-dtatABCE, N169-dtatABC, N169-dtatB, N169-dtatC, and N169-dtatE (data not shown). Flagellum synthesis and motility It has been reported that tat mutants lose motility and their flagellum synthesis is impaired .