Figure 3

Tle1^AHis a phospholipase effector secreted by T6SS ofA. hydrophilaNJ-35. A T6SS-dependent secretion of Tle1^AH was confirmed by Western blot on whole cells and supernatants of A. hydrophila NJ-35 and the ∆clpV strain. ClpV, which encodes a putative ATPase required for T6SS function, was deleted to construct the T6SS⁻ strain (∆clpV). The anti-His antibody was used to measure the production of Tle1^AH and anti-GroEL antibody served as an internal reference. GroEL: heat shock protein Hsp60. B Growth of E. coli TOP10 producing peri-Tle1^AH and peri-Tle1^AHS303A in LB broth. pBAD/His was used for construction of the expression vectors for tle1^AH and its point mutant tle1^AHS303A (the catalysis site of Tle1^AH at position 303 mutated from serine to alanine). To achieve periplasmic localization, the PelB leader sequence was fused in front of the tle1^AH and tle1^AHS303A. Cultures were induced by l-arabinose (l-Ara) at the indicated time by the arrow. A growth curve was drawn by measuring the OD₆₀₀ every 30 min. Data are presented as the mean ± standard deviation (error bars) of three independent experiments. The expression of peri-Tle1^AH and peri-Tle1^AHS303A was detected in E. coli TOP10 by Western blot using anti-His antibody.