FIG 2.

LptA41 mutations and lptA41 suppressor phenotypes. (A) Ribbon diagram of E. coli LptA. Residues mutated by site-directed mutagenesis (I36, I38, R76, and K83) are indicated in red. The intragenic suppressor (lptA42 quintuple-mutant allele) encodes an additional amino acid change at the position indicated in green (M112). (B) OM permeability assay of the lptA41 strain and suppressor mutants. Serial 10-fold dilutions of stationary-phase cultures of AM604 (wild-type reference strain); PS001 (wild-type lptA control strain ectopically expressing lptAB); PS003 (ectopically expressing lptA41 lptB); and PS101, PS102, and PS103 (PS003 derivative suppressor mutants) were replicated on LD (for AM604) or LD-ampicillin agar plates supplemented with bacitracin (50 μg/ml), novobiocin (10 μg/ml), and rifampin (2.5 μg/ml), as indicated. (C) Cell lysates from AM604, PS001, PS003, PS101, PS102, and PS103 were analyzed by Western blotting with anti-LptA and anti-LPS antibodies as described in Materials and Methods. Culture samples with equal OD₆₀₀ values were processed and loaded into each lane. The different electrophoretic mobility of LptA41 relative to LptA can be explained by the different net charge of the mutant protein. A nonspecific band was used as a loading control (LC).