Figure 3

CSK29544_02616 influences the structural composition of the outer membrane. (a) Comparison of outer membrane protein profiles between WT (lane 1) and ΔCSK29544_02616 (lane 2) strains. Outer membrane fractions equivalent to 10 μg of proteins were analyzed by SDS-PAGE in parallel with molecular-weight (MW) size markers (M). Arrowheads indicate proteins with different abundance between the two strains. (b) LPS profiles in DOC-PAGE. LPS fractions were extracted by hot phenol-water method, separated on DOC-PAGE, and compared between WT (lane 1) and ΔCSK29544_02616 mutant (lane 2) strains. Lane C shows LPS from Salmonella Typhimurium LT2 as a positive control. (c) Increases in PL production in the absence of CSK29544_02616. Membrane PLs were extracted from equivalent bacterial cells of WT (lane 1) and CSK29544_02616 (lane 2) strains and separated by TLC analysis (left). Each constituent of membrane PL, separated by TLC analysis, and total membrane PLs before TLC analysis were quantified in duplicate using malachite green (right). Lane S indicates purified standard PLs, which were used to identify spots of each PL species. (d) Abolished motility caused by the loss of CSK29544_02616. C. sakazakii strains including WT, ΔCSK29544_02616, and the mutant complemented with pSK02 were injected into semi-solid agar plates (left panel). Plasmid pSK02 was designed to produce His-CSK29544_02616 protein using an arabinose-inducible promoter. Bacterial morphologies of WT and ΔCSK29544_02616 strains were analyzed by transmission electron microscopy. Arrowheads indicate flagella (right panel). Full-length images were represented in Supplementary Figure S10.