Substrate Specificity Classes and the Recognition Signal for Salmonella Type III Flagellar Export

Multicopy effects of rod-type proteins. The results of assays for determination of the swarming ability of the wild-type strain SJW1103 transformed with pET22b-, pTrc99AFF4-, and pTrc99AET-based plasmids are shown. The individual panels correspond to plasmids encoding the proteins shown at the right. V, vector alone. (In the third column, pTrc99A, not pTrc99AET, was used). Cells were inoculated onto soft tryptone agar plates containing 0.1 mM IPTG and incubated for 6 h at 30°C. Bottom panel: intracellular levels of FlgG produced in the same hosts as in the upper panels. A fixed number of cells was serially diluted at 1/1, 1/3, and 1/10 through to 1/300 and subjected to SDS-polyacrylamide gel electrophoresis and immunoblotting with polyclonal anti-FlgG antibody.

Immunoblotting (using anti-FlgD antibody) of the culture supernatants from SJW1353 (flgE) cells transformed with pTrc99AET-based plasmids and grown at 37°C in the presence (+) and absence (−) of 0.1 mM IPTG. The proteins produced by the various plasmids are shown above each lane. V, vector alone (pTrc99A). The positions of FlgD are indicated by arrowheads. Molecular mass markers (in kilodaltons) are shown to the left.

Immunoblotting (using anti-FlgG [A and B], anti-FlgD [C and D], and anti-FlgL [E and F] antibodies) of the cells and culture supernatants of SJW1353 (flgE) carrying pMM202 (FlgG) (A and B), SJW1353 (flgE) carrying pRCD1 (FlgD) (C and D), and SJW2177 (flgK) carrying pMM1903 (FlgL) (E and F), respectively, in the presence (+) and absence (−) of 100 μg of spectinomycin/ml. The positions of FlgG, FlgD, and FlgL are indicated by arrowheads. The high-molecular-mass bands in the left panels of Fig. 4A, B, and C represent either aggregates or a cross-reacting species. Molecular mass markers (in kilodaltons) are shown to the left.