Components of the Salmonella flagellar export apparatus and classificationof export substrates

Until now, identification of components of the flagellar protein export app aratus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components cou ld translocate export substrates across the cytoplasmic membrane into the p eriplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede ho ok-type protein export and therefore that both types of proteins belong to a single export class, the rod/hook-type class, which is distinct from the filament-type class. Hook-capping protein (FlgD) and hook protein (FlgE) re quired FlhA, FlhB, FliH, FliI, FliO, FliP, FliQ, and FliR for their export to the periplasm, In the case of flagellin as an export substrate, because of the phenomenon of hook-to-filament switching of export specificity, it w as necessary to use temperature-sensitive mutants and establish whether fla gellin could be exported to the cell exterior following a shift from the pe rmissive to the restrictive temperature. Again, FlhA, FlhB, FliH, FliI, and FliO were required for its export, No suitable temperature-sensitive fliQ or fliR mutants were available. FliP appeared not to be required for flagel lin export, but we suspect that the temperature sensitive FliP protein cont inued to function at the restrictive temperature if incorporated at the per missive temperature, Thus, we conclude that these eight proteins are genera l components of the flagellar export pathway. FliJ was necessary for export of hook-type proteins (FlgD and FlgE); we were unable to test whether FliJ is needed for export of filament-type proteins, We suspect that FliJ may b e a cytoplasmic chaperone for the hook-type proteins and possibly also for FliE and the rod proteins. FlgJ was not required for the export of the hook -type proteins; again, because of lack of a suitable temperature-sensitive mutant, we were unable to test whether it was required for export of filame nt-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the oute r membrane by the rod and nascent hook, the latter process being of course necessary for passage of export substrates into the external medium. During the brief transition stage from completion of rod assembly and initiation of hook assembly, the L ring and perhaps the capping protein FlgD can be re garded as bona fide export components,,vith the L ring being in a formal se nse the equivalent of the outer membrane secretin structure of type III vir ulence factor export systems.