Components of the Salmonella flagellar export apparatus and classification of export substrates - PubMed (original) (raw)

Components of the Salmonella flagellar export apparatus and classification of export substrates

T Minamino et al. J Bacteriol. 1999 Mar.

Abstract

Until now, identification of components of the flagellar protein export apparatus has been indirect. We have now identified these components directly by establishing whether mutants defective in putative export components could translocate export substrates across the cytoplasmic membrane into the periplasmic space. Hook-type proteins could be exported to the periplasm of rod mutants, indicating that rod protein export does not have to precede hook-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) required 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 was necessary to use temperature-sensitive mutants and establish whether flagellin could be exported to the cell exterior following a shift from the permissive 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 flagellin export, but we suspect that the temperature-sensitive FliP protein continued to function at the restrictive temperature if incorporated at the permissive temperature. Thus, we conclude that these eight proteins are general 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 be 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 filament-type proteins. Finally, it was established that there is an interaction between the processes of outer ring assembly and of penetration of the outer 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 regarded as bona fide export components, with the L ring being in a formal sense the equivalent of the outer membrane secretin structure of type III virulence factor export systems.

PubMed Disclaimer

Figures

FIG. 1

FIG. 1

Immunoblot, using polyclonal anti-FlgD antibody, of fractionated whole-cell material from various mutants as indicated above the lanes. c, whole cells; p, periplasmic fraction; s, culture supernatant. Positions of molecular mass markers are shown in kilodaltons to the left; the position of FlgD (whose deduced molecular mass is 24 kDa) is indicated at the right. Bands at higher apparent molecular mass are nonspecific cross-reacting material; the band at lower apparent molecular mass is probably a degradation product of FlgD.

FIG. 2

FIG. 2

Immunoblot, using polyclonal anti-FlgD antibody, of the periplasmic fraction from various mutants as indicated above the lanes. Positions of molecular mass markers are shown in kilodaltons to the left; the position of FlgD is indicated at the right. The bands at lower apparent molecular mass are probably degradation products of FlgD.

FIG. 3

FIG. 3

Autoradiogram of sample precipitated with polyclonal anti-FliC antibody of the supernatant fraction from the wild type (except for the flgK::Tn_10_ mutation; wt) and various mutants in temperature shift experiments with temperature-sensitive mutants (see the text). In addition to the mutation of interest, all strains contain a flgK::Tn_10_ mutation to prevent filament assembly. The position of FliC is indicated.

FIG. 4

FIG. 4

Immunoblot, using polyclonal anti-FlgD antibody, of fractionated whole-cell material from a flgG (rod) mutant and a fliE mutant. c, whole cells; p, periplasmic fraction; s, culture supernatant. Molecular mass markers are shown in kilodaltons to the left; the position of FlgD is indicated at the right. The gel used contained 15% acrylamide.

FIG. 5

FIG. 5

Immunoblot, using polyclonal anti-FlgD antibody, of the periplasmic (p) and supernatant (s) fractions from various mutants with defects in outer ring production and from the wild-type control (wt). Positions of molecular mass markers are shown in kilodaltons to the left; the position of FlgD is indicated at the right.

FIG. 6

FIG. 6

Hypothetical model for the flagellar protein export apparatus (see the text for further details). This study has shown that FlhA, FlhB, FliO, FliP, FliQ, FliR, FliH, and FliI are components of this apparatus. FlhA, FlhB, FliO, FliP, FliQ, and FliR are all integral membrane proteins. Of these, FlhA, FliP, and FliR (shown in boldface) have been shown to be physically associated with the flagellar basal body (6, 23); FlhB, FliO, and FliQ may be also. It is suggested that these proteins may be located in a pore within the basal body MS ring. Two soluble components, FliH and FliI (an ATPase) may receive the export substrates from cytoplasmic chaperones (such as FliJ?) and deliver them in an energy-dependent process to the membrane-associated structure, which then translocates them to the channel or lumen in the nascent structure. FliK is involved in control of the length of the flagellar hook. FliE appears to be needed for the export of other substrates, but it is not known whether it is itself an export substrate. FlgJ is needed for assembly of the rod but does not appear to be involved in the export process. The representation of the rod and hook structures is not to scale and is highly schematic.

Similar articles

Cited by

References

    1. Amann E, Ochs B, Abel K-J. Tightly regulated tac promoter vectors useful for the expression of unfused and fused proteins in Escherichia coli. Gene. 1988;69:301–314. - PubMed
    1. Charkowski A O, Huang H-C, Collmer A. Altered localization of HrpZ in Pseudomonas syringae pv. syringae hrp mutants suggests that different components of the type III secretion pathway control protein translocation across the inner and outer membranes of gram-negative bacteria. J Bacteriol. 1997;179:3866–3874. - PMC - PubMed
    1. Dijkstra A J, Keck W. Peptidoglycan as a barrier to transenvelope transport. J Bacteriol. 1996;178:5555–5562. - PMC - PubMed
    1. Doi, H., and K. Kutsukake. Unpublished data.
    1. Emerson S U, Tokuyasu K, Simon M I. Bacterial flagella: polarity of elongation. Science. 1970;169:190–192. - PubMed

Publication types

MeSH terms

Substances

LinkOut - more resources