Genetic Structure and Function Analysis of the Conserved Integral Membrane Components (FliOPQR) of the Flagellar Type III Secretion Apparatus of Salmonella enterica

Abstract

Bacteria swim in liquid environment by rotation of an unique nanomachine, the flagellum. For assembly of the flagellum, a flagellar-specific type III secretion apparatus is essential. The conserved integral membrane components FliOPQR of Salmonella enterica serovar Typhimurium were previously proposed to be part of the type III secretion apparatus. It has been previously shown that FliOPQR are essential for export of flagellar substrates. However, virtually nothing is known about the function and localization of the proteins.<br />The first part of this work reports the growth and purification of the bacteriophage Chi, who only attacks motile bacteria. Furthermore, the virulence of phage Chi in S. typhimurium strains expressing antigenically distinct flagellin variants is examined. Afterwards, the construction of tetracycline resistance cassette insertions, as well as the construction of clean deletions of fliOPQR is described. It is shown in this chapter that fliO, fliP, fliQ and fliR are required for motility and that a clean deletion of each gene can be complemented by introduction of the respective wildtype gene.<br />The next part of this work reports conserved domains and topology predictions of FliOPQR based on a homology search. Afterwards, the topology prediction is confirmed by construction of C-terminal beta-galactosidase and alkaline phosphatase fusion proteins. Non-motile fliO and fliP mutants are examined by DNA sequencing analysis and subsequently motile revertant mutants of the revealed fliO point mutations (V72G and L91R) are isolated and characterized. In addition, a C-terminal YPF (yellow fluorescent protein) fusion to FliO is constructed and the localization of the FliO-YFP fusion construct is analyzed by fluorescence microscopy. It is found that FliO is probably not localized within the flagellar basal body structure, contrary to previous suggestions. The construction of a S. typhimurium strain, chromosomally expressing FliM-CFP (cyan fluorescent protein) as well as FliO-YFP constructs, confirms this finding by the observation that FliM-CFP and FliO-YFP are not colocalized.<br />In the last part of this work the energization of flagellar type III secretion is analyzed. The flagellar type III secretion apparatus transports most proteins necessary for the assembly of the flagellum across the inner membrane in an ATP-dependent manner. This work shows that flagellar type III secretion is also dependent on the proton motive force. Abolishment of both the proton gradient deltapH and the membrane potential deltaPsi; using the ionophore carbonyl cyanide m-chlorophenylhydrazone (CCCP), prevented secretion of the anti-Sigma28 factor FlgM.<br />Secretion of FlgM could be restored by growth in medium lacking CCCP. Furthermore, it is shown that the secretion of FlgM is inhibited at pH 5 in the presence of 34 mM acetate, indicating an important role of the proton gradient deltapH and/or the intracellular proton concentration. Addition of the ionophore CCCP resulted in an immediate growth arrest, but not in a significant decrease of cytoplasmic ATP levels, thus demonstrating that both the flagellar type III secretion ATPase FliI and the proton motive force are necessary for the export of flagellar type III secretion substrates.