FliZ Is a posttranslational activator of FlhD4C2-dependent flagellar gene expression

Abstract

Flagellar assembly proceeds in a sequential manner, beginning at the base and concluding with the filament. A critical aspect of assembly is that gene expression is coupled to assembly. When cells transition from a nonflagellated to a flagellated state, gene expression is sequential, reflecting the manner in which the flagellum is made. A key mechanism for establishing this temporal hierarchy is the sigma(28)-FlgM checkpoint, which couples the expression of late flagellar (P(class3)) genes to the completion of the hook-basal body. In this work, we investigated the role of FliZ in coupling middle flagellar (P(class2)) gene expression to assembly in Salmonella enterica serovar Typhimurium. We demonstrate that FliZ is an FlhD(4)C(2)-dependent activator of P(class2)/middle gene expression. Our results suggest that FliZ regulates the concentration of FlhD(4)C(2) posttranslationally. We also demonstrate that FliZ functions independently of the flagellum-specific sigma factor sigma(28) and the filament-cap chaperone/FlhD(4)C(2) inhibitor FliT. Furthermore, we show that the previously described ability of sigma(28) to activate P(class2)/middle gene expression is, in fact, due to FliZ, as both are expressed from the same overlapping P(class2) and P(class3) promoters at the fliAZY locus. We conclude by discussing the role of FliZ regulation with respect to flagellar biosynthesis based on our characterization of gene expression and FliZ's role in swimming and swarming motility.

FIG. 1.

FliZ enhances P_class2 and P_class3 activity. A comparison of P_class2 (flgA, flgB, flhB, and fliE), P_class2/3 (fliD), and P_class3 (flgK and fliC) promoter activities in the wild-type, ΔfliZ, and ΔfliZ pFliZ strains is shown. In the last strain, FliZ is constitutively expressed from the P_LtetO-1 promoter on a plasmid. Error bars indicate standard deviations.

FIG. 2.

Dynamics of flagellar gene expression in wild-type and ΔfliZ strains. Gene expression measurements were made every 10 min after induction following dilution of strains in fresh medium (32). Note that strains were grown at 30°C in the kinetic experiments in order to obtain better resolution and temporal ordering between P_class2 and P_class3 promoter activities. Identical experiments were also performed at 37°C with similar results except that the separation between class 2 and 3 gene expression was far less pronounced (results not shown). Error bars indicate standard deviations.

FIG. 3.

Relative effects of FliZ and σ²⁸ expression on P_class2 activity. A comparison of flagellar promoter activities in a ΔfliAZ mutant where fliA (σ²⁸) or fliZ is expressed from an aTc-inducible promoter on a plasmid is shown. Note that tetR is also expressed from this plasmid in order to achieve aTc-inducible expression. Error bars indicate standard deviations.

FIG. 4.

Comparison of flagellar promoter activities for wild-type, ΔfliZ, ΔfliA, ΔfliAZ, ΔfliT, ΔfliZ ΔfliT, ΔfliA ΔfliT, and ΔfliAZ ΔfliT strains in a P_flhDC⁺ (A) or a P_flhDC::tetRA (B) background. P_class2 (flhB) and P_class3 (fliC) activities were measured. Error bars indicate standard deviations.

FIG. 5.

FliZ's effect on flagellar promoter activity is independent of SPI1, the SPI1 activator, rtsA, and the P_flhDC repressor, rtsB. A comparison of flagellar promoter activities in ΔSPI1 ΔfliZ and ΔSPI1 ΔfliZ ΔrtsAB mutants is shown. Error bars indicate standard deviations.

FIG. 6.

FliZ is responsible for increased levels of FlhC protein independent of flhDC transcription. The amount of FlhC protein was measured by Western blotting using anti-FLAG antibody targeting chromosomally 3× FLAG-tagged FlhC protein. Lanes: A, wild type; B, ΔfliZ::FRT; C, P_flhDC::tetRA; D, P_flhDC::tetRA ΔfliZ::FRT; E, wild type without 3× FLAG tag.

FIG. 7.

FliZ enhances both swimming and swarming motilities. (A) Swimming on tryptone broth plates with 0.3% agar. (B) Swarming on LB plates with 0.6% agar and 0.5% glucose. (C) Swarming on tryptone broth plates with 0.6% agar and 0.02% Tween 80. The first column shows the results for the wild type, the second for the ΔfliZ mutant, the third for the ΔfliZ mutant complemented with the fliZ gene under the control of its native promoter on a plasmid, and the fourth the ΔfliZ mutant complemented with the fliZ gene under the control of a constitutive promoter on a plasmid. For all motility and swarming experiments, the plates were incubated at 37C for 8 h.