Sensing wetness: a new role for the bacterial flagellum

Abstract

We have uncovered a new role for the bacterial flagellum in sensing external wetness. An investigation into why mutants in the chemotaxis signaling pathway of Salmonella typhimurium exhibit fewer and shorter flagella than wild-type when propagated on a surface, first showed that the mutants downregulate only a small set of genes on swarm media--class 3 or 'late' motility genes, and genes associated with the pathogenicity island SPI-1 TTSS (type three secretion system). Based on observations that swarm colonies of the mutants appear less hydrated, we tested a model in which the flagellum itself is a sensor: suboptimal external hydration interferes with secretion of flagellin subunits, inhibiting filament growth and blocking normal export of the class 3 transcription inhibitor FlgM. We provide strong experimental support for the model. In addition, the data show that the flagellar and SPI-1 TTSS are coupled via regulatory proteins. These studies implicate the flagellum, a bacterial organ for motility, in sensing the external environment to modulate not only its own biogenesis but other physiological functions as well.

Figure 1

Flagellar filament visualization. Cells growing for indicated times in broth (B) or swarm plates (P) were stained with filament antibodies as described in Materials and methods. A color version of this figure is available at The EMBO Journal Online.

Figure 2

Relative flagellin amounts in strains grown on swarm plates. Cells from exponentially growing broth cultures were used as inoculum for plates (0 h). Samples were normalized to an OD₆₀₀ of 0.6 prior to electrophoresis, and Western blots developed using anti-FljB antibodies as described in Materials and methods. Signal intensities are plotted as a function of total OD₆₀₀, and relative to wild-type at 4.5 h.

Figure 3

Lawn morphologies of wild-type and che mutants propagated on swarm agar. (A) At indicated times, plates were photographed with a DP-12 digital camera attached to an Olympus BH-2 microscope. Magnification, × 25. (B) Left: comparison of fluid retention in 3 h-lawns of indicated strains by the capillary-drop method, as described in Materials and methods. Two capillaries are shown for each strain. Height of capillary in mm is indicated on the y-axis (center). Right: data from triplicate plates measured on the same day; error bars indicate standard variation.

Figure 4

The flagellum as an environment sensor, and FlgM as a dual developmental checkpoint. (A) The FlhCD regulator activates class 2 transcription (only relevant genes shown). Class 2 FlgM inhibits FliA while HBB construction is in progress (Chilcott and Hughes, 2000). (B) Completion of HBB allows FlgM export, lifting FliA inhibition and allowing class 3 expression (e.g. filament genes fljB/fliC and class 3 flgM). (C) Class 3 FlgM is primarily secreted during filament biogenesis (Karlinsey et al, 2000). (D) An external block to filament growth interferes with FlgM secretion and re-establishes repression of FliA. CM, cytoplasmic membrane; OM, outer membrane; X, block to transcription (A, D) or filament growth (D); wavy lines, transcripts. Nascent flagellin subunits in purple.

Figure 5

(A) FlgM levels of wild-type and che mutants in broth versus on swarm plates 3 h postinoculation. EC, extracellular; IC, intracellular. fliM affects formation of the initial C-ring, and serves as a negative control for extracellular FlgM (Macnab, 2003). (B) EC FlgM in WT versus cheY during a 5 h time course. (C) EC FlgM in a cheY mutant at indicated times after hydration (+) of a lawn grown for 2 h prior. (−) is a nonhydrated control at the 1.5 h time point equivalent.

Figure 6

Flagellar staining following hydration and restoration of swarming motility to a cheY lawn. The mutant was propagated on swarm plates for 2 h (0 h time point) before spraying with a fine mist of water as described in Materials and methods. Cells stained for flagella 1 and 2 h after spraying are shown. A color version of this figure is available at The EMBO Journal Online.

Figure 7

Swarming and swimming morphology of wild-type, flgM, cheY and cheY flgM strains.

Figure 8

Flagellar staining of wild-type, flgM and cheY flgM strains propagated on swarm plates for 3 h. A color version of this figure is available at The EMBO Journal Online.