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. 2006 Jul;188(14):5196-203.
doi: 10.1128/JB.00299-06.

sigma28-dependent transcription in Salmonella enterica is independent of flagellar shearing

Valentina Rosu  1 , Kelly T Hughes
Affiliations

sigma28-dependent transcription in Salmonella enterica is independent of flagellar shearing

Valentina Rosu et al. J Bacteriol. 2006 Jul.

Abstract

The FlgM anti-sigma28 factor is secreted in response to flagellar hook-basal body completion to allow sigma28-dependent transcription of genes needed late in flagellar assembly, such as the flagellin structural gene, fliC. A long-standing hypothesis was that one role of FlgM secretion was to allow rapid expression of flagellin in response to shearing. We tested this hypothesis by following FlgM secretion and fliC transcription in response to flagellar shearing. Experiments showed that the level of FlgM inside the cell was unchanged after shearing whereas the extracellular FlgM levels increased in the growth medium as time passed. Identical results were obtained with cells that were not exposed to shear forces: internal FlgM levels remained constant while external FlgM levels rose with time at rates similar to those for the sheared culture. Consistent with this find, FlgM/sigma28-dependent class 3 gene expression was unaffected by flagellar shearing but was affected by the growth phase of the cell. Regardless of exposure to shear forces, flagellar class 3 transcription rose sharply and then declined. These results demonstrate that flagellar regrowth following shearing is independent of FlgM secretion.

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Figures

FIG. 1.
FIG. 1.
Flagellar-filament visualization with FITC-conjugated anti-FliC antibody. (A) Immediately after shearing (t = 0), cells from a log-phase culture showed an average of one to two very short flagella. (B) Immediately after shearing (t = 0), only a few cells from a stationary-phase culture showed short flagella; one cell having this phenotype is shown on the left. (C) As a positive control, we used a sample of unblended cells (picture on the left). As a negative control, a nonflagellated strain was used (picture on the right).
FIG. 2.
FIG. 2.
Detection of cellular and supernatant FlgE hook subunit protein. Western blots of IC and EC extracts made from cells of strain TH6232 were prepared using anti-FlgE antibody. Supernatant Western blots were prepared using anti-FlgE antibody and protein present in the growth medium from the same cell culture. Cells were washed two times in saline and resuspended in fresh LB broth. The absence of FlgE in the supernatant suggests that the hook subunit is not broken by blending treatment. According to Aizawa and Kubori, if a flagellum is broken somewhere on the basal body, a filament does not regrow; instead, a new filament is synthesized (1). The arrows indicate the positions of FlgE in the gel. Pre, preblending.
FIG. 3.
FIG. 3.
Quantification of FlgM protein inside (IC) and outside (EC) the cells after flagellar shearing. Immunoblot analysis of TH6223 was performed in triplicate using log-phase cells washed in saline and resuspended in fresh LB; quantification was performed with ImageQuant for Macintosh, v1.2 (Molecular Dynamics). Pre, preblending.
FIG. 4.
FIG. 4.
Detection of IC and EC FlgM protein. (A) IC Western blots were prepared using anti-FlgM antibody and extract made from stationary-phase cells of strain TH8223. EC Western blots were prepared using anti-FlgM antibody and protein present in the growth medium. Cells were washed two times in saline and resuspended in ΔflgM-spent media without inducer; samples were taken at different time points after flagellar shearing. Pre, preblending. (B) Data from panel A presented in graph format. FlgM levels are presented relative to that of preblended IC FlgM, set at 100.
FIG. 5.
FIG. 5.
Flagellar-filament regeneration over time on log-phase cells. Flagellar filaments were visualized at different time points after blending treatment of log-phase cells (OD600, ∼0.6). Cell membranes and bacterial DNA were stained with the dyes FM4-64 and DAPI, respectively. Flagellar filaments were detected by using the anti-FliC primary antibody and the secondary antibody conjugated with the fluorescence molecule FITC. (Panel 1) Unblended cells. Individual cells showed an average of 6 ± 2 flagella of different lengths (see time points 0, 5, and 10). (Panel 2) Blended cells. Cells monitored at time zero, immediately after shearing, showed an average of one to two short flagella per cell. Flagellar length rapidly increased on subsequent incubations at 5 and 10 minutes after shearing, while a gradual increase in flagellar number was detected at 30 and 60 minutes after shearing.
FIG. 6.
FIG. 6.
Flagellar-filament visualization of stationary-phase cells. (Panel 1) Unblended cells. Cell membranes were stained with the FM4-64 dye. Flagellar filaments were detected on stationary-phase cells (OD600, ∼2) by using the anti-FliC primary antibody and the secondary antibody conjugated with the fluorescence molecule FITC. (Panel 2) Blended cells. Cells were stained similarly to the unblended controls, except that the chromosomal DNA was also stained with DAPI. Pictures were taken immediately after blending treatment (t = 0) and 60 min later (t = 60).
FIG. 7.
FIG. 7.
β-Galactosidase assays for the TH6232 ataA::[P22 PfliC-lac] construct. Log-phase, flagellated cells carrying a PfliC-lac reporter construct were assayed for β-galactosidase activity before and at different time points after shearing. PRE, preblending.
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References

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