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. 2013 Feb;195(4):823-32.
doi: 10.1128/JB.02024-12. Epub 2012 Dec 7.

Activity of Proteus mirabilis FliL is viscosity dependent and requires extragenic DNA

Yi-Ying Lee  1 , Julius Patellis, Robert Belas
Affiliations

Activity of Proteus mirabilis FliL is viscosity dependent and requires extragenic DNA

Yi-Ying Lee et al. J Bacteriol. 2013 Feb.

Abstract

Proteus mirabilis is a urinary tract pathogen and well known for its ability to move over agar surfaces by flagellum-dependent swarming motility. When P. mirabilis encounters a highly viscous environment, e.g., an agar surface, it differentiates from short rods with few flagella to elongated, highly flagellated cells that lack septa and contain multiple nucleoids. The bacteria detect a surface by monitoring the rotation of their flagellar motors. This process involves an enigmatic flagellar protein called FliL, the first gene in an operon (fliLMNOPQR) that encodes proteins of the flagellar rotor switch complex and flagellar export apparatus. We used a fliL knockout mutant to gain further insight into the function of FliL. Loss of FliL results in cells that cannot swarm (Swr(-)) but do swim (Swm(+)) and produces cells that look like wild-type swarmer cells, termed "pseudoswarmer cells," that are elongated, contain multiple nucleoids, and lack septa. Unlike swarmer cells, pseudoswarmer cells are not hyperflagellated due to reduced expression of flaA (the gene encoding flagellin), despite an increased transcription of both flhD and fliA, two positive regulators of flagellar gene expression. We found that defects in fliL prevent viscosity-dependent sensing of a surface and viscosity-dependent induction of flaA transcription. Studies with fliL cells unexpectedly revealed that the fliL promoter, fliL coding region, and a portion of fliM DNA are needed to complement the Swr(-) phenotype. The data support a dual role for FliL as a critical link in sensing a surface and in the maintenance of flagellar rod integrity.

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Figures

Fig 1
Fig 1
Phenotypic characterization of fliL strain YL1003. (A) Swarming motility on LB agar. (B). Swimming motility through semisolid Mot agar. (C) Phase-contrast micrographs of cells obtained from LB broth after 2.75 h of incubation. Scale bar, 10 μm. WT, wild type.
Fig 2
Fig 2
Comparison of fliL pseudoswarmer cells to wild-type swimmer and swarmer cells. (A) BB2000 swimmer cells. (B) BB2000 swarmer cell. (C) YL1003 pseudoswarmer cell. From left to right, membrane (FM1-43; green), nucleoids (DAPI; blue), and composite with flagellum immunostaining in red. Scale bar, 7 μm.
Fig 3
Fig 3
Immunoblot with polyvalent antibodies to flagellin (FlaA). BB2000 (WT) and YL1003 (fliL) cells were harvested from LB broth or agar.
Fig 4
Fig 4
Expression of flagellar genes in fliL cells. Values are a comparison of expression of the target gene in fliL cells (mt) to the same gene in wild-type (wt) cells. A value of >1-fold change indicates that expression of the gene is greater in the fliL cells than in the wild-type cells, while a value of <1 means that expression decreases in fliL cells. Growth media are as indicated on the figure. Means and standard deviations for three independent measurements from three biological samples are shown.
Fig 5
Fig 5
Motility and flaA expression in fliL cells (YL1003) and wild-type (WT) cells (BB2000) as a function of the viscosity of the medium. (A) Swimming (0.3% agar) or swarming (1.0 to 2.5% agar) as the distance (mm) the cells moved after 5 h. (B) flaA (flagellin) expression, measured as β-galactosidase activity from a PflaA::lacZ transcriptional fusion. Means ± standard deviations are shown.
Fig 6
Fig 6
Complementation of fliL cells by plasmids bearing deletions of the fliL operon. (A) Phase-contrast microscopy to detect pseudoswarmer cells. Scale bar, 20 μm. (B) Swarming motility on LB agar. (C) Sequential deletion of the fliL operon resulted in six plasmids, each harboring one less gene starting from the 3′ end of the operon. This yielded pYL47 (fliL to fliR), pYL60 (fliL to fliP), pYL61 (fliL to fliO), pYL62 (fliL to fliN), pYL63 (fliL to fliM), and pYL64 (fliL). All plasmids contain the native fliL promoter (PfliL; arrows in front of fliL). HindIII sites are indicated by H.
Fig 7
Fig 7
Immunoblotting with polyvalent antibodies to FliM. BB2000 (WT) and YL1003 (fliL) cells were harvested from LB broth or agar. The antiserum used was directed against E. coli FliM and cross-reacts with P. mirabilis FliM.
Fig 8
Fig 8
Immunoblot measuring the abundance of detached and attached flagella in fliL cells complemented with fliL+ or fliL+ fliM+. Wild-type and fliL cells were harvested after 4.5 h of incubation on LB agar. Rabbit polyvalent antiserum to FlaA was used, as described in the legend of Fig. 3.
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