Figure 1.
FlhG controls polar flagellar numbers in C. jejuni.
(A and B) Electron micrographs of negatively stained (A) wild-type C. jejuni and (B) C. jejuni ΔflhG. Bars = 1 µm (A) and 2 µm (B). (C) Quantification of flagellar numbers of wild-type C. jejuni and ΔflhG mutant populations. Individual bacteria were analyzed for the number of flagella produced at each pole. Wild-type C. jejuni and C. jejuni ΔflhG were complemented with vector alone, or vector expressing wild-type flhG. The data are reported as the percentage of the bacterial population with the following flagellar numerical patterns: >2 flagella, producing two or more flagella at least at one pole (purple); wild-type flagella, producing a single flagellum at one or both poles (grey); and 0 flagella, aflagellated bacteria (black). Data represent the average of two experiments. Bars represent standard errors.
Figure 2.
Analysis of the minicell phenotype upon deletion of flhG in C. jejuni.
(A-D) Electron micrographs of negatively stained C. jejuni ΔflhG cell bodies and minicells. Bars = 2 µm (A and B), 0.2 µm (C), and 1 µm (D). Red arrows indicate minicells next to normal size bacteria (A) or forming at a pole of a bacterium (B). (E) Quantification of lengths of cell bodies of wild-type C. jejuni and ΔflhG mutant populations. The lengths of the cell bodies of bacteria were measured. Wild-type C. jejuni and C. jejuni ΔflhG mutant were complemented with vector alone, or vector expressing wild-type flhG. The data are reported as the percentage of bacterial populations with the following cell lengths:<0.5 µm, minicells (red); 0.5–1 µm (blue); 1–2 µm (yellow); and >2 µm (brown). The data represent the average of two experiments. Bars represent standard errors.
Table 1.
Minicell production in C. jejuni ΔflhG upon exposure with cephalexin.
Figure 3.
The elongated cell phenotype of C. jejuni flhGD61A.
Electron micrographs of wild-type C. jejuni (A) or C. jejuni flhGD61A (B-F) after negative staining. Bars = 2 µm (A, C, D, and F), 10 µm (B), and 1 µm (E).
Table 2.
Analysis of the effects of FtsZ overexpression on minicell and elongated cell phenotypes of wild-type C. jejuni and C. jejuni flhG D61A.
Figure 4.
The polar flagellar number and minicell phenotypes of C. jejuni ΔflhG upon complementation in trans with flhG or minD orthologs.
(A and B) C. jejuni ΔflhG was complemented in trans with vector alone (-), or flhG or minD from C. jejuni (Cj), H. pylori (Hp), V. cholerae (Vc), or E. coli (Ec). (A) Quantification of flagellar numbers of wild-type C. jejuni and C. jejuni ΔflhG upon complementation. Individual bacteria were analyzed for the number of flagella produced at each pole. The data are reported as the percentage of bacterial populations with the following flagellar numerical patterns: >2 flagella, producing two or more flagella at least at one pole (purple); wild-type flagella, producing a single flagellum at one or both poles (grey); and 0 flagella, aflagellated bacteria (black). The data represent the average of two experiments. Bars represent standard errors. (B) Quantification of lengths of the cell bodies of wild-type C. jejuni and C. jejuni ΔflhG upon complementation. The lengths of the cell bodies of bacteria were measured. Wild-type C. jejuni and C. jejuni ΔflhG mutant were complemented with vector alone, or vector expressing wild-type flhG. The data are reported as the percentage of bacterial populations with the following cell lengths:<0.5 µm, minicells (red); 0.5–1 µm (blue); 1–2 µm (yellow); and >2 µm (brown). The data represent the average of two experiments. Bars represent standard errors.
Figure 5.
Analysis of cellular localization of FlhG-GFP.
C. jejuni ΔflhG containing plasmids to express wild-type FlhG with a C-terminal GFP (A) or GFP alone (B) were stained with FM4-64 to visualize membranes. Shown are images visualizing FlhG-GFP or GFP alone (green), FM4-64 stained membranes alone (red), and merged images. Bar = 2 µm.
Figure 6.
Analysis of the minicell phenotype of C. jejuni flagellar mutants.
(A) Quantification of the lengths of the cell bodies of wild-type C. jejuni and mutants lacking a motility gene. (B) Quantification of the lengths of the cell bodies of mutants lacking FlhF, FliF, FliM or FliN after treatment with cephalexin. Strains were grown in liquid broth in the absence (-) or presence (+) of 15 µg/ml cephalexin for 6 h. The lengths of the cell bodies were then measured. (C) Quantification of the lengths of the cell bodies of wild-type C. jejuni or mutants lacking FlhF, FliF, FliM, and FliN upon overexpression of flhG in trans. The mutants were complemented with either vector alone (-) or a plasmid to overexpress wild-type flhG (+ FlhG). (D). Quantification of the lengths of the cell bodies of wild-type C. jejuni, C. jejuni ΔflhG, and C. jejuni mutants lacking flhG and either fliF, fliM, or fliN. For (A-D), the lengths of the cell bodies of individual bacteria were measured. The data are reported as the percentage of the population with the following cell lengths:<0.5 µm, minicells (red); 0.5–1 µm (blue); 1–2 µm (yellow); and >2 µm (brown). Data represents the average of two experiments. Bars represent standard errors.