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. 2007 Oct 31;26(21):4433-44.
doi: 10.1038/sj.emboj.7601877. Epub 2007 Oct 11.

Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus

Simone Leonardy  1 , Gerald Freymark, Sabrina Hebener, Eva Ellehauge, Lotte Søgaard-Andersen
Affiliations

Coupling of protein localization and cell movements by a dynamically localized response regulator in Myxococcus xanthus

Simone Leonardy et al. EMBO J. .

Erratum in

  • EMBO J. 2009 Apr 22;28(8):1192

Abstract

Myxococcus xanthus cells harbor two motility machineries, type IV pili (Tfp) and the A-engine. During reversals, the two machineries switch polarity synchronously. We present a mechanism that synchronizes this polarity switching. We identify the required for motility response regulator (RomR) as essential for A-motility. RomR localizes in a bipolar, asymmetric pattern with a large cluster at the lagging cell pole. The large RomR cluster relocates to the new lagging pole in parallel with cell reversals. Dynamic RomR localization is essential for cell reversals, suggesting that RomR relocalization induces the polarity switching of the A-engine. The analysis of RomR mutants shows that the output domain targets RomR to the poles and the receiver domain is essential for dynamic localization. The small GTPase MglA establishes correct RomR polarity, and the Frz two-component system regulates dynamic RomR localization. FrzS localizes with Tfp at the leading pole and relocates in an Frz-dependent manner to the opposite pole during reversals; FrzS and RomR localize and oscillate independently. The Frz system synchronizes these oscillations and thus the synchronous polarity switching of the motility machineries.

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Figures

Figure 1
Figure 1
The romR locus and the RomR protein. (A) Organization of the romR locus. Arrows indicate the direction of transcription of romR and the flanking ORFs. Coordinates are relative to the start codon of romR. The deduced proteins encoded by the flanking ORFs have the following characteristics: MXAN4460 is similar to Val-tRNA synthethases; MXAN4462 contains two CheW domains; MXAN4463 is a response regulator with a GGDEF output domain. (B) Plasmids used in this work. Coordinates are relative to the romR start codon. Light gray boxes indicate gfp and the dark gray box indicates mDsRed. (C) Scheme of RomR domain structure. (D) Alignment of N-terminal receiver domain of RomR with characterized receiver domains. Asterisks indicate conserved signature residues of receiver domains (Stock et al, 2000). Residues shaded black are 100% identical, and those shaded gray are 60–100% conserved. (E) Primary sequence of RomR output domain. Pro residues in the Pro-rich region are shaded gray. The Glu-rich region is underlined, and Glu residues are in bold and italic.
Figure 2
Figure 2
RomR is required for A-motility and localizes in a bipolar, asymmetric pattern. (A) Motility phenotype of romR mutant. Cells were incubated at 32°C for 24 h on 1.5% agar supplemented with 0.5% CTT medium and visualized with a Leica MZ8 stereomicroscope (upper row) and a Leica IMB/E inverted microscope (lower row). Scale bars: upper row, 5 mm; lower row, 50 μm. (B) Developmental phenotype of romR mutant. Cells were starved on CF agar for 72 h and visualized with a Leica MZ8 stereomicroscope. Scale bar: 50 μm. (C) RomR and RomR-GFP accumulation. Cells from steady-state cultures were harvested, and total protein was separated by SDS–PAGE (1 μg of protein per lane) and analyzed by immunoblotting. Strains used (left to right): DK1622, SA1128, SA2272, and SA2271. The blot on the left was probed with rabbit anti-RomR antibodies and the blot on the right with monoclonal anti-GFP antibodies. RomR and Rom-GFP proteins are indicated. Migration of molecular size markers is indicated on the left. (D) Localization of RomR-GFP. Cells were transferred from a steady-state culture to a thin agar pad on a microscope slide and imaged by fluorescence and phase-contrast microscopy. Depicted are overlays of fluorescence and phase-contrast images. Scale bar: 10 μm. (E) Localization of RomR by immunofluorescence microscopy. Cells were harvested from 1.5% agar supplemented with 1% CTT, fixed, reacted with affinity-purified anti-RomR antibodies, and imaged by fluorescence and phase-contrast microscopy. Depicted are overlays of fluorescence and phase-contrast images. (F) The large RomR-GFP cluster localizes to the pole opposite to that containing Tfp. SA2271 cells were grown as in (E), stained with Cy3, and inspected by fluorescence microscopy to visualize Tfp (Cy3, arrow points to Tfp) and RomR-GFP (GFP, arrow points to large RomR-GFP cluster) and by phase-contrast microscopy (Ph). (G) The large RomR-GFP cluster localizes to the lagging pole. Cells of SA2271 were grown as in (D), transferred to a thin agar pad on a microscope slide, and imaged by fluorescence and phase-contrast microscopy at 30-s intervals. Shown is a representative cell that did not reverse. Depicted are overlays of fluorescence and phase-contrast images recorded at the indicated time points in minutes. Arrows indicate the direction of movement. (H) Quantitative analysis of polar fluorescence signals. Relative fluorescence intensities (arbitrary units) of each pole in the cell in (D) were measured and plotted as a function of time. Filled squares, lagging pole; open circles, leading pole. (I) RomR localization is dynamic. Cells of SA2271 were grown and visualized as in (G). Shown is a representative cell that underwent one reversal. Depicted are overlays of fluorescence and phase-contrast images recorded at the indicated time points in minutes. Arrows indicate the direction of movement. From 1:30 to 2:00, the cell did not move. From 2:00 to 2:30, the cell reversed. (J) Quantitative analysis of polar fluorescence signals. Relative fluorescence intensities (arbitrary units) of each pole in the cell in (I) were measured and plotted as a function of time. Filled squares, initial lagging pole; open circles, initial leading pole. Time intervals with a stop or reversal (Rev.) are indicated by double-headed arrows.
Figure 3
Figure 3
The RomR output domain is sufficient for bipolar, asymmetric localization. (A) Motility phenotype of romR mutant complemented with different romR alleles. Cells were incubated at 32°C for 24 h on 0.5% CTT medium/1.5% agar and visualized with a Leica MZ8 stereomicroscope. Scale bar: 5 mm. (B) Motility phenotype of romR mutant complemented with different romR-gfp alleles and localization of the corresponding GFP fusion proteins. For the experiments shown in the upper row, cells were incubated and visualized as in (A). For the experiments shown in the lower rows, cells were transferred from a steady-state culture to an agar pad on a slide and imaged by fluorescence and phase-contrast microscopy. Depicted are overlays of fluorescence and phase-contrast images, except for the strain containing the receiver-GFP construct for which the images are shown separately. (C) Immunoblots of accumulated mutant RomR and RomR-GFP proteins. Cells were grown as in (B) and harvested, and total protein (1 μg per lane) was separated by SDS–PAGE and analyzed by immunoblotting. Strains used in the left blot (left to right): SA2059, SA2244, SA2256, SA2063, SA2061, SA2058, SA2259, SA2260, SA2062, and SA2060. Strains used in the right blot (left to right): SA2058, SA2259, SA2260, SA2062, and SA2060. The left blot was probed with rabbit anti-RomR antibodies, and the right blot was probed with monoclonal anti-GFP antibodies. The different RomR and RomR-GFP proteins are indicated. The migration of molecular size markers is indicated on the left.
Figure 4
Figure 4
The Frz two-component system regulates RomR localization. Localization of RomR proteins in an frz mutant. Cells were transferred from a steady-state culture to an agar pad on a microscope slide and imaged by fluorescence and phase-contrast microscopy. Depicted are overlays of fluorescence and phase-contrast images.
Figure 5
Figure 5
The MglA GTPase regulates RomR localization. (A) Localization of RomR-GFP in an mglA mutant. Cells from steady-state cultures were transferred to an agar pad on a microscope slide and imaged by fluorescence and phase-contrast microscopy. Depicted are overlays of fluorescence and phase-contrast images. (B) The RomR-GFP cluster localizes to the cell pole containing Tfp in the mglA mutant. SA2042 cells were harvested from 1% CTT/1.5% agar, stained with Cy3, and inspected by fluorescence microscopy to visualize Tfp (Cy3; black arrows point to Tfp) and RomR-GFP (GFP; white arrows point to large RomR-GFP cluster) and by phase-contrast microscopy (Ph).
Figure 6
Figure 6
RomR and FrzS localize independently and relocate synchronously. (A) RomR-GFP localizes independently of FrzS. Cells were transferred from steady-state cultures to an agar pad on a microscope slide and imaged by fluorescence and phase-contrast microscopy. Depicted are overlays of fluorescence and phase-contrast images. (B) FrzS-GFP localizes independently of RomR. Cells were treated and imaged as in (A). Depicted are overlays of fluorescence and phase-contrast images. (C) FrzS-GFP and RomR-mDsRed relocate synchronously. Cells were treated as in (A) and imaged by fluorescence and phase-contrast microscopy at 30-s intervals. Shown is a representative cell that underwent one reversal. Depicted are fluorescence images (upper and middle panels) and phase-contrast images (lower panel). Arrows indicate the direction of movement. From 1:30 to 2:00, the cell stopped, and from 2:00 to 2:30, it reversed. (D) Quantitative analysis of polar fluorescence signals. The relative fluorescence intensity (arbitrary units) of each pole in the cell in (C) was measured and plotted as a function of time. Squares, FrzS-GFP signals; circles, RomR-mDsRed signals; filled symbols, initial lagging pole; open symbols, initial leading pole. Time intervals with a stop and a reversal (Rev.) are indicated by double-headed arrows.
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