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Contact- and Protein Transfer-Dependent Stimulation of Assembly of the Gliding Motility Machinery in Myxococcus xanthus.

Jakobczak B, Keilberg D, Wuichet K, Søgaard-Andersen L - PLoS Genet. (2015)

Bottom Line: Conversely, incorporation of AglZ and AglQ into the gliding motility complex depends on CglC, GltB, GltA and GltC.Remarkably, physical transfer of the OM lipoprotein CglC to a ΔcglC recipient stimulates assembly of the gliding motility complex in the recipient likely by facilitating the OM integration of GltB and GltA.These data provide evidence that the gliding motility complex in M. xanthus includes OM proteins and suggest that this complex extends from the cytoplasm across the cell envelope to the OM.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

ABSTRACT
Bacteria engage in contact-dependent activities to coordinate cellular activities that aid their survival. Cells of Myxococcus xanthus move over surfaces by means of type IV pili and gliding motility. Upon direct contact, cells physically exchange outer membrane (OM) lipoproteins, and this transfer can rescue motility in mutants lacking lipoproteins required for motility. The mechanism of gliding motility and its stimulation by transferred OM lipoproteins remain poorly characterized. We investigated the function of CglC, GltB, GltA and GltC, all of which are required for gliding. We demonstrate that CglC is an OM lipoprotein, GltB and GltA are integral OM β-barrel proteins, and GltC is a soluble periplasmic protein. GltB and GltA are mutually stabilizing, and both are required to stabilize GltC, whereas CglC accumulate independently of GltB, GltA and GltC. Consistently, purified GltB, GltA and GltC proteins interact in all pair-wise combinations. Using active fluorescently-tagged fusion proteins, we demonstrate that GltB, GltA and GltC are integral components of the gliding motility complex. Incorporation of GltB and GltA into this complex depends on CglC and GltC as well as on the cytoplasmic AglZ protein and the inner membrane protein AglQ, both of which are components of the gliding motility complex. Conversely, incorporation of AglZ and AglQ into the gliding motility complex depends on CglC, GltB, GltA and GltC. Remarkably, physical transfer of the OM lipoprotein CglC to a ΔcglC recipient stimulates assembly of the gliding motility complex in the recipient likely by facilitating the OM integration of GltB and GltA. These data provide evidence that the gliding motility complex in M. xanthus includes OM proteins and suggest that this complex extends from the cytoplasm across the cell envelope to the OM. These data add assembly of gliding motility complexes in M. xanthus to the growing list of contact-dependent activities in bacteria.

No MeSH data available.


GltB and GltA are incorporated into gliding motility complexes.(A) Localization of GltB-mCherry, GltA-mCherry GltC-mCherry in the indicated genetic backgrounds. Cells were transferred from exponentially growing cultures to a thin agar pad on a microscope slide and imaged by fluorescence microscopy. For the z-sections, the z positions are indicated by a barred circle. The localization patterns observed are indicated in the schematics and numbers represent % of cells with that pattern; n > 100. Scale bar, 2μm. (B) Time-lapse microscopy of cells containing GltB-mCherry, GltA-mCherry or GltC-mCherry. Cells of the indicated genotypes were treated as in (A) and imaged by time-lapse DIC and fluorescence microscopy at 60 s intervals. Same colored arrowheads indicate position of motility complex during cell movement. Left panel, fluorescence microscopy images; right panel, merged DIC and fluorescence microscopy images. (C) Fluorescence microscopy images and line scans of cells expressing GltB-mCherry or GltA-mCherry and AglZ-YFP. In the line scans, red lines refer to GltB/GltA-mCherry while green lines refer to AglZ-YFP. Cells were treated as in A. Scale bar, 2μm. (D) Time-lapse microscopy of cells containing GltB-mCherry or GltA-mCherry and AglZ-YFP. Cells containing the indicated fusions were treated as in (A) and imaged by time-lapse fluorescence microscopy at 60 s intervals. Same colored triangles indicate position of motility complex during cell movement. The corresponding linescans are shown in S4 Fig.
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pgen.1005341.g006: GltB and GltA are incorporated into gliding motility complexes.(A) Localization of GltB-mCherry, GltA-mCherry GltC-mCherry in the indicated genetic backgrounds. Cells were transferred from exponentially growing cultures to a thin agar pad on a microscope slide and imaged by fluorescence microscopy. For the z-sections, the z positions are indicated by a barred circle. The localization patterns observed are indicated in the schematics and numbers represent % of cells with that pattern; n > 100. Scale bar, 2μm. (B) Time-lapse microscopy of cells containing GltB-mCherry, GltA-mCherry or GltC-mCherry. Cells of the indicated genotypes were treated as in (A) and imaged by time-lapse DIC and fluorescence microscopy at 60 s intervals. Same colored arrowheads indicate position of motility complex during cell movement. Left panel, fluorescence microscopy images; right panel, merged DIC and fluorescence microscopy images. (C) Fluorescence microscopy images and line scans of cells expressing GltB-mCherry or GltA-mCherry and AglZ-YFP. In the line scans, red lines refer to GltB/GltA-mCherry while green lines refer to AglZ-YFP. Cells were treated as in A. Scale bar, 2μm. (D) Time-lapse microscopy of cells containing GltB-mCherry or GltA-mCherry and AglZ-YFP. Cells containing the indicated fusions were treated as in (A) and imaged by time-lapse fluorescence microscopy at 60 s intervals. Same colored triangles indicate position of motility complex during cell movement. The corresponding linescans are shown in S4 Fig.

Mentions: In the WT background, GltB-mCherry localized to multiple clusters along the cell length in the majority of cells and in the ΔgltB background 48% of cells contained these clusters (Fig 6A) suggesting that GltB-mCherry is more efficiently incorporated into these clusters in the presence of native GltB. GltA-mCherry localized to multiple clusters along the cell length in the majority of cells in the WT as well as in the ΔgltA background (Fig 6A). GltC-mCherry also localized to multiple clusters along the cell length and did so more efficiently in the ΔgltC background (Fig 6A). In microscopy z-stacks of these cells, the GltB-mCherry, GltA-mCherry and GltC-mCherry clusters were only visible when the focal plane was focused close to the substratum (Fig 6A). In time-lapse fluorescence microscopy, all three proteins behaved similarly and formed fixed clusters that remained stationary with respect to the substratum as cells moved, assembled towards the leading cell pole, and disassembled towards the lagging cell pole (Fig 6B) suggesting that all three proteins are incorporated into the gliding motility complex.


Contact- and Protein Transfer-Dependent Stimulation of Assembly of the Gliding Motility Machinery in Myxococcus xanthus.

Jakobczak B, Keilberg D, Wuichet K, Søgaard-Andersen L - PLoS Genet. (2015)

GltB and GltA are incorporated into gliding motility complexes.(A) Localization of GltB-mCherry, GltA-mCherry GltC-mCherry in the indicated genetic backgrounds. Cells were transferred from exponentially growing cultures to a thin agar pad on a microscope slide and imaged by fluorescence microscopy. For the z-sections, the z positions are indicated by a barred circle. The localization patterns observed are indicated in the schematics and numbers represent % of cells with that pattern; n > 100. Scale bar, 2μm. (B) Time-lapse microscopy of cells containing GltB-mCherry, GltA-mCherry or GltC-mCherry. Cells of the indicated genotypes were treated as in (A) and imaged by time-lapse DIC and fluorescence microscopy at 60 s intervals. Same colored arrowheads indicate position of motility complex during cell movement. Left panel, fluorescence microscopy images; right panel, merged DIC and fluorescence microscopy images. (C) Fluorescence microscopy images and line scans of cells expressing GltB-mCherry or GltA-mCherry and AglZ-YFP. In the line scans, red lines refer to GltB/GltA-mCherry while green lines refer to AglZ-YFP. Cells were treated as in A. Scale bar, 2μm. (D) Time-lapse microscopy of cells containing GltB-mCherry or GltA-mCherry and AglZ-YFP. Cells containing the indicated fusions were treated as in (A) and imaged by time-lapse fluorescence microscopy at 60 s intervals. Same colored triangles indicate position of motility complex during cell movement. The corresponding linescans are shown in S4 Fig.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4488436&req=5

pgen.1005341.g006: GltB and GltA are incorporated into gliding motility complexes.(A) Localization of GltB-mCherry, GltA-mCherry GltC-mCherry in the indicated genetic backgrounds. Cells were transferred from exponentially growing cultures to a thin agar pad on a microscope slide and imaged by fluorescence microscopy. For the z-sections, the z positions are indicated by a barred circle. The localization patterns observed are indicated in the schematics and numbers represent % of cells with that pattern; n > 100. Scale bar, 2μm. (B) Time-lapse microscopy of cells containing GltB-mCherry, GltA-mCherry or GltC-mCherry. Cells of the indicated genotypes were treated as in (A) and imaged by time-lapse DIC and fluorescence microscopy at 60 s intervals. Same colored arrowheads indicate position of motility complex during cell movement. Left panel, fluorescence microscopy images; right panel, merged DIC and fluorescence microscopy images. (C) Fluorescence microscopy images and line scans of cells expressing GltB-mCherry or GltA-mCherry and AglZ-YFP. In the line scans, red lines refer to GltB/GltA-mCherry while green lines refer to AglZ-YFP. Cells were treated as in A. Scale bar, 2μm. (D) Time-lapse microscopy of cells containing GltB-mCherry or GltA-mCherry and AglZ-YFP. Cells containing the indicated fusions were treated as in (A) and imaged by time-lapse fluorescence microscopy at 60 s intervals. Same colored triangles indicate position of motility complex during cell movement. The corresponding linescans are shown in S4 Fig.
Mentions: In the WT background, GltB-mCherry localized to multiple clusters along the cell length in the majority of cells and in the ΔgltB background 48% of cells contained these clusters (Fig 6A) suggesting that GltB-mCherry is more efficiently incorporated into these clusters in the presence of native GltB. GltA-mCherry localized to multiple clusters along the cell length in the majority of cells in the WT as well as in the ΔgltA background (Fig 6A). GltC-mCherry also localized to multiple clusters along the cell length and did so more efficiently in the ΔgltC background (Fig 6A). In microscopy z-stacks of these cells, the GltB-mCherry, GltA-mCherry and GltC-mCherry clusters were only visible when the focal plane was focused close to the substratum (Fig 6A). In time-lapse fluorescence microscopy, all three proteins behaved similarly and formed fixed clusters that remained stationary with respect to the substratum as cells moved, assembled towards the leading cell pole, and disassembled towards the lagging cell pole (Fig 6B) suggesting that all three proteins are incorporated into the gliding motility complex.

Bottom Line: Conversely, incorporation of AglZ and AglQ into the gliding motility complex depends on CglC, GltB, GltA and GltC.Remarkably, physical transfer of the OM lipoprotein CglC to a ΔcglC recipient stimulates assembly of the gliding motility complex in the recipient likely by facilitating the OM integration of GltB and GltA.These data provide evidence that the gliding motility complex in M. xanthus includes OM proteins and suggest that this complex extends from the cytoplasm across the cell envelope to the OM.

View Article: PubMed Central - PubMed

Affiliation: Department of Ecophysiology, Max Planck Institute for Terrestrial Microbiology, Marburg, Germany.

ABSTRACT
Bacteria engage in contact-dependent activities to coordinate cellular activities that aid their survival. Cells of Myxococcus xanthus move over surfaces by means of type IV pili and gliding motility. Upon direct contact, cells physically exchange outer membrane (OM) lipoproteins, and this transfer can rescue motility in mutants lacking lipoproteins required for motility. The mechanism of gliding motility and its stimulation by transferred OM lipoproteins remain poorly characterized. We investigated the function of CglC, GltB, GltA and GltC, all of which are required for gliding. We demonstrate that CglC is an OM lipoprotein, GltB and GltA are integral OM β-barrel proteins, and GltC is a soluble periplasmic protein. GltB and GltA are mutually stabilizing, and both are required to stabilize GltC, whereas CglC accumulate independently of GltB, GltA and GltC. Consistently, purified GltB, GltA and GltC proteins interact in all pair-wise combinations. Using active fluorescently-tagged fusion proteins, we demonstrate that GltB, GltA and GltC are integral components of the gliding motility complex. Incorporation of GltB and GltA into this complex depends on CglC and GltC as well as on the cytoplasmic AglZ protein and the inner membrane protein AglQ, both of which are components of the gliding motility complex. Conversely, incorporation of AglZ and AglQ into the gliding motility complex depends on CglC, GltB, GltA and GltC. Remarkably, physical transfer of the OM lipoprotein CglC to a ΔcglC recipient stimulates assembly of the gliding motility complex in the recipient likely by facilitating the OM integration of GltB and GltA. These data provide evidence that the gliding motility complex in M. xanthus includes OM proteins and suggest that this complex extends from the cytoplasm across the cell envelope to the OM. These data add assembly of gliding motility complexes in M. xanthus to the growing list of contact-dependent activities in bacteria.

No MeSH data available.