Fig 1.
Subcellular localization of proteins required for gliding motility.
Synonyms for proteins required for gliding motility are indicated on the left. Proteins shown on a grey, brown or purple background in the left panel and in grey, brown or purple in the right panel are encoded together in the genome [17]; proteins in white are not encoded near other proteins shown here. Proteins outlined in red have been shown by fluorescence microscopy to localize in clusters along the cell body [here; [15–18]]; proteins that interact based on pull down experiments using M. xanthus cell extracts are indicated in italics [15]. CglB is an OM lipoprotein [29] and CglD is predicted to be an OM lipoprotein [20]. It is not known if they face towards the periplasm or are exposed on the cell surface. They are shown on the cell surface because they contain a von Willebrand domain (VWA_2), which is often involved in cell adhesion [30].
Fig 2.
CglC, GltB, GltA and GltC are required for gliding motility.
(A) Genetic organization of cglC, gltB, gltA and gltC region. Numbers below diagram indicates distances between start and stop codons of flanking genes. Orange box indicates the intergenic region containing the divergent cglC and gltBAC promoters. The four complementation constructs with the native promoter (orange) are indicated below. (B, C) Motility phenotypes of the indicated mutants (B) and complementation strains (C). Cells were incubated at 32°C for 24 h on 0.5% and 1.5% agar to score for type IV pili-dependent and gliding motility, respectively. In-frame deletion mutants are shown in (B) and the corresponding complementation strains are shown in (C). In the rows labeled Pnat and PpilA, the complementing genes were expressed from the native promoter (marked orange in panel A) and the pilA promoter, respectively. Scale bar, 50μm on 1.5% agar and 2 mm on 0.5% agar. (D) Accumulation of CglC, GltB, GltA and GltC. Total cell lysates from exponentially growing cultures were separated by SDS-PAGE (proteins from 7×107 cells loaded per lane) and analyzed by immunoblotting using specific antibodies as indicated. In the left and right panels, the complementation strains express the relevant gene from the native and the pilA promoter, respectively. Arrowheads indicate the relevant protein with the calculated molecular mass without signal peptides in brackets. Molecular mass markers are indicated in the leftmost lane of the two panels. Note that GltC has a calculated molecular mass of 73.3 kDa but is consistently observed to run as a higher molecular mass protein by SDS-PAGE. (E) Domain structure of CglC, GltB, GltA and GltC.
Fig 3.
Subcellular localization of CglC, GltB, GltA and GltC.
(A) GltB, GltA and GltC localize to the OM and GltC is a soluble protein. Total WT cell extracts were separated into soluble and membrane fractions. Outer membrane vesicles (OMVs) were isolated from the cell free supernatant. Fractions were analyzed by immunoblotting using the indicated antibodies. Molecular mass markers are indicated to the left. The relevant proteins are indicated. (B) CglC is facing towards the periplasm. WT cells were treated with Proteinase K (PK) at the indicated concentrations for 10 min, followed by SDS-PAGE and immunoblotting. Lanes labeled Δ contain total cell extracts from the relevant in-frame deletion mutant. (C) CglC is important for OM incorporation of GltB and GltA. Left panel, total cell extracts from ΔcglC cells were separated into soluble and membrane fractions. Right panel, OMVs were isolated as in (A) from the indicated strains. All fraction were analyzed by immunoblotting using the indicated antibodies. Lanes labeled Δ contain total cell extracts from the relevant in-frame deletion mutants.
Fig 4.
GltB and GltA are mutually stabilizing and stabilize GltC.
(A) GltB and GltA are mutually stabilizing and stabilize GltC. Total cell extracts from cells grown as described in Fig 2D were isolated from strains of the indicated genotypes and analyzed by immunoblotting using specific antibodies as indicated. Molecular mass markers are indicated to the left. The relevant proteins are indicated. (B) Summary of observed effects on protein stability. Green and red indicate no effect or negative effect, respectively.
Fig 5.
GltB, GltA and GltC interact directly.
(A) GltC interacts with GltB and GltA. Purified MalE, MalE-CglC20-172, MalE-GltB20-275 or MalE-GltA22-256 were mixed with an equal amount of GltC25-673-His6, loaded on an amylose matrix, the matrix was washed and then bound proteins were eluted with 10 mM maltose. Samples before loading (L), from the flow through (FT), the washing step (W) and elution (E) were analyzed by immunoblotting using antibodies against GltC and MalE. In the upper row, arrowheads indicate GltC25-673-His6 with its calculated molecular mass and in the lower row, the individual MalE proteins. Molecular mass markers are indicated to the left. (B) GltB interacts with GltA. Purified MalE, MalE-CglC20-172 or MalE-GltA22-256 were mixed with an equal amount of GST-GltB20-275 and analyzed as described in A. Blots are marked as in A. (C) Summary of direct protein interactions. Green lines indicate interaction detected, red lines indicate interactions tested but not detected; grey line indicates interaction not tested.
Fig 6.
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.
Fig 7.
GltB and GltA incorporation into motility complexes depends on other gliding motility proteins.
Cells were treated and analyzed as in Fig 6A. Note that all strains tested for GltB-mCherry and GltA-mCherry localization are gltB+ and gltA+, respectively. Scale bar, 2μm.
Fig 8.
AglZ-YFP and AglQ-mCherry incorporation into motility complexes depends on CglC, GltB, GltA and GltC.
Cells were treated and analyzed as in Fig 6A. Scale bar, 2μm.
Fig 9.
CglC stimulates formation of motility complexes after transfer.
(A) Motility and stimulation phenotypes of the indicated strains and strain mixtures. Cells were incubated at 32°C for 48 h on 1.5% agar to score for gliding motility. Scale bar, 50μm. (B) Localization of GltB-mCherry, GltA-mCherry, AglZ-YFP and AglQ-mCherry in the ΔcglC mutant background incubated alone or in the presence of the non-motile CglC donor DK6204. Recipient and donor were mixed in a 1:1 ratio and incubated as described in (A), transferred to a thin agar pad on a microscope slide and imaged by fluorescence microscopy. Scale bar, 2μm.
Table 1.
M. xanthus strains used in this work1.