A multi-protein complex from Myxococcus xanthus required for bacterial gliding motility

Abstract

Myxococcus xanthus moves by gliding motility powered by Type IV pili (S-motility) and a second motility system, A-motility, whose mechanism remains elusive despite the identification of approximately 40 A-motility genes. In this study, we used biochemistry and cell biology analyses to identify multi-protein complexes associated with A-motility. Previously, we showed that the N-terminal domain of FrzCD, the receptor for the frizzy chemosensory pathway, interacts with two A-motility proteins, AglZ and AgmU. Here we characterized AgmU, a protein that localized to both the periplasm and cytoplasm. On firm surfaces, AgmU-mCherry colocalized with AglZ as distributed clusters that remained fixed with respect to the substratum as cells moved forward. Cluster formation was favoured by hard surfaces where A-motility is favoured. In contrast, AgmU-mCherry clusters were not observed on soft agar surfaces or when cells were in large groups, conditions that favour S-motility. Using glutathione-S-transferase affinity chromatography, AgmU was found to interact either directly or indirectly with multiple A-motility proteins including AglZ, AglT, AgmK, AgmX, AglW and CglB. These proteins, important for the correct localization of AgmU and AglZ, appear to be organized as a motility complex, spanning the cytoplasm, inner membrane and the periplasm. Identification of this complex may be important for uncovering the mechanism of A-motility.

Figure 1. The two TPR clusters of AgmU interact with the N-terminus of FrzCD

A) Schematic representation of the different AgmU fragments used in the in vitro cross-linking shown in (C). Each protein is fused to a N-terminal His₆ tag. The first 60 amino acids of AgmU are shown in the inset. The possible recognition sites for the Tat secretion pathway (twin arginines) are shown in bold characters. B) Schematic representation of the N-terminal and C-terminal regions of FrzCD used in the in vitro cross-linking shown in (C). Each protein is fused to a N-terminal His₆ tag. C) Anti-FrzCD immunoblotting of the in vitro formaldehyde cross-linking experiments with AgmU and FrzCD fragments. Purified FrzCD N-terminal (FrzCD-N) or C-terminal (FrzCD-C) regions and/or purified AgmU fragments (Roman numerals indicate AgmU domains shown in (A) incubated in the presence of the cross-linker (10 mM formaldehyde). Lanes 1–6, FrzCD or AgmU proteins incubated alone; lanes 7–10, FrzCD-N co-incubated with different AgmU fragments; lanes 11–14, FrzCD-C co-incubated with different AgmU fragments. The arrowheads indicate bands only seen after co-incubating FrzCD-N with full length AgmU or the TPR clusters of AgmU and cross-linker.

Figure 2. A-motility analysis of M xanthus strains DZ2 (wt), frzCD, aglZ and agmU mutants

In the S⁻ background (pilA), single cells swarming out from the edge of colonies on 1.5% agar (or agarose) were monitored as an indicator of A-motility (Hodgkin, 1979). Cells (10 µl), at a concentration of 4 × 10⁹ cfu ml⁻¹, were spotted on CYE plates containing 1.5% (w/v) agar (or agarose), incubated at 32°C and photographed after 48 h with a WTI charge-coupled device (CCD)-72 camera, using a Nikon Labphot-2 microscope. Scale bar, 40 µm.

Figure 3. AgmU-mCherry shows two distinct localization patterns

A) In large cell groups on 1.5% (w/v) agar (or agarose), AgmU-mCherry concentrates near the cell envelope. Images were taken with an Olympus IX70 DeltaVision microscope. B) Statistical analysis of the trans-section fluorescence scans in the same condition as panel A gives two fluorescence peaks at the location of the membrane. Twenty individual cells were scanned with ImageQuant software (GE healthcare) and the highest fluorescence density of each scan was normalized to 255 (same below). The inset shows a typical scan position in panel A. C) fluorescence scan along the long axis of one typical cell in panel A, shows the relative high concentration of AgmU-mCherry in the posterior half of the cell. D) In small groups or isolated cells on 1.5% (w/v) agar (or agarose), AgmU-mCherry shows two localization patterns. Besides the envelope-associated localization, protein clusters distributed along the cells were also seen. E) Statistical analysis of the scans between the clusters in the condition of panel D. F) Statistical analysis of the scans across clusters in the condition panel D. G) When the signal sequence of AgmU is deleted, AgmU_Δ8--41-mCherry only forms cytoplasmic clusters. H) Statistical analysis of the scans between clusters in the condition of panel G. I) Statistical analysis of across the clusters in the condition of panel G. J) On 1.5% agar (or agarose), no clusters formed by AgmUΔ809-1218-mCherry are evident in either grouped or isolated cells, while the periplasmic localization is retained in this mutant. K) Statistical analysis of the scans in the condition of panel J. Scale bars, 5 µm.

Figure 4. AgmU localizes to both the periplasm and cytoplasm

Western immunoblots of cell fractions following osmotic shock show that wild type AgmU, AgmU-mCherry and AgmU_Δ809-1218-mCherry (AgmU C-terminus deletion) localize in both cytoplasmic and periplasmic fractions. While the protein with the hypothetical signal sequence deleted (AgmU_Δ8--41-mCherry), only localizes in the cytoplasm. The cytoplasmic protein FrzE and lipoprotein MbhA are also shown as controls.

Figure 5. The clusters of AgmU identify the same “focal adhesion” sites as AglZ

A) The localization of AgmU-mCherry clusters overlaps with those of AglZ-GFP in vivo. B) Fluorescence scans along the cell length in panel A. mCherry and GFP signals are scanned separately with ImageQuant (GE Healthcare). The highest fluorescence density of each scan was normalized to 255. The inset shows the 2-fold magnification of the cell scanned in panel A. C) AgmU-mCherry clusters localize differently from that of FrzCD-GFP in vivo. D) A fluorescence scan along the cell length in panel C. The inset shows the two-fold magnification of the cell scanned in panel C. E) When cells move forward, AgmU-mCherry clusters remain fixed relative to the substratum. The fluorescent signal of mCherry in a single cell on 1.5% (w/v) agar (or agarose) is monitored with an Olympus DeltaVision IX70 microscope and recorded every 30 s. The positions of each cluster during a 390 s time course are marked with arrows in different colors. Scale bars, 5 µm.

Figure 6. The formation of AgmU clusters is regulated by the hardness of the substratum

A) AgmU never formed clusters in 1% (w/v) methylcellulose. B) On 5% (w/v) agar (or agarose), AgmU-mCherry formed clusters even in large cell groups. C) Super-sized AgmU-mCherry clusters are found when cells are placed on a glass surface. Four individual cells are shown, but more than 50 cells were observed. D) AgmU^Δ8–41-mCherry did not form clusters in 1% (w/v) methylcellulose. Scale bars, 5 µm.

Figure 7. The genes that encode proteins interacting with AgmU are all required for A-motility

A) agmU is the first gene of a large A-motility related gene cluster. B) A-motility analysis of the parental strain agmU∷mCherry aglZ-gfp∷kan, the additional deletion mutants of the genes downstream of agmU, and the additional deletion mutants of cglB and aglW. The movement of (pilA) cells that lack S-motility were monitored as an indicator of A-motility on 1.5% agar (or agarose) (Hodgkin, 1979). Samples were cultured and photographed as described in Figure 2. Note that the strains aglT agmU-mCherry∷kan and aglT aglZ-gfp∷kan have similar phenotypes and only the phenotype of aglT agmU-mCherry∷kan is shown. Scale bar, 40 µm. C) The products of a series of genes effect the localization of AgmU and AglZ. All the gene products regulate the localization of AglZ while only AglT, PglI and AgmK affect the localization of AgmU. In the aglT background, almost no AgmU-mCherry is detectable (see Figure S2). Scale bar, 5 µm.

Figure 8. A working model for the A-motility complex

The proteins identified in this report and previous studies are summarized in this model. The outer membrane, pepidoglycan layer and the inner membrane near the “focal adhesion” site are shown in fragments. Previously, FrzCD was shown to interact with both AgmU and AglZ (Mauriello et al., 2009b); AglZ was shown to interact with the cytoskeleton protein MreB and the GTPase MglA (Hartzell & Kaiser, 1991, Mauriello et al., 2010). Data from E. coli TolB protein (AglW in this study) suggest an interaction of this protein with peptidoglycan-associated lipoprotein PAL (Bonsor et al., 2009), whose homologue in M. xanthus is MXAN_4581. Note that the proteins and structures in this model are not represented proportionally to their actual sizes.