Combinatorial control of type IVa pili formation by the four polarized regulators MglA, SgmX, FrzS, and SopA

Abstract

Type IVa pili (T4aP) are widespread and enable bacteria to translocate across surfaces. T4aP engage in cycles of extension, surface adhesion, and retraction, thereby pulling cells forward. Accordingly, the number and localization of T4aP are critical to efficient translocation. Here, we address how T4aP formation is regulated in Myxococcus xanthus, which translocates with a well-defined leading and lagging cell pole using T4aP at the leading pole. This localization is orchestrated by the small GTPase MglA and its downstream effector SgmX that both localize at the leading pole and recruit the PilB extension ATPase to the T4aP machinery at this pole. Here, we identify the previously uncharacterized protein SopA and show that it interacts directly with SgmX, localizes at the leading pole, stimulates polar localization of PilB, and is important for T4aP formation. We corroborate that MglA also recruits FrzS to the leading pole, and FrzS stimulates SgmX recruitment. In addition, FrzS and SgmX separately recruit SopA. Precise quantification of T4aP-formation and T4aP-dependent motility in various mutants supports a model whereby the main pathway for stimulating T4aP formation is the MglA/SgmX pathway. FrzS stimulates this pathway by recruiting SgmX and SopA. SopA stimulates the MglA/SgmX pathway by stimulating the function of SgmX, likely by promoting the SgmX-dependent recruitment of PilB to the T4aP machinery. The architecture of the MglA/SgmX/FrzS/SopA protein interaction network for orchestrating T4aP formation allows for combinatorial regulation of T4aP levels at the leading cell pole resulting in discrete levels of T4aP-dependent motility.

Importance: Type IVa pili (T4aP) are widespread bacterial cell surface structures with important functions in translocation across surfaces, surface adhesion, biofilm formation, and virulence. T4aP-dependent translocation crucially depends on the number of pili. To address how the number of T4aP is regulated, we focused on M. xanthus, which assembles T4aP at the leading cell pole and is a model organism for T4aP biology. Our results support a model whereby the four proteins MglA, SgmX, FrzS, and the newly identified SopA protein establish a highly intricate interaction network for orchestrating T4aP formation at the leading cell pole. This network allows for combinatorial regulation of the number of T4aP resulting in discrete levels of T4aP-dependent motility.

Keywords: FrzS; MglA; Myxococcus xanthus; SgmX; bacterial cell biology; bacterial motility; polarity; type IV pili.

Fig 1

SopA is important for T4aP-dependent motility. (A) sopA locus and SopA domain architecture. Upper panel, sopA locus; numbers in arrows, MXAN locus tags (in the NCBI Reference Sequence NC_008095.1, MXAN_0370, and MXAN_0372 are reannotated as MXAN_RS01820 and MXAN_RS01830, respectively); numbers in black indicate the first and last nucleotide in start and stop codons, respectively, relative to +1, the transcriptional start site of sopA (62). Kinked arrows, transcriptional start sites. Light red bar labeled P_nat indicates the 500 bp fragment upstream of the sopA start codon used for ectopic expression of sopA. Lower panel, conserved regions of SopA homologs are indicated in light red using the coordinates of SopA of M. xanthus. (B) SopA is important for T4aP-dependent motility in population-based assay. T4aP-dependent motility and gliding were analyzed on 0.5% and 1.5% agar supplemented with 0.5% CTT, respectively. Numbers indicate the colony expansion in 24 h as mean ± SD (n = 3 biological replicates). *P < 0.05, two-tailed Student’s t-test for samples with equal variances. In the complementation strain, sopA was expressed from its native promoter from a plasmid integrated in a single copy at the Mx8 attB site. Scale bars, 1 mm (left, middle), 100 µm (right). (C) SopA is important for T4aP-dependent motility in single-cell-based motility assay. T4aP-dependent motility was measured for cells on a polystyrene surface covered with 1% methylcellulose and gliding on 1.5% agar supplemented with 0.5% CTT. Individual data points from three biological replicates indicated in three different colors and with the number of cells per replicate indicated in the corresponding colors. The mean is shown for each experiment, and the mean for all experiments ± SD is shown in black. *P < 0.05, two-tailed Student’s t-test for samples with equal variances, ns, not significant, NA, not applicable because cells are non-motile.

Fig 2

SopA is important for T4aP extension and polar PilB localization. (A) SopA is important for T4aP formation. T4aP sheared off from 5 mg cells were separated by SDS-PAGE and probed with α-PilA antibodies (top rows). Middle row, protein from total cell extracts of 10⁸ cells was separated by SDS-PAGE and probed with α-PilA antibodies (middle rows), and after stripping, with α-PilC antibodies as a loading control (bottom rows). Numbers below blots indicate PilA levels as the mean ± SD from four biological replicates relative to WT. *, P < 0.05, two-tailed Student’s t-test for samples with equal variances. (B) SopA is important for T4aP extension. Experiment was done, presented, and analyzed as in A. For better comparison, only 10% of T4aP sheared from the hyper-piliated ∆pilT strains (#) were loaded. * (black, green), P < 0.05 compared to WT and the ∆pilT mutant, respectively. (C) Accumulation of mCherry-PilM, PilB-mCherry, and mCherry-PilT in the presence and absence of SopA. Protein from total cell extracts of 10⁸ cells was separated by SDS-PAGE and probed with α-mCherry antibodies (top) and after stripping with α-PilC antibodies as a loading control (bottom). All fusion proteins were synthesized from their native locus. (D) Quantification of the polar localization of mCherry-PilM, PilB-mCherry, and mCherry-PilT in the presence and absence of SopA by fluorescence microscopy. Scale bar, 5 µm. In the scatter plots, the percentage of total fluorescence at pole 2 is plotted against the percentage of total fluorescence at pole 1 for all cells with polar cluster(s). Pole 1 is per definition the pole with the highest fluorescence. Individual data points from three independent experiments are shown in three different colors and with the number of cells per replicate indicated in the corresponding colors. Bright green dot, mean fraction of fluorescence at the poles based on all three experiments and including cells with and without clusters. Numbers in the upper right corners, the mean percentage of total cytoplasmic fluorescence based on all three experiments and including cells with and without clusters. Black lines are symmetry lines. For all cells with cluster(s), an asymmetry index, ω, was calculated as indicated; based on ω values, localization patterns were binned into three categories as indicated; diffuse localization was determined when no polar signal was detected. Bar diagrams to the right, the percentage of cells with a polar localization pattern and diffuse localization according to the color code.

Fig 3

Polar localization of mVenus-SopA, SgmX-mVenus, and FrzS-GFP in the presence and absence of MglA, SopA, SgmX, and/or FrzS. (A) mVenus-SopA is dynamically localized with a large cluster at the leading cell pole. Cells were imaged by time-lapse fluorescence microscopy every 30 seconds. Scale bar, 5 µm. (B–D) Quantification of the polar localization of mVenus-SopA, SgmX-mVenus, and FrzS-GFP. Experiments were done and are presented as in Fig. 2D. All fusion proteins were synthesized from their native locus. Schematics below each row, summarize effects observed. In the schematics, the protein being analyzed for localization is indicated by black circle. (E) Model of protein interaction network for polar localization of MglA, SgmX, FrzS, and SopA. Gray circle surrounding MglA-GTP indicates the polar recruitment of MglA-GTP by the RomR/RomX complex of the polarity module.

Fig 4

Combinatorial effect of SgmX, FrzS, and SopA on T4aP-dependent motility and T4aP formation. (A) Effect of SgmX, FrzS, and/or SopA on T4aP-dependent motility. Cells were incubated on 0.5% agar supplemented with 0.5% CTT. Scale bar, 1 mm. Numbers, colony expansion in millimeter in 24 h as mean ± SD from three biological replicates; * (black, red, and purple) P < 0.05, two-tailed Student’s t-test for samples with equal variances compared to WT, the ΔsopA mutant, and the ΔfrzS mutant, respectively. (B and C) Effect of SgmX, FrzS, and/or SopA on T4aP formation. Experiments were done and data presented as in Fig. 2A and B, except that in B T4aP sheared off from 7.5 mg cells were loaded. * (black, red, blue, and green), P < 0.05, two-tailed Student’s t-test for samples with equal variances compared to WT, the ΔsopA mutant, the ΔfrzS mutant, and the ∆pilT mutant, respectively. (D) FrzS is important for polar localization of PilB-mCherry. Experiment was done and data presented as in Fig. 2D.

Fig 5

BACTH assay for SgmX, FrzS, and SopA interactions. Full-length SgmX, FrzS, and SopA were fused to the C-terminus of T25 and T18. Lower left corner, T25-Zip + T18 Zip positive control.

Fig 6

Model of protein interaction network for combinatorial regulation of T4aP-formation and T4aP-dependent motility in M. xanthus. The box shown by stippled lines indicates interactions that stimulate polar recruitment of proteins; gray circle surrounding MglA-GTP indicates the polar recruitment of MglA-GTP by the RomR/RomX complex of the polarity module.