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.2015 Nov 16;198(3):510-20.
doi: 10.1128/JB.00548-15. Print 2016 Feb 1.

MglC, a Paralog of Myxococcus xanthus GTPase-Activating Protein MglB, Plays a Divergent Role in Motility Regulation

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MglC, a Paralog of Myxococcus xanthus GTPase-Activating Protein MglB, Plays a Divergent Role in Motility Regulation

Anna L McLoon et al. J Bacteriol..

Abstract

In order to optimize interactions with their environment and one another, bacteria regulate their motility. In the case of the rod-shaped cells of Myxococcus xanthus, regulated motility is essential for social behaviors. M. xanthus moves over surfaces using type IV pilus-dependent motility and gliding motility. These two motility systems are coordinated by a protein module that controls cell polarity and consists of three polarly localized proteins, the small G protein MglA, the cognate MglA GTPase-activating protein MglB, and the response regulator RomR. Cellular reversals are induced by the Frz chemosensory system, and the output response regulator of this system, FrzZ, interfaces with the MglA/MglB/RomR module to invert cell polarity. Using a computational approach, we identify a paralog of MglB, MXAN_5770 (MglC). Genetic epistasis experiments demonstrate that MglC functions in the same pathway as MglA, MglB, RomR, and FrzZ and is important for regulating cellular reversals. Like MglB, MglC localizes to the cell poles asymmetrically and with a large cluster at the lagging pole. Correct polar localization of MglC depends on RomR and MglB. Consistently, MglC interacts directly with MglB and the C-terminal output domain of RomR, and we identified a surface of MglC that is necessary for the interaction with MglB and for MglC function. Together, our findings identify an additional member of the M. xanthus polarity module involved in regulating motility and demonstrate how gene duplication followed by functional divergence can add a layer of control to the complex cellular processes of motility and motility regulation.

Importance: Gene duplication and the subsequent divergence of the duplicated genes are important evolutionary mechanisms for increasing both biological complexity and regulation of biological processes. The bacterium Myxococcus xanthus is a soil bacterium with an unusually large genome that carries out several social processes, including predation of other bacterial species and formation of multicellular, spore-filled fruiting bodies. One feature of the large M. xanthus genome is that it contains many gene duplications. Here, we compare the products of one example of gene duplication and divergence, in which a paralog of the cognate MglA GTPase-activating protein MglB has acquired a different and opposing role in the regulation of cellular polarity and motility, processes critical to the bacterium's social behaviors.

Copyright © 2016 McLoon et al.

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Figures

FIG 1
FIG 1
MglC is an orphan homolog of MglB. (A) Identification of the MglB_orphan1 subfamily. A phylogenetic tree was constructed from a multiple-sequence alignment of 328 orphan MglB sequences identified in a previous study (46). The innermost ring around the tree (blue) shows the sequences encoded in genomes that also encode group 1 MglA sequences, and the ring in gray shows the sequences encoded in genomes that encode group 2, 3, 4, or 5 MglA sequences. The blue branches identify the conserved MglB_orphan1 clade. (B) A phylogenetic tree built from a multiple-sequence alignment of members of the MglB_orphan1 clade and the MglB_coupled1 clade. Branches and ring colors identify the sequences from the MglB_orphan1 clade found in panel A (blue) and the MglB_coupled1 sequences that are genomically coupled to group 1 MglA (black). (C) Sequence logo corresponding to the clade composed entirely of coupled sequences and the clade composed of primarily orphan sequences (MglB_orphan1 sequences plus nine MglB_coupled1 sequences, as described in the text) were generated from the multiple-sequence alignment used in panel B that includes residues 15 to 129 of MglB (MXAN_1926) or residues 3 to 120 of MglC (MXAN_5770). Arrows below the orphan sequence logo indicate residues of MglC targeted for mutagenesis: F25, D26, and I28. In the logos, the letter size at each position represents the relative frequency of the given amino acid at that position.
FIG 2
FIG 2
MglC is important for regulation of T4P-dependent motility and gliding motility. Strains of the indicated genotypes were incubated on 0.5% agar–0.5% CTT plates (A) or 1.5% agar–0.5% CTT plates (B) for 24 h. Images within the black rectangle in panels A and B represent double mutants. In the ΔmglC mutant that is complemented by a WT copy ofmglC, the complementing copy is expressed from thepilA promoter at the Mx8attB site. Colonies on 1.5% agar–0.5% CTT plates were incubated for 96 h and imaged (C), and the colony diameter was measured (D). Bars represent the average diameter of at least four colonies, and error bars represent the standard deviation. *,P < 0.05 from a two-samplet test between WT and the indicated mutant. Scale bars represent 2 mm (A) or 100 μm (B). (D) MglC is necessary for timely reversals. Fifty representative cells of the indicated genotype were imaged at 30-s intervals for 15 min, and the number of reversals per cell were manually quantified and plotted. Dashed lines represent the mean, the solid lines represent the median, the boxes denote quartiles, whiskers indicate 10% and 90% quantiles, and circles represent outliers.
FIG 3
FIG 3
Asymmetric polar localization of MglC depends on MglB and RomR. Cells from exponentially growing liquid cultures of the ΔmglC YFP-mglC, ΔmglA ΔmglC YFP-mglC, ΔmglB ΔmglC YFP-mglC, or ΔromR ΔmglC YFP-mglC strain were spotted onto TPM agar pads and imaged by fluorescence microscopy. (A and C) Dynamic localization of YFP-MglC in moving cells; individual images were captured every 30 s. Numbers indicate time in minutes, and the cartoons below indicate the fluorescence localization patterns and direction of cell movement. The cell in panel A reversed at 5 min, and the cell in panel C reversed at 6 min.
FIG 4
FIG 4
MglC interacts with itself, MglB, and the C-terminal Glu-rich region of RomR. (A) Full-length MglB, MglA, MglC, FrzZ, the C-terminal Glu-rich region of RomR, the Pro-rich linker of RomR, or the N-terminal receiver domain of RomR was fused to the indicated variant of the Bordetella pertussis adenylate cyclase and coexpressed in the indicated combinations in E. coli BTH101. The negative control demonstrates that there is no interaction between the T25 and T18 adenylate cyclase fragments in the absence of bait proteins. +, blue colonies and protein interactions; −, white colonies and no protein-protein interactions. (B) Purified MglC does not inhibit GAP activity of MglB and does not stimulate GTPase activity of MglAin vitro. In vitro GTPase assays were carried out as described previously (45) using 2 μM (each) indicated purified protein combinations. Aliquots of each reaction were sampled and quenched with activated charcoal at the indicated time points. Each data point represents the average of two separate experiments, except for MglA 20 min and MglA + MglB 20 min, which only represent one replicate. Error bars represent standard deviations.
FIG 5
FIG 5
The FDI surface of MglC is necessary for MglB interaction and functionin vivo. (A) A structural model of an MglC dimer based on the MglB structures from T. thermophilus andS. avermitilis. Substituted residues on the FDI surface are highlighted in red. (B) MglCF25A D26A I28A interacts less efficiently with MglB than the MglC WT protein. BACTH analyses were performed as described in the legend to Fig. 4A. (C) MglCF25A D26A I28A does not complement the ΔmglC mutant. Motility assays on 0.5% agar, favoring T4P-dependent motility, and on 1.5% agar, favoring gliding motility, were performed as described in the legend to Fig. 2A and B. (D) YFP-MglCF25A D26A I28A shows aberrant localization. Cells were imaged as described in the legend to Fig. 3.
FIG 6
FIG 6
Model of regulation of motility polarity in M. xanthus. Schematic of protein localization in a cell moving in the direction indicated by the arrow. The size of the colored regions corresponds to the relative amounts of protein at the indicated pole or cellular location. Yellow, MglA; red, MglB; green, RomR; orange, MglC. The small clusters of MglA-GTP along the cell length indicate MglA-GTP associated with gliding motility complexes.
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References

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