Effects of site-directed mutagenesis of mglA on motility and swarming of Myxococcus xanthus

Abstract

Background: The mglA gene from the bacterium Myxococcus xanthus encodes a 22kDa protein related to the Ras superfamily of monomeric GTPases. MglA is required for the normal function of A-motility (adventurous), S-motility (social), fruiting body morphogenesis, and sporulation. MglA and its homologs differ from all eukaryotic and other prokaryotic GTPases because they have a threonine (Thr78) in place of the highly conserved aspartate residue of the consensus PM3 (phosphate-magnesium binding) region. To identify residues critical for MglA function or potential protein interactions, and explore the function of Thr78, the phenotypes of 18 mglA mutants were characterized.

Results: Nine mutants, with mutations predicted to alter residues that bind the guanine base or coordinate magnesium, did not produce detectable MglA. As expected, these mutants were mot- dev- because MglA is essential for these processes. Of the remaining nine mutants, seven showed a wild-type distribution pattern for MglA but fell into two categories with regard to function. Five of the seven mutants exhibited mild phenotypes, but two mutants, T78D and P80A, abolished motility and development. The localization pattern of MglA was abolished in two mutants that were mot- spo- and dev-. These two mutants were predicted to alter surface residues at Asp52 and Thr54, which suggests that these residues are critical for proper localization and may define a protein interaction site. Improving the consensus match with Ras at Thr78 abolished function of MglA. Only the conservative serine substitution was tolerated at this position. Merodiploid constructs revealed that a subset of alleles, including mglAD52A, were dominant and also illustrated that changing the balance of MglA and its co-transcribed partner, MglB, affects A-motility.

Conclusion: Our results suggest that GTP binding is critical for stability of MglA because MglA does not accumulate in mutants that cannot bind GTP. The threonine in PM3 of MglA proteins represents a novel modification of the highly conserved GTPase consensus at this position. The requirement for a hydroxyl group at this position may indicate that MglA is subject to modification under certain conditions. Proper localization of MglA is critical for both motility and development and likely involves protein interactions mediated by residues Asp52 and Thr54.

Figure 1

A. In silico model of MglA with GPPNHP in the predicted active site; B. MglA model without docked nucleotide. A three-dimensional representation of MglA was constructed with SWISS-MODEL using the crystal structure of Sar1p as a template [24-26] and the result is shown here as generated by PyMOL [27]. All mutations made in MglA were between residues 18 and 145. In both panels, targeted residues are colored as follows: P-loop (PM1), yellow; PM3, green; D52/T54, red; G2 motif, purple; leucine rich repeat (LRR), orange. Thr78 corresponds to the conserved aspartate residue characteristic of the Ras-superfamily, and is located at the end of the α-helix shown in green. Side-chains are shown for residues that were targets of study through site-directed mutagenesis. A: A GTP analog was docked with MglA to identify residues in or near the active site that might directly interact with either the guanine base or the phosphates. B: The MglA apoenzyme is shown with residues indicated. G21 denotes the location of the PM1 region, the N114 residue shown is in the G2 motif. Both D52A and L124 are predicted surface residues on opposite faces of the protein.

Figure 2

Mutants in the P-loop fail to complement the motility defect of ΔmglBA. Mutations were generated in conserved residues Gly19 (MxH2445), Lys25 (MxH2430) and Thr26 (MxH2410) that define the γ-phosphate interacting region of MglA (P-loop). A. Graphic representation of the MglA protein, showing the relative position of PM1 (dark box). Residues mutated are indicated with an arrow head. B. (upper) Relative swarming of each strain on 1.5% CTPM agar; (lower) relative swarming of each strain on 0.3% CTPM agar. The WT M. xanthus strain DK1622 and ΔmglBA strain DK6204 are shown as the first and second bars respectively. The third bar (B⁺A⁺) shows the complemented control MxH2419 (ΔmglBA+pKD100). C. Colony edge morphology of isolated colonies on 1.5% CTPM agar at 100× magnification. Bar = 25 μm. D. Immunoblot showing production of MglA in each strain. PVDF membranes were probed with α-MglA (1:1000) and goat α-rabbit IgG tagged with Alexa Fluor 800 (1:2500).

Figure 3

Immunofluorescence of MglA demonstrates a change in localization in some MglA mutants. Mutant mglA-containing strains were probed with an anti-MglA antibody after fixation as described in Methods. A. WT cells probed with anti-MglA antibody reveal a punctate distribution throughout the cell. B. ΔmglBA strain probed with anti-MglA antibody. No background fluorescence is observed. C. T54A cells probed with anti-MglA antibody. A diffuse fluorescence is observed with no punctate localization. D. L22V cells probed with anti-MglA antibody. Localization similar to that of the WT can be observed.

Figure 4

Mutants that fail to produce MglA display increased transcript levels relative to the WT. cDNA was produced from mRNA harvested from the WT, ΔmglBA mutant and complementing strains as described in Methods and analyzed by qRT-PCR (Applied Biosystems). Background fluorescence was subtracted using the no-template control (NTC), resulting in the data shown. The data (n = 6) shown are relative to the normalized WT (value = 1). In order, the bars represent the WT, DK6204, MxH2432 (T78D) and MxH2406 (T54A) as positive controls as mutants that make MglA in amounts detectable by Western blot, and the mutants G19A, K25A, T26N, Q82A, Q82R, L117/120A, N141A, K142A and D144A respectively. MglA: (+) = made MglA, (--) = did not make MglA.

Figure 5

G2 mutations fail to complement the motility defect of ΔmglBA. MglA alleles with mutations in residues Asn141, Lys142 and Asp144, which are predicted to interact with the guanine base of GTP fail to complement the deletion phenotype. Mutations shown in this panel are from the G2 region: MxH2338 (N141A), MxH2365 (K142A) and MxH2367 (D144A). The first two bars represent the ΔmglBA parent and control respectively. See Figure 2 legend.

Figure 6

Mutants with activating mutations display defects in one or both motility systems. MglA alleles which were made to resemble activating mutations in Ras displayed decreased or absent motility in a complementing strain. Mutations shown in this figure include MxH2361 (G21V), MxH2359 (L22V), MxH2357 (P80A), MxH2320 (Q82A) and MxH2319 (Q82R). See Figure 2 legend.

Figure 7

Mutations predicted to alter surface residues abolish function of MglA. Residues predicted to exist on the surface of MglA either failed to complement the deletion phenotype or partially restored the activity of both motility systems. Strains in this panel include MxH2408 (D52A), MxH2406 (T54A), MxH2339 (L117/120A) and MxH2279 (L124K). See Figure 2 legend.

Figure 8

MglA represents a new family of monomeric GTPases in prokaryotes. Shown is the alignment of the predicted sequences of MglA from M. xanthus with Deinococccus radiodurans, Thermus thermophilus, Bdellovibrio bacteriovorus, and Geobacter metallireducens. Conserved sequence elements (PM1, PM3 and G2) for GTP binding are boxed. Consensus: Upper case letter = conserved in all five proteins listed; lower case letter = conserved in at least 3 of 5 proteins; * = conservative substitution; + = semi-conservative substitution; . = no conservation.

Figure 9

Mutations in T78 demonstrate the requirement of a novel PM3 substitution. This panel shows the phenotypes of strains MxH2247 (T78A), MxH2432 (T78D) and MxH2248 (T78S). See Figure 2 legend.

Figure 10

Some MglA point mutations give a dominant-negative phenotype. Addition of a second copy of the mgl locus depressed the motility phenotypes of merodiploids. A: Linear model of MglA. B: Swarming on 1.5% CTPM agar (top graph) and 0.3% CTPM agar (bottom graph). Bars are colored with respect to location within a conserved motif (red for PM1, green for PM3 and purple for G2), matching the colors used in Figure 10A. Yellow bars represent the mgl merodiploids MxH2375 (WT+mglBA) and MxH2391 (WT+mglB) respectively. The dashed lines provide comparison to merodiploid control, while the dotted line in the upper panel provides comparison to MxH2391. Strains which make detectable mutant MglA in complementing strains are circled. C. Colony edge morphology of selected merodiploids relative to WT and merodiploid controls. Pictures were obtained from isolated colonies on 1.5% CTPM agar at 100× magnification. Bar = 25 μm.

Figure 11

MglA mutations abolish or alter fruiting body formation. Fruiting body formation of mglA mutants was compared with the WT strain on TPM starvation medium containing 1.5% agar as described in Methods. a) Wild type DK1622(mgl+). b) DK6204 (mgl-) c. MxH2278 (mglA + mglA-L124K merodiploid). d). MxH2279 (mglA- + mglA-L124K). e). MxH2336 (mglA + mglA-N141A merodiploid). f). MxH2338 (mglA- + mglA-N141A). g). MxH2360 (mglA + mglA-G21V merodiploid). h). MxH2361 (mglA- + mglA-G21V). i). MxH2358 (mglA + mglA-L22V merodiploid). j). MxH2359 (mglA- + mglA-L22V). k). MxH2425 (mglA + mglA-T78A merodiploid). l). MxH2247 (mglA- + mglA-T78A). m). MxH2428 (mglA + mglA-T78D merodiploid). n). MxH2432 (mglA- + mglA-T78D). Photographs were taken with a Nikon FXA microscope at 100× magnification. Bar = 50 μm.

Figure 12

Summary of mutations in MglA and their corresponding phenotypes with regard to M. xanthus motility. Sixteen residues on WT MglA were targeted to make 18 point mutants. Nine mutants made MglA protein and were divided into groups based on phenotype and distribution of MglA (mot- (nonmotile), swm- (do not swarm), dev- (do not develop) and spo- (do not sporulate). Mutants that showed near WT phenotypes were scored as mot+ dev+. Those that showed only partial restoration of a characteristic were scored as (+). Those showed restoration of motility are called class I mutants, those that did not show a full restoration of motility are class II mutants. A subset of class II mutants which include the surface mutants D52A and T54A fail to localize correctly as identified using immunofluorescence microscopy. The remaining class II mutants localize correctly, but do not restore motility. The remaining nine point mutants failed to accumulate detectable amounts of MglA and are classified as class III mutants, which are mot- and dev-. Localization patterns are shown for each motility phenotype and mutant class.