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J Bacteriol. Mar 2005; 187(5): 1716–1723.
PMCID: PMC1064013

Divergent Regulatory Pathways Control A and S Motility in Myxococcus xanthus through FrzE, a CheA-CheY Fusion Protein

Abstract

Myxococcus xanthus moves on solid surfaces by using two gliding motility systems, A motility for individual-cell movement and S motility for coordinated group movements. The frz genes encode chemotaxis homologues that control the cellular reversal frequency of both motility systems. One of the components of the core Frz signal transduction pathway, FrzE, is homologous to both CheA and CheY from the enteric bacteria and is therefore a novel CheA-CheY fusion protein. In this study, we investigated the role of this fusion protein, in particular, the CheY domain (FrzECheY). FrzECheY retains all of the highly conserved residues of the CheY superfamily of response regulators, including Asp709, analogous to phosphoaccepting Asp57 of Escherichia coli CheY. While in-frame deletion of the entire frzE gene caused both motility systems to show a hyporeversal phenotype, in-frame deletion of the FrzECheY domain resulted in divergent phenotypes for the two motility systems: hyperreversals of the A-motility system and hyporeversals of the S-motility system. To further investigate the role of FrzECheY in A and S motility, point mutations were constructed such that the putative phosphoaccepting residue, Asp709, was changed from D to A (and was therefore never subject to phosphorylation) or E (possibly mimicking constitutive phosphorylation). The D709A mutant showed hyperreversals for both motilities, while the D709E mutant showed hyperreversals for A motility and hyporeversal for S motility. These results show that the FrzECheY domain plays a critical signaling role in coordinating A and S motility. On the basis of the phenotypic analyses of the frzE mutants generated in this study, a model is proposed for the divergent signal transduction through FrzE in controlling and coordinating A and S motility in M. xanthus.

Surface translocation in the gram-negative soil bacterium Myxococcus xanthus is accomplished through two distinct types of gliding motility, which are termed adventurous (A) motility and social (S) motility (9). The two motility systems are, for the most part, genetically separate; A motility powers individual-cell movement, while S motility enables coordinated group motility. These two motility systems enable M. xanthus cells to move selectively on different surfaces; A motility facilitates translocation on relatively hard and dry surfaces (22), whereas S motility enables the cells to travel across relatively soft and wet surfaces or hard surfaces coated with methylcellulose (22, 26). Genetic and functional studies showed that S motility is achieved by extension of polarly localized type IV pili (TFP), attachment of the pili to an external substrate, and pilus retraction, which pulls the cells forward in the direction of the attachment (14, 20, 23, 26, 28). In contrast, the cellular structures and mechanism of propulsion required for A motility are poorly understood, although recent evidence has suggested that directed slime extrusion may be a means to power cell movement (30).

Cells moving by A or S motility periodically reverse their direction of movement, allowing cells to reorient themselves and show directional motility. Cellular reversals in M. xanthus are controlled primarily by the Frz signal transduction system. The frz genes were first identified in mutants with altered aggregation behavior during fruiting-body development (33) but later were also shown to affect the ability of cells to swarm on rich medium and control the directional movements of individual cells (3, 12, 21). Most frz mutants (frzA, -B, -C, -E, and -F) rarely show cellular reversals (3, 5); in contrast, some FrzCD mutants (FrzCDC mutants) display a greatly elevated reversal rate (3). The Frz system controls and coordinates both the A- and S-motility systems, even though these two motility systems are fundamentally different (V. Bustamante and D. Zusman, unpublished data).

The Frz signal transduction system contains proteins that are similar to enteric chemotaxis proteins. For example, FrzCD, a methyl-accepting chemotaxis protein (MCP) homologue, can be modified by methylation (17, 18). FrzE, whose N-terminal 653 amino acids and C-terminal 124 amino acids are homologous to CheA and CheY, respectively, possesses autokinase and phosphoacceptor activities (1). The CheY-like portion of FrzE (referred to in this paper as FrzECheY) contains the highly conserved residues found in the CheY superfamily of response regulators, including Asp709, which is analogous to Asp57 of CheY, the phosphoaccepting residue during signal transduction (19). Mutational analysis of the Frz pathway has shown that FrzCD (MCP), FrzA (CheW), and FrzE (CheA and CheY) are the core components of the Frz signal transduction system (5). However, it is not known what the output signals from the pathways are and how the two motility motors are controlled. This study aims to address these questions by isolating and analyzing mutants with altered FrzECheY. These mutants provide important insights into the different mechanisms regulating cellular reversals of A and S motility.

MATERIALS AND METHODS

Bacterial strains, phage, and culture conditions.

The M. xanthus strains used in this study are listed in Table Table1.1. Cells were grown at 32°C and 225 rpm in CYE (10 g of Casitone per liter-5 g of yeast extract per liter-8 mM MgSO4 in 10 mM morpholinepropanesulfonic acid [MOPS] buffer, pH 7.6) (6). M. xanthus phage Mx4 was used for generalized transduction as described previously (6).

TABLE 1.
M. xanthus strains used in this study

Construction of frzE mutants.

The frzE mutations were introduced into wild-type M. xanthus by gene replacement with the positive-negative KG cassettes (27).

To obtain the in-frame deletion of the region encoding the CheY domain from frzE, a deletion cassette was generated by overlap extension PCR of two ~600-bp primary PCR products that correspond to upstream and downstream regions of the target deletion. Primers 5′-CGA GAA TTC TGG CCG TGG CGT GG-3′ and 5′-GCC TGC GCG AGA ACC TCC ACC ACC CGG AGG CGC TTG GCG GC-3′ and primers 5′-GTG GAG GTT CTC GCG CAG GC-3′ and 5′-ATA GGA TCC GGA ATC ATC CGC AGC ACC TGC-3′ were used to obtain the upstream and downstream PCR products, respectively. The deletion cassette was subcloned into pBJ113, with the EcoRI and BamHI restriction sites introduced in the primers, generating pVB110.

For the two point mutations, a 601-bp fragment containing the target site was PCR amplified with oligonucleotides 5′-GAA TTC CGC GAA GTG GCC G AAG-3′ and 5′-CGG ATC CCT TGC CCA CCA TGA GCA CC-3′ and cloned into pCR2.1-TOPO to generate pYL101. Inverse PCR was performed with Elongase (Invitrogen), with pYL101 as the template and a pair of mutagenesis oligonucleotides. For the D709A mutation, primers 5′-P-ACG GCC GTG CAG ATG CCC AAG CTG-3′ and 5′-P-GAG GAT GAG GTC GTA GGT GTT GTT CTG CAC-3′ were used to turn the GAC codon for aspartic acid into GCC, a codon for alanine, which also created an EagI restriction site (CG GCC G). Similarly, for the D709E mutation, primers 5′-P-ACG GAA GTG CAG ATG CCC AAG CTT GAC-3′ and 5′-P-GAG GAT GAG GTC GTA GGT GTT GTT CTG CAC-3′ were used to change the GAC codon for aspartic acid into GAA, a codon for glutamic acid. In order to facilitate mutant screening, a silent mutation changing the CTG codon of L715 into CTT was introduced to create a HindIII restriction site (AAG CTT). The original template was removed by DpnI digestion; the extension product was then ligated and transformed into E. coli. The resulting plasmids were screened for the newly created restriction sites, sequenced to verify the mutated gene, and cloned into pBJ113, generating pYL102 for the D709A mutation and pYL103 for the D709E mutation.

pVB110, pYL102, and pYL103 were transferred by electroporation into M. xanthus as previously described (11), creating strains DZ4547 (ΔfrzEcheY in a DZ2 background), DZ4567 (ΔfrzEcheY in a DK1622 background), SW901 (frzE-D709A in a DK1622 background), and SW902 (frzE-D709E in a DK1622 background). Chromosomal integration was determined by kanamycin resistance (positive selection), and removal of the vector backbone was achieved by negative selection on CYE plates containing 2.5% galactose (27). PCR analysis was used to screen the different frzE mutations. The presence of the D709A and D709E mutations was further confirmed by EagI and HindIII restriction digestion, respectively.

Phenotypic analysis.

Developmental assays were performed by spotting 5 μl of cells at 5 × 109 ml−1 (in MOPS buffer [10 mM MOPS, 8 mM MgSO4, pH 7.6]) onto 1.5% CF agar. CF contains 0.015% Casitone, 0.2% sodium citrate, 0.1% sodium pyruvate, 0.02% (NH4)2SO4, 10 mM MOPS (pH 7.6), 8 mM MgSO4, and 1 mM KH2PO4 (8). The formation of developmental aggregates and fruiting bodies was observed over a period of 48 h. Vegetative-swarming phenotypes were analyzed by spotting the same amount of cells onto CYE plates containing 1.5% (hard) or 0.3% (soft) agar. After incubation at 32°C for the indicated time, the diameters of the colonies were measured and the swarming colonies and fruiting bodies were imaged with a Leica DM IL compact inverted microscope at ×40 magnification and photographed with a SPOT digital camera (SP401-115; Diagnostic Instruments Inc.).

Reversal frequency analysis.

For analysis of motion in 1% methylcellulose, 1 μl of log-phase cells (optical density at 600 nm, ~1.0) was diluted to 10 μl in CYE and spotted into 250 μl of 1% methylcellulose (in CYE) in a 24-well polystyrene plate. For analysis on 1.5% CYE agar, the cells were further diluted 10× and spotted onto a thin layer of 1.5% agar in a 24-well polystyrene plate. Cell motility was monitored with Nikon Eclipse TE200 inverted microscope, captured with a Sony CCD-IRIS/RGB color video camera, and recorded with a Panasonic AG-6040 time-lapse video cassette recorder. Recording was set at 1/60 of real time, and the video was played back at normal speed to reveal individual-cell movement. To follow individual-cell movement, selected movie frames were captured as images and positions of a chosen cell were tracked at 1- or 2-min intervals. These positions were then connected manually in a timely order with the curve function in Microsoft PowerPoint autoshape to reflect the cell net displacement pattern over time. To determine the cellular reversal frequency, for each strain the movements of 20 isolated cells were followed for 20 reverses (or for 1 h of actual time in the nonreversing strain), and the number of reversals per hour was manually determined.

RESULTS

frzE in-frame deletion mutants differentially affect A and S motility.

Chemotaxis in enteric bacteria is mediated by a specialized two-component signal transduction pathway that includes a receptor MCP, a coupling protein (CheW), a histidine protein kinase (CheA), a response regulator (CheY), and a phosphatase (CheZ). In the absence of an attractant or in the presence of a repellant, CheA autophosphorylates and acts as the phosphodonor for the response regulator CheY. CheY~P interacts with components of the flagellar switch to affect the direction of flagellar rotation, thus altering the direction of cell movement (4). M. xanthus does not have flagella and uses two very different mechanisms for motility. Nevertheless, many of the components of the Frz system resemble the enteric chemotaxis system. However, the Frz pathway does have some important differences. For example, FrzE is a fusion protein that contains a CheA-like module at its N terminus and a CheY-like receiver domain at its C terminus. In order to determine the function of the two domains in this fusion protein, we prepared in-frame frzE deletion mutants of wild-type strain DZ2 (Fig. (Fig.1)1) and analyzed their phenotypes under vegetative conditions and during development, since both vegetative swarming and developmental aggregation require a functional Frz pathway. Figures Figures22 and and33 show that a deletion mutant lacking both domains of FrzE (DZ4481 ΔfrzE) is unable to swarm on rich medium and forms “frizzy” aggregates under fruiting conditions (CF agar). This phenotype has been documented previously for mutants with frzA-F and frzZ deleted (5). However, a mutant with an in-frame deletion in just the CheY domain of FrzE had a very different phenotype. This mutant, DZ4547, was defective in swarming on soft agar but still showed some swarming on 1.5% agar (Fig. (Fig.3).3). Furthermore, this mutant was still able to form fruiting bodies on fruiting agar, unlike DZ4481, the mutant lacking both domains of FrzE (Fig. (Fig.2C).2C). However, it should be noted that the fruiting bodies produced by the ΔfrzEcheY mutant were slightly aberrant in morphology, as they consisted of irregularly shaped aggregates. Since these results were unexpected, we also isolated the same ΔfrzEcheY in-frame deletion in strain DK1622, another wild-type strain. The new mutant, DZ4567, showed phenotypes that were similar to those of DZ4547 (Fig. (Fig.2D2D and and33).

FIG. 1.
Construction of frzE mutants. The frzE in-frame deletion mutations and point mutations that were studied are described. The numbers indicate the amino acid positions within the FrzE protein; the strain names for the corresponding mutants are indicated. ...
FIG. 2.
Developmental phenotypes of frzE mutants. Cells were deposited on CF plates for 2 days. Fruiting-body development was examined under a light microscope with a 4× objective lens and photographed with a digital camera. Strain DZ4547 contains Δ ...
FIG. 3.
Vegetative-swarming phenotype of frzE mutants. Cells were concentrated to 5.0 × 109 ml−1, and 5 μl was spotted on 0.3 or 1.5% CYE agar. The initial spot size was 7 mm. After 4 days of incubation at 32°C, the swarming diameters ...

To determine the nature of the motility defects caused by ΔfrzEcheY, the rate of colony spreading of the mutant DZ4567 was followed over a period of 96 h on 0.3 and 1.5% CYE agar, which favors S and A motility, respectively (22). Figure Figure3A3A shows that wild-type strain DK1622 exhibited a spot diameter increase of 28 mm (from 7 to 35 mm) after 96 h on 0.3% agar. In contrast, ΔfrzEcheY mutant DZ4567 only showed an increase of 4 mm (7 to 11 mm). As a control, the colony diameter of the nonswarming mglA mutant (24) increased from 7 to 10 mm (3-mm increase; data not shown). These results suggest that the mutant might be defective in S motility. Colony spreading on 1.5% CYE agar favors A motility, although S motility also contributes to spreading. Under these conditions, DK1622 spots increased from 7 to 18 mm after 96 h (11-mm increase; Fig. Fig.3A).3A). DZ4567 showed a small defect in swarming ability compared to the wild-type strain, with a spot diameter increase of 7 mm (Fig. (Fig.3A);3A); the swarming edge appeared quite different from that of the wild type as well (Fig. (Fig.3B3B).

Since the Frz system controls cell reversals, we were also interested in determining the reversal frequency of the ΔfrzEcheY mutant. In order to investigate the reversal frequency associated with A motility, isolated M. xanthus cells were observed on 1.5% agar. Table Table22 shows that DZ4567 exhibited a significant increase in reversal frequency compared to that of DK1622 (18.0 ± 7.9 reversals per h compared to 8.5 ± 4.5 reversals per h for DK1622), and an example of cell displacement pattern is shown in Fig. Fig.4A.4A. This “hyperreversal” phenotype for A motility is in contrast to the nearly nonreversing phenotype of the ΔfrzE and Tn5 insertional mutants (3, 5). Next, the S motility of individual cells was examined with the combined tethering and gliding assay developed by Sun et al. (26). This assay involves placing M. xanthus cells on a polystyrene surface covered with 1% methylcellulose in MOPS buffer. Under these conditions, cells can use their TFP to tether to the surface and exhibit S motility as individual isolated cells. Furthermore, the assay allows one to determine the reversal frequency of the cells as the duration of tethering is correlated with the cellular reversal frequency, i.e., hyporeversing FrzE mutants remain tethered for extended periods of time and hyperreversing FrzCDC mutants are tethered for very brief periods of time (26). We first examined ΔfrzE mutant strain DZ4481 with this assay and confirmed the hyporeversal S-motility phenotype, which was previously observed for the Tn5 insertion mutant (Table (Table2).2). DZ4567 (ΔfrzEcheY), when examined in the same assay, showed a similar hyporeversal S-motility phenotype (Table (Table2;2; Fig. Fig.5A).5A). This phenotype was in sharp contrast to the hyperreversal phenotype observed for the same mutant under conditions in which cells move by A motility only. This is the first time that divergent cell reversal phenotypes have been observed for the two M. xanthus motility systems.

FIG. 4.
Reversal phenotypes of the frzEcheY mutants on 1.5% MOPS agar. Cells were allowed to glide on 1.5% agar, and motility was recorded by time-lapse video microscopy. Movie frames captured at 0 and 20 min (left and middle parts) are shown. Two cells, numbered ...
FIG. 5.
Reversal phenotype of the frzEcheY mutants in 1% methylcellulose. Cell movements were recorded by time-lapse video microscopy. Movie frames captured at 0 min (left part of each panel) and 10 min (A and B, middle) or 5 min (C, middle) are shown. A 5-min ...
TABLE 2.
Reversal phenotypes of frzE mutants

Since DZ4567 (ΔfrzEcheY) contains intact A- and S-motility systems, we were concerned that our reversal assays might be subject to cross talk between the two motility systems. To eliminate cross talk, we generated A and S derivatives of DZ4567. Generalized transduction mediated by the phage Mx4 (6) was used to introduce an A-motility mutation from MxH1216 (A S+) (15) and an S-motility mutation from SW501 (A+ S) (32) into DZ4567, generating SW907 (ΔfrzEcheY A) and SW908 (ΔfrzEcheY S). When ΔfrzEcheY A mutant strain SW907 was allowed to glide in 1% methylcellulose, it rarely reversed (Table (Table2),2), confirming that the nonreversing phenotype associated with ΔfrzEcheY is due to aberrant regulation of S motility and not due to A motility. The velocity of movement generated by SW907, however, was reduced compared to that of DZ4567 (data not shown). Similarly, ΔfrzEcheY S mutant SW908 still displayed a hyperreversing phenotype (16.3 ± 5.3 reversals per h) on 1.5% MOPS agar, as observed for DZ4567 (Table (Table22).

Analysis of FrzECheY by site-directed mutagenesis.

Since FrzECheY appears to be important for regulating A and S motility in M. xanthus, we were interested in determining if its conserved putative phosphorylation domain may be responsible for its activity. Previous work showed that FrzE can autophosphorylate and that the phosphate can subsequently be transferred to FrzECheY. Phosphotransfer from FrzECheA to FrzECheY is likely to occur at highly conserved residue D709 (1, 19). In previous studies on NtrC in enteric bacteria and MrpB in M. xanthus, replacing the conserved aspartate with an alanine abolished the function of the response regulator; in contrast, changing the same residue to glutamate produced a constitutive phenotype that mimicked the phosphorylated state of the response regulator (13, 25). Accordingly, FrzE-D709A and FrzE-D709E substitutions were generated by site-specific mutagenesis, creating strains SW901 (frzE-D709A) and SW902 (frzE-D709E). SW901 and SW902 were first examined for developmental defects as described above. Figure Figure22 shows that after 48 h of development, SW901 failed to aggregate into fruiting bodies. SW902, in contrast, did form fruiting bodies, although they were somewhat irregularly shaped and appeared almost identical to the aggregates formed by DZ4567 (Fig. (Fig.2).2). When tested for vegetative swarming, both frzE point mutation strains had severely reduced spreading compared to that of the wild type (Fig. (Fig.3A).3A). Over a 96-h time period, wild-type cells spread from 7 to 35 mm on 0.3% CYE agar (28-mm diameter increase), whereas the frzE-D709A mutant SW901 only increased 3 mm (from 7 to 10 mm; Fig. Fig.3A),3A), resembling the spreading of the nonswarming mglA mutant (24) or the hyperreversing FrzCDC mutants (5). frzE-D709E mutant SW902 displayed a slightly larger diameter increase of 9 mm (from 7 to 16 mm; Fig. Fig.3A),3A), and the swarm edge was jagged (Fig. (Fig.3B),3B), resembling that of the frzE insertional and in-frame deletion mutants (5, 29). On 1.5% CYE agar, SW901 showed a severe reduction in swarming ability (2-mm diameter increase; Fig. Fig.3A);3A); in contrast, the wild-type DK1622 spot increased 11 mm (from 7 to 18 mm) during this same time period. SW902 exhibited a less drastic (8 mm) but still relevant defect in diameter expansion (Fig. (Fig.3A).3A). In addition to their slow swarming rates, the swarming edge of SW901 and SW902 also appeared different from that of the wild-type colony (Fig. (Fig.3B).3B). The phenotypes observed on 0.3 and 1.5% agar indicated that the D709 point mutation affects both A and S motility.

In order to determine the effects of the point mutations on the individual motility systems, mutations in the A- and S-motility systems were introduced into SW901 and SW902 by generalized transduction, generating SW903 (frzE-D709A A), SW904 (frzE-D709A S), SW905 (frzE-D709E A), and SW906 (frzE-D709E S). Individual-cell motility was then monitored as described above. On 1.5% MOPS agar, both SW901 (frzE-D709A) and SW902 (frzE-D709E) mutants exhibited normal gliding speed, but both had a significant increase in reversal frequency (57.4 ± 12.1 and 25.6 ± 12.9 reversals per h for SW901 and SW902, respectively). Interestingly, SW901 displayed only small variations in its moving path, resulting in little net cell displacement over time (Fig. (Fig.4B),4B), which is similar to the phenotype described for the FrzCDC frzD mutant (3). In contrast, although SW902 showed a significant increase in reversal frequency, the individual cells strayed away from the original path when reversing, resulting in net movement (Fig. (Fig.4C).4C). These motility phenotypes may explain the smooth and nonspreading edge for SW901 and the jagged edge for SW902 observed on agar plates (Fig. (Fig.3).3). Examination of the S-motility mutant derivatives of SW901 and SW902, strains SW904 and SW906 (Table (Table2),2), confirmed that the hyperreversal phenotype on 1.5% agar is indeed due to the effects of the frzE-D709A and frzE-D709E mutations on A motility.

The effects of the frzE-D709A and frzE-D709E mutations on S motility were analyzed on the basis of the tethering and gliding behavior of SW901 and SW902 in methylcellulose. A lower percentage of SW901 cells was tethered under this condition (3.6%) compared to those of wild-type strain DK1622 (11.2%). The gliding cells of SW901 had normal gliding speed but exhibited an elevated reversal frequency (Fig. (Fig.5B),5B), averaging 41.24 ± 8.5 reversals per h, or around 1.4 min per reversal (Table (Table2).2). Accordingly, cells were tethered for only a very short time, resulting in a “quick-flip” phenotype by time-lapse video microscopy, with the “flip” time averaging 1.5 min, correlating well with the reversal frequency (Movie 1 in the supplemental data). For SW902, however, a significantly higher percentage of cells was tethered (53.1%) than what was observed for the wild type, and the majority of cells were tethered for more than 60 min (Fig. (Fig.5C,5C, tethered cells) (Movie 2 in the supplemental data). Gliding SW902 cells had a normal gliding speed but rarely reversed (Fig. (Fig.5C).5C). These data indicated that the frzE-D709A and frzE-D709E mutations have opposite effects on the reversal frequency of S motility. Examination of the A-motility mutant derivatives of SW901 and SW902, strains SW903 and SW905, confirmed that the reversal phenotype on 1% methylcellulose is indeed due to S motility (Table (Table22).

DISCUSSION

The frz genes encode chemotaxis homologues that control the cellular reversal frequency, which allows cells to bias their movements. These genes are essential for vegetative swarming on nutrient-rich agar medium and for developmental aggregation on a solid starvation medium. Most of the Frz proteins are similar to their enteric counterparts. However, FrzE, which is homologous to both CheA and CheY, is a novel CheA-CheY fusion protein. In this study, we investigated the role of this fusion protein, in particular, the CheY response regulator domain. We constructed in-frame deletion mutants lacking the FrzECheY domain and point mutants with substitutions in the putative phosphorylation site of activated FrzECheY, FrzE-D709. Analysis of the mutants revealed that the FrzECheY domain regulates A and S motility in different ways. This unexpected finding also helps to explain the observed coordination of the two very different motility systems of M. xanthus.

Figure Figure6A6A summarizes the results of our cellular reversal analysis of the various mutants studied in this paper. Wild-type cells were observed to reverse their direction of gliding about every 8 min. In-frame deletion of the entire frzE gene resulted in nonreversal phenotypes for both motility systems. These findings are consistent with previous results (5). However, deletion of the FrzECheY domain led to divergent motility phenotypes for the A- and S-motility systems: while the mutation caused a nonreversal phenotype for S motility, it triggered a drastic increase in the reversal frequency for A motility (Table (Table2;2; Fig. Fig.6A).6A). This was the first indication that the regulation of both motility systems can be separated at the level of the frz chemosensory system. The D709E point mutant also caused divergent phenotypes for A and S motility: nonreversal of S motility and hyperreversal of A motility, which is similar to the phenotype observed for ΔfrzEcheY (Table (Table2).2). Since the D709E mutation also caused a swarming phenotype and aggregation morphology similar to those caused by the ΔfrzEcheY mutant (Fig. (Fig.22 and and6A),6A), we cannot determine from this experiment if this mutation merely caused FrzECheY to be nonfunctional or if the mutation caused FrzECheY to mimic the phosphorylated state of the protein. However, the phenotype differs markedly from the D709A mutation, which presumably generates an FrzECheY variant that cannot be phosphorylated (13, 25); this mutation significantly raised the reversal rate of both motility systems.

FIG. 6.
Summary of mutant phenotypes and model for FrzE signal transduction in directing A and S motility. (A) Hypothetical intramolecular phosphotransfer of FrzE for the various frz mutant strains constructed in this study and the corresponding reversal phenotypes. ...

Model to explain the phenotypes of the various mutants analyzed in this study (Fig. (Fig.6B).6B). (i) Regulation of S-motility-mediated reversals.

Since the frzE-D709A mutant is the only mutant studied that caused a dramatic increase in S-motility-mediated reversals, unphosphorylated FrzECheY is therefore the likely output signal that triggers the switching of TFP sites of extrusion from one pole to the other (S motility is mediated by pilus extension and retraction from a cell pole; hyperreversals are therefore thought to be caused by very frequent pole-to-pole switching (26). Since the FrzE-D709A mutant should be unable to participate in Frz-mediated phosphorelay, the output signal for reversing must be conveyed by physical interaction with downstream partners. If this model holds true, then lack of this physical interaction will fail to activate pilus pole-to-pole switching, leading to the hyporeversal phenotype of S motility. This is supported by the phenotypes observed for the ΔfrzE, ΔfrzEcheY, and frzE-D709E mutant strains. Since the ΔfrzE and ΔfrzEcheY mutant strains lack FrzECheY and the frzE-D709E mutant strain mimics either phosphorylated or misfolded FrzECheY, all three mutant strains are unable to perform the physical interaction required by the model. How other components known to be involved in S-motility control (e.g., FrzS) fit into this model remains to be investigated.

(ii) Regulation of A-motility-mediated reversals.

In contrast to S motility, phosphotransfer to FrzECheY is hypothesized to be important for controlling the reversal frequency of A motility. All of the FrzECheY mutants that abolished phosphotransfer from FrzECheA to FrzECheYfrzEcheY, frzE-D709A, and frzE-D709E) caused hyperreversing of A motility. In contrast, mutant strains lacking both domains of FrzE, including the FrzECheA kinase domain, were hyporeversing for A motility (Fig. (Fig.6A).6A). To explain the phenotypes observed in the FrzE mutants, we propose that an additional cognate response regulator may compete with FrzECheY (as the phosphoacceptor from FrzECheA) and carry out the downstream interactions to control reversal of the A motor (Fig. (Fig.6B,6B, pathways 1 and 2). Behavioral analysis of the various FrzE mutant strains characterized in this study supports this model. The three mutants that abolish phosphotransfer to FrzECheY (the ΔfrzEcheY, frzE-D709A, and frzE-D709E mutants) should experience a relative increase in the FrzECheA phosphorylation level and thus phosphotransfer to the additional putative response regulator. Since the phosphorylated form of this hypothetical second cognate response regulator for FrzECheA presumably triggers reversal of the A motor, the hyperreversal phenotype observed for the above mutant strains is in excellent agreement with the model proposed here (Fig. (Fig.6B).6B). Additionally, deleting this response regulator will abolish the link between FrzECheA and the A motor, resulting in the same phenotype as ΔfrzE in A motility, which is hyporeversal. In S motility, however, lack of this potential competitor for FrzECheA phosphotransfer will contribute to the phosphorylation of FrzECheY, leading to a hyporeversal phenotype (Fig. (Fig.6B).6B). FrzZ, an Frz system protein composed of two CheY-like receiver domains, has been proposed to receive signals from the FrzE kinase domain and function as an additional response regulator for FrzECheA (5). Interestingly, deletion of FrzZ did lead to hyporeversing phenotypes in both A and S motility (5), consistent with the hypothetical second response regulator proposed in Fig. Fig.6B.6B. However, further mutant analysis including ΔfrzZ ΔfrzEcheY and ΔfrzZ frzE-D709A double mutants is necessary to further elucidate this pathway. These experiments are in progress.

How are A and S motility coordinated by the Frz system? According to our model (Fig. (Fig.6B),6B), the phosphorylation state of FrzECheY determines the reversal frequency of S motility: unphosphorylated FrzECheY results in hyperreversals, and phosphorylated FrzECheY results in hyporeversals. For A motility, FrzECheY and FrzZ, the putative second response regulator, compete for phosphorylation from phospho-FrzECheA. Therefore, when FrzECheY is unphosphorylated (and S motility is hyperreversing), the phosphotransfer equilibrium is shifted toward phosphorylation of FrzZ, increasing the reversal frequency of A motility. Thus, in this case the cells will be hyperreversing with both motility systems. In contrast, when FrzECheY is phosphorylated (hyporeversal in S motility), the phosphorylation level of FrzZ will decrease, leading to a hyporeversal phenotype of A motility. Thus, in this case the cells will be hyporeversing with both motility systems. This coordinated reversal frequency control in both motilities is expected; since the cells should conform rather than have conflicting motility systems while gliding. This model thus explains the function of FrzE signal transduction in coordination of both A and S motility.

The possibility that FrzECheY acts as a phosphate sink (phosphatase) for FrzZ cannot be completely ruled out (Fig. (Fig.6B,6B, pathway 3). Bacteria that lack CheZ (which accelerates CheY dephosphorylation to enhance timely adaptation to environmental stimuli) are known to transfer phosphoryl groups to other CheY-like response regulators, which act as a phosphate sink (2). M. xanthus does not have a CheZ homologue, so it is possible that FrzECheY performs a similar function. It is particularly interesting that if FrzECheY does act as a phosphate sink, it will function to balance the phosphorylation level of FrzECheY and the putative response regulator (Fig. (Fig.6B,6B, pathway 3), thus coordinating the reversal phenotypes of A and S motility at the FrzE level.

An additional interesting observation in this study is the developmental phenotypes exhibited by the ΔfrzEcheY and frzE-D709E mutant strains. Despite their nonreversing behavior in S motility, which they share with all of the other frz mutant strains that typically form the characteristic frizzy aggregates, these mutants were able to aggregate and form fruiting bodies, although with aberrant morphologies. The difference between these particular mutants and the other frz mutants is the hyperreversing A motor. An earlier study has demonstrated that S-motility mutants lacking TFP, the S-motility motors, are also able to develop into fruiting-body-like structures, although they are not as regular as those formed by the wild type (31). It has also been shown that certain mutations in A motility can lead to defective fruiting-body formation (16). The finding in this study provided additional evidence that A motility may play roles complementing those of S motility in fruiting-body formation.

Supplementary Material

[Supplemental material]

Acknowledgments

This work was supported by National Institutes of Health grants GM54666 (W.S.) and GM20509 (D.Z.).

We thank Melissa Sondej for review of the manuscript.

Footnotes

Supplemental material for this article may be found at http://jb.asm.org/.

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