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Summary

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The Frz chemosensory system controls directed motility in Myxococcus xanthus by regulating cellular reversal frequency. M. xanthus requires the Frz system for vegetative swarming on rich media and for cellular aggregation during fruiting body formation on starvation media. The Frz signal transduction pathway is formed by proteins that share homology with chemotaxis proteins from enteric bacteria, which are encoded in the frzA-F putative operon and the divergently transcribed frzZ gene. FrzCD, the Frz system chemoreceptor, contains a conserved C-terminal module present in methyl-accepting chemotaxis proteins (MCPs); but, in contrast to most MCPs, FrzCD is localized in the cytoplasm and the N-terminal region of FrzCD does not contain transmembrane or sensing domains, or even a linker region. Previous work on the Frz system was limited by the unavailability of deletion strains. To understand better how the Frz system functions, we generated a series of in-frame deletions in each of the frz genes as well as regions encoding the N-terminal portion of FrzCD. Analysis of mutants containing these deletions showed that FrzCD (MCP), FrzA (CheW) and FrzE (CheA–CheY) control vegetative swarming, responses to repellents and directed movement during development, thus constituting the core components of the Frz pathway. FrzB (CheW), FrzF (CheR), FrzG (CheB) and FrzZ (CheY–CheY) are required for some but not all responses. Furthermore, deletion of ≈ 25 amino acids from either end of the conserved C-terminal region of FrzCD results in a constitutive signalling state of FrzCD, which induces hyper-reversals with no net cell movement. Surprisingly, deletion of the N-terminal region of FrzCD shows only minor defects in swarming. Thus, signal input to the Frz system must be sensed by the conserved C-terminal module of FrzCD and not the usual N-terminal region. These results indicate an alternative mechanism for signal sensing with this cytoplasmic MCP.


Introduction

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Myxococcus xanthus is a Gram-negative soil bacterium that exhibits complex multicellular behaviours. On nutrient-rich agar, rod-shaped cells grow vegetatively and spread outwards in organized groups referred to as swarms. When plated at high cell density on a solid surface with limiting nutrients, individual cells aggregate by coordinated and directed movements to form raised mounds called fruiting bodies, each containing ≈ 100 000 cells. Within the fruiting bodies, cells begin to differentiate into spherical spores (myxospores) that are resistant to environmental stress. When nutrients are added to the medium, the myxospores germinate and the vegetative growth cycle commences (for reviews, see Dworkin, 1996; Shimkets, 1999; Spormann, 1999).

Myxococcus xanthus cells do not have flagella, but use gliding motility to swarm over solid surfaces and to aggregate into fruiting bodies (Burchard, 1984). The cells show two patterns of gliding (Hodgkin and Kaiser, 1979a,b). The first system is called adventurous (A)-motility and involves the movement of individual cells. The second pattern is called social (S)-motility and involves the movement of cells in groups. S-motility requires type IV pili, lipopolysaccharide (LPS) O-antigen, and extracellular matrix polysaccharide (called fibrils) (Arnold and Shimkets, 1988; Wu and Kaiser, 1995; Bowden and Kaplan, 1998). S-motility is similar to twitching motility in Pseudomonas aeruginosa and has been shown to be powered by type IV pili. Pili are extruded from one cell pole where the tip of a pilus adheres to a surface or to another cell; retraction of the pilus then pulls the cell in the direction of the adhering pilus (Sun et al., 2000; Li et al., 2003). A-motility is hypothesized to be mediated by directed slime extrusion through nozzle-like structures located on the cell surface primarily near the cell poles (Wolgemuth et al., 2002). Because M. xanthus cells reverse their direction of gliding approximately every 7–8 min (Blackhart and Zusman, 1985a), reversals are proposed to result from the sites of pilus extrusion or directed slime secretion switching periodically from one cell pole to another (Sun et al., 2000; Wolgemuth et al., 2002). Cell reversals are thought to be required for directional adjustments as part of a biased random walk (Blackhart and Zusman, 1985a).

These gliding motility mechanisms, adapted for movement on solid surfaces, provide a contrast to flagellar motility exhibited by free-swimming bacteria. For example, Escherichia coli establishes a biased random walk toward attractants and away from repellents by periodically switching the direction of flagellar rotation from counterclockwise to clockwise, causing the cells to ‘run’ or ‘tumble’ respectively. E. coli contains five chemoreceptors which it uses to sense signals: Tsr, Tar, Trg, Tap and Aer. These chemoreceptors are known as methyl-accepting chemotaxis proteins (MCPs). MCPs generally contain a unique periplasmic domain, which binds specific ligands or otherwise senses signals, and a conserved cytoplasmic module which interacts with downstream components of the signalling pathway. In addition to the MCPs, six cytoplasmic proteins (CheA, a histidine protein kinase; CheW, a coupling protein; CheY, a response regulator; CheZ, a phosphatase; CheR, a methyltransferase; and CheB, a methylesterase) are needed to process and adapt to sensory information and to transmit rotational control signals to the flagellar motors (for reviews, see Armitage, 1999; Bren and Eisenbach, 2000; Bourret and Stock, 2002).

In contrast to E. coli, M. xanthus has eight chemotaxis-like pathways (J. Kirby, pers. comm.), four of which have been characterized (Ward and Zusman, 1997; Yang et al., 1998; Kirby and Zusman, 2003; Vlamakis et al., 2004). The Frz system was the first pathway to be studied and is responsible for controlling the frequency at which individual cells reverse their direction of gliding (Blackhart and Zusman, 1985a). Directional control is required for cells to move in a biased random walk during colony swarming and for aggregation during fruiting body formation. The Frz system consists of: FrzCD, a cytoplasmic chemoreceptor; FrzA and FrzB, two CheW-like proteins; FrzE, a CheA–CheY fusion protein; FrzF, a methyltransferase; FrzG, a methylesterase; and FrzZ, a CheY–CheY fusion protein (McBride et al., 1989; 1992; Ward and Zusman, 1997; 1999; Astling, 2003). All of these proteins are encoded in a putative operon containing frzA through frzF, as well as in the divergently transcribed frzZ gene (Trudeau et al., 1996). Most mutants containing transposon or plasmid insertions in the frz genes rarely reverse their direction of gliding and are defective in swarming (Blackhart and Zusman, 1985a; Shi et al., 1993; Trudeau et al., 1996). Furthermore, instead of forming fruiting bodies when starved, these frz mutants aggregate into tangled filaments, a phenotype called ‘frizzy’ (Blackhart and Zusman, 1985a,b; Kashefi and Hartzell, 1995; Trudeau et al., 1996).

In this article, we extended the previous work by constructing a series of in-frame deletions in each of the frz genes as well as regions encoding the N-terminal portion of FrzCD. The analysis of these mutants revealed that a core of the Frz system, formed by FrzCD (MCP), FrzA (CheW) and FrzE (CheA–CheY), controls vegetative swarming, responses to repellents and directed movement during development. FrzB (CheW), FrzF (CheR), FrzG (CheB) and FrzZ (CheY–CheY) are required for some but not all responses. Interestingly, deletion of ≈ 25 amino acids from either end of the conserved C-terminal region results in a constitutive signalling state of FrzCD, which induces hyper-reversals with no net cell movement. Finally, we show that FrzCD senses signals through its conserved C-terminal module and not the usual N-terminal region, which indicates an alternative mechanism for signal sensing for a cytoplasmic MCP.

Results

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Construction and characterization of mutants with in-frame deletions in the frz genes

Previous studies of the Frz system utilized transposon or plasmid insertion mutants to characterize the roles of the various genes during vegetative growth and developmental aggregation (Blackhart and Zusman, 1985a,b; Shi et al., 1993; Kashefi and Hartzell, 1995; Trudeau et al., 1996). However, most of the Frz proteins are encoded in a large putative operon containing frzA through frzF(Fig. 1A). Furthermore, the characterizations were incomplete and often in the strain background DZF1, which contains the pilQ1 allele and is partially defective in S-motility. We were particularly concerned that the transposon insertion mutants may have polar effects on downstream genes and were not ideal for evaluating the roles of individual Frz proteins. We therefore constructed a series of mutants containing in-frame deletions in frzA, frzB, frzCD, frzE, frzF, frzG and frzZ using the procedure of Ueki et al. (1996), which is described in Experimental procedures. All of the mutations were constructed in strain DZ2, a fully motile wild-type strain. The deleted codons in the various strains are listed in Table 1. Figure 1B shows a Western immunoblot analysis of whole cell extracts prepared from the different mutants using polyclonal anti-FrzCD antibodies. As seen previously, FrzCD is detected as multiple bands representing various methylated forms (McCleary et al., 1990; McBride et al., 1992). This analysis showed that expression of FrzCD is not affected by the deletion of other frz genes and that the methylated form of FrzCD (lower band) is absent in the ΔfrzF (cheR) mutant and abundant in the ΔfrzG (cheB) mutant (Fig. 1B). These results are consistent with the function of FrzF and FrzG as methyltransferase and methylesterase respectively.

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Figure 1. A. Schematic representation of the organization of the frz locus. Arrows indicate the direction of transcription of the frz genes. The number of base pairs for each intergenic region is shown above them. Genes which when mutated present the frizzy phenotype are shown in black. Protein homologues are shown between parentheses below the corresponding Frz protein. B. Western immunoblot analysis of FrzCD expression. Whole-cell extracts, prepared from the strains grown in liquid CYE media to mid-log phase, were analysed by SDS polyacrylamide gel electrophoresis and probed with a polyclonal anti-FrzCD antibody. Similar results were obtained when whole-cell extracts prepared from cells starved on CF-agar plates were used (data not shown). The arrows indicate the unmethylated and methylated forms of FrzCD.

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Table 1. Bacterial strains and plasmids used in this study.
Strain or plasmidGenotype or relevant featureSource or reference
M. xanthus strains
 DZ2Wild-type Campos and Zusman (1975)
 DZF1 (FB) sglA (pleomorphic) Dworkin (1962)
 DK1622Wild-type Kaiser (1979)
 DZ4038DZ2 frzDΩ224 (frzCD::Tn5Ω224)Laboratory collection
 DZ4478DZ2 ΔfrzA, from codons 15–145This study
 DZ4479DZ2 ΔfrzB, from codons 11–92This study
 DZ4480DZ2 ΔfrzCD, from codons 6–393This study
 DZ4481DZ2 ΔfrzE, from codons 13–766This study
 DZ4482DZ2 ΔfrzG, from codons 16–334This study
 DZ4483DZ2 ΔfrzF, from codons 13–582This study
 DZ4484DZ2 ΔfrzZ, from codons 16–280This study
 DZ4485DZ2 frzCDΔ6–130This study
 DZ4486DZ2 frzCDΔ6–153This study
 DZ4487DZ2 frzCDΔ6–182This study
 DZ4488DZ2 frzCDΔ6–130 frzDΩ224This study
 DZ4489DZ2 ΔfrzF frzCDΔ6–130This study
 DZ4490DZF1 frzCDΔ6–130This study
 DZ4491DK1622 frzCDΔ6–130This study
E. coli strains
 DH10BHost for cloningInvitrogen
Plasmids
 pBJ113Used to create deletions, galK, KmR Julien et al. (2000)
 pVB100pBJ113 with a deletion cassette for frzAThis study
 pVB101pBJ113 with a deletion cassette for frzBThis study
 pVB102pBJ113 with a deletion cassette for frzCDThis study
 pJPM3pBJ113 with a deletion cassette for frzEThis study
 pVB104pBJ113 with a deletion cassette for frzGThis study
 pVB105pBJ113 with a deletion cassette for frzFThis stud
 pVB106pBJ113 with a deletion cassette for frzZThis study
 pVB107pBJ113 with a deletion cassette for frzCDΔ6–130This study
 pVB108pBJ113 with a deletion cassette for frzCDΔ6–153This study
 pVB109pBJ113 with a deletion cassette for frzCDΔ6–182This study

Developmental phenotype of the in-frame frz deletion mutants

The different Δfrz mutants were analysed for their ability to undergo development by spotting 10 µl of cells at 4 × 109 cfu ml−1 on a starvation medium (CF-agar). After 3 days of incubation, the wild-type strain formed fruiting bodies (Fig. 2A); in contrast, the mutants containing in-frame deletions in frzZ, frzA, frzB, frzCD, frzE or frzF showed tangled, swirling aggregates, the characteristic frizzy phenotype. The mutant containing an in-frame deletion in frzG aggregated, like the wild-type strain, into fruiting bodies (Fig. 2A). This phenotype was observed previously with a Tn5 insertion mutant in strain DZF1 (McCleary et al., 1990). Thus, with the exception of FrzG (CheB), all of the Frz proteins are required for normal aggregation during development.

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Figure 2. Developmental aggregation and vegetative swarming phenotypes of the Δfrz mutants. Cells (10 µl), at a concentration of 4 × 109 cfu  ml−1, were spotted on CF-agar plates (A), or CYE plates containing a concentration of 1.5% (B) or 0.3% (C) agar, which were incubated at 32°C and photographed after 72 h. A and C show the fruiting bodies and the swarming colonies respectively. B shows the colony edges of the swarming colonies on CYE hard agar.

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Vegetative phenotype of the in-frame frz deletion mutants

To evaluate the ability of the Δfrz mutants to spread (swarm) under vegetative conditions, concentrated cells from the Δfrz mutants were spotted onto nutrient-rich CYE plates containing hard (1.5%) or soft (0.3%) agar, substrates that favour movement through the A- or S-motility systems respectively (Shi and Zusman, 1993). Figure 3 shows the diameter of the initial spots and the diameter of the expanded swarms after 3 days of incubation at 32°C. On CYE hard agar plates, all of the Δfrz mutants showed reduced swarming. Thus, all of these genes are required for directed movement away from the colony centre and towards fresh nutrient agar. Examination of the colony edges of the ΔfrzZ, ΔfrzA, ΔfrzB, ΔfrzCD, ΔfrzE and ΔfrzF mutants revealed a difference from the wild-type strain in that these Δfrz mutants contained more single cells and fewer groups of cells (Fig. 2B). The colony edges of the ΔfrzG mutant resembled the wild-type strain in that most cells were in groups (Fig. 2B). On CYE soft agar plates, the Δfrz mutants showed a severe reduction in swarming relative to the wild-type strain, although the ΔfrzG and ΔfrzF mutants swarmed better than the other Δfrz mutants (Figs 2C and 3). Previously, we described two Tn5 insertion mutants that contained insertions near the C-terminus of frzCD; this locus was originally called frzD (Blackhart and Zusman, 1985a). These mutants, now called frzCDc, form non-spreading colonies because the individual cells constitutively reverse direction (Fig. 4; Table 2). Figure 3 shows that the frzCDc mutants failed to swarm on hard or soft agar, suggesting that the Frz system controls vegetative swarming for both the A- and S-motility systems. More direct evidence for involvement of the Frz system in controlling A- and S-motility has been obtained by introducing frz mutations in pilA (A+S) or aglB1 (AS+) strains. The frz mutations affect directed motility, swarming and aggregation in both backgrounds, indicating that the Frz pathway signals to both the A- and S-motility systems (V.H. Bustamante and D.R. Zusman, unpubl.).

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Figure 3. Vegetative swarming analysis of the Δfrz mutants. Cells (10 µl), at a concentration of 4 × 109 cfu ml−1, were spotted onto CYE plates containing 1.5% or 0.3% agar, and incubated at 32°C for 72 h. The black bars represent the average diameter in millimeters for the initial spots and the white bars represent the average diameter in millimeters for the swarming colonies after 3 days. The averages were obtained from at least 10 colonies from three independent experiments. Errors bars indicate standard deviation.

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Figure 4. Mutants that display the FrzCD constitutive signalling phenotype (FrzCDc). The phenotypes were obtained as described in Fig. 2.

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Table 2. Frequency of cell reversal in wild-type and frz mutants.
StrainNo. of reversals in 30 min (SEM)Time between reversals (in min)
  1. Cells were diluted, placed on CF starvation media containing 1.5% agar that was layered on slides and filmed for 30 min periods. The number of reversals was calculated for each strain using an automated motility analysis program and a subset of cells were confirmed manually. At least 20 cells were analysed for each strain. SEM is the standard error of the mean. The reversal rates for ΔfrzA, ΔfrzB, ΔfrzCD, ΔfrzE, ΔfrzF and ΔfrzZ were statistically indistinguishable from each other and we therefore grouped those results to obtain an average reversal frequency for those strains.

Wild-type (DZ2) 4.16 (0.32) 7.21
ΔfrzA-F and ΔfrzZ 0.88 (0.09)34.1
ΔfrzG 7.23 (0.72) 4.15
frzCDΔ6–130  5.43 (0.51) 5.52
frzCDΔ6–153 21.24 (0.75) 1.41
frzCDΔ6–182 17.61 (1.28) 1.70
frzDΩ224 19.50 (1.30) 1.54

Previously, isoamyl alcohol was shown to increase cellular reversal frequency, consistent with a strong repellent response (McBride et al., 1992). To determine which Frz proteins are required for this repellent response, we analysed colony expansion for all the Δfrz mutants on CYE hard agar supplemented with 0.3% isoamyl alcohol (v/v). Figure 5 shows that after 3 days of incubation, the wild-type strain failed to show colony expansion in the presence of isoamyl alcohol; this repellent response was also observed for the ΔfrzZ, ΔfrzB, ΔfrzG and ΔfrzF mutants. In contrast, the ΔfrzA, ΔfrzCD and ΔfrzE mutants were unresponsive to the repellent and showed spreading of the colonies, presumably because of random cell movements (Fig. 5). These results suggest that FrzA (CheW), FrzCD (MCP) and FrzE (CheA–CheY) are essential for all Frz-directed signalling and constitute the core of the Frz system. These results also suggest that the Frz system uses different input pathways to sense repellents and signals that control vegetative swarming and aggregation.

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Figure 5. Repellent response analysis of the Δfrz mutants. Ten microliters of cells at a concentration of 4 × 109 cfu ml−1 were spotted on CYE plates containing a concentration of 1.5% agar and 0.3% isoamyl alcohol, incubated at 32°C for 72 h. The black bars represent the average diameter in millimeters for the initial spots and the white bars represent the average diameter in millimeters for the swarming colonies after 3 days. The averages were obtained from at least 10 colonies from three independent experiments. Errors bars indicate standard deviation.

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To determine the role of the individual Frz proteins in regulating cellular reversal frequencies, we analysed the cellular reversal frequency for each of the Δfrz mutants by following the movements of individual cells on hard agar. The ΔfrzZ, ΔfrzA, ΔfrzB, ΔfrzCD, ΔfrzE and ΔfrzF mutants all showed decreased cellular reversal frequencies. Reversal frequencies for those strains were statistically indistinguishable from each other at the 95% confidence level using an analysis of variance test followed by Tukey's multiple comparison. Whereas the wild-type strain reversed every 7.2 min, the ΔfrzZ, ΔfrzA, ΔfrzB, ΔfrzCD, ΔfrzE and ΔfrzF mutants reversed much less frequently, about every 34.1 min (Table 2). The cellular reversal frequency of the ΔfrzG mutant was 1.7 times higher than that of the wild-type strain (Table 2). Thus, all the Frz proteins are required for normal cellular reversal frequencies.

Construction and analysis of a mutant lacking the N-terminal region of FrzCD

Most characterized MCPs have a variable N-terminal region, containing a periplasmic sensing domain flanked by two transmembrane segments. The cytoplasmic C-terminal region is conserved, consisting of a linker region and a highly conserved signalling domain flanked by two methylation domains (Fig. 6A; Zhulin, 2001). FrzCD, unlike most MCPs, is a cytoplasmic protein, although it might interact with components from the membrane (McBride et al., 1992). The C-terminal region of FrzCD conforms to the general pattern and shares homology with the cytoplasmic region of MCPs (McBride et al., 1989). The conserved C-terminal region of FrzCD begins at around position 137, based on alignments between the amino acid sequence of FrzCD and others MCPs (data not shown). We therefore divided the sequence of FrzCD into an N-terminal region, from positions 1–136, and a conserved C-terminal region, from position 137–417 (Fig. 6B). The N-terminal region does not appear to contain transmembrane or sensing domains, or even a linker region. The C-terminal region of FrzCD is composed of a highly conserved signalling domain flanked by two putative methylation domains, based on sequence homologies shared with other MCPs (Fig. 6A and B).

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Figure 6. A. Domain organization of the E. coli aspartate chemoreceptor Tar, a typical MCP. B. Domain organization of FrzCD and its derivative deletions. The positions that divide the N-terminal and C-terminal regions are shown below the diagram of FrzCD. The putative methylation sites for FrzCD are represented by black circles above the diagrams. The truncated FrzCD proteins are named according to their deleted amino acids. FrzCDΔ6–153 (expressed by frzCDΔ6–153), FrzCDΔ6–182 (expressed by frzCDΔ6–182), FrzCDΔ393–417 (expressed by frzDΩ224) and FrzCDΔ6–130/Δ366–417 (expressed by frzCDΔ6–130 frzDΩ224) induce the FrzCD constitutive signalling phenotype (FrzCDc). C and D. Expression analysis for FrzCD and its derivative deletions by Western blot. A polyclonal anti-FrzCD antibody and whole-cell extracts prepared from liquid CYE cultures were used. Lanes were loaded as follow: 1 – DZ2 (WT); 2 –frzCDΔ6–130; 3 –ΔfrzF frzCDΔ6–130; 4 –frzCDΔ6–130 frzDΩ224; 5 –ΔfrzCD; 6 –ΔfrzCD; 7 –frzCDΔ6–153; and 8 –frzCDΔ6–182. Lanes 6, 7 and 8 were exposed five times longer than the rest of the blot. The asterisk indicates a cross-reacting protein. To test the response to isoamyl alcohol, mid-log phase cultures of frzCDΔ6–130 were incubated at 32°C in the presence/absence of 0.3% isoamyl alcohol, for 2 h. The arrows indicate the unmethylated, methylated and unmethylated/deamidated forms of FrzCD.

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Because the N-terminal region of MCPs is almost always important for sensing signals, we generated a mutant that expresses a FrzCD protein lacking the N-terminal region, from positions 6–130 (frzCDΔ6–130). To confirm that this protein is expressed with the correct apparent molecular weight, extracts of the wild-type strain DZ2 and the frzCDΔ6–130 deletion strain were made and analysed by polyacrylamide gel electrophoresis and Western immunoblotting using polyclonal anti-FrzCD antibodies. Multiple bands can be apparent for FrzCD because methylation or deamidation of FrzCD alters its migration in SDS–PAGE gels (McCleary et al., 1990; McBride et al., 1992). Figure 6C (lane 1) shows that the wild-type strain expressed bands centred around 45 kDa, the expected molecular weight for the FrzCD protein. Four bands, with apparent molecular weights of 26–30 kDa, were observed for frzCDΔ6–130 (Fig. 6C, lane 2) and no bands were detected in a mutant containing a deletion of the complete frzCD gene (ΔfrzCD) (Fig. 6C, lane 5). These experiments were carried out with whole-cell extracts made from vegetative cells but similar results were obtained using extracts prepared from cells starved for 20 h on CF-agar plates (data not shown). The four bands detected in frzCDΔ6–130 could be generated by different methylated forms of FrzCD or, alternatively, by degradation of the C-terminus of FrzCD. To discriminate between these two possibilities, the frzCDΔ6–130 deletion was introduced into a mutant containing an in-frame deletion of frzF, the gene that encodes the methyltransferase. Figure 6C (lane 3) shows that the double mutant, ΔfrzF frzCDΔ6–130, expressed a single band (≈ 30 kDa) consisting of the demethylated form of FrzCD lacking the N-terminal region. Thus, the multiple FrzCD forms expressed by frzCDΔ6–130, observed in the Western immunoblots, were the result of multiple forms of the methylated protein. Figure 6D shows that FrzCDΔ6–130 was not only methylated when cells were grown in rich media which contains attractants, but demethylated when exposed to the repellent isoamyl alcohol (McBride et al., 1992), as shown by a single prominent band in the cells exposed to isoamyl alcohol, that migrates more slowly than that in the cells that were not exposed to isoamyl alcohol. Together, these results indicate that the frzCDΔ6–130 mutant expresses a stable FrzCDΔ6–130 protein that lacks the N-terminal region, but is still subject to methylation/demethylation in response to attractants and repellents.

The N-terminal region of FrzCD is not required during development

To determine if signals that control development in M. xanthus are sensed by the N-terminal region of FrzCD, the wild-type strain DZ2, frzCDΔ6–130 and ΔfrzCD, were analysed for their developmental morphology by spotting 10 µl of cells at 4 × 109 cfu ml−1 on CF-agar plates. After 3 days of incubation, the frzCDΔ6–130 mutant aggregated into fruiting bodies, which were similar to those formed by the wild-type strain, DZ2 (Fig. 7). Surprisingly, the frzCDΔ6–130 mutant consistently formed a greater number of fruiting bodies than the parent strain, DZ2, as shown in Fig. 7. In contrast, the ΔfrzCD mutant formed no fruiting bodies, only the characteristic frizzy aggregates. The timing of developmental events, cell density dependence of fruiting body formation and sporulation efficiencies were identical for strains DZ2 and frzCDΔ6–130. In contrast, the sporulation efficiency showed by ΔfrzCD was only 15% of that of the wild-type strain (data not shown). Figure 6C and D shows that the FrzCDΔ6–130 protein is methylated in vegetative cells. To determine if this protein is also regulated by methylation during development, we analysed the developmental phenotype of the ΔfrzF frzCDΔ6–130 double mutant. Figure 7 shows that this double mutant displayed the frizzy phenotype, like ΔfrzCD, indicating that methylation of the FrzCDΔ6–130 protein is required to control fruiting body formation. To determine whether the frzCDΔ6–130 phenotype was strain dependent, the frzCDΔ6–130 deletion was also constructed in strains DK1622 and DZF1, with similar results (data not shown). These observations, taken together, show that the N-terminal domain of FrzCD is not the principal sensing domain for developmental signal sensing by FrzCD.

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Figure 7. FrzCD senses signals that control aggregation through its conserved C-terminal region. Developmental phenotypes were obtained as described in Fig. 2.

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The N-terminal region of FrzCD is not required during vegetative swarming

Because the Frz proteins are also required for swarming on rich media (Figs 2 and 3), we examined the phenotype of the frzCDΔ6–130 mutant under vegetative conditions. Concentrated cells from DZ2, frzCDΔ6–130 and ΔfrzCD were spotted onto CYE plates containing soft (0.3%) or hard (1.5%) agar. Figure 8 shows the diameter of the initial spots and the diameter of the expanded swarms after 3 days of incubation at 32°C. On CYE soft agar plates, strains DZ2 and frzCDΔ6–130 showed similar swarming; in contrast, swarming by the ΔfrzCD mutant was reduced by 75% (Fig. 8). On CYE hard agar plates, frzCDΔ6–130 showed a slight reduction (13%) in swarming compared with DZ2, while ΔfrzCD swarming was reduced by 38% (Fig. 8). These results indicate that the N-terminal domain of FrzCD is also not the principal sensing domain of this MCP for vegetative swarming.

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Figure 8. FrzCD senses signals for vegetative swarming through its conserved C-terminal region. The swarming was analysed as described in Fig. 3.

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To determine if the N-terminal region of FrzCD is required for the response to repellents, we analysed the spreading of spots of DZ2, frzCDΔ6–130 and ΔfrzCD on CYE hard agar supplemented with 0.3% isoamyl alcohol (v/v). Figure 9 shows that after 3 days of incubation, strains DZ2 and frzCDΔ6–130 did not spread in the presence of isoamyl alcohol, indicating that both strains were able to sense the presence of this repellent. In contrast, the ΔfrzCD mutant was unable to sense the presence of isoamyl alcohol and cells showed a similar amount of spreading as in the absence of this repellent (Fig. 9). Furthermore, the FrzCDΔ6–130 protein is demethylated in the presence of isoamyl alcohol (Fig. 6C and D). Together these results indicate that the N-terminal domain of FrzCD is also not the principal sensing domain of this MCP for sensing repellents.

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Figure 9. FrzCD senses isoamyl alcohol through its conserved C-terminal region. Ten microliters of cells at a concentration of 4 × 109 cfu ml−1 were spotted on CYE plates containing a concentration of 1.5% agar and 0.3% isoamyl alcohol, incubated at 32°C, and photographed after 72 h.

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Because FrzCD is essential for the regulation of cellular reversal frequency, we were interested in determining if the N-terminal region of FrzCD is required for this control. Table 2 shows that single cells from DZ2 and frzCDΔ6–130 showed statistically similar reversal frequencies, in contrast to ΔfrzCD, which showed a greatly reduced reversal frequency. These results indicate that the N-terminal region of FrzCD is not required for the regulation of cellular reversal frequency.

Mutations that induce a constitutive signalling state of FrzCD

Previously, it was shown that mutants containing Tn5 transposon insertions in the last 25 or 52 codons of frzCD generate truncated forms of FrzCD that induced a constitutive signalling state: individual cells reversed at high frequency and colonies failed to spread (Blackhart and Zusman, 1985a). We wondered whether the FrzCD protein lacking the N-terminal region would still show this constitutive signalling phenotype. We therefore introduced the frzCDΔ6–130 deletion into DZ2 frzDΩ224 (this strain contains a Tn5 insertion in codon number 365 of frzCD) to generate frzCDΔ6–130 frzDΩ224. The double mutant was analysed for developmental defects on CF-agar plates, as well as for swarming defects on CYE-agar plates. This double mutant had the same constitutive phenotype as frzDΩ224 (data not shown). Western immunoblot analysis revealed that the frzCDΔ6–130 frzDΩ224 mutant expressed a truncated FrzCD protein, which is detected as a single band around 26 kDa (Fig. 6C, lane 4). This is in agreement with previous results that showed that the truncated FrzCD protein expressed by frzDΩ224 is not methylated (McCleary et al., 1990) and that the FrzCD constitutive signalling phenotype shown by frzDΩ224 does not require FrzF-mediated methylation (I. Martínez-Flores and D.R. Zusman, unpubl.). Thus, analysis of the frzCDΔ6–130 frzDΩ224 double mutant shows that the N-terminal region of FrzCD is not required for the constitutive signalling phenotype.

To delimit the functional domains of the C-terminal region of FrzCD, we made two mutants containing longer N-terminal deletions, from positions 6–153 (frzCDΔ6–153) and from positions 6–182 (frzCDΔ6–182), using DZ2 as the wild-type parent strain. The resulting mutants, frzCDΔ6–153 and frzCDΔ6–182, were analysed for phenotypic defects during vegetative swarming on CYE-agar plates and development on CF-agar plates. Surprisingly, both mutants showed no colony spreading on soft or hard agar and no aggregation into fruiting bodies or even ‘frizzy’ aggregates on CF fruiting agar (Fig. 4). This phenotype was identical to that seen in the mutant frzDΩ224, the constitutive signalling phenotype (FrzCDc). Because the frzDΩ224 mutant shows a hyper-reversal frequency (Blackhart and Zusman, 1985a), we analysed the reversal frequency of single cells of the mutants frzCDΔ6–153 and frzCDΔ6–182. Table 2 shows that cells from both strains reversed at high frequency, which was statistically similar to the reversal frequency shown by cells of frzDΩ224. Additionally, like frzDΩ224, the mutants frzCDΔ6–153 and frzCDΔ6–182 expressed truncated FrzCD proteins. These were detected on Western immunoblots as single bands with the expected molecular weights for each protein, suggesting they were not methylated (Fig. 6C, lanes 7 and 8). These results confirm that the conserved C-terminal region of FrzCD begins around amino acid 137 and extends to the end of the protein. Furthermore, these results indicate that FrzCD can be constitutively activated by deleting amino acids from either end of the conserved C-terminal region.

Discussion

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

The Frz system is the central chemosensory pathway for the control of directed motility in M. xanthus. In this study we have re-examined the roles of the Frz proteins by constructing and analysing in-frame deletion mutants in each of the key frz genes. Our experiments showed the importance of each of the Frz proteins in the regulation of individual cell reversals as well as the multicellular events of aggregation, vegetative swarming and the repellent response (Table 2; Figs 2–4). Furthermore, we provide evidence that the Frz system controls both the A- and S-motility systems. The most striking finding in this study is that the N-terminal domain of FrzCD does not appear to be required for sensing signals. Indeed, deletion of the entire N-terminal domain of FrzCD, from amino acids 6–130, had little or no effect on cellular reversal frequencies, vegetative swarming, the repellent response or developmental aggregation (Table 2; Figs 7–9). Because the N-terminal domain is not required for most signal sensing, we hypothesize that somehow the C-terminus of FrzCD, which is similar to the C-terminal domains of the enteric MCPs, must be involved in both signal sensing and transmission.

Given the multitude of responses controlled by the Frz system, it is not surprising that this system shows several functional differences from the paradigm enteric chemotaxis pathways. E. coli senses external stimuli through five transmembrane MCPs, which transmit the signals to the CheA kinase via CheW, a coupling protein, that functionally links CheA to the chemoreceptors. CheA regulates cell motility by controlling the rate of phosphorylation of CheY. Phosphorylation causes an increase in the affinity of CheY for binding to the flagellar motor switch proteins, and CheY binding to the switch causes a reversal in flagellar rotation, from counterclockwise to clockwise, that generates a tumble response. Decay of the signals transmitted to the flagellar apparatus occurs by dephosphorylation of CheY, a reaction that is accelerated by CheZ. In the adaptation pathway, CheR constitutively methylates the MCPs, which increases the ability of the MCPs to stimulate CheA kinase activity. Phosphorylation mediated by CheA enhances the ability of CheB to demethylate MCPs, thus forming a negative feedback loop, which mediates a return to prestimulus CheA activity (for reviews, see Armitage, 1999; Bren and Eisenbach, 2000; Bourret and Stock, 2002).

In this study we have shown that FrzCD (MCP), FrzA (CheW) and FrzE (CheA–CheY) form the core of the Frz system, which is essential for directional signalling. Mutations in the core functions affect all of the responses mediated by the Frz system (Table 2; Figs 2, 3 and 5). In this regard, FrzCD, FrzA and FrzE seem to function in a similar way to their respective homologues from E. coli. However, as shown in this study, FrzCD is a cytoplasmic MCP that senses signals for aggregation, vegetative swarming and repellent response through its conserved C-terminal module (Figs 7–9). In Halobacterium salinarum, the cytoplasmic MCP, Car, is responsible for arginine chemotaxis (Storch et al., 1999). In Rhodobacter sphaeroides and Sinorhizobium meliloti the soluble Tlp proteins, which exhibit MCP-like motifs, mediate chemotaxis to different compounds under aerobic or photoheterotrophic conditions (Ward et al., 1995; Armitage and Schmitt, 1997; Harrison et al., 1999; Wadhams et al., 2002). However, little or nothing is known about the sensory mechanisms used by these cytoplasmic MCPs. Thus, to our knowledge FrzCD is the first example in which a cytoplasmic MCP uses its conserved C-terminal region to sense chemotaxis signals. In E. coli, the conserved C-terminal region of Tar mediates chemotaxis to changes in temperature and pH (Nara et al., 1996; Nishiyama et al., 1997; Umemura et al., 2002). Recently, in Bacillus subtilis, it was reported that the conserved region of the transmembrane chemoreceptor McpC mediates carbohydrate chemotaxis (Kristich et al., 2003). The model for McpC proposes that the methylation domains from the C-terminal module act as sensors for chemotactic stimuli. Furthermore, it was proposed that chemotactic signals are transmited to the methylation domains by interaction with EI (or Hpr), a component of the phosphotransfer system (Kristich et al., 2003). In this study we have shown that deletion of 16 amino acids from the first methylation domain of FrzCD induces a constitutive signalling state of FrzCD (Figs 4 and 6B). The constitutive signalling state of FrzCD was also induced by deleting the last 25 amino acids from the second methylation domain (Blackhart and Zusman, 1985a). Based on these results we propose that, like McpC of B. subtillis, the methylation domains from FrzCD could act as sensors for chemotactic stimuli. Thus, deletions of some amino acids could induce a conformational change in the methylation domains that resembles the conformational change induced by protein–protein interactions or a site-specific methylation that are involved in the transmission of chemotactic stimuli to FrzCD.

FrzG, the methylesterase for FrzCD, is only partially required for normal vegetative swarming, suggesting that it does not form a negative feedback loop, as does CheR in E. coli. In contrast, FrzF, the methyltransferase of FrzCD, is required for fruiting-body formation and normal vegetative swarming but not for repellent response (Figs 2, 3 and 5). In contrast to CheR from E. coli and most CheR-like proteins, FrzF has an extra C-terminal region containing three tetratricopeptide repeats (TPR) (Shiomi et al., 2002). Our recent results indicate that this TPR domain is important for the correct FrzF-mediated methylation of FrzCD (I. Martínez-Flores and D.R. Zusman, unpubl.). The TPR motifs are known to mediate protein–protein interactions in both eukaryotes and prokaryotes (Blatch and Lassle, 1999). Interestingly, using the yeast two-hybrid system, we found that the TPR motifs from FrzF show multiple interactions: with the catalytic domain of FrzF (N-terminal region), with two kinase proteins and with the CheY domain from FrzE (I. Martínez-Flores and D.R. Zusman, unpubl.). Thus, in contrast to CheR from E. coli, which is constitutively active, FrzF activity must be regulated by some of these protein–protein interactions. For example, FrzCD activity could be regulated by a site-specific, FrzF-mediated methylation. In B. subtilis chemoreceptor McpB methylation is site specific; methylation at residue 630 of the receptor increases activity of CheA and methylation at residue 637 decreases activity of CheA (Zimmer et al., 2000). Furthermore, in B. subtilis, in addition to CheR and CheB, methylation of the chemoreceptor is regulated by CheC, CheD and CheY (for a review, see Rao et al., 2004). Recently, it has been shown that mutations in the predicted methylation sites of FrzCD differentially affect swarming and aggregation, suggesting that FrzCD methylation is site specific (Astling, 2003).

The Frz system has two CheW-like proteins, FrzA, which is part of the core, and FrzB, which is required for fruiting-body formation and normal vegetative swarming but not for repellent response (Figs 2, 3 and 5). Thus, whereas FrzA seems to work like CheW in E. coli, FrzB seems to play a different role. The second chemotaxis operon (cheOp2) from R. sphaeroides encodes two CheW-like proteins. One of them is required for normal localization of McpG and for normal chemotaxis under both aerobic and photoheterotrophic conditions. The other CheW-like protein is required for localization of McpG and for normal chemotaxis but only under photoheterotrophic conditions, which suggests that these CheW-like proteins have distinct, non-redundant, but partially overlapping roles in chemotaxis (Martin et al., 2001). In B. subtilis, CheV is a CheW–CheY fusion protein that is functionally redundant to CheW, but is predicted to negatively regulate receptor activity (Karatan et al., 2001). Thus, the presence of multiple CheW-like proteins can provide additional control of MCP activity. Interestingly, a screen with the yeast two-hybrid system revealed that FrzB interacts with the conserved C-terminal regions of FrzCD, MCP3A (an MCP from the Che3 system) and one orphan MCP not encoded with other chemotaxis-like proteins (I. Martínez-Flores and D.R. Zusman, unpubl.). Thus, FrzB could link FrzCD with other chemoreceptors to receive external signals.

The Frz system has three CheY-like domains, two in FrzZ (CheY–CheY) and one in FrzE (CheA–CheY) (McBride et al., 1989; Ward and Zusman, 1997; 1999). In addition, one other CheY-like domain is present in FrzS, a protein required for S-motility, which may be part of the Frz system (Ward et al., 2000). FrzZ is required for fruiting-body formation and normal vegetative swarming but not for the response to repellents (Figs 2, 3 and 5). On the other hand, we have shown that the CheY domain from FrzE is required for vegetative swarming (V.H. Bustamante and D.R. Zusman, unpubl.). Thus, the Frz system uses multiple CheY-like domains to regulate A- and S-motility.

A model for the Frz system, based on the results from this study, is shown in Fig. 10. The cytoplasmic chemoreceptor FrzCD senses signals that control aggregation, vegetative swarming and repellent response through its conserved C-terminal module. We predict that the signals are transmitted from FrzCD (MCP) to FrzE (CheA–CheY) through the coupling protein FrzA (CheW). These three proteins form the core of the Frz system, which is essential for signalling. FrzB, the other CheW-like protein from the Frz system, could act as an input pathway to the Frz system, coupling FrzCD to other MCPs localized in the membrane. FrzCD is methylated and demethylated by FrzF (CheR) and FrzG (CheB) respectively. The activity of FrzCD could be additionally regulated by site-specific methylation mediated by FrzF, in response to protein–protein interactions with the TPR motifs present in the C-terminal region of FrzF. FrzZ (CheY–CheY) and at least one more protein containing a CheY-like domain (FrzE or FrzS) are involved in the control of cell reversal frequency mediated by the Frz system. These CheY domains are predicted to receive signals from the kinase domain from FrzE to regulate, directly or indirectly, the A- and S-motility systems.

image

Figure 10. Model for the Frz chemosensory pathway. The cytoplasmic chemoreceptor FrzCD senses chemotactic signals through its conserved C-terminal region. FrzCD, FrzA (CheW) and FrzE (CheA/CheY) form the core of the Frz system, which is essential for signalling. FrzB (CheW) may be involved in coupling other input pathways to the Frz system. FrzCD is methylated and demethylated by FrzF and FrzG respectively. FrzF has an extra C-terminal region containing three tetratricopeptide repeats (TPRs), which could mediate protein–protein interactions involved in the control of FrzCD activity by site-specific methylation. FrzE kinase is predicted to transmit the signals to the CheY domains contained in FrzZ (CheY/CheY), FrzE (CheA/CheY) and FrzS (CheY/Coiled-coil domain) to regulate, directly or indirectly, the A- and S-motility systems.

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Experimental procedures

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

Bacterial strains, plasmids and culture conditions

Bacterial strains and plasmids used are listed in Table 1. M. xanthus strains were grown vegetatively at 32°C in CYE media, which contains 1% (w/v) Casitone, 0.5% yeast extract, 10 mM MOPS (pH 7.6) and 4 mM MgSO4 (Campos et al., 1978). Development was assayed at 32°C on CF plates containing 1.5% 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 (Hagen et al., 1978).

DNA manipulations and sequence analysis

DNA manipulations were performed using standard protocols (Sambrook et al., 1989). Taq DNA polymerase (Promega) or Pfu turbo DNA polymerase (Stratagene) and FailSafeTM polymerase chain reaction (PCR) 2× PreMix J buffer (Epicentre) were used in all PCRs. Oligonucleotides were obtained from Qiagen Inc. DNA sequencing was carried out at the DNA Sequencing Facility at University of California – Berkeley. DNA sequences were analysed using DNAstar or DNA Strider programs.

Construction of mutants

The in-frame deletions were obtained by homologous recombination based on a previously reported method (Ueki et al., 1996). Briefly, deletion cassettes were generated by overlap extension PCR of two ≈ 500 bp primary PCR products that correspond to upstream and downstream regions of the target deletion. The deletion cassettes were subcloned into pBJ113, using the EcoRI and HindIII restriction sites. The resulting recombinant plasmids were transformed by electroporation into the respective M. xanthus strains to obtain plasmid insertion mutants, by selecting for kanamycin resistance. Deletions were generated in the transformants by counterselection on CYE-agar plates containing 2.5% galactose. Galactose-resistant, kanamycin-sensitive colonies were analysed by PCR to confirm the respective deletion. The frzCD deletions were further confirmed by immunoblot analysis, using polyclonal anti-FrzCD antibodies.

Immunoblot analysis

Whole-cell extracts used for the Western immunoblots were prepared from cells grown in liquid CYE media to mid-log phase, or from cells starved for 20 h on CF-agar plates. Ten micrograms of total protein was loaded into 12% or 15% SDS-polyacrylamide gels to detect expression of the complete FrzCD protein or its derivative deletions respectively. Proteins were transferred to a nitrocellulose membrane (0.45 µm) using a semidry transfer apparatus (Bio-Rad). Immunoblots were prepared by standard procedures (Sambrook et al., 1989). Polyclonal anti-FrzCD antibodies and HRP-conjugated secondary antibodies (Pierce) were diluted at 1:10 000. Detection was performed by Western lightning chemiluminescence reagent plus (PerkinElmer Life Sciences, Inc.) and Kodak Biomax films.

Phenotypic analysis

Developmental assays were performed by spotting 10 µl of cells at 4 × 109 cfu ml−1 directly onto CF 1.5% agar plates. Vegetative swarming phenotypes were analysed by spotting the same amount of cells onto CYE plates containing a concentration of 1.5% (hard) or 0.3% (soft) agar. After incubation at 32°C during the indicated time, the diameter of the colonies was measured and the swarming colonies and fruiting bodies were photographed with an MTI charge-coupled device (CCD)-72 camera, using a Nikon Labphot-2 or a Zeiss (Model 47 60 09-9901) microscopes. Images were processed in a Macintosh computer using the NIH Image and Photoshop software packages.

Motion analysis

Time-lapse motion analysis of cell movements was performed on cultures diluted to 4 × 106 cells ml−1, with 10 µl spotted over CF 1.5% agar thinly layered on microscope slides. The spots were allowed to dry, after which cells were covered with an oxygen-permeable membrane (YSI Inc.) and incubated for at least 1 h at room temperature. Isolated individual cells were filmed with an MTI CCD-72 camera coupled to a Nikon Labphot-2 microscope (40× objective), using the NIH Image software. Pictures were taken every 15 s for 30 min. Individual cell coordinates were determined using Metamorph software (Universal Imaging) and the reversal frequency of single cells was calculated using an automated program developed by David Astling in our laboratory. At least 20 cells were assayed for each strain. Statistical analysis was performed using an analysis of variance test followed by Tukey's multiple comparison at 95% confidence.

Acknowledgements

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References

We would like to thank Penelope Higgs, John Merlie and Tam Mignot for helpful discussions and critical review of this manuscript. We would also like to thank John Merlie for constructing the ΔfrzE mutant. We would also like to thank Daniel Portnoy for the use of the Metamorph software and David Astling for providing us the program to analyse the cellular reversal frequency. This research was supported by a grant from the National Institute of Health to DRZ (GM20509).

References

  1. Top of page
  2. Summary
  3. Introduction
  4. Results
  5. Discussion
  6. Experimental procedures
  7. Acknowledgements
  8. References