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Journal of Bacteriology, July 2005, p. 5029-5031, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.5029-5031.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

EspC Is Involved in Controlling the Timing of Development in Myxococcus xanthus

Bongsoo Lee,^1,2 Penelope I. Higgs,² David R. Zusman,² and Kyungyun Cho^1*

Section of Life Science, Hoseo University, Asan, 336-795 Korea,¹ University of California—Berkeley, Department of Molecular and Cellular Biology, Berkeley, California 94720-3204²

Received 12 August 2004/ Accepted 14 April 2005

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The espC null mutation caused accelerated aggregation and formation of tiny fruiting bodies surrounded by spores, which were also observed in the espA mutant and in CsgA-overproducing cells in Myxococcus xanthus. In addition, the espC mutant appeared to produce larger amounts of the complementary C-signal than the wild-type strain. These findings suggest that EspC is involved in controlling the timing of fruiting body development in M. xanthus.

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Fruiting body development of Myxococcus xanthus consists of two major processes: (i) aggregation of 10⁵ to 10⁶ cells to form raised mounds and (ii) sporulation of individual cells in the mounds (4, 8). M. xanthus normally does not sporulate outside of mounds, suggesting that it has a mechanism that inhibits sporulation when the cells are outside of the mounds and induces sporulation only when the cells are in the mounds.

Previously, we described two genes that influence the timing of development and sporulation in M. xanthus: espA and espB (2). An espA null mutation triggered early development and formation of tiny fruiting bodies surrounded by spores. In contrast, the espB null mutation caused delayed and reduced sporulation. We were interested in identifying other genes that might be related to the EspAB pathway or might also be involved in regulating the timing of development. Since espA espB double mutants show the EspA phenotype, we hypothesized that mutations in functionally related genes in the pathway, when introduced into the espB strain, would also show the EspA phenotype in early development. We therefore mutagenized the espB strain, DZ4417, with the pMycoMar plasmid carrying the magellan4 transposon, a derivative of the mariner transposon (10). Electroporants were plated on CF agar (12) containing kanamycin. Since pMycoMar cannot autonomously replicate in M. xanthus, only transformants carrying the transposon in the chromosome as a result of transposition should grow and form fruiting bodies on this medium. Among the approximately 50,000 colonies screened, nine colonies showed early development. Genomic DNA from each of the nine colonies was purified, and the site of the transposon insertion was determined as described previously (10). One of the mutants, DZ4422, had a transposon insertion in an unidentified open reading frame (ORF) encoding a histidine protein kinase that was similar to EspA. This ORF was therefore chosen for further study. Since the phenotype of the strain containing a mutation in this ORF was similar to that of the espA strain, this ORF was designated espC (for early sporulation).

Characterization of the espC locus. To verify that the transposon insertion in espC was responsible for the EspA-like phenotype of DZ4422, we created a new mutant, DZ4586, which had an insertion of pBS102 in espC, as well as an in-frame deletion mutant, DZ4588, from which amino acids 43 to 797 were deleted using pBS113 (Fig. 1). When DZ4586 and DZ4588 were placed on CF agar (12), they showed the EspA phenotype (Fig. 2). While we were characterizing the espC locus, Youderian et al. deposited the DNA sequence of the espC gene in the GenBank DNA sequence database, as a sequence adjacent to the aglR and aglS A-motility genes (15). Figure 1 shows that the espC gene is 3.7 kb downstream of aglS. Sequence analysis showed three ORFs oriented in the same direction as espC in the region between aglS and espC (orfA, orfB, and orfC in Fig. 1). To test whether there is polarity between these ORFs and espC, we created plasmid insertion mutations in orfA (DZ4594) and orfC (DZ4595). However, the resulting mutants produced normal fruiting bodies on CF agar, suggesting that orfA, orfB, and orfC are transcribed independently from espC. Since the espC mutant displayed normal motility, it is unlikely that espC is related to aglR and aglS, which are required for A-motility. orfD, the ORF downstream of the espC gene, is oriented convergently with espC, suggesting that the espC gene is monocistronic.

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FIG. 1. Physical map of the espC region. The black arrowhead indicates the site of the mariner transposon insertion. The strains and sites of plasmid insertions are listed below. The site of the in-frame deletion in DZ4588 is indicated by the depressed bar.

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FIG. 2. Developmental phenotype of the espC mutant. Cells (20-µl spots containing 5 x 109 cells ml–1) of M. xanthus strains DZ2 (A, C, and E) and DZ4588, the espC mutant (B, D, and F), were placed on CF agar plates and incubated at 32°C for 5 days. Panels A and B show the density of fruiting bodies in each spot, panels C and D show the relative size of individual fruiting bodies, and panels E and F show individual cells or spores outside of fruiting bodies.

Reverse transcription-PCR experiments showed that the level of espC mRNA increased under developmental conditions but that no espC mRNA was detected under the vegetative conditions (data not shown). This indicates that expression of espC is developmentally regulated, which is similar to what occurs with espA.

Similarity of EspC to other proteins. Sequence analysis using SMART (11) predicted that EspC contains 842 amino acids with the following domain structure: a MASE1 domain (from amino acids 25 to 300), a PAS domain (from amino acids 307 to 380), a PAC domain (from amino acids 382 to 424), a His kinase A domain (from amino acids 439 to 510), a histidine kinase-like ATPase domain (from amino acids 550 to 663), and a signal receiver domain (from amino acids 690 to 804). MASE1 is a predicted integral membrane sensory domain found in histidine kinases, diguanylate cyclases, and other bacterial signaling proteins (1). PAS/PAC domains are involved in many signaling proteins, where they are used as a sensor domain for energy or oxygen stress (14). The His kinase A domain and the histidine kinase-like ATPase domains constitute the histidine protein kinase found in many sensory kinases of two-component systems (9). This domain (from amino acids 439 to 663) was most similar to corresponding domains from MokA (6) (50% identity) and EspA (2) (49% identity) from M. xanthus.

Phenotype of the espC null mutant. The espC null mutant produced many tiny fruiting bodies under starvation conditions, which is similar to what occurs in the espA mutant (Fig. 2B). In addition, the espC mutation caused sporulation outside of fruiting bodies, which is also similar to the espA mutation (Fig. 2F). The espC mutation also caused earlier sporulation initiation than in the wild-type strain DZ2 (Fig. 3). The sporulation timing of the espC mutant, DZ4588, was very similar to that of the espA mutant, DZ4227. espC was epistatic to espB, pktA5, or pktB8, similar to what occurs in the espA mutant (2, 3). When 10⁸ cells of each double mutant, DZ4590 (espB espC), DZ4591 (pktB8 espC), and DZ4592 (pktA5 espC), were spotted onto CF plates, all of them showed an EspC-like phenotype. Sporulation defects were also rescued. As shown in Table 1, the sporulation efficiencies of DZ4225 (espB), DZ4282 (pktB8), and DZ4308 (pktA5) were increased to near wild-type level in the presence of the espC mutation in the double mutants. These results suggest that EspC is involved in controlling the timing of fruiting body development in M. xanthus.

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FIG. 3. Timing of sporulation of the espC mutant. M. xanthus DZ2 (; wild type), DZ4227 (; espA), and DZ4588 (; espC) were spotted on CF agar plates and incubated at 32°C. Cells were harvested at different time points, and the numbers of sonication-resistant refractile spores were counted.

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TABLE 1. Effect of the espC mutation on sporulation frequency

Extracellular complementation of the csgA mutation by the espA and espC mutants. Aggregation-independent early sporulation is also observed when the CsgA protein is overproduced in the cell (7). The CsgA protein is proposed to be a signal for C-signaling in M. xanthus, and the csgA null mutant is defective in fruiting body development (5, 13). Since the effects of the espA and espC null mutations mimic the overproduction of the CsgA protein, we tested whether the espA and espC mutants overproduce the extracellular signal molecules that rescue the developmental defects of the csgA mutant. When 5 x 10⁷ cells of the tetracycline-resistant csgA mutant (DZ4655) were mixed with 5 x 10⁷ cells of the espC mutant (DZ4588) and placed as 20-µl spots on CF plates, the mixed cells produced 1.2 x 10⁵ tetracycline-resistant spores per spot in 5 days (Table 2). Under the same conditions, the wild-type strain (DZ2) and the espA mutant (DZ4227) stimulated the csgA mutant to produce only 5.1 x 10³ and 1.1 x 10³ tetracycline-resistant spores, respectively. Thus, the espC mutant appeared to produce more complementing factor than the wild-type strain, stimulating the csgA mutant to produce 24-fold more spores than the wild-type strain, while the espA mutant appeared to produce less complementing factor than the wild type. This suggests that, although the developmental phenotypes of the espC and espA mutants are very similar, the functions of EspC and EspA may be different. We speculate that EspC may function in controlling C-signal generation. It is not known at this point whether the espC mutant overproduces CsgA, as observed in the CsgA-overproducing strain. It is also not clear how the espA mutant, which produces smaller amounts of the complementary C-signal than the wild-type strain, shows a developmental phenotype similar to the CsgA-overproducing strain and the espC mutant. However, since all of the espA and espC mutants and the CsgA-overproducing strain show similar altered timing in fruiting body development, further studies of EspA and EspC will help us to understand the timing mechanism of the fruiting body development in M. xanthus.

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TABLE 2. Extracellular complementation of the csgA mutation by the espA and espC mutants

 
This research was supported by grants from the Korean Ministry of Science and Technology (21C Frontier Microbial Genomics and Applications Program) (to K.C.) and from the National Institutes of Health (GM64463) (to D.Z.).

 
* Corresponding author. Mailing address: Section of Life Science, Hoseo University, Asan, 336-795 Korea. Phone: 82415504627. Fax: 82415486231. E-mail: kycho{at}office.hoseo.ac.kr.

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Journal of Bacteriology, July 2005, p. 5029-5031, Vol. 187, No. 14
0021-9193/05/$08.00+0     doi:10.1128/JB.187.14.5029-5031.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

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