JB
Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lancero, H.
Right arrow Articles by Shimkets, L. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lancero, H.
Right arrow Articles by Shimkets, L. J.

 Previous Article  |  Next Article 

Journal of Bacteriology, March 2002, p. 1462-1465, Vol. 184, No. 5
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.5.1462-1465.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.

Mapping of Myxococcus xanthus Social Motility dsp Mutations to the dif Genes

Hope Lancero,1 Jennifer E. Brofft,2 John Downard,3 Bruce W. Birren,4 Chad Nusbaum,4 Jerome Naylor,4 Wenyuan Shi,1 and Lawrence J. Shimkets2*

Molecular Biology Institute and School of Dentistry, University of California, Los Angeles, Los Angeles, California 90095-1668,1 Department of Microbiology, University of Georgia, Athens, Georgia 30602-2605,2 Department of Botany and Microbiology, University of Oklahoma, Norman, Oklahoma 73019-6131,3 Whitehead Institute/MIT Center for Genome Research, Cambridge, Massachusetts 021414

Received 8 August 2001/ Accepted 7 December 2001


   ABSTRACT
Top
 Abstract
Text
 References
 
Myxococcus xanthus dsp and dif mutants have similar phenotypes in that they are deficient in social motility and fruiting body development. We compared the two loci by genetic mapping, complementation with a cosmid clone, DNA sequencing, and gene disruption and found that 16 of the 18 dsp alleles map to the dif genes. Another dsp allele contains a mutation in the sglK gene. About 36.6 kb around the dsp-dif locus was sequenced and annotated, and 50% of the genes are novel.


   TEXT
Top
 Abstract
 Text
References
 
Many organisms live in biofilms, where they move by surface translocation mechanisms, such as twitching or gliding (28). In Myxococcus xanthus there are two different motility systems for surface translocation. Cells can move as individuals using the adventurous (A) motility system (11, 12). Cells can also move in groups using the social (S) motility system, which requires cell-cell contact (3, 13). While the molecular basis of A motility is unknown, S motility, which is analogous to twitching, is dependent on retraction of type IV pili (18, 26, 30). S motility also utilizes a matrix of extracellular protein-associated polysaccharide, known as fibrils, which mediate cell cohesion (1, 2), coordinate cell movement (29), and initiate chemotactic excitation to certain phosphatidylethanolamine species (15, 16).

Physical mapping by cotransduction. One group of S motility mutants is known as dsp (dispersed growth) mutants, and the mutations map to a large locus near Tn5 insertion {Omega}DK1407 (22, 23). The dsp mutants have a distinct phenotype; they lack fibrils and the ability to form fruiting bodies. One of the mutations is a Tn5 insertion (dsp-3119), while the remaining mutations are point mutations generated by chemical and UV mutagenesis. The approximate size of the locus was determined by examining the cotransduction frequency of {Omega}1407 with each dsp allele using bacteriophage Mx8. Cotransduction frequency may be converted to physical distance by using the equation C = (1 - t)3, where C is the cotransduction frequency and t is the fractional physical distance between the markers (27). The length of Mx8 DNA is 56 kb (17), but this length was reduced by the length of Tn5 (6 kb) to calculate physical distance (27). The predicted physical distances of the dsp mutations from {Omega}1407 range from 1.7 to 11.0 kb for the bulk of the alleles. Allele dsp-3119 is much farther away, and dsp-1689 is unlinked (Table 1). These data suggest that the dsp locus begins approximately 1.7 kb from {Omega}1407 and is approximately 9.3 kb long.


View this table:
[in this window]
[in a new window]
  		TABLE 1. Complementation and cotransduction of dsp mutants

 
Cloning and complementation. Yang et al. discovered mutants with a phenotype similar to that of dsp mutants (33). The locus which these authors described is called dif (defective in fruiting) and encodes proteins homologous to the Che chemosensory proteins of enteric bacteria. The dif locus is also linked to {Omega}1407 as determined by cotransduction, raising the possibility that dif genes are part of the dsp locus. To resolve this issue, M. xanthus DNA adjacent to {Omega}1407 was cloned by selecting for kanamycin resistance encoded by the transposon. The clone obtained (pLI209) was used to screen a cosmid library by DNA hybridization (7). A cosmid designated pREG3E1 was isolated and examined to determine whether it complemented each dsp allele. In these experiments the cosmid was introduced by specialized transduction with bacteriophage P1, and it integrated into the chromosome by homologous recombination with the native dsp locus (25). Transductants were examined for S motility (Table 1). In most cases the frequency of complementation was either 100 or 0%. In a few cases the complementation frequency was 80 to 90%, and in one case the complementation frequency was only 40% (dsp-2106). Incomplete complementation is due to gene conversion, which is a consequence of repair of the heteroduplex prior to replication. As a result, some transductants are homozygous merodiploids containing two copies of the mutant allele (25). The frequencies of gene conversion range from 0 to 33% in loci that have been studied in detail (19, 25). With the exception of dsp-2106, this range covers all of the crosses reported in Table 1. Thus, in cases in which incomplete complementation was observed, we assumed that the cosmid contained the wild-type allele. The data suggest that all of the dsp genes linked to {Omega}1407 by cotransduction were present on the cosmid. Surprisingly, the 36-kb cosmid also complemented dsp-3119, which appeared to be about 33 kb from {Omega}1407 as determined by cotransduction mapping (Table 1) and about 44 kb from {Omega}1407 as determined by pulsed-field gel electrophoresis (5). The cosmid did not complement allele dsp-1689, which showed no cotransduction with {Omega}1407 and therefore was assumed to be more than 39 kb from {Omega}1407.

DNA sequence analysis. The M. xanthus DNA in pREG3E1 is 36.6 kb long and was annotated with the aid of Artimis (The Sanger Center; http://www.sanger.ac.uk/Software/) and FramePlot 2.3.2 (National Institutes of Health; http://www.nih.go.jp/~jun/cgi-bin/frameplot.pl), which rely on third-position codon bias to identify putative protein-coding regions (14, 20). The M. xanthus DNA encodes 29 complete putative protein-coding regions, and the average gene density is about 0.8 kb-1. DNA sequencing of pLI209 was used to identify the insertion site of {Omega}1407, which follows base 23065 of the insert in pREG3E1 (data not shown). The distance from the insertion to the beginning of the first gene in the dif cluster, difA, is about 3 kb, and the distance to the end of the last gene in the dif cluster, difE, is about 10 kb, suggesting that the locus is about 7 kb long. This agrees fairly well with the calculated physical distances based on the cotransduction data for dsp (Table 1), suggesting that the dif genes are at least part of the dsp locus. However, it is formally possible that the dsp point mutations and the dif genes are located on opposite sides of the Tn5 insertion. On the side of {Omega}1407 opposite the dif side are several intriguing candidates for motility genes, including an mglA homolog and two pilT homologs. MglA is a GTPase that is essential for both A motility and S motility (8, 9). PilT is essential for S motility (32).

Gene disruption. Systematic gene disruption was used to determine whether dsp-like genes are located on the other side of the transposon insertion or anywhere else on the cosmid. Gene disruption was accomplished by integrating an internal fragment of a gene by homologous recombination into the wild-type (DK1622) genome. Each disruption mutant was confirmed Southern blotting and then examined for agglutination, fruiting body development, sporulation, and S motility, which are uniformly defective in dsp strains. Agglutination measures fibril-dependent cohesion and is completely absent in dsp strains, which lack fibrils (22). Fruiting body development and sporulation were examined following starvation. The dsp mutants failed to form fruiting bodies and produced only 1% of the wild-type levels of spores, as determined by direct counting (23). S motility was assayed on Casitone-yeast extract plates containing 0.3% agar, which favors movement of S+ strains (21).

The coordinates of the fragments causing gene disruption relative to the cosmid sequence are shown in Table 2, along with the phenotypes of the disrupted mutants. Insertions in each of the dif genes generated a dsp phenotype, as expected. While the results suggest that all five dif genes are essential for S motility, we cannot rule out the possibility that the insertions are polar on difE, which is known to be essential for S motility. Disruption of the mglA homolog or one of the pilT homologs did not have a deleterious effect on motility or development. With the exception of the mutants with dif gene disruptions, the disruption mutants were not severely defective in development or S motility. Disruption of open reading frame 3E1-22, which encodes a putative polypeptide with 28% amino acid identity to protein alanine N-acetyl transferase, resulted in delayed fruiting body development but normal motility.


View this table:
[in this window]
[in a new window]
  		TABLE  2. Positions of the putative protein-coding regions, their homologies, and the phenotypes of disruption mutants

 
Cosmid pREG3E1 complements dsp-3119, which was generated by Tn5 insertion {Omega}3119. The cotransduction data (Table 1) and physical mapping using pulsed-field gel electrophoresis accurately predicted that this gene is not represented on the cosmid despite the complementation results (5, 10). Complemented cells contain fibrils, as determined by the use of fibril-specific dyes and the agglutination assay (6). To explore this puzzle in more detail, DNA surrounding insertion {Omega}3119 was cloned into plasmid pLJS107 and sequenced to identify the gene containing the insertion. The insertion is located in sglK, a gene that has been described previously, and the phenotype of an sglK null mutant is similar to that of a dsp-3119 mutant in that fibrils are not produced and fruiting body development is inhibited (31, 34). SglK belongs to the DnaK family of chaperone proteins. Although it is possible that a chaperone-like gene on pREG3E1 can substitute for sglK at the higher copy number of the merodiploid, none of the putative proteins encoded by the cosmid belong to the DnaK family. Fibrils do not appear to be part of the S motility motor but rather regulate motility and coordinate cell behavior (6). The restoration of both S motility and fibril formation implies that multiple copies of a gene(s) located on the cosmid bypass the mutational block imposed by {Omega}3119.

Features of the genome. The piece of the DNA sequenced in this study is the largest piece of DNA that has been sequenced for M. xanthus, which has one of the largest prokaryotic genomes (the genome of M. xanthus is 9.5 Mb long) (4, 24). Perhaps the most striking feature of the cosmid sequence is the number of novel genes (defined by using a cutoff value of 25% amino acid identity), which represent about one-half the total number of genes (Table 2). All of the genes, novel or otherwise, have a third-position codon bias typical of high-G+C-content organisms and indicative of M. xanthus. Whether these genes were imported from other as-yet-unsequenced genomes or evolved as novel structures in M. xanthus should become clearer as more genome sequences are added to the database.

In conclusion, using genetic complementation, DNA sequencing, and systematic mutagenesis, we found that the dsp locus is composed of dif genes, which are the only S motility genes near {Omega}1407. Based on these findings, it is likely that all of the dsp alleles shown in Table 1 except dsp-3119 and dsp-1689 are mutations in dif genes. The dif genes are chemotaxis homologs located in the cytoplasm and the inner membrane, whereas fibrils are located on the cell surface. Apparently, additional genes are required for the biosynthesis, transport, and assembly of fibrils. The sglK gene and the gene represented by the dsp-1689 allele may be two examples of such genes. It would be interesting to identify the gene products involved in fibril biosynthesis and to study their interaction with the Dif gene products.

The GenBank accession number of the pREG3E1 sequence is AF449411.


   ACKNOWLEDGMENTS
 
We are grateful to the following individuals who generously contributed to this project. David Morandi and Dale Kaiser provided the dsp alleles used in this analysis. Shengfeng Li isolated clone pLI209. Ron Gill provided the cosmid library. Many members of the Whitehead Institute/MIT Center for Genome Research contributed to the sequencing of the cosmid.

This work was supported by grant MCG0090946 from the National Science Foundation to L.J.S. and by grant GM54666 from NIH to W.S.


   FOOTNOTES
 
* Corresponding author. Mailing address: Department of Microbiology, University of Georgia, Athens, GA 30602-2605. Phone: (706) 542-2681. Fax: (706) 542-2674. E-mail: shimkets{at}arches.uga.edu. Back


   REFERENCES
Top
 Abstract
 Text
 References
 

  1. Arnold, J. W., and L. J. Shimkets. 1988. Cell surface properties correlated with cohesion in Myxococcus xanthus. J. Bacteriol. 170:5771-5777.[Abstract/Free Full Text]
  2. Arnold, J. W., and L. J. Shimkets. 1988. Inhibition of cell-cell interactions in Myxococcus xanthus by Congo red. J. Bacteriol. 170:5765-5770.[Abstract/Free Full Text]
  3. Burchard, R. P. 1970. Gliding motility mutants of Myxococcus xanthus. J. Bacteriol. 104:940-947.[Abstract/Free Full Text]
  4. Chen, H.-W., I. Keseler, and L. J. Shimkets. 1990. Genome size of Myxococcus xanthus determined by pulsed-field gel electrophoresis. J. Bacteriol. 172:4206-4213.[Abstract/Free Full Text]
  5. Chen, H.-W., A. Kuspa, I. Keseler, and L. J. Shimkets. 1991. Physical map of the Myxococcus xanthus chromosome. J. Bacteriol 173:2109-2115.[Abstract/Free Full Text]
  6. Dana, J. R., and L. J. Shimkets. 1993. Regulation of cohesion-dependent cell interactions in Myxococcus xanthus. J. Bacteriol. 175:3636-3647.[Abstract/Free Full Text]
  7. Hager, E., H. Tse, and R. E. Gill. 2001. Identification and characterization of spdR mutations that bypass the BsgA protease-dependent regulation of developmental gene expression in Myxococcus xanthus. Mol. Microbiol. 39:765-780.[CrossRef][Medline]
  8. Hartzell, P., and D. Kaiser. 1991. Function of MglA, a 22-kilodalton protein essential for gliding in Myxococcus xanthus. J. Bacteriol. 173:7615-7624.[Abstract/Free Full Text]
  9. Hartzell, P. L. 1997. Complementation of sporulation and motility defects in a prokaryote by a eukaryotic GTPase. Proc. Natl. Acad. Sci. USA 94:9881-9886.[Abstract/Free Full Text]
  10. He, Q., H. Chen, A. Kuspa, Y. Cheng, D. Kaiser, and L. J. Shimkets. 1994. A physical mpa of the Myxococcus xanthus chromosome. Proc. Natl. Acad. Sci. USA 91:9584-9587.[Abstract/Free Full Text]
  11. Hodgkin, J., and D. Kaiser. 1977. Cell-to-cell stimulation of movement in nonmotile mutants of Myxococcus. Proc. Natl. Acad. Sci. USA 74:2938-2942.[Abstract/Free Full Text]
  12. Hodgkin, J., and D. Kaiser. 1979. Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): genes controlling movement of single cells. Mol. Gen. Genet. 171:167-176.[CrossRef]
  13. Hodgkin, J., and D. Kaiser. 1979. Genetics of gliding motility in Myxococcus xanthus (Myxobacterales): two gene systems control movement. Mol. Gen. Genet. 171:177-191.[CrossRef]
  14. Ishikawa, J., and K. Hotta. 1999. FramePlot: a new implementation of the Frame analysis for predicting protein-coding regions in bacterial DNA with a high G+C content. FEMS Microbiol. Lett. 174:251-253.[CrossRef][Medline]
  15. Kearns, D. B., B. D. Campbell, and L. J. Shimkets. 2000. Myxococcus xanthus fibril appendages are essential for excitation by a phospholipid attractant. Proc. Natl. Acad. Sci. USA 97:11505-11510.[Abstract/Free Full Text]
  16. Kearns, D. B., A. Venoit, P. J. Bonner, B. Stevens, G.-J. Boons, and L. J. Shimkets. 2001. Identification of a developmental chemoattractant in Myxococcus xanthus through metabolic engineering. Proc. Natl. Acad. Sci. USA 98:13990-13994.[Abstract/Free Full Text]
  17. Martin, S., E. Sodergren, T. Masuda, and D. Kaiser. 1978. Systematic isolation of transducing phages for Myxococcus xanthus. Virology 88:44-53.[CrossRef][Medline]
  18. Merz, A., M. So, and M. P. Sheetz. 2000. Pilus retraction powers bacterial twitching motility. Nature 407:98-101.[CrossRef][Medline]
  19. O'Connor, K. A., and D. R. Zusman. 1983. Coliphage P1-mediated transduction of cloned DNA from Escherichia coli to Myxococcus xanthus: use for complementation and recombinational analyses. J. Bacteriol. 155:317-329.[Abstract/Free Full Text]
  20. Rutherford, K., J. Parkhill, J. Crook, T. Horsnell, P. Rice, M.-A. Rajandream, and B. Barrell. 2000. Artemis: sequence visualisation and annotation. Bioinformatics 16:944-945.[Abstract/Free Full Text]
  21. Shi, W., and D. R. Zusman. 1993. The two motility systems of Myxococcus xanthus show different selective advantages on various surfaces. Proc. Natl. Acad. Sci. USA 90:3378-3382.[Abstract/Free Full Text]
  22. Shimkets, L. J. 1986. Correlation of energy-dependent cell cohesion with social motility in Myxococcus xanthus. J. Bacteriol. 166:837-841.[Abstract/Free Full Text]
  23. Shimkets, L. J. 1986. Role of cell cohesion in Myxococcus xanthus fruiting body formation. J. Bacteriol. 166:842-848.[Abstract/Free Full Text]
  24. Shimkets, L. J. 1997. Structure and sizes of genomes of the Archaea and Bacteria, p. 5-11. In F. J. de Bruijn, J. R. Lupski, and G. M. Weinstock (ed.), Bacterial genomes: physical structure and analysis. Chapman & Hall, New York, N.Y.
  25. Shimkets, L. J., R. E. Gill, and D. Kaiser. 1983. Developmental cell interactions in Myxococcus xanthus and the spoC locus. Proc. Natl. Acad. Sci. USA 80:1406-1410.[Abstract/Free Full Text]
  26. Skerker, J. M., and H. C. Berg. 2001. Direct observation of extension and retraction of type IV pili. Proc. Natl. Acad. Sci. USA 98:6901-6904.[Abstract/Free Full Text]
  27. Sodergren, E., Y. Cheng, L. Avery, and D. Kaiser. 1983. Recombination in the vicinity of insertions of transposon Tn5 in Myxococcus xanthus. Genetics 105:281-291.[Abstract/Free Full Text]
  28. Spormann, A. M. 1999. Gliding motility in bacteria: Insights from studies of Myxococcus xanthus. Microbiol. Mol. Biol. Rev. 63:621-641.[Abstract/Free Full Text]
  29. Sun, H., Z. Yang, and W. Shi. 1999. Effect of filamentation on adventurous and social gliding motility of Myxococcus xanthus. Proc. Natl. Acad. Sci. USA 96:15178-15183.[Abstract/Free Full Text]
  30. Sun, H., D. R. Zusman, and W. Shi. 2000. Type IV pilus of Myxococcus xanthus is a motility apparatus controlled by the frz chemosensory system. Curr. Biol. 10:1143-1146.[CrossRef][Medline]
  31. Weimer, R. M., C. Creighton, A. Stassinopoulos, P. Youderian, and P. Hartzell. 1998. A chaperone in the HSP70 family controls production of extracellular fibrils in Myxococcus xanthus. J. Bacteriol. 180:5357-5368.[Abstract/Free Full Text]
  32. Wu, S. S., J. Wu, and D. Kaiser. 1997. The Myxococcus xanthus pilT locus is required for social gliding motility although pili are still produced. Mol. Microbiol. 23:109-121.[CrossRef][Medline]
  33. Yang, Z., Y. Geng, D. Zu, H. B. Kaplan, and W. Shi. 1998. A new set of chemotaxis homologues is essential for Myxococcus xanthus social motility. Mol. Microbiol. 30:1123-1130.[CrossRef][Medline]
  34. Yang, Z., Y. Geng, and W. Shi. 1998. A DnaK homolog in Myxococcus xanthus is involved in social motility and fruiting body formation. J. Bacteriol. 180:218-224.[Abstract/Free Full Text]
  35. Yang, Z., X. Ma, T. Leming, H. B. Kaplan, L. J. Shimkets, and W. Shi. 2000. Myxococcus xanthus dif genes are required for biogenesis of cell surface fibrils essential for social gliding motility. J. Bacteriol. 182:5793-5798.[Abstract/Free Full Text]

Journal of Bacteriology, March 2002, p. 1462-1465, Vol. 184, No. 5
0021-9193/02/$04.00+0     DOI: 10.1128/JB.184.5.1462-1465.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:


This Article
Right arrow Abstract Freely available
Right arrow Full Text (PDF)
Right arrow Alert me when this article is cited
Right arrow Alert me if a correction is posted
Services
Right arrow Similar articles in this journal
Right arrow Similar articles in PubMed
Right arrow Alert me to new issues of the journal
Right arrow Download to citation manager
Right arrowReprints and Permissions
Right arrow Copyright Information
Right arrow Books from ASM Press
Right arrow MicrobeWorld
Citing Articles
Right arrow Citing Articles via HighWire
Right arrow Citing Articles via Google Scholar
Google Scholar
Right arrow Articles by Lancero, H.
Right arrow Articles by Shimkets, L. J.
Right arrow Search for Related Content
PubMed
Right arrow PubMed Citation
Right arrow Articles by Lancero, H.
Right arrow Articles by Shimkets, L. J.

Home Help [Feedback] [For Subscribers] [Archive] [Search] [Contents]
Appl. Environ. Microbiol. Infect. Immun. Eukaryot. Cell
Mol. Cell. Biol. J. Virol. Microbiol. Mol. Biol. Rev.
ALL ASM JOURNALS