Abstract
Mutations in the tgl locus inactivate social gliding motility in Myxococcus xanthus and block production of pili. The tgl locus is distinctive among the genes for social motility because social gliding and pili can be restored transiently to tgl mutant cells by mixing them with tgl+ cells, a process known as stimulation. The tgl locus was cloned with a linked insertion of transposon Tn5 by using the kanamycin resistance encoded by that transposon. A 16-kb segment of chromosomal DNA complemented the social motility defect when introduced into tgl mutant cells to form a tandem duplication tgl+/tgl heterozygote. To delimit the autonomous tgl transcription unit, subfragments of this 16-kb piece were integrated at the ectopic Mx8 prophage attachment site. A 1.7-kb DNA fragment was identified which, when integrated at the Mx8 site, simultaneously rescued social motility and pilus production. The ability to stimulate tgl mutants was also rescued by the 1.7-kb fragment. Because rescue of stimulation from an mgl-deficient donor strain which cannot swarm was observed, this demonstrates that a stimulation donor requires a tgl+ allele but does not require the capacity to swarm actively. The nucleotide sequence of the 1.7-kb fragment revealed two protein coding regions, open reading frame A and open reading frame B (ORFB). ORFB is the tgl gene, because a 613-bp DNA fragment which includes 75% of ORFB rescues tgl-1, -2, and -3 mutants and because disruption of ORFB by deletion or insertion of transposon Tn5lac constitutes a tgl mutation.
Full Text
The Full Text of this article is available as a PDF (356.2 KB).
Selected References
These references are in PubMed. This may not be the complete list of references from this article.
- Avery L., Kaiser D. Construction of tandem genetic duplications with defined endpoints in Myxococcus xanthus. Mol Gen Genet. 1983;191(1):110–117. doi: 10.1007/BF00330897. [DOI] [PubMed] [Google Scholar]
- Avery L., Kaiser D. In situ transposon replacement and isolation of a spontaneous tandem genetic duplication. Mol Gen Genet. 1983;191(1):99–109. doi: 10.1007/BF00330896. [DOI] [PubMed] [Google Scholar]
- Bankier A. T., Weston K. M., Barrell B. G. Random cloning and sequencing by the M13/dideoxynucleotide chain termination method. Methods Enzymol. 1987;155:51–93. doi: 10.1016/0076-6879(87)55009-1. [DOI] [PubMed] [Google Scholar]
- Bibb M. J., Findlay P. R., Johnson M. W. The relationship between base composition and codon usage in bacterial genes and its use for the simple and reliable identification of protein-coding sequences. Gene. 1984 Oct;30(1-3):157–166. doi: 10.1016/0378-1119(84)90116-1. [DOI] [PubMed] [Google Scholar]
- Gill R. E., Cull M. G., Fly S. Genetic identification and cloning of a gene required for developmental cell interactions in Myxococcus xanthus. J Bacteriol. 1988 Nov;170(11):5279–5288. doi: 10.1128/jb.170.11.5279-5288.1988. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Goebl M., Yanagida M. The TPR snap helix: a novel protein repeat motif from mitosis to transcription. Trends Biochem Sci. 1991 May;16(5):173–177. doi: 10.1016/0968-0004(91)90070-c. [DOI] [PubMed] [Google Scholar]
- Griff I. C., Schekman R., Rothman J. E., Kaiser C. A. The yeast SEC17 gene product is functionally equivalent to mammalian alpha-SNAP protein. J Biol Chem. 1992 Jun 15;267(17):12106–12115. [PubMed] [Google Scholar]
- Hartzell P., Kaiser D. Function of MglA, a 22-kilodalton protein essential for gliding in Myxococcus xanthus. J Bacteriol. 1991 Dec;173(23):7615–7624. doi: 10.1128/jb.173.23.7615-7624.1991. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hase T., Riezman H., Suda K., Schatz G. Import of proteins into mitochondria: nucleotide sequence of the gene for a 70-kd protein of the yeast mitochondrial outer membrane. EMBO J. 1983;2(12):2169–2172. doi: 10.1002/j.1460-2075.1983.tb01718.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- He Q., Chen H., Kuspa A., Cheng Y., Kaiser D., Shimkets L. J. A physical map of the Myxococcus xanthus chromosome. Proc Natl Acad Sci U S A. 1994 Sep 27;91(20):9584–9587. doi: 10.1073/pnas.91.20.9584. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Henikoff S. Unidirectional digestion with exonuclease III creates targeted breakpoints for DNA sequencing. Gene. 1984 Jun;28(3):351–359. doi: 10.1016/0378-1119(84)90153-7. [DOI] [PubMed] [Google Scholar]
- Hirano T., Hiraoka Y., Yanagida M. A temperature-sensitive mutation of the Schizosaccharomyces pombe gene nuc2+ that encodes a nuclear scaffold-like protein blocks spindle elongation in mitotic anaphase. J Cell Biol. 1988 Apr;106(4):1171–1183. doi: 10.1083/jcb.106.4.1171. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hodgkin J., Kaiser D. Cell-to-cell stimulation of movement in nonmotile mutants of Myxococcus. Proc Natl Acad Sci U S A. 1977 Jul;74(7):2938–2942. doi: 10.1073/pnas.74.7.2938. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Hodgson D. A. Light-induced carotenogenesis in Myxococcus xanthus: genetic analysis of the carR region. Mol Microbiol. 1993 Feb;7(3):471–488. doi: 10.1111/j.1365-2958.1993.tb01138.x. [DOI] [PubMed] [Google Scholar]
- Ikeda H., Tomizawa J. I. Transducing fragments in generalized transduction by phage P1. I. Molecular origin of the fragments. J Mol Biol. 1965 Nov;14(1):85–109. doi: 10.1016/s0022-2836(65)80232-7. [DOI] [PubMed] [Google Scholar]
- Kaiser D. Social gliding is correlated with the presence of pili in Myxococcus xanthus. Proc Natl Acad Sci U S A. 1979 Nov;76(11):5952–5956. doi: 10.1073/pnas.76.11.5952. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Koelle M. R., Talbot W. S., Segraves W. A., Bender M. T., Cherbas P., Hogness D. S. The Drosophila EcR gene encodes an ecdysone receptor, a new member of the steroid receptor superfamily. Cell. 1991 Oct 4;67(1):59–77. doi: 10.1016/0092-8674(91)90572-g. [DOI] [PubMed] [Google Scholar]
- Koerner T. J., Hill J. E., Myers A. M., Tzagoloff A. High-expression vectors with multiple cloning sites for construction of trpE fusion genes: pATH vectors. Methods Enzymol. 1991;194:477–490. doi: 10.1016/0076-6879(91)94036-c. [DOI] [PubMed] [Google Scholar]
- Kroos L., Kaiser D. Construction of Tn5 lac, a transposon that fuses lacZ expression to exogenous promoters, and its introduction into Myxococcus xanthus. Proc Natl Acad Sci U S A. 1984 Sep;81(18):5816–5820. doi: 10.1073/pnas.81.18.5816. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Laemmli U. K. Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 1970 Aug 15;227(5259):680–685. doi: 10.1038/227680a0. [DOI] [PubMed] [Google Scholar]
- Lamb J. R., Michaud W. A., Sikorski R. S., Hieter P. A. Cdc16p, Cdc23p and Cdc27p form a complex essential for mitosis. EMBO J. 1994 Sep 15;13(18):4321–4328. doi: 10.1002/j.1460-2075.1994.tb06752.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Legrain P., Choulika A. The molecular characterization of PRP6 and PRP9 yeast genes reveals a new cysteine/histidine motif common to several splicing factors. EMBO J. 1990 Sep;9(9):2775–2781. doi: 10.1002/j.1460-2075.1990.tb07465.x. [DOI] [PMC free article] [PubMed] [Google Scholar]
- McCollum D., Monosov E., Subramani S. The pas8 mutant of Pichia pastoris exhibits the peroxisomal protein import deficiencies of Zellweger syndrome cells--the PAS8 protein binds to the COOH-terminal tripeptide peroxisomal targeting signal, and is a member of the TPR protein family. J Cell Biol. 1993 May;121(4):761–774. doi: 10.1083/jcb.121.4.761. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Mizusawa S., Nishimura S., Seela F. Improvement of the dideoxy chain termination method of DNA sequencing by use of deoxy-7-deazaguanosine triphosphate in place of dGTP. Nucleic Acids Res. 1986 Feb 11;14(3):1319–1324. doi: 10.1093/nar/14.3.1319. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Nicolet C. M., Craig E. A. Isolation and characterization of STI1, a stress-inducible gene from Saccharomyces cerevisiae. Mol Cell Biol. 1989 Sep;9(9):3638–3646. doi: 10.1128/mcb.9.9.3638. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Ordway R. W., Pallanck L., Ganetzky B. A TPR domain in the SNAP secretory proteins. Trends Biochem Sci. 1994 Dec;19(12):530–531. doi: 10.1016/0968-0004(94)90052-3. [DOI] [PubMed] [Google Scholar]
- Rhee S. K., Icho T., Wickner R. B. Structure and nuclear localization signal of the SKI3 antiviral protein of Saccharomyces cerevisiae. Yeast. 1989 May-Jun;5(3):149–158. doi: 10.1002/yea.320050304. [DOI] [PubMed] [Google Scholar]
- Rodriguez-Soto J. P., Kaiser D. Identification and localization of the Tgl protein, which is required for Myxococcus xanthus social motility. J Bacteriol. 1997 Jul;179(13):4372–4381. doi: 10.1128/jb.179.13.4372-4381.1997. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Samejima I., Yanagida M. Bypassing anaphase by fission yeast cut9 mutation: requirement of cut9+ to initiate anaphase. J Cell Biol. 1994 Dec;127(6 Pt 1):1655–1670. doi: 10.1083/jcb.127.6.1655. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sanger F., Nicklen S., Coulson A. R. DNA sequencing with chain-terminating inhibitors. Proc Natl Acad Sci U S A. 1977 Dec;74(12):5463–5467. doi: 10.1073/pnas.74.12.5463. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Schlossmann J., Dietmeier K., Pfanner N., Neupert W. Specific recognition of mitochondrial preproteins by the cytosolic domain of the import receptor MOM72. J Biol Chem. 1994 Apr 22;269(16):11893–11901. [PubMed] [Google Scholar]
- Schultz J., Carlson M. Molecular analysis of SSN6, a gene functionally related to the SNF1 protein kinase of Saccharomyces cerevisiae. Mol Cell Biol. 1987 Oct;7(10):3637–3645. doi: 10.1128/mcb.7.10.3637. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Shimkets L. J., Asher S. J. Use of recombination techniques to examine the structure of the csg locus of Myxococcus xanthus. Mol Gen Genet. 1988 Jan;211(1):63–71. doi: 10.1007/BF00338394. [DOI] [PubMed] [Google Scholar]
- Shimkets L. J., Gill R. E., Kaiser D. Developmental cell interactions in Myxococcus xanthus and the spoC locus. Proc Natl Acad Sci U S A. 1983 Mar;80(5):1406–1410. doi: 10.1073/pnas.80.5.1406. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Short J. M., Fernandez J. M., Sorge J. A., Huse W. D. Lambda ZAP: a bacteriophage lambda expression vector with in vivo excision properties. Nucleic Acids Res. 1988 Aug 11;16(15):7583–7600. doi: 10.1093/nar/16.15.7583. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Sikorski R. S., Boguski M. S., Goebl M., Hieter P. A repeating amino acid motif in CDC23 defines a family of proteins and a new relationship among genes required for mitosis and RNA synthesis. Cell. 1990 Jan 26;60(2):307–317. doi: 10.1016/0092-8674(90)90745-z. [DOI] [PubMed] [Google Scholar]
- Sikorski R. S., Michaud W. A., Wootton J. C., Boguski M. S., Connelly C., Hieter P. TPR proteins as essential components of the yeast cell cycle. Cold Spring Harb Symp Quant Biol. 1991;56:663–673. doi: 10.1101/sqb.1991.056.01.075. [DOI] [PubMed] [Google Scholar]
- Sodergren E., Kaiser D. Insertions of Tn5 near genes that govern stimulatable cell motility in Myxococcus. J Mol Biol. 1983 Jun 25;167(2):295–310. doi: 10.1016/s0022-2836(83)80337-4. [DOI] [PubMed] [Google Scholar]
- Stellwag E., Fink J. M., Zissler J. Physical characterization of the genome of the Myxococcus xanthus bacteriophage MX-8. Mol Gen Genet. 1985;199(1):123–132. doi: 10.1007/BF00327521. [DOI] [PubMed] [Google Scholar]
- Van der Leij I., Franse M. M., Elgersma Y., Distel B., Tabak H. F. PAS10 is a tetratricopeptide-repeat protein that is essential for the import of most matrix proteins into peroxisomes of Saccharomyces cerevisiae. Proc Natl Acad Sci U S A. 1993 Dec 15;90(24):11782–11786. doi: 10.1073/pnas.90.24.11782. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Wu S. S., Kaiser D. Genetic and functional evidence that Type IV pili are required for social gliding motility in Myxococcus xanthus. Mol Microbiol. 1995 Nov;18(3):547–558. doi: 10.1111/j.1365-2958.1995.mmi_18030547.x. [DOI] [PubMed] [Google Scholar]
- Wu S. S., Wu J., Kaiser D. The Myxococcus xanthus pilT locus is required for social gliding motility although pili are still produced. Mol Microbiol. 1997 Jan;23(1):109–121. doi: 10.1046/j.1365-2958.1997.1791550.x. [DOI] [PubMed] [Google Scholar]
- Yee T., Inouye M. Reexamination of the genome size of myxobacteria, including the use of a new method for genome size analysis. J Bacteriol. 1981 Mar;145(3):1257–1265. doi: 10.1128/jb.145.3.1257-1265.1981. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Zusman D. R., McBride M. J. Sensory transduction in the gliding bacterium Myxococcus xanthus. Mol Microbiol. 1991 Oct;5(10):2323–2329. doi: 10.1111/j.1365-2958.1991.tb02077.x. [DOI] [PubMed] [Google Scholar]