Current Lab Members
Dale Kaiser, Yvonne Cheng, Ellen Licking, Lisa Gorski, Dan Wall, Thomas Gronewold, Anthony Garza, Marta Fontes (sabbatical), Todd Harrington (undergraduate), and Eric Nudleman, (undergraduate).
Departments of Biochemistry and of Developmental Biology
B300 Beckman Center, Stanford University School of Medicine
Stanford, CA 94305-5427
+Present address: Department of Pathology, Palo Alto Medical Foundation,
Palo Alto, California 94301
J. Bact. 179, 4361-4371
Mutations in the tgl locus inactivate social gliding motility in M. 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 using the kanamycin resistance encoded by that transposon. A 16 kb segment of chromosomal DNA complemented the social motility defect when introduced into tgl- cells, to form a tandem duplication heterozygote tgl+/tgl-. 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 was observed from a mgl-deficient donor strain, which cannot swarm, 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 (ORFA and 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 Tn5 lac constitutes a tgl mutation.
+Present address: Department of Pathology
Palo Alto Medical Foundation
Palo Alto, CA 94301
J. Bact. 179:4372-4381.
Tgl protein is required for production of the type IV pili found at the pole of an M. xanthus cell. These pili are essential for social motility. Evidence is presented that Tgl is a membrane protein, based on reaction with polyclonal antibody specific for Tgl that was raised against the fusion proteins b -galactosidase:Tgl and TrpE:Tgl. Immunoaffinity purified antibody reacted with a protein in M. xanthus having an apparent molecular weight of 27.5 kDa by SDS-polyacrylamide gel electrophoresis, while the sequence of the tgl gene translates into a polypeptide of 27 kDa. although these numbers are close, it is likely that the primary tgl translation product is processed and modified in M. xanthus. The N-terminus has a signal peptidase II recognition sequence cleavage of which is expected to remove 19 amino acid residues. When the tgl gene is expressed in E. coli, the protein product consistently migrates faster in the gel than mature Tgl in M. xanthus, suggesting a second modification by addition which slows migration of the protein from M. xanthus.
Tgl, as detected by its specific antibody, sediments with the membrane fraction of cells. It can be extracted with detergents but not with salt or the addition of chelators for divalent cations. In an equilibrium gradient, Tgl bands at the buoyant density of membranes and with the NADH-oxidase activity. Intact cells failed to bind anti-Tgl antibody, and less than 2% of the total Tgl is released in soluble form from the periplasm. Yet, cells that had been osmotically shocked and treated with paraformaldehyde were able to react with the specific antibody -- a reaction absent from cells deleted for the tgl transcription unit. Assuming that osmotic shock disrupts the outer membrane, the fractionation and localization data imply that Tgl is attached to the inner or outer membranes, from which it is exposed to the intermembranous space. Tgl is necessary for synthesis of pili in M. xanthus, and is the only pilus protein that can be donated by other cells ("stimulation"). Tgl contains six tandem copies of the tetratrico peptide repeat structural motif. Its membrane localization, capacity for stimulation and content of tetratrico structural repeats together suggest that Tgl may be necessary for the assembly of pilin subunits into filaments.
+Present address: Department of Medicine, Rm. 32-115 CHS, UCLA Medical Center, Los Angeles, CA 90095
J. Bacteriol. 179, 7748-7758.
Type IV pili are required for social gliding motility in Myxococcus xanthus. In this work, the expression of pilin (the pilA gene product) is examined during vegetative growth and fruiting body development. A polyclonal antibody against the pilA gene product (prepilin) was prepared, along with a pilA-lacZ fusion, and used to assay expression of pilA in M. xanthus in different mutant backgrounds. pilA expression required the response regulator pilR, but was negatively regulated by the putative sensor kinase pilS. pilA expression did not require pilB, pilC, or pilT. pilA was also autoregulated; a mutation which altered an invariant glutamate five residues from the presumed prepilin processing site eliminated this autoregulation, as did a deletion of the pilA gene. Primer extension and S1 nuclease analysis identified a sigma-54 promoter upstream of pilA, consistent with the homology of pilR to the NtrC family of response regulators. Expression of pilA was found to be developmentally regulated, but the timing of this expression pattern was not entirely dependent on pilS or pilR. Finally, pilA expression was induced by high nutrient concentrations, an effect that was also not dependent on pilS or pilR.
Contact Stimulation of Tgl and Type IV Pili in Myxococcus xanthus
J. Bacterial. 180, 759-761.
Myxococcus xanthus tgl mutant lack social motilty and type IV pili but can be transiently stimulated to swarm and to make pili by contacting tgl+ cells. The absence of pili in tgl mutants is shown not to be due to the absence of pilin. The rate of pilus elongation after Tgl stimulation is shown to be similar to the rate of pilus elongation in wild-type cells, using a new more rapid assay for stimulation.
PNAS 95, 3054-3058.
Social gliding motility of Myxococcus xanthus requires polar type IV pili. Tgl mutants lack pili and lack social motility. However, both defects can be rescued phenotypically, but not genotypically, when tgl+ donor and tgl recipient cells make physical contact with each other. What is the cellular and molecular basis of this transfer of phenotype, which is called stimulation? Stimulation does not occur in liquid nor in soft (0.5%) agar; however, on a more firm surface (1.0% agar) cells stimulate each other efficiently. Microscopy revealed that cells placed on 1.0% agar readily became aligned whereas they remained poorly aligned on 0.5% agar. It was observed, moreover, that stimulation is greatly reduced when donor and recipient cells lack the ability to move because of mutations in any of several different motility genes. Microscopy showed that motile cells became highly aligned, and can even align nonmotile cells. Using a stimulation assay based on the assembly of pili, a strong correlation was found between conditions that promote cell alignment and the speed or extent of stimulation. Because pili are assembled only at the end(s) of a cell, and a parallel alignment of cells in contact brings them end-to-end, we would suggest that end-to-end contacts are important for stimulation of pilus assembly.
The Myxo Sequencing committee formed at our Texas meeting has been asked to report. Dr. Drell is responsible for the D.O.E.'s microbial genome program.
Date: Thu, 19 Mar 1998 9:07:00-0500
From: "Daniel.Drell" <Daniel.Drell@mailgw.er.doe.gov.
Subject: Re: Message from Dale Kaiser
Thanks for your very interesting message about sequencing Myxobacteria. Your arguments are compelling and persuasive and if it were in my power to just say, "Let's do myxobacteria" then I'd add it to the list. I must realistically note several concerns however.
1) we are supporting TIGR to do Caulobacter crescentus, which has some alternative developmental potentials; its genome should provide at least some insights into simple differentiation steps.
2) The estimated genome size of Myxobacteria (9.5 Mbp) is at the very upper range of what we would consider feasible. E. coli (at 4.6 Mbp) took Fred Blattner many years and even with lessons learned, and greater efficiencies with modern sequencing, a 9.5 Mbp genome would take several years and (at current costs) take $4-5 million.
3) The strongest argument for Myxobacteria is its degradative abilities and this would indeed be of some interest to DOE. Our greater interest now, and this may be accompanied by some new budget next fiscal year, is greenhouse gases (CO2, H, CH4) and bacterial metabolism with them. Does Myxobacteria do anything with any of these gases? It may be worth your keeping an eye on our NABIR program (http://www.lbl.gov/NABIR) for program announcements that may be relevant here.
4) Our office would absolutely jump on any new technologies that made it possible to use the determined sequence of one microorganism to efficiently determine the sequence (without resequencing the entire genome) of a near-relative microorganism; this might be accomplished by some form of initial subtractive hybridization (to remove the common sequences) followed by identification and sequencing only of the "non-shared" sequences and then reconstruction of the unknown genome. In this way, DOE could achieve many more microbial sequences (at far lower cost) than presently and we could quickly add to the number (and distribution) of microbial sequences. As with ALL our sequencing (human, microbial, etc.) to date, the sequences would be made public quickly and without restriction. This is an absolute condition of all of our awards.
Again, thanks for your very interesting note. I cannot indicate that we will be able (or programmatically compelled) to undertake Myxobacteria soon, but I will keep your note handy and would be happy to revisit this issue in the future.
The Sequence Project needs additional input form you concerning Drell's items 3 and 4. Are your working on any of the degradative abilities that would enhance interest for DOE, or do you know someone who is? Or would you be interested in doing a pilot project? Or do you have an idea for a pilot project? Likewise the greenhouse gases? Likewise item 4? With respect to item 4, consider that C. crescentus is high GC like Myxo, and that a number of C.c. genes have provided good DNA probes for M. xanthus. If your answer to any of these questions is yes, please let me, Larry Shimkets or Lee Kroos know.