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S-motility & development (Yvonne Cheng)
tgl, pilQ and S motility (Eric Nudleman)
Development
Rippling (Roy Welch, Dale Kaiser)
dsg (Yvonne Cheng)
sigma-54 activators (Lisa Gorski, Thomas Gronewold)
devTRS (Anthony Garza)
Sporulation (Ellen Licking, Todd Herrington)
devTRS (Bryan Julien)
sigma-54 and Tn5-lac 4521 (Ingrid Keseler)
Pili and social motility (Samuel Wu)
tgl, pilQ and S motility(Dan Wall)
Development
Rippling (Dale Kaiser, Roy Welch)
Nascent fruiting body mounds grow as more cells enter them. Meanwhile, in the neighborhood of these enlarging aggregates, other cells are moving in a highly organized, periodic pattern of waves. Equally spaced ridges appear to move as trains of traveling waves, or ripples, which were first described by Hans Reichenbach. The wave crests are heaps of cells, and at least those cells at the base of a wave are oriented with their long axes in the direction of wave progression. Evidently, these cells are prepared to glide in the direction that the waves travel.
As cells migrate into the fruiting bodies, the waves disappear. However, by adjusting the nutrient level, one can suppress fruiting body aggregation, and cover an entire culture dish with waves that continue for days. Such cultures have revealed two striking peculiarities of the traveling waves. 1) Despite continued wave travel over several days, no accumulation of cells either at the head or foot of a wave train, nor for that matter anywhere in these cultures has been detected. Evidently the waves are not contributing to net transport of cells. 2) Two waves appear to pass through each other without any interference, either destructive or constructive. But interpenetration of two such waves without interference seems physically impossible. Cells occupy space; how can two heaps of densely packed cells simply pass through each other without distorting the shape of the heaps? Nevertheless the perception of wave interpenetration is strong, because when colliding wave fronts are curved or bent, and each has its own unique shape, that shape is preserved through the intersection. We have suggested that the perception of interpenetration is misleading and have offered the alternative of precise reflection (Sager and Kaiser, 1994).
A-motility, S-motility, mgl, frz, and C-signaling are all essential for the maintenance of traveling waves in these cultures. With the ultimate aim of understanding the role of these motility functions in fruiting body development, we are investigating cell behavior in waves.
sigma-54 activators (Lisa Gorski, Thomas Gronewold)
M. xanthus cells guide themselves through the developmental process by the manufacture and reception of several extracellular signals, the most studied of which are the A (early development) and C (mid-development) signals. In studying the regulation of genes that respond to these signals, it has been shown that at least two developmentally regulated genes have promoters that employ sigma-54, a sigma factor that is both structurally and functionally distinct from sigma-70. One of those distinctions is the requirement for at least one gene-specific activator protein for transcription. This requirement builds in a source of regulation for these genes that may be related to the extracellular signals. We have been creating potentially null mutations in sigma-54 activator proteins in M. xanthus by inserting a plasmid into target genes using a collection of 500 bp PCR fragments (made in collaboration with Tracy Nixon and co-workers) generated for the internal, heavily conserved ATP binding region common to these activator proteins.
Analysis of mutants in 13 genes has revealed five that have developmental phenotypes. Two are inhibited or delayed in development due to a motility defect, but three, which have normal motility, arrest development at specific stages. One arrests early in pre-aggregation; another stops in the middle, aggregation phase; and the third arrests late during spore formation. The early and middle activators could be involved with the A and C extracellular signaling processes respectively. Current efforts are focused on the mutant that arrests in mid aggregation.
dsg (Yvonne Cheng)
dsg was originally named for the D-signalling mutants, one of four groups of developmentally defective M. xanthus strains which could complement each other in mixing experiments. The sole representative of this group, the dsg429 mutant is delayed in fruiting body formation but can sporulate to wildtype levels after five days in development. This developmental defect can be rescued by mixing with wildtype or csg- cells, by increasing the density of the starving dsg cells.
The dsg gene is essential for cell viability and shares sequence homology with infC (Cheng et al, 1994), a gene which encodes translation initiation factor IF3 in E. coli and B. stearothermophilus. We have found that the Dsg and IF3 proteins are not only antigenically related, but probably functionally related as well. In E. coli, IF3 autoregulates its own translation from infC. Expression of Dsg in E. coli infC mutants restores regulation of IF3 to the same extent that expression of E. coli IF3 does (Kalman et al, 1994). This evidence suggests that dsg codes for a corresponding translation initiation factor IF3 in M. xanthus.
Recent studies have shown that the dsg mutant produces lower quantities of A-factor in development, perhaps due to reduced expression of the asg genes. Addition of A-factor amino acids rescues the developmental defect of the dsg mutant, so it is likely that the dsg gene is involved in the A-signalling pathway, rather than producing a separate D-signal. The dsg mutant also exhibits a myriad of other phenotypes, such as reduced yellow pigmentation, reduced secretion and decreased adventurous motility. Our continuing work is focused on determining how mutations in Dsg (i.e., IF3) cause defects in development.
Sporulation (Ellen Licking, Todd Herrington)
Within the confines of the fruiting body, M. xanthus cells transform into environmentally resistant, dormant myxospores. In the process, the cells spores change their shape, from rods to ovoids, and begin to express new proteins. In addition, metabolic activities in the spores cease. This differentiation from rod to spore is regulated by specific genes which are expressed prior to spore formation. I am interested in identifying and characterizing genes which regulate the onset of sporulation in Myxococcus xanthus. Using a genetic approach, I have screened an existing collection of Tn5-lac insertion mutants for strains blocked specifically in sporulation. 2 of 300 independent mutations resulted in severe sporulation defects. From restriction mapping and Southern analyses, these two mutations, Tn5-lac-7536 and Tn5-lac-7587, are not related to each other. Chromosomal DNA upstream of both insertions has been cloned, and the region associated with Tn5-lac-7536 has been partially sequenced.