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S-motility & development (Yvonne Cheng)
tgl, pilQ and S motility (Eric Nudleman)
Rippling (Roy Welch, Dale Kaiser)
dsg (Yvonne Cheng)
sigma-54 activators (Lisa Gorski, Thomas Gronewold)
devTRS (Anthony Garza)
Sporulation (Ellen Licking, Todd Herrington)
Former lab members
devTRS (Bryan Julien)
sigma-54 and Tn5-lac 4521 (Ingrid Keseler)
Pili and social motility (Samuel Wu)
tgl, pilQ and S motility (Dan Wall)
devTRS (Bryan Julien, Anthony Garza)
During development, M. xanthus activates transcription of many genes that are required for fruiting body development and sporulation. Many such genes have been identified using the transposable promoter probe Tn5-lac (Kroos et al, 1986). One of these Tn5-lac insertions, omega-4414, identifies a genetic locus required for proper fruiting body formation and for the production of wild-type levels of spores. Sequence analysis has shown that omega-4414 is inserted in a gene which has been designated devR (Thöny-Meyer and Kaiser, 1993). The devR gene is part of an operon that may contain four additional open reading frames (ORFs) designated orf1, orf2, devT and devS. The regulation of this operon appears to be complex. Expression begins around 6 hours and peaks at 28. Full expression requires both the A and C signals. The operon is negatively autoregulated at the level of RNA accumulation, probably by the devS gene product. It is also regulated by the number of cells present at starvation: low cell densities result in high dev expression whereas high cell densities results in low levels of dev expression. In addition to its temporal regulation, the dev locus may be regulated spatially. We are currently making in-frame deletions of each gene within the operon to identify their roles in development and to identify the gene responsible for the negative autoregulation. In addition, we are trying to locate the promoter region and to identify any cis acting elements required for regulating the dev operon.
sigma-54 and Tn5lac 4521 (Ingrid Keseler)
A-signaling plays an essential role in the early stages of M. xanthus fruiting body development. Expression of the 4521 gene, which is regulated at the level of RNA accumulation, depends on starvation and on A-signaling. Mapping of the 4521 transcription start site has revealed a region with sequence similarity to the sigma-54 family of promoters and to the developmentally regulated mbhA promoter of M. xanthus. Although mutational analysis provided further evidence of the similarity between the 4521 core promoter and the mbhA and sigma-54 promoters, the regulation of the 4521 and mbhA genes was found to differ with respect to timing of expression and requirement for a solid surface and extracellular signals. (Keseler and Kaiser, 1995). Deletion analysis has identified a positive regulatory element downstream of the 4521 transcription start site, but it is unclear whether it regulates 4521 expression at the transcriptional or posttranscriptional level.
To assist in the identification of regulatory elements upstream of the core promoter region, genes were sought whose regulation resembles that of 4521. From a collection of Tn5lac insertion strains, seven whose beta-galactosidase expression was increased more than 3-fold early in development were identified for further study. Three of these Tn5lac elements were inserted in the 4521 locus, while the remaining four were inserted in different and distinct transcription units. One of them, omega-7557, was regulated like omega-4521 in all tested aspects. The transcription start sites of omega-7515, omega-7557, and omega-7757 have been mapped by S1 nuclease digestion; the core promoter regions of these genes can now be mapped more precisely and further compared with the 4521 promoter. Comparison of the promoter regions of these genes to those of 4521 and mbhA may allow experimental confirmation of a model in which different activator proteins are responsible for regulating the expression of the genes.
Only indirect evidence indicated that the 4521 promoter was transcribed by a sigma-54-containing RNA polymerase holoenzyme. To obtain more direct evidence, the gene encoding the transcription factor sigma-54, rpoN, was cloned and sequenced. Various attempts to construct a strain containing a null mutation in rpoN have been unsuccessful, although partial diploids of rpoN+/rpoN null were viable. The product of the rpoN gene, sigma-54, therefore appears to be essential for growth in M. xanthus. (Keseler and Kaiser, 1997).
Pili
and social motility (Samuel Wu)
Gliding motility in M. xanthus is controlled by two distinct genetic systems. One of these systems, (S)ocial motility, has long been associated with the presence of polar pili (Kaiser, 1979). These pili are generally 1-2 cell lengths (5-10 um) long, are less than 10 nm thick, and are found on the ends of cells (see picture at right).
A number of S motility mutations have been discovered to map within one region of the chromosome, near the transposons designated Tn5-3163 and Tn5-3188. Several genes have been cloned from this region, revealing that the pili of M. xanthus have genetic as well as morphologic homology with, and should therefore be classified as, Type IV pili. So far, we have published the sequences of six genes from this region: pilA, pilB, pilC, pilR, pilS and pilT (see figure below).
pilA appears to code for pilin, the major subunit of pili. pilR and pilS appear to code for a two component regulatory system similar to the NtrB/NtrC system for nitrogen regulation in other bacteria; based on homology to corresponding genes in Pseudomonas aeruginosa, it is predicted that pilR and pilS should regulate expression of pilA (Wu and Kaiser, 1995). However, a deletion of pilS does not eliminate pilus formation or social motility (Wu and Kaiser, 1996).
pilBand pilC are required for pilus biogenesis and are probably involved in pilus assembly or secretion. pilT is unique among these genes in that it is required for social motility, but not for pilus biogenesis. It was nevertheless designated a pil gene because of its homology to pilT in P. aeruginosa, and its position contiguous with other pil genes (between pilB and pilC) (Wu et al, 1997).
Ongoing work is directed toward clarifying the roles of some of these genes with respect to expression of pilA and pili. We also expect that further study of pilT may shed light on how pilus function to either stimulate or produce social motility.