Characterization of the regulatory region of a cell interaction-dependent gene in Myxococcus xanthus.
ABSTRACT: omega 4403 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C-signaling, are required for expression of Tn5 lac omega 4403. We have cloned DNA upstream of the omega 4403 insertion site, localized the promoter, and identified a potential open reading frame. From the deduced amino acid sequence, the gene disrupted by Tn5 lac omega 4403 appears to encode a serine protease that is dispensable for development. The gene begins to be expressed between 6 and 12 h after starvation initiates development, as determined by measuring mRNA or beta-galactosidase accumulation in cells containing Tn5 lac omega 4403. The putative transcriptional start site was mapped, and sequences centered near -10 and -35 bp relative to this site show some similarity to the corresponding regions of promoters transcribed by Escherichia coli sigma70 RNA polymerase. However, deletions showed that an essential promoter element lies between -80 and -72 bp, suggesting the possible involvement of an upstream activator protein. DNA downstream of -80 is sufficient for C-signal-dependent activation of this promoter. The promoter is not fully expressed when fusions are integrated at the Mx8 phage attachment site in the chromosome. Titration of a limiting factor by two copies of the regulatory region (one at the attachment site and one at the native site) can, in part, explain the reduced expression. We speculate that the remaining difference may be due to an effect of chromosomal position. These results provide a basis for studies aimed at identifying regulators of C-signal-dependent gene expression.
Project description:Omega4400 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn5 lac omega4400. The DNA upstream of the omega4400 insertion has been cloned, the promoter has been localized, and a partial open reading frame has been identified. From the deduced amino acid sequence of the partial open reading frame, the gene disrupted by Tn5 lac omega4400 may encode a protein with an ATP- or GTP-binding site. Expression of the gene begins 6 to 12 h after starvation initiates development, as measured by beta-galactosidase production in cells containing Tn5 lac omega4400. The putative transcriptional start site was mapped, and deletion analysis has shown that DNA downstream of -101 bp is sufficient for C-signal-dependent, developmental activation of this promoter. A deletion to -76 bp eliminated promoter activity, suggesting the involvement of an upstream activator protein. The promoter may be transcribed by RNA polymerase containing a novel sigma factor, since a mutation in the M. xanthus sigB or sigC gene did not affect Tn5 lac omega4400 expression and the DNA sequence upstream of the transcriptional start site did not match the sequence of any M. xanthus promoter transcribed by a known form of RNA polymerase. However, the omega4400 promoter does contain the sequence 5'-CATCCCT-3' centered at -49 and the C-signal-dependent omega4403 promoter also contains this sequence at the same position. Moreover, the two promoters match at five of six positions in the -10 regions, suggesting that these promoters may share one or more transcription factors. These results begin to define the cis-acting regulatory elements important for cell-cell interaction-dependent gene expression during the development of a multicellular prokaryote.
Project description:Omega4499 is the site of a Tn5 lac insertion in the Myxococcus xanthus chromosome that fuses lacZ expression to a developmentally regulated promoter. Cell-cell interactions that occur during development, including C signaling, are required for normal expression of Tn5 lac Omega4499. The DNA upstream of the Omega4499 insertion has been cloned, and the promoter has been localized. Analysis of the DNA sequence downstream of the promoter revealed one complete open reading frame and a second partial open reading frame that is interrupted by Tn5 lac Omega4499. The predicted products of these open reading frames are highly similar to reductase and oxidase components of bacterial cytochrome P-450 systems, which allow catabolism or anabolism of unusual compounds. However, the function of the gene products of the Omega4499 locus remains unclear because M. xanthus containing Tn5 lac Omega4499 exhibits no apparent defect in growth, developmental aggregation, fruiting body formation, or sporulation. Deletion analysis of the Omega4499 regulatory region showed that multiple DNA elements spanning more than 500 bp upstream of the transcriptional start site contribute to developmental promoter activity. At least two DNA elements, one downstream of -49 bp and one between -49 and -218 bp, boosted activity of the promoter in response to intercellular C signaling. Three sequences in the Omega4499 promoter region, centered at -55, -33, and -1 bp, nearly match a 7-bp sequence found in other C signal-dependent promoters. We propose that these sequences, matching the consensus sequence 5'-CAYYCCY-3', be called C box sequences, and we speculate that these sequences are cis-acting regulatory elements important for the expression of M. xanthus genes that depend upon intercellular C signaling during development.
Project description:Omega4514 is the site of a Tn5 lac insertion in the Myxococcus xanthus genome that fuses lacZ expression to a developmentally regulated promoter. DNA upstream of the insertion site was cloned, and the promoter was localized. The promoter resembles vegetative promoters in sequence, and sigma(A) RNA polymerase, the major form of RNA polymerase in growing M. xanthus, initiated transcription from this promoter in vitro. Two complete open reading frames were identified downstream of the promoter and before the Omega4514 insertion. The first gene product (ORF1) has a putative helix-turn-helix DNA-binding motif and shows sequence similarity to transcriptional regulators. ORF2 is most similar to subunit A of glutaconate coenzyme A (CoA) transferase, which is involved in glutamate fermentation. Tn5 lac Omega4514 is inserted in the third codon of ORF3, which is similar to subunit B of glutaconate CoA-transferase. An orf1 disruption mutant exhibited a mild sporulation defect, whereas neither a disruption of orf2 nor insertion Omega4514 in orf3 caused a defect. Based on DNA sequence analysis, the three genes are likely to be cotranscribed with a fourth gene whose product is similar to alcohol dehydrogenases. ORF1 delays and reduces expression of the operon during development, but relief from this negative autoregulation does not fully explain the regulation of the operon, because expression from a small promoter-containing fragment is strongly induced during development of an orf1 mutant. Also, multiple upstream DNA elements are necessary for full developmental expression. These results suggest that transcriptional activation also regulates the operon. Omega4514 is the first example of a developmentally regulated M. xanthus operon that is transcribed by the major vegetative RNA polymerase, and its regulation appears to involve both negative autoregulation by ORF1 and positive regulation by one or more transcriptional activators.
Project description: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