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Site-specific integration and expression of a developmental promoter in Myxococcus xanthus
1Department of Microbiology, University of Georgia, Athens 30602.
Abstract:
A series of intercellular signals are involved in the regulation of gene expression during fruiting body formation of Myxococcus xanthus. Mutations which block cell interactions, such as csgA (formerly known as spoC), also prevent expression of certain developmentally regulated promoters. csgA+ cells containing Tn5 lac omega DK4435, a developmentally regulated promoter fused to lacZ, began synthesizing lacZ mRNA 12 to 18 h into the developmental cycle. beta-Galactosidase specific activity increased about 12 h later. Neither lacZ mRNA nor beta-galactosidase activity was detected in a developing csgA mutant containing omega DK4435. The developmental promoter and its fused lacZ reporter gene were cloned into a pBR322-derived plasmid vector containing a portion of bacteriophage Mx8. These plasmids preferentially integrated into the M. xanthus chromosome by site-specific recombination at the bacteriophage Mx8 attachment site and maintained a copy number of 1 per chromosome. The integrated plasmids were relatively stable, segregating at a frequency of 0.0007% per generation in the absence of selection. The cloned and integrated promoter behaved like the native promoter, expressing beta-galactosidase at the proper time during wild-type development and failing to express the enzyme during development of a csgA mutant. The overall level of beta-galactosidase expression in merodiploid cells containing one native promoter and one promoter fused to lacZ was about half that of cells containing a single promoter fused to lacZ. These results suggest that the timing of developmentally regulated gene expression is largely independent of the location of this gene within the chromosome. Furthermore, they show that site-specific recombination can be a useful tool for establishing assays for promoter or gene function in M. xanthus.
Insights
Intercellular signals regulate gene expression in Myxococcus xanthus fruiting body formation. Site-specific recombination aids in studying promoter function and gene expression timing during development.
Area of Science:
- Microbiology
- Molecular Biology
- Developmental Biology
Background:
- Intercellular signals are crucial for regulating gene expression during Myxococcus xanthus fruiting body formation.
- Mutations affecting cell interactions, like csgA, disrupt the expression of developmentally regulated promoters.
Purpose of the Study:
- To investigate the role of intercellular signals in gene expression during Myxococcus xanthus development.
- To clone and characterize a developmentally regulated promoter using site-specific recombination for functional assays.
Main Methods:
- Utilized a Tn5 lac omega DK4435 reporter construct to monitor a developmentally regulated promoter fused to lacZ.
- Employed site-specific recombination with a bacteriophage Mx8-derived vector for stable integration of the promoter-reporter construct into the M. xanthus chromosome.
- Assessed beta-galactosidase expression in wild-type and csgA mutant strains under various developmental conditions.
Main Results:
- The cloned promoter, integrated via site-specific recombination, accurately reflected the timing of native promoter expression in wild-type and csgA mutant M. xanthus.
- beta-Galactosidase mRNA and activity were detected 12-24 hours into development in wild-type cells, but absent in csgA mutants.
- Integrated plasmids showed high stability, and merodiploid cells exhibited approximately half the expression of single-copy cells, suggesting gene dosage effects.
Conclusions:
- The timing of developmentally regulated gene expression in M. xanthus is largely independent of chromosomal location.
- Site-specific recombination is an effective tool for creating stable reporter gene assays to study promoter function and gene regulation in M. xanthus.