Full text loading...
29 Genetic Tools for Studying Myxococcus xanthus Biology
Ebook: Choose a downloadable PDF or ePub file. Chapter is a downloadable PDF file. File must be downloaded within 48 hours of purchase
Myxococcus xanthus has a remarkable repertoire of complex multicellular behaviors, including social gliding motility, predation, rippling, and fruiting body formation. There are a few genetic tools such as an autonomously replicating plasmid and a defined library of M. xanthus mutant strains that have yet to be developed to study these complex behaviors. This chapter contains a description of M. xanthus genetic tools and their practical applications. Generalized transducing particles form when by mistake a bacteriophage head or capsid assembles around a fragment of a donor bacterium’s chromosomal DNA or around a plasmid instead of the phage genome. When these particles infect a recipient host, the donor bacterium’s DNA is inserted into the recipient host and is free to undergo homologous recombination with the host cell chromosome. In M. xanthus, generalized transducing phages are typically used for genetic mapping and for strain constructions. The plasmids are introduced into M. xanthus cells, strains that carry the plasmid integrated into phage attachment sites in the chromosome are identified, and the appropriate assays are performed. Gene fusions have been used to assay the transcriptional/translational regulation of genes/proteins and to examine protein localization in bacterial cells. Fusions belong to one of two classes: transcriptional fusions or translational fusions. In the case of a transcriptional fusion, the reporter gene lacks a promoter, but it possesses a functional ribosome-binding site. In the case of a translational fusion, the reporter gene lacks a promoter and a functional ribosome-binding site.
Procedure for linking chromosomal mutations to Tn5 . The diagram shows a Tn5 insertion being genetically linked to a mutation (*) that causes a gliding motility defect (Mot-1), but it is applicable to any chromosomal mutation that produces a distinguishable phenotype. Open rectangles represent M. xanthus cells, and the ovals inside the rectangles represent the M. xanthus chromosome. The small, shaded rectangles denote Tn5 chromosomal insertions that impart kanamycin resistance to recipient cells. Hexagons represent the indicated phages.
Site-specific recombination at a chromosomal Mx8 phage attachment site. A plasmid carrying a gene conferring kanamycin resistanc (Kanr), the Mx8 phage attachment site attP (gray box) located within the coding sequence for the integrase gene int (stippled box), and the locus of interest (cross-hatched box) is shown. The plasmid integrates into the M. xanthus chromosomal Mx8 phage attachment site via site-specific recombination between the attP and attB sites. The integration event produces the indicated DNA arrangement in the chromosome.
Structures of Tn5 and its derivatives. (A) Tn5 containing the genes that confer resistance to kanamycin (Kanr), tetracycline (Tetr), or trimethoprim (Tmpr) flanked by IS50 elements. (B) A derivative of Tn5 used to create lacZ transcriptional fusions. In this Tn 5 derivative, a segment of the IS50 L element is replaced with a promoterless lacZ reporter gene. (C) A derivative of Tn5 used to create PhoA translational fusions. A segment of the IS50 L element is replaced with a fragment of the phoA gene. The phoA fragment lacks sequences corresponding to the PhoA translational start site and sequences needed for export of the PhoA protein across the cytoplasmic membrane.
Strategy for making targeted insertions. An internal fragment of the gene of interest is generated using PCR, the PCR fragment is cloned into a plasmid vector that confers resistance to an antibiotic such as kanamycin, and plasmid DNA is electroporated into wild-type M. xanthus cells. A single homologous crossover produces a tandem duplication of the internal fragment and incorporation of the vector into the chromosomal copy of the gene. The likely result of the crossover is an inactivated copy of the gene.