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Chapter 18 : Genetic Manipulation of Myxobacteria
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Genome sequencing of a few myxobacteria has revealed genes for the biosynthesis of unidentified secondary metabolites. Considering the need for new drugs to treat a variety of diseases, further exploration of the myxobacteria is imperative. Tools for genetic manipulation of myxobacteria are important for identifying and engineering strains to maximize production of secondary metabolites. Several developments have enabled heterologous gene expression in myxobacteria, including the construction and development of regulated promoters and the identification of strong constitutive promoters. While most genetic tools were first developed for the model myxobacterium Myxococcus xanthus, many have been applied to Sorangium cellulosum, Stigmatella aurantiaca, and the lesser-known myxobacteria Chondromyces crocatus, Cystobacter fuscus, Angiococcus disciformis, and Corallococcus macrosporus. This chapter describes these tools and hopes to facilitate the increased use of myxobacteria for applications in biotechnology and drug discovery. Electroporation has become the most common technique to introduce DNA into myxobacteria, including the species M. xanthus, S. aurantiaca, C. fuscus, A. disciformis, and C. macrosporus. Mutagenesis is fundamental to any genetic manipulation. Transposon mutagenesis is critical for myxobacterial genetics due to the ease of mutation identification. Genetic mapping and linkage analysis may be performed using generalized transduction or genomic DNA transformation by electroporation in M. xanthus. Several systems have been used for regulated gene expression in M. xanthus. The tetR-tetA regulatory system from Escherichia coli was used recently to engineer another regulated promoter in M. xanthus.
Schematic for insertional inactivation. An internal fragment of the gene to be disrupted is cloned into a nonrep-licative plasmid for integration by homologous recombination ( Table 1 ). Single homologous crossover between the fragment and the chromosome results in plasmid integration and a partial merodiploid with a 3′ and a 5′ truncation of the gene.
Diagram of allelic exchange to construct a deletion. A fragment containing the upstream and the downstream DNA of a gene (see text for details) is cloned into an allelic exchange plasmid ( Table 1 ). The first round of homologous recombination, selectable by Kanr, results in integration of the plasmid onto the chromosome. This may happen through the 5′ or the 3′ fragment. For simplicity, only integration through the 5′ end is shown here. Kanr transformants are plated for the selection of galactose resistance (negative selection) and the loss of the plasmid by a second round of recombination. Kanamycin-sensitive and galactose-resistant colonies may contain either the wild-type allele (A) or the deletion allele (B), depending on whether the second recombination occurred through the 5′ or the 3′ end, respectively.
PCR-based method for creating a deletion fragment. In the first-round PCR, primer pairs F1-R1 and F2-R2 are used to amplify the upstream and the downstream fragments individually. The 20 bases at the 5′ end of primer F2 are complementary to primer R1. The fragments are gel purified, mixed, and subjected to the second round of PCR using only F1 and R2 to produce the deletion allele. For convenience, restriction sites may be engineered into primers F1 and R2 to facilitate cloning.
PCR-based method for introducing point mutations. In the first-round PCR, primer pairs F1-R1 and F2-R2 are used to amplify the upstream and the downstream fragments individually. Primers R1 and F2 contain the desired point mutation at the center and are fully complementary. It is desirable to engineer a restriction site within R1 and F2 for screening purposes in later steps. The fragments are gel purified, mixed, and subjected to the second round of PCR using only F1 and R2. For convenience, restriction sites may be engineered into primers F1 and R2 to facilitate cloning.
Selected plasmids for myxobacteria