Thursday, August 16, 2012

Transposon mutagenesis of Planctomyces limnophilus

The EZ-Tn5™ Transposome System is now finding favor in studies of more unusual environmental microbial species. The bacterial phylum Planctomycetes is found in various environmental habitats, as well as in the gastrointestinal tract of many mammals and fish. Members of this phylum possess characteristics that make them, at first glance, unsuitable for in vivo transposition experiments to develop a model genetic system. Although genome sequencing data are available for several Planctomycetes species, the lack of molecular genetic tools has proved challenging to more in-depth study of these organisms.

Schrier et al. describe the use of the EZ-Tn5 R6Kγori/KAN-2 Transposome, co-electroporated into P. limnophilus with Epicentre's TypeOne™ Restriction Inhibitor, and successfully generated mutants in two separate experiments. Screening on kanamycin allowed the selection of transposon mutants, which were rescued using the R6Kγ origin of replication in the transposome. Recovered mutants were screened using PCR to identify those that contained an insertion within the pckA gene. The PckA gene product catalyzes the first step in the gluconeogenesis pathway; thus, a mutation in the gene's open-reading frame should result in growth inhibition in a medium not supplemented with glucose. This blockage in the mutant studied was found to be reversible, as addition of pyruvate appeared to restore wild-type growth in the absence of glucose.

The study is another example of the utility of the EZ-Tn5 Transposome system in unusual bacteria, allowing the generation and screening of mutants in specific genes to study behavior predicted from sequencing analysis.

ResearchBlogging.orgSchreier HJ et al. (2012). Transposon Mutagenesis of Planctomyces limnophilus and Analysis of a pckA Mutant. Appl Environ Microbiol PMID: 22798371

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