A Planctomyces limnophilus mutant generated using the EZ-Tn5 transposome was found to possess an insertion within pckA, encoding phosphoenolpyruvate carboxykinase. Disruption of pckA expression and elimination of enzymatic activity resulted in poor growth in glucose-free medium, demonstrating a gluconeogenic role for pckA in P. limnophilus. Bacteria belonging to the phylum Planctomycetes possess several distinctive phenotypic and molecular characteristics, including a proteinaceous cell wall that is devoid of peptidoglycan (17), reproduction by yeast-like division, and in many cases, internal cell compartmentalization resembling the eukaryotic nucleus (2, 19). They are found in aquatic and soil environments (3), as well as among microbial communities associated with the intestinal tracts of many organisms, including mammals (11) and fish (15). The Planctomycetes are significant participants in the global carbon and nitrogen cycles and contain a unique group capable of carrying out anaerobic ammonium oxidation (anammox) (5,8). While the availability of genome sequences for several Planctomycetes (cited in reference 6), as well as biochemical, physiological, and ecological studies, have provided some insight into this fascinating group of bacteria, the lack of molecular genetic tools has made it difficult to examine them in greater detail.Recently, Jogler et al. (6) described a genetic approach for studying Planctomyces limnophilus and suggested that this bacterium be used as a model species for the Planctomycetes. P. limnophilus is a chemoheterotroph that has a distinct cell cycle, with sessile cells that form stalks; its ability to form multicellular rosettes, its low salt tolerance, pinkish red pigmentation, high growth rate compared to the growth of other Planctomycetes (4), intracytoplasmic membrane structure, and the availability of a genome sequence (10) make it an ideal subject for molecular genetic studies. We have been examining the use of the Epicentre (Madison, WI) R6K␥ori/Kan-2 transposon kit (EZ-Tn5) (9) for creating P. limnophilus mutants by a procedure similar to that described by Jogler et al. (6). Here, we report the isolation of mutants obtained using this approach and describe the characterization of one mutant that contains an insertion within the gene encoding phosphoenolpyruvate carboxykinase, pckA.Transposon mutagenesis and isolation of mutants. Electrocompetent cells of P. limnophilus (DSM 3776), a gift from Naomi Ward, University of Wyoming, were prepared from a 5-day-old culture grown in 500 ml of modified 621 medium (DSMZ) (peptone and yeast extract concentrations were doubled to 0.05% each) at 28°C and 190 rpm that was washed with 10% glycerol and suspended in 1.5 ml 10% glycerol. Cells prepared in this manner were electrocompetent for several months when stored at Ϫ80°C. Electroporation was carried out by mixing 1 l of EZ-Tn5 transposome solution and 1 l of TypeOne restriction inhibitor (Epicentre) with 70-l cells in a microcentrifuge tube, incubating on ice for 5 min, and then transf...