A forward genetic screen to identify genes essential for motility in the filamentous cyanobacterium N. punctiforme

The goal of this project is to identify the genes essential for motility in the filamentous cyanobacterium Nostoc punctiforme. N. punctiforme differentiates hormogonia, motile filaments which facilitate dispersal, the establishment of nitrogen-fixing symbioses with plants and fungi, and phototaxis to seek out favorable light environments. The motor driving hormogonium motility has not been defined. Both type IV pili and polysaccharide secretion have been suggested as propulsion mechanisms but the exact role each plays is unknown. The experimental approach described here utilizes a transposon-mutagenesis screen to identify genes essential for motility. Plasmid pRL1063a, carrying a Tn5 derivative transposon, was introduced into wild-type N. punctiforme through conjugation with E. coli. Exconjugants were selected for on media containing neomycin and then screened for motility. For non-motile isolates, transposon insertion sites were identified using an inverse PCR method followed by sequencing. To date, 10,465 exconjugants have been screened and 127 non-motile strains identified, of which 125 insertion sites have been sequenced. Inactivated genes include homologs of both type IV pilus components and polysaccharide synthesis systems implying that both type IV pili and polysaccharide secretion are essential for motility. A more detailed accounting of these genes using available bioinformatics tools and previously published data is currently under way