High-throughput functional analysis of the Synechococcus elongatus PCC 7942 genome.
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abstract
Synechococcus elongatus PCC 7942 was the first cyanobacterial strain to be reliably transformed by exogenously added DNA and has become the model organism for cyanobacterial circadian rhythms. With a small genome (2.7 Mb) and well-developed genetic tools, PCC 7942 provides an exceptional opportunity to elucidate the circadian mechanism through genetics. We describe a project to create mutations in every locus of the genome, both to assay each locus for its potential contribution to the circadian clock and to archive data for the cyanobacterial community. Cosmid clones that carry inserts of PCC 7942 DNA are saturated with transposon insertions in vitro to provide sequencing templates and substrates for mutagenesis of the PCC 7942 genome via homologous recombination. We have mutagenized 53% of the chromosome from 50 chromosome-bearing cosmids and identified the positions of insertions in 31 of those cosmids and the 46 kb plasmid, pANL. PCC 7942 mutants defective for 490 different genes have been screened for circadian phenotypes. Mutagenesis of three apparently essential loci, including clpPIIclpX, resulted in circadian phenotypes. We developed an effective antisense suppression method to further the analysis of essential genes. When completed, the set of comprehensive mutations will provide the community with a unique resource whose impact will extend beyond circadian research.
