Functional genomics of the unicellular cyanobacterium Synechococcus elongatus PCC 7942

Abstract

Unicellular freshwater cyanobacterium Synechococcus elongatus PCC 7942 is the model organism for studying the circadian clock in cyanobacteria. Despite tremendous work over the last decade in identification of clock-related loci and elucidation of molecular mechanisms of the central oscillator, many details of the basic steps in generating circadian rhythms of biological processes remain unsolved and many components are still missing. A transposon-mediated mutagenesis and sequencing strategy has been adopted to disrupt essentially every locus in the genome so as to identify all of the loci that are involved in clock function. The complete genome sequence has been determined by a combination of shotgun sequences and transposon-mediated sequences. The S. elongatus PCC 7942 genome is 2,695,903 bp in length, and has a 55.5% GC content. Automated annotation identified 2,856 protein-coding genes and 51 RNA coding loci. A system for community refinement of the annotation was established. Organization and characteristic features of the genome are discussed in this dissertation. More than 95% of the PCC 7942 genome has been mutagenized and mutants affected in approximately 30% of loci have been screened for defects in circadian function. Approximately 70 new clock loci that belong to different functional categories have been discovered through a team effort. Additionally, functional analysis of insertion mutants revealed that the Type-IV pilus assembly protein PilN and the RNA chaperon Hfq are involved in transformation competence of S. elongatus cells. Functional analysis of an atypical short period kaiA insertional mutant showed that the short period phenotype is caused mainly by the truncation of KaiA by three amino acid residues. The interaction between KaiC and the truncated KaiA is weakened as shown by fluorescence anisotropy analysis. Deletion analysis of pANL, the large endogenous plasmid, implies that two toxin-antitoxin cassettes were responsible for inability to cure cells of this plasmid. In summary, the results indicate that this functional genomics project is very promising toward fulfilling our goal to assemble a comprehensive view of the cyanobacterial circadian clock. The mutagenesis reagents and dataset generated in this project will also benefit the greater scientific community.