RESUMO
The basic circadian oscillator of the unicellular fresh water cyanobacterium Synechococcus elongatus PCC 7942, the model organism for cyanobacterial circadian clocks, consists of only three protein components: KaiA, KaiB, and KaiC. These proteins, all of which are homomultimers, periodically interact to form large protein complexes with stoichiometries that depend on the phosphorylation state of KaiC. KaiA stimulates KaiC autophosphorylation through direct physical interactions. Screening a library of S. elongatus transposon mutants for circadian clock phenotypes uncovered an atypical short-period mutant that carries a kaiA insertion. Genetic and biochemical analyses showed that the short-period phenotype is caused by the truncation of KaiA by three amino acid residues at its C terminus. The disruption of a negative element upstream of the kaiBC promoter was another consequence of the insertion of the transposon; when not associated with a truncated kaiA allele, this mutation extended the circadian period. The circadian rhythm of KaiC phosphorylation was conserved in these mutants, but with some modifications in the rhythmic pattern of KaiC phosphorylation, such as the ratio of phosphorylated to unphosphorylated KaiC and the relative phase of the circadian phosphorylation peak. The results showed that there is no correlation between the phasing of the KaiC phosphorylation pattern and the rhythm of gene expression, measured as bioluminescence from luciferase reporter genes. The interaction between KaiC and the truncated KaiA was stronger than normal, as shown by fluorescence anisotropy analysis. Our data suggest that the KaiA-KaiC interaction and the circadian pattern of KaiC autophosphorylation are both important for determining the period, but not the relative phasing, of circadian rhythms in S. elongatus.
Assuntos
Proteínas de Bactérias/fisiologia , Ritmo Circadiano/genética , Synechococcus/metabolismo , Synechococcus/fisiologia , Proteínas de Bactérias/genética , Proteínas de Bactérias/metabolismo , Peptídeos e Proteínas de Sinalização do Ritmo Circadiano , Fluoresceínas/química , Polarização de Fluorescência , Regulação Bacteriana da Expressão Gênica/genética , Regulação Bacteriana da Expressão Gênica/fisiologia , Immunoblotting , Mutagênese Insercional , Fenótipo , Fosforilação , Ligação Proteica/genética , Synechococcus/genéticaRESUMO
Two endogenous plasmids are present in Synechococcus elongatus PCC 7942, a model organism for studying photosynthesis and circadian rhythms in cyanobacteria. The large plasmid, pANL, was shown previously to be involved in adaptation of S. elongatus cells to sulfur starvation, which provided the first evidence of cellular function of a cyanobacterial plasmid. Here, we report the complete sequence of pANL, which is 46,366 bp in length with 53% GC content and encodes 58 putative ORFs. The pANL plasmid can be divided into four structural and functional regions: the replication origin region, a signal transduction region, a plasmid maintenance region, and a sulfur-regulated region. Cosmid-based deletion analysis suggested that the plasmid maintenance and replication origin regions are required for persistence of pANL in the cells. Transposon-mediated mutagenesis and complementation-based pANL segregation assays confirmed that two predicted toxin-antitoxin cassettes encoded in the plasmid maintenance region, belonging to PemK and VapC families, respectively, are necessary for plasmid exclusion. The compact and efficient organization of sulfur-related genes on pANL may provide selective advantages in environments with limited sulfur.
Assuntos
Cianobactérias/genética , Plasmídeos/metabolismo , Synechococcus/genética , Proteínas de Bactérias/genética , Cosmídeos/metabolismo , Cianobactérias/metabolismo , DNA/genética , Elementos de DNA Transponíveis , Proteínas de Ligação a DNA/genética , Proteínas de Escherichia coli/genética , Glicoproteínas de Membrana/genética , Modelos Genéticos , Mutagênese Sítio-Dirigida , Hibridização de Ácido Nucleico , Fases de Leitura Aberta , Reação em Cadeia da Polimerase , Análise de Sequência de DNA , Enxofre/química , Synechococcus/metabolismoRESUMO
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.
