Expression of a gene cluster kaiABC as a circadian feedback process in cyanobacteria

Science

Volumn 281, Issue 5382, 1998, Pages 1519-1523

  Ishiura, Masahiro ^a   Kutsuna, Shinsuke ^a   Aoki, Setsuyuki ^a   Iwasaki, Hideo ^a   Andersson, Carol R ^b,d   Tanabe, Akio ^a   Golden, Susan S ^b   Johnson, Carl H ^c   Kondo, Takao ^a  

^a NAGOYA UNIVERSITY   (Japan)

^b TEXAS A AND M UNIVERSITY   (United States)

^c VANDERBILT UNIVERSITY SCHOOL OF MEDICINE   (United States)

^d SCRIPPS RESEARCH INSTITUTE   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

ARTICLE; BACTERIAL GENE; CIRCADIAN RHYTHM; CYANOBACTERIUM; GENE CLUSTER; GENE EXPRESSION REGULATION; GENE MAPPING; GENE REPRESSION; NONHUMAN; NUCLEOTIDE SEQUENCE; PRIORITY JOURNAL; PROMOTER REGION;

AMINO ACID SEQUENCE; BACTERIAL PROTEINS; BIOLOGICAL CLOCKS; CIRCADIAN RHYTHM; CLONING, MOLECULAR; CYANOBACTERIA; FEEDBACK; GENE EXPRESSION REGULATION, BACTERIAL; GENES, BACTERIAL; GENES, REPORTER; LUMINESCENCE; MODELS, BIOLOGICAL; MOLECULAR SEQUENCE DATA; MULTIGENE FAMILY; MUTATION; PROMOTER REGIONS (GENETICS); RECOMBINANT FUSION PROTEINS; TRANSCRIPTION, GENETIC;

BACTERIA (MICROORGANISMS); CYANOBACTERIA; KAIA; PROKARYOTA; SYNECHOCOCCUS; SYNECHOCOCCUS SP. PCC 6301;

EID: 0032483510     PISSN: 00368075     EISSN: None     Source Type: Journal    
DOI: 10.1126/science.281.5382.1519     Document Type: Article

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30. Genomic DNAs prepared from rescued clones were digested with Not I, circularized with T4 DNA ligase, and electroporated into E. coli DH10B.
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40. Previously, we isolated plasmid pS1K1 as a gene that appeared to complement the kaiC1 mutation (8). However, pS1K1 carries a suppressor gene for the mutation [T. Kutsuna, T. Kondo, S. Aoki, M. Ishiura., J. Bacteriol. 180, 2167 (1998)].
41. A plasmid for disruption of the kai gene cluster, pDkaiABC, carries three segments in tandem: a 0.55-kb DNA segment carrying an upstream region of the kaiA gene [nucleotides (nt) 1424 to 1975 relative to the left Eco RI site of the 4.9-kb Eco RI segment shown in Fig. 1B]; a 1.2-kb Nhe I - Bsm FI segment from pACYC177 carrying a km gene [A. C. Y. Chang and S. N. Cohen, J. Bacteriol. 134, 1141 (1978); R. E. Rose, Nucleic Acids Res. 16, 356 (1988)]; a 2.2-kb Nhe I-Pvu II segment carrying a downstream region of the kaiC gene (nt 4831 to 7071) on the pSEQΩ (M. Ishiura and S. Kutsuna, unpublished data) backbone. CR1 [a psbAI-reporter strain carrying a chloramphenicol-resistance gene as a selective marker gene (19)] was transformed with pDkaiABC and selected with kanamycin sulfate (33 u.g/ml) (CR1/ΔkaiABC cells).
42. A plasmid for disruption of the kai gene cluster, pDkaiABC, carries three segments in tandem: a 0.55-kb DNA segment carrying an upstream region of the kaiA gene [nucleotides (nt) 1424 to 1975 relative to the left Eco RI site of the 4.9-kb Eco RI segment shown in Fig. 1B]; a 1.2-kb Nhe I - Bsm FI segment from pACYC177 carrying a km gene [A. C. Y. Chang and S. N. Cohen, J. Bacteriol. 134, 1141 (1978); R. E. Rose, Nucleic Acids Res. 16, 356 (1988)]; a 2.2-kb Nhe I-Pvu II segment carrying a downstream region of the kaiC gene (nt 4831 to 7071) on the pSEQΩ (M. Ishiura and S. Kutsuna, unpublished data) backbone. CR1 [a psbAI-reporter strain carrying a chloramphenicol-resistance gene as a selective marker gene (19)] was transformed with pDkaiABC and selected with kanamycin sulfate (33 u.g/ml) (CR1/ΔkaiABC cells).
43. A plasmid for disruption of the kai gene cluster, pDkaiABC, carries three segments in tandem: a 0.55-kb DNA segment carrying an upstream region of the kaiA gene [nucleotides (nt) 1424 to 1975 relative to the left Eco RI site of the 4.9-kb Eco RI segment shown in Fig. 1B]; a 1.2-kb Nhe I - Bsm FI segment from pACYC177 carrying a km gene [A. C. Y. Chang and S. N. Cohen, J. Bacteriol. 134, 1141 (1978); R. E. Rose, Nucleic Acids Res. 16, 356 (1988)]; a 2.2-kb Nhe I-Pvu II segment carrying a downstream region of the kaiC gene (nt 4831 to 7071) on the pSEQΩ (M. Ishiura and S. Kutsuna, unpublished data) backbone. CR1 [a psbAI-reporter strain carrying a chloramphenicol-resistance gene as a selective marker gene (19)] was transformed with pDkaiABC and selected with kanamycin sulfate (33 u.g/ml) (CR1/ΔkaiABC cells).
44. pTS2CkaiABC is a targeting plasmid carrying a kaiABC cassette in the Bst EII site in NSII. The cassette is composed of a 3.0-kb Dra I-Nhe I segment carrying the kai gene cluster and the 2.0-kb Hind III Ω fragment (15). CR1/ΔkaiABC cells (20) were transformed with pTS2CkaiABC and selected with spectinomycin dihydrochloride (40 μg/ml).
45. A 1.5-kb Hpa I-Bcl I segment of p60N (another plasmid carrying the kaiABC gene cluster) (M. Ishiura, S. Kutsuna, T. Kondo, unpublished data) was replaced with the 2.1-kb Bam HI Ω segment (pDkaiC) and introduced into CR1 cells to disrupt kaiC in the genome. A nonsense mutation (TAA) was introduced by PCR into the fourth codon CAA of kaiA or the fifth codon AAA of kaiB in pSEQkaiABC, which carries a 3.7-kb Sma I - Bss HII fragment from p44N on the pSEQ1 backbone (13). pCkaiABC is a derivative of pSEQkaiABC, which carries the 2.0-kb Hind III Ω segment in the Nhe I site just downstream of the kaiC gene. CR1/ΔkaiABC cells (20) were transformed with derivatives of pCkaiABC that carried an inactivated kaiA or kaiB gene to reintroduce the modified kai gene cluster into the original kai locus in the genome. Transformants were selected with spectinomycin. Insertion of a nonsense mutation into an upstream gene may result in reduction of the transcription of a downstream gene if these two genes are organized as an operon, although the extent of the polar effect is unpredictable. Nevertheless, it is evident that the kaiB gene has an important role in the clock function because point mutations in the kaiB gene affect rhythmicity (Fig. 2B).
46. pkaiABC::lux cames a 2.8-kb Pvu II segment carrying the kaiC gene and its downstream region on the pSEQ1K (M. Ishiura and S. Kutsuna, unpublished data ) backbone, in which segment a 2.7-kb Hind III-Eco47 III promoterless segment of a luxAB gene set from Vibrio harveyi [ T. O. Baldwin et al., Biochemistry 23, 3663 (1984)] and the 2.0-kb Hind III Ω fragment (15) were inserted at the Nhe I site of the segment. WT Synechococcus cells were transformed with pkaiABC::lux and selected with spectinomycin .
47. pkaiABC::lux cames a 2.8-kb Pvu II segment carrying the kaiC gene and its downstream region on the pSEQ1K (M. Ishiura and S. Kutsuna, unpublished data ) backbone, in which segment a 2.7-kb Hind III-Eco47 III promoterless segment of a luxAB gene set from Vibrio harveyi [ T. O. Baldwin et al., Biochemistry 23, 3663 (1984)] and the 2.0-kb Hind III Ω fragment (15) were inserted at the Nhe I site of the segment. WT Synechococcus cells were transformed with pkaiABC::lux and selected with spectinomycin .
48. kaiC::lux carry a DNA segment including each upstream region of the kaiA (an 859-base pair Sma I-Xho I segment from p44N), kaiB (nt 1984 to 2920; the 5′-end nucleotide of the 4.9-kb Eco RI segment carrying the kaiABC gene cluster is numbered +1), and kaiC (nt 2961 to 3278) genes, respectively, on the pTS2Slux plasmid (19). To target these reporter constructs into the Bst EII site of NSII in the genome, we transformed WT Synechococcus cells with the plasmids and selected the cells with spectinomycin.
49. 730 was maintained between 0.27 and 0.45 by dilution with fresh BG-11 medium. The culture was exposed to 12 hours of darkness to synchronize the circadian clock, and then returned to LL. At 3-hour intervals in LL, cells were harvested, immediately frozen, and stored at -80°C. RNA was extracted from each frozen sample as described [A. Mohamed and C. Jansson, Plant Mol. Biol. 13, 693 (1989)]. RNA was subjected to electrophoresis, blotted onto nylon membranes (Boehringer GmbH, Mannheim, Germany), and hybridized with digoxigenin-labeled kaiA, kaiB, and kaiC ORF probes. The bioluminescence rhythm of the culture was confirmed by measuring the bioluminescence at each time point. Experiments were carried out three times and typical data are shown in Fig. 3, D and E.
50. kaiB::lux were targeted into NSM in the nonbioluminescent derivatives of the mutants as described (24).
51. kaiB::lux were targeted into NSM in the nonbioluminescent derivatives of the mutants as described (24).
52. M. Ishiura, S. Kutsuna, T. Kondo, unpublished data.
53. trc (79). The start codon GTG of the kaiA gene was modified to an ATG codon. Cells were transformed with these plasmids and selected with kanamycin. Twenty to 100 colonies carrying each construct were allowed to develop on solid medium. After 24 hours in LL, IPTG was added under the agar medium at a final concentration of 1 mM. For pulse administration of IPTG, colonies were grown on nitrocellulose membranes placed on solid medium, and the filter was transferred onto solid medium containing 1 mM IPTG for 6 hours and then returned to unsupplemented solid medium. Other conditions were the same as described in the legend to Fig. 4.
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55. T. Kondo et al., ibid. 275, 224 (1997).
56. Homologies to and phylogeny of the kaiB and kaiC genes in cyanobacteria and Archaea will be reported elsewhere (M. Ishiura et al., in preparation).
57. Cloning of the kai genes was carried out at the National Institute for Basic Biology (Okazaki, Japan) and further studies at Nagoya University with M.I., S.K., H.I., A.T., and T.K. We are grateful to H. Shinagawa (Osaka University), H. Aiba (Nagoya University), and S. Itoh (National Institute for Basic Biology) for helpful discussions; to S. Ishikawa (Instrument Development Center of School of Science, Nagoya University) for help in developing LDM; and to T. Suto and H. Kondo for technical assistance. This work was supported by grants from the Japanese Ministry of Education, Science and Culture (08454244, 08877053, 07558103, 08404053, 07554045), the lshida Foundation, the Nissan Foundation, the Yamada Foundation, the Chiba-Geigy Foundation for the Promotion of Science, the Kurata Research Grant, grants from the Shimadzu Foundation, Research for the Future program of Japan Society for the Promotion of Science (JSPS) (JSPS-RFTF96L00601), the competitive research grant from the Agency of Industrial Science and Technology, Ministry of International Trades and Industry and the Mitsubishi Foundation (to T.K.), and from the U.S. NSF (MCB-9311352 and MCB-9513367 to S.S.G. and MCB-9633267 to C.H.J.), the U.S. National Institute of Mental Health (MH01179 to C.H.J.), and the Human Frontier Science Program. S.K. and H.I. were supported by the Research Fellowships of the JSPS for Young Scientists.