Skip to main content
SpringerLink
Log in

Construction of insertion mutants of Synechocystis sp. PCC 6803: evidence for an essential function of subunit IV of the cytochrome b6/f complex

  • Original Papers
  • Published:
Archives of Microbiology Aims and scope Submit manuscript

Abstract

The gene encoding subunit IV of the cytochrome b6/f complex (petD) has been isolated from a genomic library of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 480 nucleotides and can code for a polypeptide with a molecular weight of 17.5 kDa. The deduced amino acid sequence shows high identity with the corresponding sequences of both the photoautotrophic prokaryote Nostos sp. PCC 7906 as well as of lower and higher photoautotrophic eukaryotes (e.g. Chlorella protothecoides, Nicotiana tabacum). Transformation of Synechocystis sp. PCC 6803 with a plasmid containing the cloned petD gene in which the coding sequence is interrupted by the aminoglycoside 3′-phosphotransferase gene (aph) from Tn903 resulted in the formation of km resistant transformants. The molecular analysis of independent transformants revealed that all clones were merodiploid containing both uninterrupted wild-type as well as interrupted mutant petD copies. Approaches to segregate these two genomes were unsuccessful implying an essential function of the petD gene product in Synechocystis sp. PCC 6803.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Similar content being viewed by others

Abbreviations

aph :

aminoglycoside 3′-phosphotransferase

cpDNA :

chloroplast DNA

km :

kanamycin

PSI :

photosystem I

PSII :

photosystem II

References

  • Bennoun P (1982) Evidence for a respiratory chain in the chloroplast. Proc Natl Acad Sci USA 79: 4352–4356

    Google Scholar 

  • Benton WD, Davis RW (1977) Screening lambda gt recombinant clones by hybridization to single plaques in situ. Science 196: 180–182

    Google Scholar 

  • Burnap RL, Sherman LA (1991) Deletion mutagenesis in Synechocystis sp. PCC 6803 indicates that the Mn-stabilizing protein of photosystem II is not essential for O2 evolution. Biochemistry 30: 440–446

    Google Scholar 

  • Chauvat F, Rouet P, Bottin H, Boussac A (1989) Mutagenesis by random cloning of the Escherichia coli kanamycin resistance gene into the genome of the cyanobacterium Synechocystis PCC 6803: selection of mutants defective in photosynthesis. Mol Gen Genet 216: 51–59

    Google Scholar 

  • Chitnis PR, Reilly PA, Miedel MC, Nelson N (1989) Structure and targeted mutagenesis of the gene encoding 8-kDa subunit of photosystem I from the cyanobacterium Synechocystis sp. PCC 6803. J Biol Chem 264: 18374–18380

    Google Scholar 

  • Debus RJ, Barry BA, Babcock GT, McIntosh L (1988) Site-directed mutagenesis identifies a tyrosine radical involved in the photosynthetic oxygen-evolving system. Proc Natl Acad Sci USA 85: 427–430

    Google Scholar 

  • DiRago J-P, Colson A-M (1986) DNA sequence analysis of diuron-resistant mutations in the mitochondrial cytochrome b gene of Saccharomyces cerevisiae. FEBS Lett 208: 208–211

    Google Scholar 

  • DiRago JP, Colson A-M (1988) Molecular basis for resistance to antimycin and diuron, Q-cycli inhibitors acting at the Qisite in the mitochondrial ubiquinol-cytochrome c reductase in Saccharomyces cerevisiae. J Biol Chem 263: 12564–12570

    Google Scholar 

  • Gabellini N, Sebald W (1986) Nucleotide sequence and transcription of the fbc operon from Rhodopseudomonas sphaeroides. Eur J Biochem 154: 569–579

    Google Scholar 

  • Grigorieva G, Shestakov S (1982) Transformation in the cyanobacterium Synechocystis sp. 6803. FEMS Microbiol Lett 13: 367–370

    Google Scholar 

  • Jansson C, Debus RJ, Osiewacz HD, Gurevitz M, McIntosh L (1987) Construction of an obigate photoheterotrophic mutant of the cyanobacterium Synechocystis 6803. Plant Physiol 85: 1021–1025

    Google Scholar 

  • Kallas T, Spiller S, Malkin R (1989) Characterization of two operons encoding the cytochrome b6/f complex of the cyanobacterium Nostoc PCC 7906. J Biol Chem 263: 14334–14342

    Google Scholar 

  • Karn I, Brenner S, Barnett L, Cesareni G (1980) Novel bacteriophage cloning vector. Proc Natl Acad Sci USA 77: 5172–5176

    Google Scholar 

  • Kück U (1989) The intron of a plastid gene from a green alga contains an open reading frame for a reverse transcriptase-like enzyme. Mol Gen Genet 218: 257–265

    Google Scholar 

  • Kück U, Osiewacz HD, Schmidt U, Kappelhoff B, Schulte E, Esser K (1985) The onset of senescence is affected by DNA rearrangements of a discontinuous mitochondrial gene in Podospora anserina. Curr Genet 9: 373–382

    Google Scholar 

  • Kuhsel MG, Strickland R, Palmer JD (1990) An ancient group I intron shared by eubacteria and chloroplasts. Science 250: 1570–1573

    Google Scholar 

  • Labarre J, Chauvat F, Thuriaux P (1989) Insertional mutagenesuis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis PCC 6803. J Bacteriol 171: 3449–3457

    Google Scholar 

  • Majewski C, Trebst A (1990) The pet genes of Rhodospirillum rubrum: cloning and sequencing of the genes for the cytochrome bc1-complex. Mol Gen Genet 224: 373–382

    Google Scholar 

  • Meunier B, Colson A-M, (1989) Increased diuron resistance in the joint expression of mutations located at the DIU2, DIU3 and DIU4 loci of Saccharomyces cerevisiae. Curr Genet 15: 121–127

    Google Scholar 

  • Murphy RC, Gasparich GE, Bryant DA, Porter RD (1990) Nucleotide sequence and further characterization of Synechococcus sp. strain PCC 7002 recA gene: complementation of a cyanobacterial recA mutation by the Escherichia coli recA gene. J Bacteriol 172: 967–976

    Google Scholar 

  • Ohyama K, Fukuzawa H, Kohchi T, Shirai H, Sano T, Sano S, Umesono K, Shiki Y, Takeuchi M, Chang Z, Aota S, Inokuchi H, Ozeki H (1986) Chloroplast gene organization deduced from complete sequence of liverwort Marchantia polymorpha chloroplast DNA. Nature 322: 572–574

    Google Scholar 

  • Reimann A, Kück U (1989) Nucleotide sequence of the plastid genes for apocytochrome b6 (petB) and subunit IV of the cytochrome b6/f complex (petD) from the green alga Chlorella protothecoides: lack of introns. Plant Mol Biol 13: 255–256

    Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual. Cold Spring Harbor Laboratory, Cold Spring Harbor

    Google Scholar 

  • Sandmann G, Malkin R (1984) Light inhibition of respiration is due to a dual function of cytochrome b6/f complex and the plastocyanin/cytochrome c-553 pool in Aphanocapsa. Arch Biochem Biophys 234: 105–111

    Google Scholar 

  • Sanger F, Nicklen S, Coulson AR (1977) DNA sequencing with chain-termination inhibitors. Proc Natl Acad Sci USA 74: 5463–5467

    Google Scholar 

  • Scherer S (1990) Do photosynthetic and respiratory electron transport chains share redox protein? TIBS 15: 458–462

    Google Scholar 

  • Shinozaki K, Ohme M, Tanaka M, Wakasugi T, Hayashida N, Matsubayashi T, Zaita N, Chunwongse J, Obokata J, Yamaguchi-Shinozaki K, Ohto C, Torozawa K, Meng BY, Sugita M, Deno H, Kamogashira T, Yamada K, Kusuda J, Takaiwa F, Kato A, Tohdoh N, Shimada H, Sugiura M (1986) The complete nucleotide sequence of the tobacco chloroplast genome: its gene organization and expression. EMBO J 5: 2043–2049

    Google Scholar 

  • Turmel M, Boulanger J, Bergeron A (1989) Nucleotide sequence of the chloroplast petD gene of Chlamydomonas eugametos. Nucleic Acids Res 17: 3593

    Google Scholar 

  • Vermaas WFJ, Williams JGK, Arntzen CJ (1987) Sequencing and modification of psbB, the gene encoding the CP47 protein of photosystem II, in the cyanobacterium Synechocystis 6803. Plant Mol Biol 8: 317–326

    Google Scholar 

  • Vermaas WFJ, Rutherford AW, Hansson Ö (1988) Site-directed mutagenesis in photosystem II of the cyanobacterium Synechocystis sp. PCC 6803: donor D is a tyrosine residue in the D2 protein. Proc Natl Acad Sci USA 85: 8477–8481

    Google Scholar 

  • Widger WR, Cramer WA, Herrmann RG, Trebst A (1984) Sequence homology and structural similarity between cytochrome b of mitochondrial complex III and the chloroplast b6/f complex: Position of the cytochrome b hemes in the membrane. Proc Natl Acad Sci USA 81: 674–678

    Google Scholar 

  • Willeford KO, Gombos Z, Gibbs M (1989) Evidence for chloroplastic succinate dehydrogenase participation in the chloroplastic respiratory and photosynthetic electron transport chains of Chlamydomonas reinhardtii. Plant Physiol 90: 1084–1087

    Google Scholar 

  • Williams JGK (1988) Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803. Meth Enzymol 167: 766–778

    Google Scholar 

  • Xu M-Q, Kathe SD, Goodrich-Blairt H, Nierzwicki-Bauer SA, Shub DA (1990) Bacterial origin of a chloroplast intron: conserved self-splicing group I introns in cyanobacteria. Science 250: 1566–1569

    Google Scholar 

Download references

Author information

Author notes

  1. Heinz D. Osiewacz

    Present address: Institut für Virusforschung, Angewandte Tumorvirologie, Projekt: Molekularbiologie der Alterungsprozesse, Deutsches Krebsforschungszentrum, Im Neuenheimer Feld 506, W-6900, Heidelberg, FRG

Authors and Affiliations

  1. Lehrstuhl für Allgemeine Botanik, Ruhr-Universität, Postfach 1021 48, W-4630, Bochum, Germany

    Heinz D. Osiewacz

Authors
  1. Heinz D. Osiewacz
    View author publications

    You can also search for this author in PubMed Google Scholar

Rights and permissions

Reprints and permissions

About this article

Cite this article

Osiewacz, H.D. Construction of insertion mutants of Synechocystis sp. PCC 6803: evidence for an essential function of subunit IV of the cytochrome b6/f complex. Arch. Microbiol. 157, 336–342 (1992). https://doi.org/10.1007/BF00248678

Download citation

  • Received:

  • Accepted:

  • Issue Date:

  • DOI: https://doi.org/10.1007/BF00248678

Key words

Search

Navigation