ELSEVIER Biochimica et BiophysicaActa 1188 (1994) 443-446 BB Biochi~mic~a et Biophysica AFta Short Sequence-Paper Nucleotide sequence of the petB gene encoding cytochrome b 6 from the mesophilic cyanobacterium Synechocystis PCC 6803: implications for evolution and function Jochen Kruip a, Peter J. Nixon h, Heinz D. Osiewacz c, Matthias R6gner a,* a Institute of Botany, UniversityofMiinster, Schlossgarten 3, D-48149Miinster, Germany b Department of Biochemistry, Imperial Collegeof Science, Technologyand Medicine, London SW7 2AY, UK c FSPAngewandte Tumorvirologie,Abteilung:Molekularbiologieder Alterungsprozesse, Im Neuenheimer Feld 242, D-69120Heidelberg, Germany Received 1 September 1994 Abstract The gene encoding the cytochrome b6 subunit (petB) of the cytochrome b6f complex has been isolated, cloned and sequenced by nonradioactive methods from genomic DNA of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 666 nucleotides, coding for a polypeptide with a molecular mass of 25.02 kDa. In contrast to higher plant petB sequences an aminoterminal extension of seven amino acids occurs. Aminoterminal sequencing of the isolated protein excludes - different from higher plants - the existence of an intron after the first amino acids but indicate the posttranslational removal of three amino acids from the amino terminus. The aminoterminal extension - found only in non-nitrogen-fixing, unicellular cyanobacteria - shows a high degree of homology between different species. Keywords: Cyanobacterium; Cytochrome b6f complex; Evolution; Nucleotide sequence; petB; Photosynthesis The cytochrome b6f complex is a plasto- quinol:plastocyanin oxidoreductase and serves as a link between Photosystem II and Photosystem I in oxygenic photosynthesis [1]. A better understanding of the relationship between its structure and function is of primary interest since this step is the rate limiting step of the photosynthetic electron transport chain [2]. Moreover, the cytochrome b6f complex seems to be the crosspoint between photosynthesis and respiration in cyanobacteria [3]. This complex is multisubunit and transmembrane, consisting of at least four proteins: cytochrome f (petA gene product), the Rieske protein (petC gene product), subunit IV (petD gene product) and cytochrome b 6 (petB gene product) [4]. In all organisms .studied so far petB and petD are closely linked and are cotranscribed [5-16]. The nucleotide sequence data reported will appear in the EMBL, GenBank and DDBJ nucleotide sequence databases under the acces- sion number 7_31580. * Corresponding author. E-mail: rogner@dmswwula.uni-muen- ster.de. Fax: +49 251 833823. 0005-2728/94/$07.00 © 1994 Elsevier Science B.V. All rights reserved SSDI 0005-2728(94)00163-4 In this work the petB gene of the mesophilic cyano- bacterium Synechocystis sp. PCC 6803 was amplified from genomic DNA by PCR using two synthetic oligo- nucleotides, specific for the 3' (qTCCGTAAATCGG- GATC) and 5' (CCACCCTTGGTCAAACC) flanking regions of the petB gene ([17] and Osiewacz, unpub- lished results). The resulting 948 bp PCR fragment was cloned into the SrfI site of the pCR-Script SK(+) vector (Stratagene, Germany). The nucleotide se- quence of three clones was determined using the dideoxynucleotide chain termination method in combi- nation with direct blotting (Hoefer, USA) and chemilu- minescent detection (Tropix, USA). In addition, a ge- nomic copy of the gene was partially sequenced by conventional radioactive methods. The DNA sequence contains two open reading frames (ORFs) which code for cytochrome b 6 and parts of subunit IV, respectively (Fig. 1). The first ORF is homologous (70-80% iden- tity) with reported petB genes and predicts a highly hydrophobic 25019.9 Da protein. The second ORF, with only the 5' part being present, corresponds to the petD gene according to [17], thus showing that the