Sequence of the two operons encoding the four core subunits of the cytochrome b(6)f complex from the thermophilic Cyanobacterium synechococcus elongatus

The genes encoding cytochrome f (petA), cytochrome b(6) (petB), the Rieske FeS-protein (petC), and subunit IV (petD) of the cytochrome b(6)f complex from the thermophilic cyanobacterium Synechococcus elongatus were cloned and sequenced. Similar to other cyanobacteria, the structural genes are arranged in two short, single-copy operons, petC/petA and petB/petD, respectively. In addition, five open reading frames with homology to known orfs from the cyanobacterium Synechocystis PCC 6803 were identified in the immediate vicinity of these two operons.     

The cloning and sequencing of Synechococcus sp. PCC 7002 petCA operon: Implications for the cytochrome c-553 binding domain of cytochrome f

The genes encoding the Rieske iron-sulfur protein and cytochrome f from a unicellular, naturally transformable, photoheterotrophic cyanobacterium, Synechococcus sp. PCC 7002, formerly Agmenellum quadruplicatum, have been isolated and sequenced. The two genes were found to be on a single operon, petCA.The Synechococcus sp. PCC 7002 iron-sulfur protein contains 181 amino acids, the conserved putative iron-binding domains CTHLGCV, residues 108–114, and CPCHGS, residues 128–133, no presequence and has a 73% sequence identity to the Nostoc PCC 7906 iron-sulfur protein. The 325 amino acid apocytochrome f sequence contains a 42 amino acid presequence, a CANCH heme binding domain, residues 20–24 from the presumed start of the mature protein, and a predicted hydrophobic membrane-spanning domain, residues 250–269. The mature cytochrome f sequence has a 71.5% sequence identity with Nostoc PCC 7906 cytochrome f and possesses a large (-14) negative charge and low calculated pI of 4.47 compared to higher plant chloroplast sequences. Nine separate domains showing differences in charged residues among cyanobacteria and plants have been identified and the possibility that these domains are involved in the ionic interactions with plastocyanin or cytochrome c-553 is discussed.The sequences reported in this paper have been deposited in the EMBL/Genbank data base (IntelliGenetics, Mountain View, CA, and Eur. Mol. Biol. Lab., Heidelberg) (accession no. M74514).     

Homology of the N-terminal domain of the petH gene product from Anabaena sp. PCC 7119 to the CpcD phycobilisome linker polypeptide

The complete nucleotide sequence of the petH gene encoding ferredoxin-NADP⁺ reductase from the nitrogen-fixing cyanobacterium Anabaena sp. PCC 7119 has been determined. The encoded polypeptide is 136 amino acids longer than the enzyme obtained after purification to homogeneity. The extended N-terminal domain consists of 80 amino acids which shows homology to the CpcD phycobilisome linker polypeptide, through which FNR might be anchored to the thylakoid-bound phycobilisomes. A 56 amino acid interdomain fragment is found which could be a target for proteolysis.     

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

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.Abbreviations aph aminoglycoside 3-phosphotransferase - cpDNA chloroplast DNA - km kanamycin - PSI photosystem I - PSII photosystem II     

Cloning and transcription analysis of the ndh(A-I-G-E) gene cluster and the ndhD gene of the cyanobacterium Synechocystis sp. PCC6803

The plastid DNA of higher plants contains eleven reading frames that are homologous to subunits of the mitochondrial NADH-ubiquinone oxidoreductase (complex I). The genes are expressed, but a plastid NAD(P)H dehydrogenase has not yet been isolated and the function of the enzyme in plastid metabolism is unknown. Cyanobacteria also contain a NADH dehydrogenase that is homologous to the mitochondrial complex I. The enzyme is sensitive to rotenone and is located on the cytoplasmic and the thylakoid membrane. We report here the sequence of five subunits (ndhA, -I, G, -E and -D) of the NADH dehydrogenase from the unicellular cyanobacterium Synechocystis sp. PCC6803. As in plastid DNA, the genes ndh(A-I-G-E) are clustered and probably constitute an operon. The ndhD gene is associated with a gene encoding an iron-sulphur protein of photosystem I (psaC) as in plastid DNA. In contrast to the situation in plastids, psaC and ndhD are not cotranscribed but transcribed from opposite strands. The deduced amino acid sequence of the cyanobacterial polypeptides is more similar to the corresponding plastid (40-68% identity) than to the corresponding mitochondrial subunits (17-39% identity). Thus, the cyanobacterial NADH-dehydrogenase provides a prokaryotic model system which is more suitable to genetic analysis than the enzyme of plastids.     

Cloning and sequence analysis of the glucose-6-phosphate dehydrogenase gene from the cyanobacterium Synechococcus PCC 7942

The glucose-6-phosphate dehydrogenase (EC 1.1.1.49) gene (zwf) of the cyanobacterium Synechococcus PCC 7942 was cloned on a 2.8 kb Hind III fragment. Sequence analysis revealed an ORF of 1572 nucleotides encoding a polypeptide of 524 amino acids which exhibited 41% identity with the glucose-6-phosphate dehydrogenase of Escherichia coli.     

Nucleotide sequence and homology comparison of two genes of the sulfate transport operon from the cyanobacterium Synechocystis sp. PCC 6803

The genome of Synechocystis sp. PCC 6803 contains an operon with homology to the sulfate permease of other prokaryotes. We used antibodies raised against cytoplasmic membrane protein to find three genes with strong homology to sbpA, orf81 and cysT genes of the cyanobacterium Synechococcus sp. PCC 7942, Escherichia coli, Salmonella typhymurium and Marchantia polymorpha. It is likely that the permease genes are expressed and the proteins are inserted into the cytoplasmic membrane.     

Structure of a cyanobacterial gene encoding the 50S ribosomal protein L9

The rplI gene encoding the ribosomal protein L9 was found 4 kbp downstream from the desA gene, but on the opposite strand, in the genome of the cyanobacterium Synechocystis PCC6803. The deduced amino acid sequence is homologous to the sequences of the L9 proteins from Escherichia coli and chloroplasts of Arabidopsis and pea. The gene is present as a single copy in the chromosome and is transcribed as a mRNA of 0.64 kb. An open reading frame of unknown function (ORF291) was found in the upstream region of the rplI gene.     

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.Non-standard abbreviations bp base pairs - kb kilobase pairs - kd kilodaltons     

A homologous expression system for obtaining engineered cytochrome ba3 from Thermus thermophilus HB8

Cytochrome ba3 is an integral membrane protein that serves as a terminal oxidase of the respiratory chain in some prokaryotes. We have cloned the complete cba operon of Thermus thermophilus HB8 in an Escherichia coli/T. thermophilus shuttle vector. The ba3-encoding operon, cba, was eliminated from the chromosome of T. thermophilus strain MT111 using the pyrE system of Yamagishi and co-workers. Expression of functional cytochrome ba3 occurred in cells grown at reduced dioxygen levels. A hepta-histidine tag was placed at the N-terminus of subunit I, and a purification method for this form of the enzyme was developed. Growth conditions were investigated for moderate sized cultures (2L) with typical yields of approximately 2 mg of highly pure enzyme per liter of culture medium. The physical properties and enzymatic activities of these recombinant enzymes were compared with those of native enzyme. Recombinant enzyme lacking the histidine tag is spectrally identical to wild-type enzyme. Histidine-tagged cytochrome ba3 shows minor differences from wild-type, and it appears be somewhat less active as a cytochrome c552 oxidase. Exemplary mutants were also produced and compared to native protein. Tyrosine I-237, previously found to be covalently bonded to I-His-233, was changed to phenylalanine (I-Y237F) and to histidine (I-Y237H) in the hepta-histidine tagged cytochrome ba3. The Y to F mutant is devoid of enzyme activity whereas the Y to H mutant possesses approximately 5% wild-type oxidase activity; their properties are compared with those of wild-type enzyme. The above versions of the histidine-tagged enzyme have been crystallized, and our analysis of a 2.3 angstrom resolution electron-density map will be discussed elsewhere.