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D A Bryant

Publications and source records attributed to D A Bryant.

At least 73 records · Page 4Linked to original sources
PubMed1991

Molecular cloning and transcriptional analysis of the cpeBA operon of the cyanobacterium Pseudanabaena species PCC7409.

The cpeBA operon of the Group III chromatically adapting cyanobacterium Pseudanabaena species PCC 7409 was cloned, sequenced and characterized. The cpeBA genes are transcribed in green-light-grown cells as an abundant 1400-nucleotide mRNA which initiates 69 nucleotides upstream from the cpeB translation start. Extensive sequence identity, extending 70 nucleotides 5' to the transcription start, occurs among cpeBA promoters of Group II and III chromatic adapters. Cell extracts of green-light-grown Calothrix species PCC 7601 contain an activity which specifically binds a restriction fragment containing the Pseudanabanea species PCC 7409 cpeBA promoter. Green-light-dependent cpeBA transcription in Group II and III chromatically adapting cyanobacteria is suggested to be similarly controlled by a transcriptional activator.

Amino Acid Sequence↗
PubMed1990

Reconstitution of electron transport in photosystem I with PsaC and PsaD proteins expressed in Escherichia coli.

A fusion protein, denoted PsaC1, which contains an amino-terminal extension of five amino acids (MEHSM...) and is derived from an in vitro modified form of the psaC gene of Synechococcus sp. PCC 7002, has been over-expressed in Escherichia coli. The product of the psaD gene of Nostoc sp. PCC 8009 has similarly been over-expressed. The PsaC1 and PsaD proteins can be combined with the photosystem I core protein of Synechococcus sp. PCC 6301 to reconstitute electron transport from P700 to the terminal FA/FB acceptors. Reconstitution was found to be absolutely dependent on reinsertion of the iron-sulfur clusters in the PsaC1 apoprotein and on the presence of the PsaD protein. This implies that the PsaC1 holoprotein does not bind solely to the PsaA/PsaB heterodimer but rather that its interaction with these proteins is mediated through the PsaD protein.

Amino Acid Sequence↗
PubMed1990

Genetic analysis of a 9 kDa phycocyanin-associated linker polypeptide.

The gene encoding LR9, a 9 kDa phycocyanin-associated linker polypeptide, was cloned from the cyanobacterium Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR-6). This gene, termed cpcD, was located immediately 3' to cpcC, a gene which encodes another phycocyanin-associated linker, LR33. Mutation of cpcD by insertion led to the loss of LR9 as the only detectable change in phycobilisome composition. Cells and isolated phycobilisomes from the cpcD- strain did not detectably differ from the wild-type in absorption or steady-state fluorescence emission. Purified phycobilisomes from the wild-type and cpcD- strains were compared by electron microscopy. The number of phycocyanin discs in the rod substructures of the mutant was more variable than in the wild-type. Hence, one function of LR9 may be to minimize the heterogeneity of rod length, possibly by binding to the core-distal face of phycocyanin-LR33 complexes to prevent the tandem joining of such units. A mutant in which cpcD and cpcC-cpcD intergenic sequences are deleted shows a partial loss of LR33. Inverted repeats in this intergenic region may be required for optimal stability of the cpcC transcript.

Amino Acid Sequence↗
PubMed1990

The cyanelle genome of Cyanophora paradoxa encodes ribosomal proteins not encoded by the chloroplasts genomes of higher plants.

The rpl35, rpl20, rpl5, rps8, and a portion of the rpl6 genes of the cyanelle genome of Cyanophora paradoxa have been cloned, mapped and sequenced. Homologs of the rpl35, rpl5, and rpl6 genes are not found in the chloroplasts of higher plants. The rpl35 genes most likely form a dicistronic operon which is located upstream from the apcE-apcA-apcB locus of the cyanelle and which is divergently transcribed from this locus. The rpl5, rpl8, and rpl6 genes probably form a part of a larger cluster of genes encoding components of the cyanellar ribosomes. These genes are organized in a fashion similar to that observed in all procaryotes examined to date, with the exception that the rps14 gene is not found between the rpl5 and rps8 coding sequences. Hypotheses concerning the origins of cyanelles and chloroplasts are discussed.

Amino Acid Sequence↗
PubMed1990

Structure and mutation of a gene encoding a Mr 33,000 phycocyanin-associated linker polypeptide.

The gene encoding a phycocyanin-associated linker polypeptide of Mr 33,000 from the cyanobacterium Synechococcus sp. PCC 7002 was found to be located adjacent and 3' to the genes encoding the alpha and beta subunits of phycocyanin. The identity of this gene, designated cpcC, was proven by matching the amino-terminal sequence of the authentic polypeptide with that predicted by the nucleotide sequence. A cpcC mutant strain of this cyanobacterium was constructed. The effect of the mutation was to prevent assembly of half the total phycocyanin into phycobilisomes. By electron microscopy, phycobilisomes from this mutant were shown to contain rod substructures composed of a single disc of hexameric phycocyanin, as opposed to two discs in the wild type. It was concluded that the Mr 33,000 linker polypeptide is required for attachment of the core-distal phycocyanin hexamer to the core-proximal one. Using absorption spectra of the wild type, CpcC-, and phycocyanin-less phycobilisomes, the in situ absorbances expected for specific phycocyanin-linker complexes were calculated. These data confirm earlier findings on isolated complexes regarding the influence of linkers on the spectroscopic properties of phycocyanin.

Amino Acid Sequence↗
PubMed1990

Structural and compositional analyses of the phycobilisomes of Synechococcus sp. PCC 7002. Analyses of the wild-type strain and a phycocyanin-less mutant constructed by interposon mutagenesis.

The phycobilisomes and phycobiliproteins of Synechococcus sp. PCC 7002 wild-type strain PR6000 have been isolated and characterized. The hemidiscoidal phycobilisomes of strain PR6000 are composed of eleven different polypeptides: phycocyanin alpha and beta subunits; allophycocyanin alpha and beta subunits; alpha subunit of allophycocyanin B; the allophycocyanin beta-subunit-like polypeptide of Mr 18,000; the linker phycobiliprotein of Mr 99,000; and non-chromophore-carrying linker polypeptides of Mr 33,000, 29,000, 9000, and 8000. Several of these polypeptides were purified to homogeneity and their amino acid compositions and amino-terminal amino acid sequences were determined. Analyses of the phycobiliproteins of Synechococcus sp. PCC 7002 were greatly facilitated by comparative studies performed with a mutant strain, PR6008, constructed to be devoid of the phycocyanin alpha and beta subunits by recombinant DNA techniques and transformation of strain PR6000. The absence of phycocyanin did not greatly affect the allophycocyanin content of the mutant strain but caused the doubling time to increase 2-7-fold depending upon the light intensity at which the cells were grown. Although intact phycobilisome cores could not be isolated from this mutant, it is probable that functionally intact cores do exist in vivo.

Amino Acid Sequence↗
PubMed1990

Nucleotide sequence and further characterization of the Synechococcus sp. strain PCC 7002 recA gene: complementation of a cyanobacterial recA mutation by the Escherichia coli recA gene.

The nucleotide sequence and transcript initiation site of the Synechococcus sp. strain PCC 7002 recA gene have been determined. The deduced amino acid sequence of the RecA protein of this cyanobacterium is 56% identical and 73% similar to the Escherichia coli RecA protein. Northern (RNA) blot analysis indicates that the Synechococcus strain PCC 7002 recA gene is transcribed as a monocistronic transcript 1,200 bases in length. The 5' endpoint of the recA mRNA was mapped by primer extension by using synthetic oligonucleotides of 17 and 27 nucleotides as primers. The nucleotide sequence 5' to the mapped endpoint contained sequence motifs bearing a striking resemblance to the heat shock (sigma 32-specific) promoters of E. coli but did not contain sequences similar to the E. coli SOS operator recognized by the LexA repressor. An insertion mutation introduced into the recA locus of Synechococcus strain PCC 7002 via homologous recombination resulted in the formation of diploids carrying both mutant and wild-type recA alleles. A variety of growth regimens and transformation procedures failed to produce a recA Synechococcus strain PCC 7002 mutant. However, introduction into these diploid cells of the E. coli recA gene in trans on a biphasic shuttle vector resulted in segregation of the cyanobacterial recA alleles, indicating that the E. coli recA gene was able to provide a function required for growth of recA Synechococcus strain PCC 7002 cells. This interpretation is supported by the observation that the E. coli recA gene is maintained in these cells when antibiotic selection for the shuttle vector is removed.

Amino Acid Sequence↗
PubMed1989

Spectroscopic studies of cyanobacterial phycobilisomes lacking core polypeptides.

Synechococcus sp. PCC 7002 (Agmenellum quadruplicatum PR6) genes encoding two highly conserved phycobilisome core polypeptides, a small linker polypeptide (LC8, apcC) and the allophycocyanin-B alpha-subunit (alpha APB, apcD), respectively, were interrupted by insertion of restriction fragments carrying the neomycin phosphotransferase gene of Tn5. The interrupted genes were used to transform Synechococcus sp. PCC 7002 to kanamycin resistance. The apcC- mutant assembled phycobilisomes lacking the LC8 polypeptide and the apcD- mutant assembled phycobilisomes lacking alpha APB. No other differences between the compositions of the mutant and wild-type phycobilisomes were detected. The apcC- strain grew about 25% more slowly than the wild-type, and its phycobilisomes dissociated more rapidly in 0.33 M Na/K-PO4 (pH 8.0) or in 0.75 M Na/K-PO4 at pH 8.0, at 40 degrees C, than did those of the wild-type. The phycobilisomes of this mutant were indistinguishable from those of the wild-type with respect to absorption and circular dichroism spectra, as well as time-resolved fluorescence emission. Steady-state emission spectra indicate a small decrease in long wavelength (680 nm) emission from the apcC- phycobilisomes and a complementary increase in shorter wavelength (665 nm) emission, relative to wild-type phycobilisomes. Strain apcD- phycobilisomes appear to be functionally indistinguishable from those of the wild-type, in spite of the absence of the two alpha APB subunits which bear terminal acceptor bilins. The only spectroscopic difference was seen in the steady-state fluorescence emission, for which the emission of the mutant was about 15% higher than that of the wild-type and was slightly blue-shifted. A phenotype has yet to be found for the apcD- mutation.

Cloning, Molecular↗
PubMed1989

State transitions in a phycobilisome-less mutant of the cyanobacterium Synechococcus sp. PCC 7002.

State transitions were investigated in the cyanobacterium Synechococcus sp. PCC 7002 in both wild-type cells and mutant cells lacking phycobilisomes. Preillumination in the presence of DCMU induced State 1 and dark-adaptation induced State 2 in both wild-type and mutant cells as determined by 77 K fluorescence emission spectroscopy. Light-induced transitions were observed in the wild-type after preferential excitation of phycocyanin (State 2) or preferential excitation of Chl a (State 1). Light-induced transitions were also observed in the phycobilisome-less mutant after preferential excitation of short-wavelength Chl a (State 2) or carotenoids and long-wavelength Chl a (State 1). We conclude that the mechanism of the light-state transition in cyanobacteria does not require the presence of the phycobilisome. Our results contradict proposed models for the state transition, which require phosphorylation of, and an active role for, the phycobilisome.

Chlorophyll↗
PubMed1988

In vitro attachment of bilins to apophycocyanin. I. Specific covalent adduct formation at cysteinyl residues involved in phycocyanobilin binding in C-phycocyanin.

Expression of cloned alpha and beta subunit genes of Synechococcus sp. PCC7002 C-phycocyanin in Escherichia coli led to the production of large amounts of apophycocyanin. The apophycocyanin was purified to homogeneity and shown to be an alpha beta monomer. The reactivity of the apoprotein toward a number of open chain and cyclic tetrapyrroles was examined. Phycocyanobilin (PCB), phycoerythrobilin, and biliverdin all formed covalent adducts with apophycocyanin in 50 mM sodium phosphate buffer at pH 7.0. Mesobiliverdin, bilirubin, PCB dimethyl ester, protoporphyrin IX, and hemin did not react with the apoprotein. None of these tetrapyrroles reacted with 2 mM 2-mercaptoethanol, cysteine, or reduced glutathione under the same conditions. The adduct with PCB was investigated in greater detail. Its visible absorption spectrum, with a maximum at 646 nm, is more similar to that of allophycocyanin than phycocyanin. Two PCBs are bound per alpha beta monomer when the reaction is performed with excess bilin. While tryptic digestion of the adduct generates numerous bilin peptides, amino acid analysis of these chromopeptides revealed that PCB reacted specifically at alpha-Cys-84 and beta-Cys-82, two of the three cysteinyl residues that serve as the attachment sites for PCB in native phycocyanin. The major bilin peptides arising from in vitro adduct formation at each of these sites differed both in chromatographic behavior and in spectroscopic properties from the corresponding PCB peptides isolated from tryptic digests of native C-phycocyanin.

Apoproteins↗
PubMed1987

Functional expression of plastid allophycocyanin genes in a cyanobacterium.

In Cyanophora paradoxa, the allophycocyanin apoprotein subunits, alpha and beta, are encoded in the cyanelle (plastid) genome. These genes were transferred to the cyanobacterium Synechococcus sp. PCC 7002 on a plasmid replicon. Phycobilisomes isolated from transformed cyanobacteria were found to contain C. paradoxa allophycocyanin subunits. Thus, these plastid genes are expressed in the cyanobacterium as polypeptides which become linked to a chromophore and are incorporated into the light-harvesting apparatus.

Cloning, Molecular↗
PubMed1987

Molecular cloning and characterization of the recA gene from the cyanobacterium Synechococcus sp. strain PCC 7002.

The recA gene of Synechococcus sp. strain PCC 7002 was detected and cloned from a lambda gtwes genomic library by heterologous hybridization by using a gene-internal fragment of the Escherichia coli recA gene as the probe. The gene encodes a 38-kilodalton polypeptide which is antigenically related to the RecA protein of E. coli. The nucleotide sequence of a portion of the gene was determined. The translation of this region was 55% homologous to the E. coli protein; allowances for conservative amino acid replacements yield a homology value of about 74%. The cyanobacterial recA gene product was proficient in restoring homologous recombination and partial resistance to UV irradiation to recA mutants of E. coli. Heterologous hybridization experiments, in which the Synechococcus sp. strain PCC 7002 recA gene was used as the probe, indicate that a homologous gene is probably present in all cyanobacterial strains.

Amino Acid Sequence↗
PubMed1986

Green light induces transcription of the phycoerythrin operon in the cyanobacterium Calothrix 7601.

Phycobilisomes, the major light-harvesting complexes of cyanobacteria are multimolecular structures made up of chromophoric proteins called phycobiliproteins and non chromophoric linker polypeptides. We report here the isolation and nucleotide sequence of the genes, cpeA and cpeB, which in Calothrix PCC 7601 encode the alpha and beta subunits of phycoerythrin, one of the major phycobiliproteins. In Calothrix PCC 7601, modulation of the polypeptide composition of the phycobilisomes occurs in response to changes of the light wavelength, a phenomenon known as complementary chromatic adaptation. Under green illumination, cells synthesize phycoerythrin and its two specifically associated linker polypeptides (LR35 and LR36), while under red illumination none of these proteins are detected. Using specific probes, a single transcript (1450 nucleotide long) corresponding to the cpe genes was detected but only in green-light-grown cells, establishing the occurrence of transcriptional regulation for the expression of this operon in response to light wavelength changes. The size of this transcript excludes the possibility that the phycoerythrin-associated LR35 and LR36 could be cotranscribed with the cpeA and cpeB genes.

Amino Acid Sequence↗
PubMed1986

Organization and nucleotide sequence of genes encoding core components of the phycobilisomes from Synechococcus 6301.

Cyanobacteria possess specialized organelles, called phycobilisomes, which collect and transfer light energy to the reaction centres of photosystem II, in the photosynthetic membrane. Phycobilisomes consist of a central core, mainly composed of allophycocyanin, from which six rods radiate. We report here the isolation, for the first time, of three genes that encode core components of cyanobacterial phycobilisomes. The genes coding for the alpha- and beta-subunit apoproteins of allophycocyanin (apcA and apcB) were cloned from Synechococcus PCC 6301 and subjected to nucleotide sequence analysis. Dowstream of apcB, we found a third open reading frame (apcC) which, by comparison with known amino acid sequences, was assigned to L7.8c, a linker polypeptide associated with phycobiliproteins within the core of the phycobilisomes. Homologies between amino acid sequences deduced from the nucleotide sequence of the Synechococcus PCC 6301 apc genes and the amino acid sequences published for corresponding proteins either from cyanobacteria or chloroplast-like organelles of eukaryotic organisms, are 75% or more. The genetic organization of this photosynthetic gene cluster relative to that observed in the cyanelle genome of the flagellate Cyanophora paradoxa is discussed.

Amino Acid Sequence↗
PubMed1985

Molecular cloning and nucleotide sequence of a developmentally regulated gene from the cyanobacterium Calothrix PCC 7601: a gas vesicle protein gene.

Since the gas vesicle protein (GVP) is highly conserved among the different gas-vacuolate prokaryotes, a 29-mer oligonucleotide corresponding to a portion of the Anabaena flos-aquae GVP gene was synthesized and used to isolate the GVP structural gene from Calothrix PCC 7601 (= Fremyella diplosiphon). Gas vacuole production in this filamentous cyanobacterium is restricted to hormogonia which occur at a specific stage during the developmental cell cycle. The GVP gene (gvpA) was localized on a 709 bp HindIII-HincII fragment. Nucleotide sequence analysis revealed a 213 bp open reading frame whose deduced amino-acid sequence shows a very high homology with that of the Anabaena flos-aquae GVP. Assuming that the first methionine residue is proteolytically processed, the molecular mass of the Calothrix GVP is 7375 daltons. Sequences resembling the Escherichia coli consensus promoter were found upstream from the gvpA gene. The initiator codon of the gvpA gene is preceded by a polypurine sequence assumed to be the ribosome binding site. Southern hybridizations with a probe specific for the gvpA gene indicated that this gene is not plasmid-borne, and that another homologous gene is present in the Calothrix genome.

Amino Acid Sequence↗