Identification of a nifDK-Like Gene (ORF467) Involved in the Biosynthesis of Chlorophyll in the Cyanobacterium Plectonema boryanum

The frxC gene, which is found in chloroplast DNA (ctDNA) andin cyanobacteria, encodes a protein that is required for thelight-independent reduction of protochlorophyllide (Pchlide)to chlorophyllide a (Chlide). A DNA fragment downstream of frxCin the filamentous cyanobacterium Plectonema boryanum was clonedand analyzed. Sequencing of the DNA fragment revealed an openreading frame (ORF) that encoded a protein of 467 amino acidresidues (designated ORF467), which showed extensive homologyto the proteins encoded by genes on ctDNAs (ORF465 in liverwort,gidA in pine and chlN in Chlamydomonas reinhardtii) and to ORF469protein of the cyanobacterium Synechocystis sp. strain PCC 6803.We isolated a targeted mutant YFM6D-3 in which ORF467 was inactivatedby the insertion of a kanamycin-resistance gene into the codingregion. YFM6D-3 exhibited a phenotype similar to that of YFC1004,an frxC-disrupted mutant, which did not synthesize chlorophyll(Chl) and accumulated Pchlide, a precursor to Chl, in the dark.These phenotypic characteristics of YFM6D-3 indicate that thelight-independent reduction of Pchlide requires not only theFrxC protein but also the ORF467 protein. The amino acid sequencesof the homologues of ORF467 exhibit low but significant similarityto those of the and ß subunits of nitrogenase MoFe-protein,suggesting a phylogenetic relationship between the light-independentPchlide reductase and nitrogenase, as is observed between theFrxC protein and the Fe-protein of nitrogenase. ¹Institute for Protein Research, Osaka University, Suita, Osaka,565 Japan     

Stabilizing Effect of Lipopolysaccharide on the l-Glycerol 3-Phosphate Acyltransferase of Escherichia coli Membrane

A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase. Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known. Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469–0RF248) was isolated from the cyanobacterium Synechocystis PCC6803. In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively. Synechocystis frxC showed 31% amino acid sequence identity with nifHl from Clostridium pasteurianum. Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C. pasteurianum nifK product, which is the β subunit of a molybdenum-iron protein of a nitrogenase complex. Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution.     

Chloroplast chlB gene is required for light-independent chlorophyll accumulation in Chlamydomonas reinhardtii

Light-independent chlorophyll synthesis occurs in some algae, lower plants, and gymnosperms, but not in angiosperms. We have identified a new chloroplast gene, chlB, that is required for the light-independent accumulation of chlorophyll in the green alga Chlamydomonas reinhardtii. The chlB gene was cloned, sequenced, and then disrupted by performing particle gun-mediated chloroplast transformation. The resulting homoplasmic mutant was unable to accumulate chlorophyll in the dark and thus exhibited a yellow-in-the-dark phenotype. The chlB gene encodes a polypeptide of 688 amino acid residues, and is distinct from two previously characterized chloroplast genes (chlN and chlL) also required for light-independent chlorophyll accumulation in C. reinhardtii. Three unidentified open reading frames in chloroplast genomes of liverwort, black pine, and Chlamydomonas moewusii were also identified as chlB genes, based on their striking sequence similarities to the C. reinhardtii chlB gene. A chlB-like gene is absent in chloroplast genomes of tobacco and rice, consistent with the lack of light-independent chlorophyll synthesis in these plants. Polypeptides encoded by the chloroplast chlB genes also show significant sequence similarities with the bchB gene product of Rhodobacter capsulatus. Comparisons among the chloroplast chlB and the bacterial bchB gene products revealed five highly conserved sequence areas that are interspersed by four stretches of highly variable and probably insertional sequences.     

Chloroplast DNA of black pine retains a residual inverted repeat lacking rRNA genes: nucleotide sequences of trnQ, trnK, psbA, trnI and trnH and the absence of rps16

Summary A physical map of black pine (Pinus thunbergii) chloroplast DNA (120 kb) was constructed and two separate portions of its nucleotide sequence were determined. One portion contains trnQ-UUG, ORF510, ORF83, trnK-UUU (ORF515 in the trnK intron), ORF22, psbA, trnI-CAU (on the opposing strand) and trnH-GUG, in that order. Sequence analysis of another portion revealed the presence of a 495 by inverted repeat containing trnI-CAU and the 3 end of psbA but lacking rRNA genes. The position of trnI-CAU is unique because most chloroplast DNAs have no gene between psbA and trnH (trnI-CAU is usually located further downstream). Black pine chloroplast DNA lacks rps16, which has been found between trnQ and trnK in angiosperm chloroplast DNAs, but possesses ORF510 instead. This ORF is highly homologous to ORF513 found in the corresponding region of liverwort chloroplast DNA and ORF563 located downstream from trnT in Chlamydomonas moewusii chloroplast DNA. A possible pathway for the evolution of black pine chloroplast DNA is discussed.     

Identification of a novel nifH-like (frxC) protein in chloroplasts of the liverwort Marchantia polymorpha

The frxC gene, one of the unidentified open reading frames present in liverwort chloroplast DNA, shows significant homology with the nifH genes coding for the Fe protein, a component of the nitrogenase complex (Ohyama et al., 1986, Nature 322: 572–574). A truncated form of the frxC gene was designed to be over-expressed in Escherichia coli and an antibody against this protein was prepared using the purified product as an antigen. This antibody reacted with a protein in the soluble fraction of liverwort chloroplasts, which had an apparent molecular weight of 31 000, as revealed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis, in good agreement with a putative molecular weight of 31945 deduced from the DNA sequence of the frxC gene. In a competitive inhibition experiment, the antigenicity of this protein was indicated to be similar to that of the over-expressed protein in E. coli. Therefore, we concluded that the frxC gene was expressed in liverwort chloroplasts and that its product existed in a soluble form. The molecular weight of the frxC protein was approximately 67 000, as estimated by gel filtration chromatography, indicating that the frxC protein may exist as a dimer of two identical polypeptides analogous to the Fe protein of nitrogenase. The results obtained from affinity chromatography supported the possibility that the frxC protein, which possesses a ATP-binding sequence in its N-terminal region that is conserved among various other ATP-binding proteins, has the ability to bind ATP.     

<Emphasis Type="Italic">SulP</Emphasis>, a nuclear gene encoding a putative chloroplast-targeted sulfate permease in<Emphasis Type="Italic"> Chlamydomonas reinhardtii</Emphasis>

Genomic, proteomic, phylogenetic and evolutionary aspects of a novel gene encoding a putative chloroplast-targeted sulfate permease of prokaryotic origin in the green alga Chlamydomonas reinhardtii are described. This nuclear-encoded sulfate permease gene (SulP) contains four introns, whereas all other known chloroplast sulfate permease genes lack introns and are encoded by the chloroplast genome. The deduced amino acid sequence of the protein showed an extended N-terminus, which includes a putative chloroplast transit peptide. The mature protein contains seven transmembrane domains and two large hydrophilic loops. This novel prokaryotic-origin gene probably migrated from the chloroplast to the nuclear genome during evolution of C. reinhardtii. The SulP gene, or any of its homologues, has not been retained in vascular plants, e.g. Arabidopsis thaliana, although it is encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). A comparative structural analysis and phylogenetic origin of chloroplast sulfate permeases in a variety of species is presented.     

Chloroplast sulfate transport in green algae – genes,proteins and effects

This review summarizes evidence at the molecular genetic, protein and regulatory levels concerning the existence and function of a putative ABC-type chloroplast envelope-localized sulfate transporter in the model unicellular green alga Chlamydomonas reinhardtii. From the four nuclear genes encoding this sulfate permease holocomplex, two are coding for chloroplast envelope-targeted transmembrane proteins (SulP and SulP2), a chloroplast stroma-targeted ATP-binding protein (Sabc) and a substrate (sulfate)-binding protein (Sbp) that is localized on the cytosolic side of the chloroplast envelope. The sulfate permease holocomplex is postulated to consist of a SulP–SulP2 chloroplast envelope transmembrane heterodimer, flanked by the Sabc and the Sbp proteins on the stroma side and the cytosolic side of the inner envelope, respectively. The mature SulP and SulP2 proteins contain seven transmembrane domains and one or two large hydrophilic loops, which are oriented toward the cytosol. The corresponding prokaryotic-origin genes (SulP and SulP2) probably migrated from the chloroplast to the nuclear genome during the evolution of Chlamydomonas reinhardtii. These genes, or any of its homologues, have not been retained in vascular plants, e.g. Arabidopsis thaliana, although they are encountered in the chloroplast genome of a liverwort (Marchantia polymorpha). The function of the SulP protein was probed in antisense transformants of C. reinhardtii having lower expression levels of the SulP gene. Results showed that cellular sulfate uptake capacity was lowered as a consequence of attenuated SulP gene expression in the cell, directly affecting rates of de novo protein biosynthesis in the chloroplast. The antisense transformants exhibited phenotypes of sulfate-deprived cells, displaying slow rates of light-saturated oxygen evolution, low levels of Rubisco in the chloroplast and low steady-state levels of the Photosystem II D1 reaction center protein. The role of the chloroplast sulfate transport in the uptake and assimilation of sulfate in Chlamydomonas reinhardtii is discussed along with its impact on the repair of Photosystem II from a frequently occurring photo-oxidative damage and H₂-evolution related metabolism in this green alga.     

Chloroplast genes encoding subunits of the H+-ATPase complex of Chlamydomonas reinhardtii are rearranged compared to higher plants: sequence of the atpE gene and location of the atpF and atpI genes

Summary The chloroplast gene for the epsilon subunit (atpE) of the CF₁/CF₀ ATPase in the green alga Chlamydomonas reinhardtii has been localized and sequenced. In contrast to higher plants, the atpE gene does not lie at the 3 end of the beta subunit (atpB) gene in the chloroplast genome of C. reinhardtii, but is located at a position 92 kb away in the other single copy region. The uninterrupted open reading frame for the atpE gene is 423 bp, and the epsilon subunit exhibits 43% derived amino acid homology to that from spinach. Codon usage for the atpE gene follows the restricted pattern seen in other C. reinhardtii chloroplast genes.The genes for the CF₀ subunits I (atpF) and IV (atpI) of the ATPase complex have also been mapped on the chloroplast genome of C. reinhardtii. The six chloroplast ATPase genes in C. reinhardtii are dispersed individually between the two single copy regions of the chloroplast genome, an organization strikingly different from the highly conserved arrangement in two operon-like units seen in chloroplast genomes of higher plants.Abbreviations bp base pairs - CF₁ chloroplast coupling factor 1 - CF₀ chloroplast coupling factor 0 - F₁ coupling factor 1 - F₀ coupling factor 0 - kb kilobase pairs     

Conserved gene clusters in the highly rearranged chloroplast genomes of Chlamydomonas moewusii and Chlamydomonas reinhardtii

We have extended to about 75 the number of genes mapped on the Chlamydomonas moewusii and Chlamydomonas reinhardtii chloroplast DNAs (cpDNAs) by partial sequencing of the very closely related C. eugametos and C. moewusii cpDNAs and by hybridizations with Chlamydomonas chloroplast gene-specific sequences. Only four of these genes (tscA and three reading frames) have not been identified in any other algal cpDNAs and thus may be specific to Chlamydomonas. Although the C. moewusii and C. reinhardtii cpDNAs differ by complex sequence rearrangements, 38 genes scattered throughout the genome define 12 conserved clusters of closely linked loci. Aside from the rRNA operon, four of these gene clusters share similarity to evolutionarily primitive operons found in other cpDNAs, representing in fact remnants of these operons. Our results thus indicate that most of the ancestral bacterial operons that characterize the chloroplast genome organization of land plants and early-diverging photosynthetic eukaryotes have been disrupted before the emergence of the polyphyletic genus Chlamydomonas. All gene rearrangements between the C. moewusii and C. reinhardtii cpDNAs, with the exception of those accounting for the relocations of atpA, psbI and rbcL, occurred within corresponding regions of the genome. One of these rearrangements seems to have led to disruption of the ancestral region containing rpl23, rpl2, rps19, rpl16, rpl14, rpl5, rps8 and the psaA exon 1. This gene cluster, which bears striking similarity to the Escherichia coli S10 and spc operons, spans a continuous DNA segment in C. reinhardtii, while it maps to two separate fragments in C. moewusii.     

Construction of a Chlamydomonas reinhardtii mutant with an intronless psbA gene

Efficient chloroplast transformation systems now available allow the manipulation of the evolutionarily highly conserved psbA gene in the eucaryotic organism Chlamydomonas reinhardtii. Two copies of this gene in the inverted repeat region of the chloroplast genome contain four large group I introns. To analyse possible functions of these introns and to generate a mutant for simplified psbA gene manipulations, a psbA cDNA fragment was introduced into a psbA deletion mutant using the biolistic transformation method. A transformant with no introns in the psbA gene has been obtained and represents the first example of the removal of a complete set of introns from a chloroplast gene. The newly generated strain is photosynthetically competent and contains no detectable recipient genome copies. The loss of all four introns appears to be phenotypically silent.     

The recombination and expression of the allophycocyanin beta subunit gene in the chloroplast of Chlamydomonas reinhardtii

Summary A Chlamydomonas reinhardtii (C. reinhardtii) chloroplast expression vector, papc-B, containing the apc-B gene that encodes the beta subunit of the light-harvesting antenna protein allophycocyanin (APC) of cyanobacteria, was constructed and transferred to the chloroplast genome of C. reinhardtii by the biolistic method. The transformants were identified by Southern blot, Western blot and ELISA assays after selection on resistant medium. The recombinant APC beta subunit was expressed in the C. reinhardtii chloroplast and accounted for up to 2–3% (w/w) of the total soluble protein (TSP), suggesting a promising prospect of using C. reinhardtii chloroplasts to produce functional plant-derived proteins.     

Molecular and genetic analysis of the chloroplast ATPase of chlamydomonas

Summary We have carried out a molecular and genetic analysis of the chloroplast ATPase in Chlamydomonas reinhardtii. Recombination and complementation studies on 16 independently isolated chloroplast mutations affecting this complex demonstrated that they represent alleles in five distinct chloroplast genes. One of these five, the ac-u-c locus, has been positioned on the physical map of the chloroplast DNA by deletion mutations. The use of cloned spinach chloroplast ATPase genes in heterologous hybridizations to Chlamydomonas chloroplast DNA has allowed us to localize three or possibly four of the ATPase genes on the physical map. The beta and probably the epsilon subunit genes of Chlamydomonas CF₁ lie within the same region of chloroplast DNA as the ac-u-c locus, while the alpha and proteolipid subunit genes appear to map adjacent to one another approximately 20 kbp away. Unlike the arrangement in higher plants, these two pairs of genes are separated from each other by an inverted repeat.     

A chloroplast gene is required for the light-independent accumulation of chlorophyll in Chlamydomonas reinhardtii.

The light-independent pathway of chlorophyll synthesis which occurs in some lower plants and algae is still largely unknown. We have characterized a chloroplast mutant, H13, of Chlamydomonas reinhardtii which is unable to synthesize chlorophyll in the dark and is also photosystem I deficient. The mutant has a 2.8 kb deletion as well as other rearrangements of its chloroplast genome. By performing particle gun mediated chloroplast transformation of H13 with defined wild-type chloroplast DNA fragments, we have identified a new chloroplast gene, chlN, coding for a 545 amino acid protein which is involved in the light-independent accumulation of chlorophyll, probably at the step of reduction of protochlorophyllide to chlorophyllide. The chlN gene is also found in the chloroplast genomes of liverwort and pine, but is absent from the chloroplast genomes of tobacco and rice.     

Using bacteria to analyze sequences involved in chloroplast gene expression

The complete nucleotide sequence of chloroplast DNA from a liverwort, Marchantia polymorpha has made clear the entire gene organization of the chloroplast genome. Quite a few genes encoding components of photosynthesis and protein synthesis machinery have been identified by comparative computer analysis. Other genes involved in photosynthesis, respiratory electron transport, and membrane-associated transport in chloroplasts were predicted by the amino acid sequence homology and secondary structure of gene products. Thirty-three open reading frames in the liverwort chloroplast genome remain unidentified. However, most of these open reading frames are also conserved in the chloroplast genomes of two species, a liverwort, Marchantia polymorpha, and tobacco, Nicotiana tabacum, indicating their active functions in chloroplasts.Abbreviations bp base pair - kDa kilodalton - IR inverted repeat - ORF open reading frame - DALA -aminolevulinate     

Structure, evolution and expression of the mitochondrial ADP/ATP translocator gene from Chlamydomonas reinhardtii

Summary The first AUG in the Chlamydomonas reinhardtii ADP/ATP translocator (CRANT) mRNA initiates an open reading frame (ORF) which is very similar (51–79% amino acid identity) to other ANT proteins. In contrast to higher plants, no evidence for a long amino-terminal extension was obtained. The 5 non-transcribed region of the single-copy CRANT gene contains sequence motifs present in other C. reinhardtii nuclear genes. Four introns, whose positions are not conserved in other ANT genes, interrupt the protein coding region. A short heat shock specifically reduces CRANT mRNA levels. CRANT mRNA levels were unaffected by a mutation in photosynthesis. In a dark/light regime CRANT mRNA levels are high in the dark phase and low in the early light phase. Data on translation initiation sites, splice junctions and the codon preferences of C. reinhardtii nuclear genes were compiled. With the exception of two rare codons, ACA and GGA, the CRANT gene exhibits the biased codon usage of C. reinhardtii nuclear genes that are highly expressed during normal vegetative growth.     

The apocytochrome b gene of Chlamydomonas smithii contains a mobile intron related to both Saccharomyces and Neurospora introns.

Summary The mitochondrial DNA of the two interfertile algal species Chlamydomonas smithii and Chlamydomonas reinhardtii are co-linear with the exception of ca. 1 kb insertion (the a insert) present in C. smithii DNA only. In vegetative diploids resulting from interspecific crosses, mitochondrial genomes are transmitted biparentally except for the a insert which is transmitted to all C. reinhardtii molecules in a manner reminiscent of the intron-mediated conversion event that occurs at the omega locus in yeast mitochondria, under the action of the I-SceI endonuclease. Here we report that the insert corresponds to a typical group I intron of 1075 bp, inserted within the gene for apocytochrome b and containing a 237 codon open reading frame (ORF). We also report the complete sequence of the apocytochrome b gene of C. smithii. Comparison with the sequence of the same gene in C. reinhardtii reveals the precise intron insertion site. These data, together with the previous genetic data provide the first example of intron mobility in mitochondria of the plant kingdom. The product of the intronic ORF shows 36% amino acid identity with the I-SceI endonuclease whereas the intron ribozyme shows a 60% identity at the nucleotide level with the Neurospora crassa cob · 1 intron. The possibility of a recent horizontal transfer of introns between fungi and algae is discussed.     

Cloning and nucleotide sequence of a frxC-ORF469 gene cluster of Synechocystis PCC6803: conservation with liverwort chloroplast frxC-ORF465 and nif operon.

A gene, frxC, which is unique to the chloroplast genome of the liverwort Marchantia polymorpha, has sequence similarity to nifH, the product of which is an iron protein of a nitrogenase. Although frxC is expressed to produce a protein in liverwort chloroplasts, its function is not known. Using a probe of liverwort chloroplast DNA, a 10.1-kb region containing a gene cluster consisting of open reading frames (ORF278-frxC-ORF469-ORF248) was isolated from the cyanobacterium Synechocystis PCC6803. In this region, frxC and ORF469 showed sequence similarities to liverwort chloroplast frxC (83%) and immediately downstream ORF465 (74%), respectively. Synechocystis frxC showed 31% amino acid sequence identity with nifH1 from Clostridium pasteurianum. Additionally, Synechocystis ORF469 showed a sequence similarity (19% identity) to C. pasteurianum nifK product, which is the beta subunit of a molybdenum-iron protein of a nitrogenase complex. Conservation of the gene arrangement between liverwort and Synechocystis suggests that the liverwort chloroplast frxC-ORF465 cluster may have evolved from an ancestor common to Synechocystis, and that these two genes may have been transferred to the nuclear genome in tobacco and rice during evolution.     

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii share features with both mitochondrial and higher plant chloroplast presequences

Chloroplast transit peptides from the green alga Chlamydomonas reinhardtii have been analyzed and compared with chloroplast transit peptides from higher plants and mitochondrial targeting peptides from yeast, Neurospora and higher eukaryotes. In terms of length and amino acid composition, chloroplast transit peptides from C. reinhardtii are more similar to mitochondrial targetting peptides than to chloroplast transit peptides from higher plants. They also contain the potential amphiphilic α-helix characteristic of mitochondrial presequences. However, in similarity with chloroplast transit peptides from higher plants, they contain a C-terminal region with the potential to form an amphiphilic β-strand. As in higher plants, transit peptides that route proteins to the thylakoid lumen consist of an N-tenninal domain similar to stroma-targeting transit peptides attached to a C-terminal apolar domain that share many characteristics with secretory signal peptides.     

Successful expression of heterologous egfp gene in the mitochondria of a photosynthetic eukaryote Chlamydomonas reinhardtii

The efficient expression of exogenous gene in mitochondria of photosynthetic organism has been an insurmountable problem. In this study, the pBsLPNCG was constructed by inserting the egfp gene into a site between TERMINVREP-Left repeats and the cob gene in a fragment of mitochondrial DNA of Chlamydomonas reinhardtii CC-124 and introduced into the mitochondria of respiratory deficient dum-1 mutation of C. reinhardtii CC-2654. Sequencing and DNA Southern analyses revealed that egfp gene had been integrated into the mitochondrial genome of transgenic algae as expected and no other copy of egfp existed in their nucleic genome. Both the fluorescence detection and Western blot analysis confirmed the presence of eGFP protein in the transgenic algae; it indicated that the egfp gene was successfully expressed in the mitochondria of C. reinhardtii.