Niji Ohta

Genome sequence of the ultrasmall unicellular red alga Cyanidioschyzon merolae 10D

Small, compact genomes of ultrasmall unicellular algae provide information on the basic and essential genes that support the lives of photosynthetic eukaryotes, including higher plants. Here we report the 16,520,305-base-pair sequence of the 20 chromosomes of the unicellular red alga Cyanidioschyzon merolae 10D as the first complete algal genome. We identified 5,331 genes in total, of which at least 86.3% were expressed. Unique characteristics of this genomic structure include: a lack of introns in all but 26 genes; only three copies of ribosomal DNA units that maintain the nucleolus; and two dynamin genes that are involved only in the division of mitochondria and plastids. The conserved mosaic origin of Calvin cycle enzymes in this red alga and in green plants supports the hypothesis of the existence of single primary plastid endosymbiosis. The lack of a myosin gene, in addition to the unexpressed actin gene, suggests a simpler system of cytokinesis. These results indicate that the C. merolae genome provides a model system with a simple gene composition for studying the origin, evolution and fundamental mechanisms of eukaryotic cells.

Nuclear Encoding of a Chloroplast RNA Polymerase Sigma Subunit in a Red Alga

A chloroplast RNA polymerase sigma factor is encoded by a nuclear gene, sigA, in the red alga Cyanidium caldarium RK-1. The encoded protein functions as an RNA polymerase sigma factor in vitro and it is localized to the chloroplast in vivo. SigA shows high sequence similarity to the sigma factors of cyanobacteria, which is indicative of the ancestral endosymbiotic event and subsequent transfer of the sigA gene to the nuclear genome.

Molecular Characterization of the PEND Protein, a Novel bZIP Protein Present in the Envelope Membrane That Is the Site of Nucleoid Replication in Developing Plastids

Plastid nucleoids are known to bind to the envelope membrane in developing chloroplasts. Here, plastid DNA is extensively replicated. We previously detected a DNA binding protein in the inner envelope membranes of developing plastids in pea and named it PEND (for plastid envelope DNA binding) protein. In this study, we report on the structure and molecular characterization of a cDNA for the PEND protein. As a result of screening cDNA libraries in λgt11 with one of the target sequences of the PEND protein as a probe, we obtained a clone (PD2) for a novel DNA binding protein consisting of 633 amino acid residues. Analysis of the N-terminal sequence of the purified PEND protein indicated that the transit peptide is just 16 residues long. The PEND protein was detected specifically in the plastid envelope membrane of young unopened leaf buds by immunoblot analysis. The PEND protein consists of a basic region plus zipper region, an unprecedented sextuple repeat region, and a putative membrane-spanning region. The basic region with a zipper region seems to have diverged from that of other plant transcription factors. In addition, the PEND protein could be a distant homolog of the trans-Golgi network integral membrane proteins. The PEND protein is therefore a novel type of DNA binding protein that binds to the membrane as an intrinsic membrane protein.

Detection and localization of a chloroplast-encoded HU-like protein that organizes chloroplast nucleoids

Chloroplast DNA (cpDNA) is packed into discrete structures called chloroplast nucleoids (cp-nucleoids). The structure of cpDNA is thought to be important for its maintenance and regulation. In bacteria and mitochondria, histone-like proteins (such as HU and Abf2, respectively) are abundant and play important roles in DNA organization. However, a primary structural protein has yet to be found in cp-nucleoids. Here, we identified an abundant DNA binding protein from isolated cp-nucleoids of the primitive red alga Cyanidioschyzon merolae. The purified protein had sequence homology with the bacterial histone-like protein HU, and it complemented HU-lacking Escherichia coli mutants. The protein, called HC (histone-like protein of chloroplast), was encoded by a single gene (CmhupA) in the C. merolae chloroplast genome. Using immunofluorescence and immunoelectron microscopy, we demonstrated that HC was distributed uniformly throughout the entire cp-nucleoid. The protein was expressed constitutively throughout the cell and the chloroplast division cycle, and it was able to condense DNA. These results indicate that HC, a bacteria-derived histone-like protein, primarily organizes cpDNA into the nucleoid.

Reexamination of phylogenetic relationships within the colonial Volvocales (Chlorophyta): An analysis of atpB and rbcL gene sequences

The chloroplast‐encoded atpB gene was sequenced from 33 strains representing 28 species of the colonial Volvocales (the Volvocaceae and its relatives) to reexamine phylogenetic relationships as previously deduced by morphological data and rbcL gene sequence data.1128 base pairs in the coding regions of the atpB gene were analyzed by MP, NJ, and ML analyses. Although supported with relatively low bootstrap values (75% and 65% in the NJ and ML analyses, respectively), three anisogamous/oogamous volvocacean genera—Eudorina, Pleodorina, and Volvox, excluding the section Volvox (=Euvolvox, illegitimate name), constituted a large monophyletic group (Eudorina group). Outside the Eudorina group, a robust lineage composed of three species of Volvox sect. Volvox was resolved as in the rbcL gene trees, rejecting the hypothesis of the previous cladistic analysis based on morphological data that the genus Volvox is monophyletic. In addition, the NJ and ML trees suggested that Eudorina is a nonmonophyletic genus as inferred from the morphological data and rbcL gene sequences. Although phylogenetic status of the genus Gonium is ambiguous in the rbcL gene trees and the paraphyly of this genus is resolved in the cladistic analysis based on morphological data, the atpB gene sequence data suggest monophyly of Gonium with relatively low bootstrap values (56–61%) in the NJ and ML trees. On the basis of the combined sequence data (2256 base pairs) from atpB and rbcL genes, Gonium was resolved as a robust monophyletic genus in the NJ and ML trees (with 68–86% bootstrap values), and Eudorina elegans Ehrenberg represented a paraphyletic species positioned most basally within the Eudorina group. However, phylogenetic status and relationships of the families of the colonial Volvocales were still almost ambiguous even in the combined analysis.

Isolation of the cell-nuclear, mitochondrial, and chloroplast DNA from the ultra-small eukaryoteCyanidioschyzon merolae

SummaryThe amounts of cell-nuclear DNA (cl-DNA), mitochondrial DNA (mt-DNA) and chloroplast DNA (cp-DNA) inCyanidioschyzon merolae were estimated by using a video-intensified microscope (VIM) system.C. merolae had the smallest amount of cell-nuclear DNA among eukaryotes. The results show that a cell-nucleus, a mitochondrion and a chloroplast contain an average 8.0×103kbp, 1.6×103kbp, and 5.0×103kbp, respectively. To confirm these results, cl-DNA, mt-DNA, and cp-DNA were isolated from cells by density centrifugation on Hoechst 33258/CsCl after density centrifugation on ethidium bromide/CsCl. The amounts of cl-DNA, mt-DNA, and cp-DNA obtained from the bands supported the data shown by the VIM-system. The cytochemical and biochemical characteristics were compared with those ofCyanidium caldarium RK-1 andC. caldarium Forma A. The ρ values of cl-DNA and cp-DNA ofC. merolae were about 1.716 and 1.709, respectively. The order in density was different from that ofC. caldarium Forma A but very similar to that ofC. caldarium RK-1. However, the restriction patterns of cp-DNA inC. merolae differed from those ofC. caldarium RK-1.

Reducing Sludge Production and the Domination of Comamonadaceae by Reducing the Oxygen Supply in the Wastewater Treatment Procedure of a Food-Processing Factory

Sludge production was reduced remarkably by reducing the dissolved oxygen supply to less than 1 mg/l in the conventional wastewater treatment procedure of a food-processing factory that produced 180 m3 of wastewater of biochemical oxygen demand (BOD) of about 1,000 mg/l daily. DNA was extracted from the sludge and subjected to PCR amplification. The PCR product was cloned into a plasmid and sequenced. Estimation of the resident bacterial distribution by 16S rDNA sequences before and after improvement of the system suggested a remarkable gradual change in the major bacterial population from Anaerolinaeceae (15.6%) to Comamonadaceae (52.3%), members of denitrifying bacteria of Proteobacteria. Although we did not directly confirm the ability of denitrification of the resulting sludge, a change in the major final electron acceptors from oxygen to nitrate might explain the reduction in sludge production in a conventional activated sludge process when the oxygen supply was limitted.

TOWARD A NATURAL SYSTEM OF SPECIES IN CHLOROGONIUM (VOLVOCALES, CHLOROPHYTA): A COMBINED ANALYSIS OF MORPHOLOGICAL AND rbcL GENE SEQUENCE DATA

The taxonomy of species of Chlorogonium (Volvocales, Chlorophyta) was studied based on comparative light and electron microscopy and DNA sequence data of 23 strains from five major algal culture collections. All of the 23 strains showed pyrenoids under photoautotrophic conditions, but 17 of the 23 exhibited marked reduction in size of pyrenoids, or pyrenoids were absent under photoheterotrophic conditions. The strains could clearly be delineated into six species, C. euchlorum, C. elongatum, C. fusiforme, C. capillatum, C. neglectum, and C. kasakii on the basis of differences in cell shape, number of contractile vacuoles, number and stability of pyrenoids, and ultrastructure of pyrenoids and stigmata. This distinction of species based on morphology was also supported by analyses of rbcL gene sequences. The later strongly showed that each species, C. euchlorum (seven strains), C. elongatum (three strains), and C. capillatum (10 strains), forms a robust clade. Although some morphological differences were noted within different strains of C. euchlorum and C. capillatum, these features were regarded as strain‐specific because they were not reflected in the rbcL gene phylogenies. In addition, the rbcL gene trees strongly suggested that C. neglectum and C. kasakii are closely related to each other, consistent with the similarity of the ultrastructure of pyrenoids and stigmata between the two species. However, C. kasakii can be distinguished clearly from C. neglectum by its multiple pyrenoids in the chloroplast and acute anterior and posterior ends in the vegetative cell.

Analysis of the Cluster of Ribosomal Protein Genes in the Plastid Genome of a Unicellular Red Alga Cyanidioschyzon merolae: Translocation of the str Cluster as an Early Event in the Rhodophyte-Chromophyte Lineage of Plastid Evolution

Abstract. The nucleotide sequence of a cluster of ribosomal protein genes in the plastid genome of a unicellular red alga, Cyanidioschyzon merolae, which has been supposed to be the most primitive alga, was determined. The phylogenetic tree inferred from the amino acid sequence of ribosomal proteins of two rhodophytes, a chromophyte, a glaucophyte, two chlorophytes (land plants), a cyanobacterium, and three eubacteria suggested a close relationship between the cyanobacterium Synechocystis PCC6803 and the plastids of various species in the kingdom Plantae, which is consistent with the hypothesis of the endosymbiotic origin of plastids. In this tree, the two species of rhodophytes were grouped with the chromophyte, and the glaucophyte was grouped with the chlorophytes. Analysis of the organization of the genes encoding the ribosomal proteins suggested that the translocation of the str cluster occurred early in the lineage of rhodophytes and chromophytes after these groups had been separated from chlorophytes and glaucophytes.

Analysis of a plastid gene cluster reveals a close relationship betweenCyanidioschyzon andCyanidium

Cyanidioschyzon merolae andCyanidium caldarium are representative species among of the most primitive algae, although the two species are distinctly different in various morphological traits. We determined the nucleotide sequence of therbcL gene and a flanking 8-kb region in the plastid genome of each of these algae. In both algae, 12 genes were identified in this region, in an identical order. This gene order is not conserved in the plastid genomes of other species of the kingdom Plantae that have been sequenced to data. An additional unidentified open reading frame was also found in the two algae that we analyzed, which has not been described in any other species of algae includingPorphyra purpurea. Comparison of the amino acid sequences of selected genes also supported the conclusion thatCyanidioschyzon merolae andCyanidium caldarium are closely related and that they are distinct from other rhodophytes.