We report studies of the fission yeast fimbrin-like protein Fim1, which contains two EF-hand domains and two actin-binding domains (ABD1 and ABD2). Fim1 is a component of both F-actin patches and the F-actin ring, but not of F-actin cables. Fim1 cross-links F-actin in vitro, but a Fim1 protein lacking either EF-hand domains (Fim1A12) or both the EF-hand domains and ABD1 (Fim1A2) has no actin cross-linking activity. Overexpression of Fim1 induced the formation of F-actin patches throughout the cell cortex, whereas the F-actin patches disappear in cells overexpressing Fim1A12 or Fim1A2. Thus, the actin cross-linking activity of Fim1 is probably important for the formation of F-actin patches. The overexpression of Fim1 also excluded the actin-depolymerizing factor Adf1 from the F-actin patches and inhibited the turnover of actin in these structures. Thus, Fim1 may function in stabilizing the F-actin patches. We also isolated the gene encoding Acp1, a subunit of the heterodimeric F-actin capping protein. fim1 acp1 double null cells showed more severe defects in the organization of the actin cytoskeleton than those seen in each single mutant. Thus, Fim1 and Acp1 may function in a similar manner in the organization of the actin cytoskeleton. Finally, genetic studies suggested that Fim1 may function in cytokinesis in cooperation with Cdc15 (PSTPIP) and Rng2 (IQGAP), respectively.

DNA introduction into cells is an essential technique for molecular genetic analysis. Here, we show that DNA is easily introduced into cells of the unicellular red alga Cyanidioschyzon merolae by a polyethylene glycol (PEG)-mediated protocol. In this study, the beta-tubulin gene of C. merolae was cloned on a plasmid and a hemagglutinin (HA) tag then added at the C-terminus. This plasmid was then introduced into C. merolae cells by a PEG-mediated transformation protocol. At 24 h after PEG-mediated transformation, intracellular localization of the tagged protein was detected by anti-HA immunocytochemistry, indicating the utility of this transient expression system for molecular genetic analyses.

Cyanidioschyzon merolae is a unicellular red alga living in acid hot springs, which is able to grow on ammonium, as well as nitrate as sole nitrogen source. Based on the complete genome sequence, proteins for nitrate utilization, nitrate transporter (NRT) and nitrate reductase (NR), were predicted to be encoded by the neighboring nuclear genes CMG018C and CMG019C, respectively, but no typical nitrite reductase (NiR) gene was found by similarity searches. On the other hand, two candidate genes for sulfite reductase (SiR) were found, one of which (CMG021C) is located next to the above-noted nitrate-related genes. Given that transcripts of CMG018C, CMG019C and CMG021C accumulate in nitrate-containing media, but are repressed by ammonium, and that SiR and NiR are structurally related enzymes, we hypothesized that the CMG021C gene product functions as an NiR in C. merolae. To test this hypothesis, we developed a method for targeted gene disruption in C. merolae. In support of our hypothesis, we found that a CMG021G null mutant in comparison with the parental strain showed decreased cell growth in nitrate-containing but not in ammonium-containing media. Furthermore, expression of CMG021C in the nirA mutant of a cyanobacterium, Leptolyngbya boryana (formerly Plectonema boryanum), could genetically complement the NiR defect. Immunofluorescent analysis indicated the localization of CMG021C in chloroplasts, and hence we propose an overall scheme for nitrate assimilation in C. merolae.

In chloroplast division, the plastid-dividing (PD) ring is a main structure of the PD machinery and is a universal structure in the plant kingdom. However, the components and formation of the PD ring have been enigmatic. By proteomic analysis of PD machineries isolated from Cyanidioschyzon merolae, we identified the glycosyltransferase protein plastid-dividing ring 1 (PDR1), which constructs the PD ring and is widely conserved from red alga to land plants. Electron microscopy showed that the PDR1 protein forms a ring with carbohydrates at the chloroplast-division site. Fluorometric saccharide ingredient analysis of purified PD ring filaments showed that only glucose was included, and down-regulation of PDR1 impaired chloroplast division. Thus, the chloroplasts are divided by the PD ring, which is a bundle of PDR1-mediated polyglucan filaments.

Extreme thermophiles produce two types of unusual polyamine: long linear polyamines such as caldopentamine and caldohexamine, and branched polyamines such as quaternary ammonium compounds [e.g. tetrakis(3-aminopropyl)ammonium]. To clarify the physiological roles of long linear and branched polyamines in thermophiles, we synthesized them chemically and tested their effects on the stability of ds (double-stranded) and ss (single-stranded) DNAs and tRNA in response to thermal denaturation, as measured by differential scanning calorimetry. Linear polyamines stabilized dsDNA in proportion to the number of amino nitrogen atoms within their molecular structure. We used the empirical results to derive formulae that estimate the melting temperature of dsDNA in the presence of polyamines of a particular molecular composition. ssDNA and tRNA were stabilized more effectively by tetrakis(3-aminopropyl)ammonium than any of the other polyamines tested. We propose that long linear polyamines are effective to stabilize DNA, and tetrakis(3-aminopropyl)ammonium plays important roles in stabilizing RNAs in thermophile cells.

Plant cells sense environmental nitrogen levels and alter their gene expression accordingly to survive; however, the underlying regulatory mechanisms still remains to be elucidated. Here, we identified and characterized a transcription factor that is responsible for expression of nitrogen assimilation genes in a unicellular red alga Cyanidioschyzon merolae . DNA microarray and Northern blot analyses revealed that transcript of the gene encoding CmMYB1, an R2R3-type MYB transcription factor, increased 1 h after nitrogen depletion. The CmMYB1 protein started to accumulate after 2 h and reached a peak after 4 h after nitrogen depletion, correlating with the expression of key nitrogen assimilation genes, such as CmNRT , CmNAR , CmNIR , CmAMT , and CmGS . Although the transcripts of these nitrogen assimilation genes were detected in nitrate-grown cells, they disappeared upon the addition of preferred nitrogen source such as ammonium or glutamine, suggesting the presence of a nitrogen catabolite repression (NCR) mechanism. The nitrogen depletion-induced gene expression disappeared in a CmMYB1 -null mutant, and the mutant showed decreased cell viability after exposure to the nitrogen-depleted conditions compared with the parental strain. Chromatin immunoprecipitation analysis demonstrated that CmMYB1 specifically occupied these nitrogen-responsive promoter regions only under nitrogen-depleted conditions, and electrophoretic mobility shift assays using crude cell extract revealed specific binding of CmMYB1, or a complex containing CmMYB1, to these promoters. Thus, the presented results indicated that CmMYB1 is a central nitrogen regulator in C. merolae .

In the extreme thermophile Thermus thermophilus, a disruption mutant of a gene homologous to speB (coding for agmatinase ‫؍‬ agmatine ureohydrolase) accumulated N 1 -aminopropylagmatine (N 8 -amidino-1,8-diamino-4-azaoctane, N 8 -amidinospermidine), a new compound, whereas all other polyamines produced by the wild-type strain were absent from the cells. Double disruption of speB and speE (polyamine aminopropyltransferase) resulted in the disappearance of N 1 -aminopropylagmatine and the accumulation of agmatine. These results suggested the following. 1) N 1 -Aminopropylagmatine is produced from agmatine by the action of an enzyme coded by speE. 2) N 1 -Aminopropylagmatine is a metabolic intermediate in the biosynthesis of unique polyamines found in the thermophile. 3) N 1 -Aminopropylagmatine is a substrate of the SpeB homolog. They further suggest a new biosynthetic pathway in T. thermophilus, by which polyamines are formed from agmatine via N 1 -aminopropylagmatine. To confirm our speculation, we purified the expression product of the speB homolog and confirmed that the enzyme hydrolyzes N 1 -aminopropylagmatine to spermidine but does not act on agmatine.Polyamines play important roles in cell proliferation and cell differentiation. Common polyamines such as putrescine, spermidine, and spermine are distributed ubiquitously in cells and tissues at relatively high concentrations (1, 2).Thermus thermophilus, of which the genome project was completed using two strains, HB8 and HB27 (Structural-Biological Whole Cell Project at www.srg.harima.riken.go.jp/ thermus/j_index.htm and see Ref. 3, respectively), produces a variety of polyamines including unusually long polyamines and branched ones (4) (see Fig. 9C). These long and branched polyamines have a marked effect of protecting and stabilizing nucleic acids (5, 6) and of activating cell-free polypeptide synthesis at high temperature (7-9).In many organisms, such as bacteria, yeast, animals, and plants, the first step of polyamine biosynthesis is production of putrescine by decarboxylation of L-ornithine (see Fig. 9A). An additional or alternative pathway of putrescine biosynthesis that is often seen in plants and sometimes in bacteria is decarboxylation of L-arginine followed by hydrolysis of agmatine. Agmatine ureohydrolase or agmatinase, coded by the speB gene, catalyzes this second reaction. The next step is production of spermidine and spermine by the addition of an aminopropyl group to putrescine and spermidine, respectively. This reaction is catalyzed by spermidine or spermine synthase (putrescine/spermidine aminopropyltransferase) coded by the speE gene (1).To investigate the polyamine biosynthetic pathway in T. thermophilus, we constructed a disruption strain of the speB gene homolog of T. thermophilus. Disruption of the speB gene homolog resulted in drastic reduction of triamines, longer and branched polyamines without accumulation of agmatine, and in accumulation of an unknown compound. Double disruption of speB and speE gene homologs resulted in disappear...

The unicellular red alga Cyanidioschyzon merolae is an emerging model organism for studying organelle division and inheritance: the cell is composed of an extremely simple set of organelles (one nucleus, one mitochondrion and one chloroplast), and their genomes are completely sequenced. Although a fruitful set of cytological and biochemical methods have now been developed, gene targeting techniques remain to be fully established in this organism. Thus far, only a single selection marker, URACm-Gs, has been available that complements the uracil-auxotrophic mutant M4. URACm-Gs, a chimeric URA5.3 gene of C. merolae and the related alga Galdieria sulphuraria, was originally designed to avoid gene conversion of the mutated URA5.3 allele in the parental strain M4. Although an early example of targeted gene disruption by homologous recombination was reported using this marker, the genome structure of the resultant transformants had never been fully characterized. In the current study, we showed that the use of the chimeric URACm-Gs selection marker caused multicopy insertion at high frequencies, accompanied by undesired recombination events at the targeted loci. The copy number of the inserted fragments was variable among the transformants, resulting in high yet uneven levels of transgene expression. In striking contrast, when the authentic URA5.3 gene (URACm-Cm) was used as a selection marker, efficient single-copy insertion was observed at the targeted locus. Thus, we have successfully established a highly reliable and reproducible method for gene targeting in C. merolae. Our method will be applicable to a number of genetic manipulations in this organism, including targeted gene disruption, replacement and tagging.