Cyanidioschyzon

{{Short description|Species of alga}} {{Speciesbox | image = Cyanidioschyzon merolae 10D.jpg | image_caption = | genus = Cyanidioschyzon | species = merolae | authority = P.De Luca, R.Taddei & L.Varano, 1978<ref>«Cyanidioschyzon merolae»: a new alga of thermal acidic environments. P De Luca, R Taddei and L Varano, Webbia, 1978</ref> }}

'''''Cyanidioschyzon merolae''''' is a small (2μm), club-shaped, unicellular haploid red alga adapted to high sulfur acidic hot spring environments (pH 1.5, 45 °C).<ref name="De Luca et al 1978">{{cite journal|author = De Luca P | author2 = Taddei R | author3 = Varano L | title=''Cyanidioschyzon merolae'' »: a new alga of thermal acidic environments | journal=Journal of Plant Taxonomy and Geography | date=1978 | volume=33 | issue=1 | pages=37–44|issn=0083-7792|doi=10.1080/00837792.1978.10670110| doi-access=free }}</ref><ref name="Matsuzaki et al 2004">{{cite journal|author=Matsuzaki M|author2=Misumi O|author3=Shin-i T|author4=Maruyama S|author5=Takahara M|author6=Miyagishima S|author7=Mori T|author8=Nishida K|author9=Yagisawa F|author10=Nishida K|author11=Yoshida Y|author12=Nishimura Y|author13=Nakao S|author14=Kobayashi T|author15=Momoyama Y|author16=Higashiyama T|author17=Minoda A|author18=Sano M|author19=Nomoto H|author20=Oishi K|author21=Hayashi H|author22=Ohta F|author23=Nishizaka S|author24=Haga S|author25=Miura S|author26=Morishita T|author27=Kabeya Y|author28=Terasawa K|author29=Suzuki Y|author30=Ishii Y|author31=Asakawa S|author32=Takano H|author33=Ohta N|author34=Kuroiwa H|author35=Tanaka K|author36=Shimizu N|author37=Sugano S|author38=Sato N|author39=Nozaki H|author40=Ogasawara N|author41=Kohara Y|author42=Kuroiwa T|title=Genome sequence of the ultrasmall unicellular red alga ''Cyanidioschyzon merolae'' 10D|journal=Nature|date=2004|volume=428|issue=6983|pages=653–657|doi=10.1038/nature02398|pmid=15071595|doi-access=free}}</ref> The cellular architecture of ''C. merolae'' is extremely simple, containing only a single chloroplast and a single mitochondrion and lacking a vacuole and cell wall.<ref name="Lee1999">{{cite book|author = Robert Edward Lee | title = Phycology |url = https://archive.org/details/phycology00robe |url-access = registration | date = 1999 | publisher = Cambridge University Press}}</ref> In addition, the cellular and organelle divisions can be synchronized. For these reasons, ''C. merolae'' is considered an excellent model system for study of cellular and organelle division processes, as well as biochemistry and structural biology.<ref name="Kuroiwa et al 1998">{{cite journal|author=Kuroiwa T|author2=Kuroiwa H|author3=Sakai A|author4=Takahashi H|author5=Toda K|author6=Itoh R|title=The division apparatus of plastids and mitochondria|journal=Int. Rev. Cytol.|date=1998|volume=181|pages=1–41|doi=10.1016/s0074-7696(08)60415-5|series=International Review of Cytology|pmid=9522454|isbn=978-0-12-364585-2}}</ref><ref name="Kuroiwa 1998">{{cite journal|author=Kuroiwa|title=The primitive red algae ''Cyanidium caldarium'' and ''Cyanidioschyzon merolae'' as model system for investigating the dividing apparatus of mitochondria and plastids|journal=BioEssays|volume=20|issue=4|pages=344–354|doi=10.1002/(sici)1521-1878(199804)20:4<344::aid-bies11>3.0.co;2-2|year=1998}}</ref><ref name="Minoda et al 2004"/> The organism's genome was the first full algal genome to be sequenced in 2004;<ref name="Matsuzaki2004">{{cite journal|date=2004|title=Genome sequence of the ultrasmall unicellular red alga ''Cyanidioschyzon merolae'' 10D|journal=Nature|volume=428|issue=6983|pages=653–657|doi=10.1038/nature02398|pmid=15071595|author=Matsuzaki, M.|display-authors=etal|doi-access=free}}</ref> its plastid was sequenced in 2000 and 2003, and its mitochondrion in 1998.<ref>{{cite journal | author = Barbier, Guillaume | title = Comparative Genomics of Two Closely Related Unicellular Thermo-Acidophilic Red Algae, ''Galdieria sulphuraria'' and ''Cyanidioschyzon merolae'', Reveals the Molecular Basis of the Metabolic Flexibility of Galdieria sulphuraria and Significant Differences in Carbohydrate Metabolism of Both Algae | journal = Plant Physiology | volume = 137 | issue = 2 | pages = 460–474 | doi = 10.1104/pp.104.051169 | pmid = 15710685 | date = 2005 | pmc = 1065348|display-authors=etal}}</ref> The organism has been considered the simplest of eukaryotic cells for its minimalist cellular organization.<ref name="Kobayashi et al 2010"/>

[[File:C merolae culture.jpg|thumb|Growing the red alga ''C. merolae'' in flasks and a {{convert|10|l|adj=on}} carboy. Although classified as a red alga, ''C. merolae'' is blue-green: it makes little if any of the red pigment phycoerythrin,<ref name="Castenholz and McDermott 2010">{{cite book|author=Castenholz RW|author2=McDermott TR|title=Red Algae in the Genomic Age|chapter=The Cyanidiales: Ecology, Biodiversity, and Biogeography|date=2010|editor=Seckbach J|editor2=Chapman DJ|pages=357–371}}</ref> and hence only displays the second red-algal pigment, phycocyanin, and the green pigment chlorophyll.<ref name="Castenholz and McDermott 2010"/>]]

== Isolation and growth in culture == Originally isolated by De Luca in 1978 from the solfatane fumaroles of Campi Flegrei (Naples, Italy),<ref name="De Luca et al 1978"/> ''C. merolae'' can be grown in culture in the laboratory in Modified Allen's medium (MA)<ref name="Minoda et al 2004">{{cite journal|author=Minoda A|author2=Sakagami R|author3=Yagisawa F|author4=Kuroiwa T|author5=Tanaka K|title=Improvement of culture conditions and evidence for nuclear transformation by homologous recombination in a red alga, ''Cyanidioschyzon merolae'' 10D|journal=Plant Cell Physiol|date=2004|volume=45|issue=6|pages=667–671|doi=10.1093/pcp/pch087|pmid=15215501|doi-access=free}}</ref> or a modified form with twice the concentration of some elements called MA2.<ref name="Kobayashi et al 2010">{{cite journal|author=Kobayashi Y|author2=Ohnuma M|author3=Kuroiwa T|author4=Tanaka K|author5=Hanaoka M|title=The basics of cultivation and molecular genetic analysis of the unicellular red alga ''Cyanidioschyzon merolae''|journal=Journal of Endocytobiosis and Cell Research|date=2010|volume=20|pages=53–61}}</ref><ref name="Ohnuma et al 2008">{{cite journal|author=Ohnuma M|author2=Yokoyama T|author3=Inouye T|author4=Sekine Y|author5=Tanaka K|title=Polyethylene Glycol (PEG)-Mediated Transient Gene Expression in a Red Alga, ''Cyanidioschyzon merolae'' 10D|journal=Plant Cell Physiol|date=2008|volume=49|issue=1|pages=117–120|doi=10.1093/pcp/pcm157|pmid=18003671|doi-access=free}}</ref> Using MA medium, growth rates are not particularly fast, with a doubling time (the time it takes a culture of microbes to double in cells per unit volume) of approximately 32 hours.<ref name="Minoda et al 2004"/> By using the more optimal medium MA2, this can be reduced to 24 hours.<ref name="Minoda et al 2004"/> Culturing is done at {{convert|42|C}} under white fluorescent light with an approximate intensity of 50 μmol photons m−2 s−1 (μE).<ref name="Kobayashi et al 2010"/> However, under a higher light intensity of 90 μE with 5% CO2 applied through bubbling, the growth rate of ''C. merolae'' can be further increased, with a doubling time of approximately 9.2 hours.<ref name="Minoda et al 2004"/> Higher light is not necessarily beneficial, as above 90 μE the growth rate begins to decrease.<ref name="Minoda et al 2004"/> This may be due to photodamage occurring to the photosynthetic apparatus. ''C. merolae'' can also be grown on gellan gum plates for purposes of colony selection or strain maintenance in the laboratory.<ref name="Minoda et al 2004"/> ''C. merolae'' is an obligate oxygenic phototroph, meaning it is not capable of taking up fixed carbon from its environment and must rely on oxygenic photosynthesis to fix carbon from CO2.<ref name="Kobayashi et al 2010"/>

== Genome == The 16.5 megabase pair genome of ''C. merolae'' was sequenced in 2004.<ref name="Matsuzaki et al 2004"/> The reduced, extremely simple, compact genome is made up of 20 chromosomes and was found to contain 5,331 genes, of which 86.3% were found to be expressed and only 26 contain introns, which contained strict consensus sequences.<ref name="Matsuzaki et al 2004"/> Strikingly, the genome of ''C. merolae'' contains only 30 tRNA genes and an extremely minimal number of ribosomal RNA gene copies,<ref name="Matsuzaki et al 2004"/> as shown in the [http://www.nature.com/nature/journal/v428/n6983/fig_tab/nature02398_T1.html genome comparison table]. The reduced nature of the genome has led to several other unusual features. While most eukaryotes contain 10 or so copies of the dynamins required for pinching membranes to separate dividing compartments, ''C. merolae'' only contains two,<ref name="Matsuzaki et al 2004"/> a fact that researchers have taken advantage of when studying organelle division.

Although possessing a small genome,<ref name="Matsuzaki2004 " /> the chloroplast genome of ''C. merolae'' contains many genes not present in the chloroplast genomes of other algae and plants.<ref>{{Cite journal | pmid = 12755171| year = 2003| last1 = Ohta| first1 = N| title = Complete sequenced analysis of the plastid genome of the unicellular red alga Cyanidioschyzon merolae| journal = DNA Research| volume = 10| issue = 2| pages = 67–77 | last2 = Matsuzaki | first2 = M | last3 = Misumi | first3 = O | last4 = Miyagishima | first4 = S. Y. | last5 = Nozaki | first5 = H | last6 = Tanaka | first6 = K | last7 = Shin-i | first7 = T | last8 = Kohara | first8 = Y | last9 = Kuroiwa | first9 = T | doi=10.1093/dnares/10.2.67 | doi-access = free }}</ref> Most of its genes are intronless.<ref name="Matsuzaki2004 " />

== Molecular biology == As is the case with most model organisms, genetic tools have been developed in ''C. merolae''. These include methods for the isolation of DNA and RNA from ''C. merolae'', the introduction of DNA into ''C. merolae'' for transient or stable transformation, and methods for selection including a uracil auxotroph that can be used as a selection marker.

===DNA isolation === Several methods, derived from cyanobacterial protocols, are used for the isolation of DNA from ''C. merolae''.<ref name="Kobayashi et al 2010"/><ref name="Imamura et al 2003">{{cite journal|author=Imamura S|author2=Yoshihara S|author3=Nakano S|author4=Shiozaki N|author5=Yamada A|author6=Tanaka K|author7=Takahashi H|author8=Asayama M|author9=Shirai M|title=Purification, characterization, and gene expression of all sigma factors of RNA polymerase in a cyanobacterium|journal=J. Mol. Biol.|date=2003|volume=325|issue=5|pages=857–872|doi=10.1016/s0022-2836(02)01242-1|pmid=12527296}}</ref> The first is a hot phenol extraction, which is a quick extraction that can be used to isolate DNA suitable for DNA amplification polymerase chain reaction (PCR),<ref name="Kobayashi et al 2010"/><ref name="Kobayashia et al 2009">{{cite journal|author=Kobayashia Y|author2=Kanesakia Y|author3=Tanakab A|author4=Kuroiwac H|author5=Kuroiwac T|author6=Tanaka K|title=Tetrapyrrole signal as a cell-cycle coordinator from organelle to nuclear DNA replication in plant cells|journal=Proc. Natl. Acad. Sci.|date=2009|volume=106|issue=3|pages=803–807|doi=10.1073/pnas.0804270105|pmid=19141634|pmc=2625283|doi-access=free}}</ref> wherein phenol is added to whole cells and incubated at 65 °C to extract DNA.<ref name="Kobayashi et al 2010"/> If purer DNA is required, the Cetyl trimethyl ammonium bromide (CTAB) method may be employed. In this method, a high-salt extraction buffer is first applied and cells are disrupted, after which a chloroform-phenol mixture is used to extract the DNA at room temperature.<ref name="Kobayashi et al 2010"/>

===RNA isolation=== Total RNA may be extracted from ''C. merolae'' cells using a variant of the hot phenol method described above for DNA.<ref name="Kobayashi et al 2010"/>

===Protein extraction=== As is the case for DNA and RNA, the protocol for protein extraction is also an adaptation of the protocol used in cyanobacteria.<ref name="Kobayashi et al 2010"/><ref name="Imamura et al 2008">{{cite journal|author=Imamura S|author2=Hanaoka M|author3=Tanaka K|title=The plant‐specific TFIIB related protein, PBRP, is a general transcription factor for RNA polymerase I|journal=EMBO J.|date=2008|volume=27|issue=17|pages=2317–2327|doi=10.1038/emboj.2008.151|pmid=18668124|pmc=2529366}}</ref> Cells are disrupted using glass beads and vortexing in a 10% glycerol buffer containing the reducing agent DTT to break disulfide bonds within proteins.<ref name="Kobayashi et al 2010"/> This extraction will result in denatured proteins, which can be used in SDS-PAGE gels for Western blotting and Coomassie staining.

===Transformant selection and uracil auxotrophic line=== ''C. merolae'' is sensitive to many antibiotics commonly used for selection of successfully transformed individuals in the laboratory, but it resistant to some, notably ampicillin and kanamycin.<ref name="Minoda et al 2004"/><ref name="Yagisawa et al 2004">{{cite journal|author=Yagisawa F|author2=Nishida K|author3=Okano Y|author4=Minoda A|author5=Tanaka K|author6=Kuroiwa T|title=Isolation of cycloheximide-resistant mutants of ''Cyanidioschyzon merolae''|journal=Cytologia|date=2004|volume=69|pages=97–100|doi=10.1508/cytologia.69.97|doi-access=free}}</ref>

A commonly used selection marker for transformation in ''C. merolae'' involves a uracil auxotroph (requiring exogenous uracil). The mutant was developed by growing ''C. merolae'' in the presence of a compound, 5-FOA, which in and of itself is non-toxic but is converted to the toxic compound 5-Fluorouracil by an enzyme in the uracil biosynthetic pathway, orotidine 5'-monophosphate (OMP) decarboxylase, encoded by the ''Ura5.3'' gene.<ref name="Minoda et al 2004"/> Random mutation led to several loss-of-function mutants in ''Ura5.3'', which allowed cells to survive in the presence of 5-FOA as long as uracil was provided.<ref name="Minoda et al 2004"/> By transforming this mutant with a PCR fragment carrying both a gene of interest and a functional copy of ''Ura5.3'', researchers can confirm that the gene of interest has been incorporated into the ''C. merolae'' genome if it can grow without exogenous uracil.

===Polyethylene glycol (PEG) mediated transient expression=== While chromosomal integration of genes creates a stable transformant, transient expression allows short-term experiments to be done using labeled or modified genes in ''C. merolae''. Transient expression can be achieved using a polyethylene glycol (PEG) based method in protoplasts (plant cells with the rigid cell wall enzymatically eliminated), and because ''C. merolae'' lacks a cell wall, it behaves much as a protoplast would for transformation purposes.<ref name="Ohnuma et al 2008"/> To transform, cells are briefly exposed to 30% PEG with the DNA of interest, resulting in transient transformation.<ref name="Ohnuma et al 2008"/> In this method, the DNA is taken up as a circular element and is not integrated into the genome of the organism because no homologous regions exist for integration.

===Gene targeting=== To create a stable mutant line, gene targeting can be used to insert a gene of interest into a particular location of the ''C. merolae'' genome via homologous recombination. By including regions of DNA several hundred base pairs long on the ends of the gene of interest that are complementary to a sequence within the ''C. merolae'' genome, the organism's own DNA repair machinery can be used to insert the gene at these regions.<ref name="Fujiwara et al 2013">{{cite journal|author=Fujiwara T|author2=Ohnuma M|author3=Yoshida M|author4=Kuroiwa T|author5=Hirano T|title=Gene targeting in the red alga ''Cyanidioschyzon merolae'': single- and multi-copy insertion using authentic and chimeric selection markers|journal=PLOS ONE|date=2013|volume=8|issue=9|article-number=e73608|doi=10.1371/journal.pone.0073608|pmid=24039997|pmc=3764038|doi-access=free}}</ref> The same transformation procedure as is used for transient expression can be used here, except with the homologous DNA segments to allow for genome integration.<ref name="Fujiwara et al 2013"/>

[[File:C merolae EM (Ursula Goodenough).jpg|thumb|Freeze fracture deep etch electron microscopy image of ''C. merolae'', showing two cells, one in which the plastid has begun to divide. Courtesy of Prof. Ursula Goodenough.]]

==Studying cell and organelle divisions== The extremely simple divisome, simple cell architecture, and ability to synchronize divisions in ''C. merolae'' makes it the perfect organism for studying mechanisms of eukaryotic cell and organelle division.<ref name="Matsuzaki et al 2004"/><ref name="Kuroiwa 1998"/> Synchronization of the division of organelles in cultured cells can be very simple and usually involves the use of light and dark cycles. The chemical agent aphidicolin can be added to easily and effectively synchronize chloroplast division.<ref name="Teriu et al 1995">{{cite journal|author=Terui S|author2=Suzuki K|author3=Takahiashi H|author4=Itoh R|author5=Kuroiwa T|title=High synchronization of chloroplast division in the ultramicro-alga ''Cyanidioschyzon merolae'' by treatment with both light and aphidicolin|journal=J. Phycol.|date=1995|volume=31|pages=958–961|doi=10.1111/j.0022-3646.1995.00958.x|s2cid=84124611}}</ref> The peroxisome division mechanism was first ascertained using ''C. merolae'' as a system,<ref name="Imoto et al 2013">{{cite journal|author=Imoto Y|author2=Kuroiwa H|author3=Yoshida Y|author4=Ohnuma M|author5=Fujiwara T|author6=Yoshida M|author7=Nishida K|author8=Yagisawa F|author9=Hirooka S|author10=Miyagishima S|author11=Misumi O|author12=Kawano S|author13=Kuroiwa T|title=Single-membrane–bounded peroxisome division revealed by isolation of dynamin-based machinery|journal=Proc. Natl. Acad. Sci.|date=2013|volume=110|issue=23|pages=9583–9588|doi=10.1073/pnas.1303483110|pmid=23696667|pmc=3677435|doi-access=free}}</ref> where peroxisome division can be synchronized using the microtubule-disrupting drug oryzalin in addition to light-dark cycles.<ref name="Imoto et al 2013"/>

==Photosynthesis research== ''C. merolae'' is also used in researching photosynthesis. Notably, the subunit composition of the photosystems in ''C. merolae'' has some significant differences from that of other related organisms.<ref name="Nilsson et al 2014">{{cite journal|author=Nilsson H|author2=Krupnik T|author3=Kargul J|author4=Messinger J|title=Substrate water exchange in photosystem II core complexes of the extremophilic red alga ''Cyanidioschyzon merolae''|journal=Biochimica et Biophysica Acta (BBA) - Bioenergetics|date=2014|volume=1837|issue=8|pages=1257–1262|doi=10.1016/j.bbabio.2014.04.001|pmid=24726350|doi-access=free}}</ref><ref name="Bricker et al 2012">{{cite journal|author=Bricker TM|author2=Roose JL|author3=Fagerlund RD|author4=Frankel LK|author5=Eaton-Rye JJ|title=The extrinsic proteins of photosystem II|journal=Biochim. Biophys. Acta|date=2012|volume=1817|issue=1|pages=121–142|doi=10.1016/j.bbabio.2011.07.006|pmid=21801710|doi-access=free}}</ref> Photosystem II (PSII) of ''C. merolae'', as might be expected, has a particularly unusual pH range in which it can function.<ref name="Nilsson et al 2014"/><ref name="Krupnik et al 2013">{{cite journal|author=Krupnik T|author2=Kotabova E|author3=van Bezouwen LS|author4=Mazur R|author5=Garstka M|author6=Nixon PJ|author7=Barber J|author8=Kana R|author9=Boekema EJ|author10=Kargul J|title=A reaction center-dependent photoprotection mechanism in a highly robust photosystem II from an extremophilic red alga, ''Cyanidioschyzon merolae''|journal=J. Biol. Chem.|date=2013|volume=288|issue=32|pages=23529–23542|doi=10.1074/jbc.m113.484659|pmid=23775073|pmc=5395030|doi-access=free}}</ref> Despite the fact that the mechanism of PSII requires protons to be quickly released, and lower pH solutions should alter the ability to do this, ''C. merolae'' PSII is capable of exchanging and splitting water at the same rate as other related species.<ref name="Nilsson et al 2014"/>

== See also == * ''Galdieria sulphuraria'' * Red algae

== External links == * [http://merolae.biol.s.u-tokyo.ac.jp/ ''Cyanidioschyzon merolae'' Genome Project] {{AlgaeBase species|name=Cyanidioschyzon merolae|id=36733}}

== References == {{Reflist}}

Category:Cyanidiophyceae Category:Species described in 1978