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==RNA Polymerase and Sigma Factors==
==RNA Polymerase and Sigma Factors==


[[RNA Polymerase] (RNAP)] and [[sigma factors]] are necessary proteins for [[transcription]] of DNA into [[messenger RNA] (mRNA)]. Eubacterial RNAP [[holoenzymes]] consist of a core with four major subunits α2 ββ’. In cyanobacteria, β’ is formed from two smaller subunits (у and β’) , which corresponds to RNAPs in plant [[chloroplasts]].<ref name=imamura>{{cite journal|last1=Imamura|first1=S.|last2=Asayama|first2=M.|title=Sigma Factors for Cyanobacterial Transcription|journal=Gene Regulation and Systems Biology|date=2009|volume=3|pages=65-87}}</ref> The [[DNA clamp|beta subunits]] are responsible for binding the RNAP to the DNA, preventing premature dissociation. In Escherichia coli, the beta “clamp” binds loosely originally and tightens as the RNAP approaches the start codon (AUG). In cyanobacteria, the beta clamp binds tightly at initial binding. The effect of this difference is that synthetic repressible promoters do not function as expected in ''Synechocystis'' sp. PCC6803. In E. coli, a repressor binds the DNA operon and dislodges RNAP due to the loosely bound beta clamp. In ''Synechocystis'', the RNAP is tightly bound leading the reverse phenomenon where the repressor is knocked off the DNA. Thus the gene is not effectively repressed.(Camsund, Heidorn, & Lindblad, 2014)
[[RNA Polymerase]] (RNAP) and [[sigma factor|sigma factors]] are necessary proteins for [[transcription]] of DNA into [[messenger RNA]] (mRNA). Eubacterial RNAP [[holoenzymes]] consist of a core with four major subunits α2 ββ’. In cyanobacteria, β’ is formed from two smaller subunits (у and β’) , which corresponds to RNAPs in plant [[chloroplasts]].<ref name=imamura>{{cite journal|last1=Imamura|first1=S.|last2=Asayama|first2=M.|title=Sigma Factors for Cyanobacterial Transcription|journal=Gene Regulation and Systems Biology|date=2009|volume=3|pages=65-87}}</ref> The [[DNA clamp|beta subunits]] are responsible for binding the RNAP to the DNA, preventing premature dissociation. In Escherichia coli, the beta “clamp” binds loosely originally and tightens as the RNAP approaches the start codon (AUG). In cyanobacteria, the beta clamp binds tightly at initial binding. The effect of this difference is that synthetic repressible promoters do not function as expected in ''Synechocystis'' sp. PCC6803. In E. coli, a repressor binds the DNA operon and dislodges RNAP due to the loosely bound beta clamp. In ''Synechocystis'', the RNAP is tightly bound leading the reverse phenomenon where the repressor is knocked off the DNA. Thus the gene is not effectively repressed.<ref>{{cite journal|last1=Camsund|first1=Daniel|last2=Heidorn|first2=Thorsten|last3=Lindblad|first3=Peter|title=Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium|journal=Journal of Biological Engineering|date=2014|volume=8|issue=1|pages=4|doi=10.1186/1754-1611-8-4}}</ref>
''Synechocystis'' possesses the 70s sigma factor (σ70), which can be divided into three groups. Group 1 sigma factors are critical for cell viability. Group 2 is not essential for cell vitality, but is similar in structure to group 1. Group 3 is structurally different and involved with survival under stress conditions. Synechocystis 6803 lacks the σN factor found in other organisms, such as Escherichia coli, although it is able to metabolize nitrogen. The nitrogen sigma factor is involved with transcribing genes related to nitrogen.
''Synechocystis'' possesses the 70s [[sigma factor]] (σ70), which can be divided into three groups. Group 1 [[sigma factor|sigma factors]] are critical for cell viability. Group 2 is not essential for cell vitality, but is similar in structure to group 1. Group 3 is structurally different and involved with survival under stress conditions. ''Synechocystis'' sp. PCC6803 lacks the σN factor found in other organisms, such as Escherichia coli, although it is able to metabolize nitrogen. The nitrogen sigma factor is involved with transcribing genes related to nitrogen.<ref name=imamura />


==References==
==References==

Revision as of 21:16, 20 March 2015

Synechocystis sp. PCC6803
Scientific classification
Kingdom:
Bacteria
Phylum:
Cyanobacteria
Order:
Chroococcales
Genus:
Synechocystis
Species:
S. sp. PCC6803
Binomial name
Synechocystis sp.

Synechocystis is a genus of unicellular, freshwater cyanobacteria primarily represented by the strain Synechocystis sp. PCC6803. Synechocystis sp. PCC6803 is capable of both phototrophic growth by oxygenic photosynthesis in sunlight and heterotrophic growth by glycolysis and oxidative phosphorylation during dark periods.[1] It is able to effectively anticipate transitions of light and dark phases by using a circadian clock.[2]

Evolutionary History

Cyanobacteria are photosynthetic prokaryotes that have existed on Earth for the past 3 billion years. The ability of cyanobacteria to produce oxygen is what began the transition from a planet consisting of high levels of carbon dioxide and little oxygen, to what has been title the Great Oxygenation Event where large amounts of oxygen gas were produced. [3] Cyanobacteria have colonized a wide diversity of habitats, from fresh and salt water ecosystems, and most land environments.[4] Phylogenetically, Synechocystis branched off later in the cyanobacterial evolutionary tree.[5] Synechocystis, which is non-diazotrophic, is closely related to another model organism, Cyanothece ATCC 51442, which is a diazotroph.[6] Thus, is has been proposed that Synechocystis originally possessed the ability to fix nitrogen gas, but lost the genes required for a fully functioning nitrogen fixation (nif) gene cluster.[7]

Growth and use as a model organism

Cyanobacteria are model microorganisms for the study of photosynthesis, carbon and nitrogen assimilation, evolution of plant plastids, and adaptability to environmental stresses. Synechocystis sp. PCC6803 is one of the most highly studied types of cyanobacteria as it can grow both autotrophically or heterotrophically in the absence of light. It was isolated from a freshwater lake in 1968 and grows best between 32 and 38 degrees Celsius.[8] Synechocystis sp. PCC6803 can readily take up exogenous DNA, in addition to up taking DNA via electroporation, ultrasonic transformation and conjugation.[9] The photosynthetic apparatus is very similar to the one found in plants. This organism also exhibits phototactic movement.

Synechocystis sp. PCC6803 can be grown on either agar plates or in liquid culture. The most widely used culture medium is a BG-11 salt solution(Williams, 1988). The ideal pH is between 7 and 8.5.[1] A light intensity of 50 μmol photons m-2 s-1 leads to best growth.[1] Bubbling with carbon dioxide enriched air (1–2% CO2) can increase the growth rate, but may require the addition of a buffer to maintain pH[1]

Selection is typically performed by antibiotic resistance genes. Heidorn et al. 2011 experimentally determined in Synechocystis sp. PCC6803 the ideal concentrations of kanamycin, spectinomycin, streptomycin, chloramphenicol, erythromycin, and gentamycin.[1] Cultures can be kept on agar plates for approximately 2 weeks and re-streaked indefinitely(Williams, 1988). For long term storage, liquid cell culture should be store in a 15% glycerol solution at -80 degrees Celsius.[10]

Genome

The genome of Synechocystis sp. PCC6803 is contained within approximately 12 copies of a single chromosome (3.57 megabases), three small plasmids: pCC5.2 (5.2 kb) pCA2.4 (2.4 kb), and pCB2.4 (2.4 kb) and four large plasmids: pSYSM (120 kb), pSYSX (106 kb), pSYSA (103kb), and pSYSG (44 kb).[11][12]

Light-activated heterotrophy

Synechocystis sp. PCC6803 can live completely heterotrophically in the dark, but for yet unknown reasons requires a minimum of 5 to 15 minutes (blue) light per day. This regulatory role of light is intact in both PSI and PSII deficient strains.[13]

NDH-2 is a regulatory quinone:NAD(P)H oxidoreductase[14] global analysis of circadian gene expression indicates that translation genes are expressed at the early subjective day.[15]

Some glycolytic genes are regulated by sll1330 under light and glucose-supplemented conditions. One of the most important glycolytic genes is fructose-1,6-bisphosphate aldolase (fbaA). The mRNA level of fbaA is increased under light and glucose-supplemented conditions. But in Δsll1330, fbaA is not increased under same conditions.[16]

Additional Strains

The primary strain of Synechocystis sp. is PCC6803. Further modifications of the parent PCC6803 strain have been created, such as one lacking photosystem 1 (PSI).[17] The other widely used version of Synechocystis sp. is a glucose tolerant strain, ATCC 27184.[18]

Native CRISPR-Cas System

The CRISPR-Cas (Clustered Regularly Interspaced Short Palindrome Repeats – CRISPR associated proteins) system provides adaptive immunity in archaea and bacteria. Synechocystis sp. PCC6803 contains three different CRISPR-Cas systems: type I-D, and two versions of type III. All three CRISPR-Cas systems are localize on the pSYSA plasmid. All cyanobacteria are lacking the type II system, which has been widely adapted for genetic engineering purposes across many species.[19]

RNA Polymerase and Sigma Factors

RNA Polymerase (RNAP) and sigma factors are necessary proteins for transcription of DNA into messenger RNA (mRNA). Eubacterial RNAP holoenzymes consist of a core with four major subunits α2 ββ’. In cyanobacteria, β’ is formed from two smaller subunits (у and β’) , which corresponds to RNAPs in plant chloroplasts.[20] The beta subunits are responsible for binding the RNAP to the DNA, preventing premature dissociation. In Escherichia coli, the beta “clamp” binds loosely originally and tightens as the RNAP approaches the start codon (AUG). In cyanobacteria, the beta clamp binds tightly at initial binding. The effect of this difference is that synthetic repressible promoters do not function as expected in Synechocystis sp. PCC6803. In E. coli, a repressor binds the DNA operon and dislodges RNAP due to the loosely bound beta clamp. In Synechocystis, the RNAP is tightly bound leading the reverse phenomenon where the repressor is knocked off the DNA. Thus the gene is not effectively repressed.[21] Synechocystis possesses the 70s sigma factor (σ70), which can be divided into three groups. Group 1 sigma factors are critical for cell viability. Group 2 is not essential for cell vitality, but is similar in structure to group 1. Group 3 is structurally different and involved with survival under stress conditions. Synechocystis sp. PCC6803 lacks the σN factor found in other organisms, such as Escherichia coli, although it is able to metabolize nitrogen. The nitrogen sigma factor is involved with transcribing genes related to nitrogen.[20]

References

  1. ^ a b c d e Heidorn, T.; Camsund, D.; Huang, H.; Lindberg, P.; Oliveria, P.; Stensjo, K.; Lindblad, P. (2011). "Chapter Twenty-Four - Synthetic Biology in Cyanobacteria: Engineering and Analyzing Novel Functions". Methods in Enzymology. 497. Academic Press: 539–579. doi:10.1016/B978-0-12-385075-1.00024-X.
  2. ^ Dong, Guogang; Golden, Susan S (December 2008). "How a cyanobacterium tells time". Current Opinion in Microbiology. 11 (6): 541–546. doi:10.1016/j.mib.2008.10.003.
  3. ^ Wang, M.; Jiang, Y.-Y.; Kim, K. M.; Qu, G.; Ji, H.-F.; Mittenthal, J. E.; Zhang, H.-Y.; Caetano-Anolles, G. (30 August 2010). "A Universal Molecular Clock of Protein Folds and Its Power in Tracing the Early History of Aerobic Metabolism and Planet Oxygenation". Molecular Biology and Evolution. 28 (1): 567–582. doi:10.1093/molbev/msq232.
  4. ^ Whitton, B.A.; Potts, M. (2012). Ecology of Cyanobacteria II. pp. 1–13.
  5. ^ Bandyopadhyay, A.; Elvitigala, T.; Welsh, E.; Stockel, J.; Liberton, M.; Min, H.; Sherman, L. A.; Pakrasi, H. B. (4 October 2011). "Novel Metabolic Attributes of the Genus Cyanothece, Comprising a Group of Unicellular Nitrogen-Fixing Cyanobacteria". mBio. 2 (5): e00214-11–e00214-11. doi:10.1128/mBio.00214-11.
  6. ^ Bandyopadhyay, A.; Elvitigala, T.; Welsh, E.; Stockel, J.; Liberton, M.; Min, H.; Sherman, L. A.; Pakrasi, H. B. (4 October 2011). "Novel Metabolic Attributes of the Genus Cyanothece, Comprising a Group of Unicellular Nitrogen-Fixing Cyanobacteria". mBio. 2 (5): e00214-11–e00214-11. doi:10.1128/mBio.00214-11.
  7. ^ Turner, S.; Huang, T-C.; Chaw, S-M. (2001). "Molecular phylogeny of nitrogen-fixing unicellular cyanobacteria". Botanical Bulletin of Academia Sinica. 42: 181–186.
  8. ^ Červený, Jan; Sinetova, Maria; Zavřel, Tomáš; Los, Dmitry (2 March 2015). "Mechanisms of High Temperature Resistance of Synechocystis sp. PCC 6803: An Impact of Histidine Kinase 34". Life. 5 (1): 676–699. doi:10.3390/life5010676.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  9. ^ Marraccini, Pierre; Bulteau, St�phane; Cassier-Chauvat, Corinne; Mermet-Bouvier, Pierre; Chauvat, Franck (November 1993). "A conjugative plasmid vector for promoter analysis in several cyanobacteria of the genera Synechococcus and Synechocystis". Plant Molecular Biology. 23 (4): 905–909. doi:10.1007/BF00021546. {{cite journal}}: replacement character in |first2= at position 3 (help)
  10. ^ Williams, J.G.K. (1988). "Construction of specific mutations in photosystem II photosynthetic reaction center by genetic engineering methods in Synechocystis 6803". Methods in Enzymology. 167. Academic Press: 766–778.
  11. ^ Labarre, J.; Chauvat, F.; Thuriaux, P. (1989). "Insertional mutagenesis by random cloning of antibiotic resistance genes into the genome of the cyanobacterium Synechocystis strain PCC 6803". Journal of Bacteriology. 171: 3449–3457.
  12. ^ Kaneko, T. (1 January 2003). "Structural Analysis of Four Large Plasmids Harboring in a Unicellular Cyanobacterium, Synechocystis sp. PCC 6803". DNA Research. 10 (5): 221–228. doi:10.1093/dnares/10.5.221.
  13. ^ Anderson SL and McIntosh L (May 1991). "Light-activated heterotrophic growth of the cyanobacterium Synechocystis sp. strain PCC 6803: a blue-light-requiring process". J Bacteriol. 173 (9): 2761–2767. PMC 207855. PMID 1902208.
  14. ^ Howitt CA, Udall PK, and Vermaas WF (July 1999). "Type 2 NADH dehydrogenases in the cyanobacterium Synechocystis sp. strain PCC 6803 are involved in regulation rather than respiration". J Bacteriol. 181 (13): 3994–4003. PMC 93889. PMID 10383967.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  15. ^ Kucho K, Okamoto K, Tsuchiya Y, Nomura S, Nango M, Kanehisa M, and Ishiura M (March 2005). "Global analysis of circadian expression in the cyanobacterium Synechocystis sp. strain PCC 6803". J Bacteriol. 187 (6): 2190–2199. doi:10.1128/JB.187.6.2190-2199.2005. PMC 1064041. PMID 15743968.{{cite journal}}: CS1 maint: multiple names: authors list (link)
  16. ^ Yusuke Tabei, Katsuhiko Okada and Mikio Tsuzuki (April 2007). "Sll1330 controls the expression of glycolytic genes in Synechocystis sp. PCC 6803". Biochem. Biophys. Res. Commun. 355 (4): 1045–1050. doi:10.1016/j.bbrc.2007.02.065. PMID 17331473.
  17. ^ Shen, G.; Boussiba, S.; Vermaas, W.F. (1993). "Synechocystis sp PCC 6803 strains lacking photosystem I and phycobilisome function". The Plant Cell. 5 (12): 1853–1863.
  18. ^ Huang, Hsin-Ho; Lindblad, Peter (2013). "Wide-dynamic-range promoters engineered for cyanobacteria". Journal of Biological Engineering. 7 (1): 10. doi:10.1186/1754-1611-7-10.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  19. ^ Scholz, Ingeborg; Lange, Sita J.; Hein, Stephanie; Hess, Wolfgang R.; Backofen, Rolf; de Crécy-Lagard, Valerie (18 February 2013). "CRISPR-Cas Systems in the Cyanobacterium Synechocystis sp. PCC6803 Exhibit Distinct Processing Pathways Involving at Least Two Cas6 and a Cmr2 Protein". PLoS ONE. 8 (2): e56470. doi:10.1371/journal.pone.0056470.{{cite journal}}: CS1 maint: unflagged free DOI (link)
  20. ^ a b Imamura, S.; Asayama, M. (2009). "Sigma Factors for Cyanobacterial Transcription". Gene Regulation and Systems Biology. 3: 65–87.
  21. ^ Camsund, Daniel; Heidorn, Thorsten; Lindblad, Peter (2014). "Design and analysis of LacI-repressed promoters and DNA-looping in a cyanobacterium". Journal of Biological Engineering. 8 (1): 4. doi:10.1186/1754-1611-8-4.{{cite journal}}: CS1 maint: unflagged free DOI (link)

Databases

  • SynechoNET: integrated protein-protein interaction database of a model cyanobacterium Synechocystis sp. PCC 6803. SynechoNET is a specialized cyanobacterial protein-protein interaction database. It shows feasible cyanobacterial domain-domain interactions, as well as their protein level interactions using the model cyanobacterium, Synechocystis sp. PCC 6803. Additionally, SynechoNET provides transmembrane topology and domain information, as well as the interaction networks in graphical web interfaces.
  • CyanoBase: Cyanobacteria carry a complete set of genes for oxygenic photosynthesis, which is the most fundamental life process on the earth. This organism is also interesting from an evolutional viewpoint, for it was born in a very ancient age and has survived in various environments. Chloroplast is believed to have evolved from cyanobacterial ancestors which developed an endosymbiontic relationship with a eukaryotic host cell. CyanoBase provides an easy way of accessing the sequences and all-inclusive annotation data on the structures of the cyanobacterial genomes. This database was originally developed by Makoto Hirosawa, Takakazu Kaneko and Satoshi Tabata, and the current version of CyanoBase has been developed and maintained by Yasukazu Nakamura, Takakazu Kaneko, and Satoshi Tabata at Kazusa DNA Research Institute.
  • STRING: STRING is a database of known and predicted protein-protein interactions.The interactions include direct (physical) and indirect (functional) associations; they are derived from four sources: Genomic Context, High-throughpot Experiments, (Conserved) Coexpression, and Previous Knowledge. The database currently contains 1,513,782 proteins in 373 species. Especially, the database provides interactions for Synechocystis sp. PCC 6803.
  • cTFbase: cTFbase contains 1288 putative trascription factors (TFs) identified from 21 fully sequenced cyanobacterial genomes. Through its user-friendly interactive interface, users can employ various criteria to retrieve all TF sequences and their detailed annotation information, including sequence features, domain architecture and sequence similarity against the linked databases. Furthermore, cTFbase also provides phylogenetic trees of individual TF family, multiple sequence alignments of the DNA-binding domain and ortholog identification from any selected genomes.
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