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CyanobacteriaSynechococcus sp. PCC 7002 projectBackground BackgroundThe organismThe cyanobacterium Synechococcus sp. PCC 7002 (formerly known as Agmenellum quadruplicatum strain PR-6; American Type Culture Collection strain 27167) was originally isolated in 1961 by Chase Van Baalen from an onshore, marine mud sample derived from fish pens on Magueyes Island, La Parguera, Puerto Rico (Van Baalen, 1962; Rippka et al., 1979).
It grows in brackish (euryhaline) and/or marine water. The organism is unicellular or forms short filaments of two to four cells at temperatures near the optimal temperature for growth, 38˚C. The cells are 1.5-2.5 µm in size and lack phycoerythrin or phycoerythrocyanin. Synechococcus sp. PCC 7002 is facultatively photoheterotrophic with glycerol as substrate, has an obligate requirement for vitamin B12, and is naturally transformable. It is among the fastest growing of all cyanobacteria, with a doubling time under optimal conditions (light intensity =~ 250 µE m-2 s-1, nitrogen source is urea, 2% (v/v) CO2) of about 3.5 hours. The strain is extremely tolerant of high light intensities and has been grown at light intensities as high as 5000 µE m-2 s-1.
The Photosynthetic ApparatusA schematic diagram of the photosynthetic apparatus of Synechococcus sp. PCC 7002 is shown below. The phycobilisomes of this species contain only phycocyanin and allophycocyanin. Although predominantly associated with Photosystem II, energy transfer to Photosystem I also occurs and is very important under certain light regimes. A comprehensive analysis of the genes for components of the photosynthetic and respiratory electron transport chains indicates that the electron transport chains of Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 are similar, although a few specific differences are found. For example, Synechococcus sp. PCC 7002 does not contain a cytochrome bd-type quinol oxidase or plastocyanin as does Synechocystis sp. PCC 6803. Nevertheless, Synechococcus sp. PCC 7002 has complete photosynthetic and respiratory electron transport chains housed in its thylakoid membranes. Genome Sequencing and AnnotationGenome AssemblyThe sequencing and assembly of the genome of this organism is now complete. Primary sequence data were collected from a combination of 45,935 reads from whole-genome shotgun sequencing, fosmid end sequences, reads from an interposon mutant library, and reads from targeted sequencing of cosmid and BAC libraries. Several methods, including fosmid end-sequence mapping, primer walking, T-linker PCR and BlastX-based open reading frame (ORF) analyses, were used to close gaps between contigs in assembly. The completed genome of Synechococcus sp. PCC 7002 is composed of one circular chromosomal DNA molecular (3008047bp) and six circular plasmids: pAQ1 (4,809 bp), pAQ3 (16,076 bp), pAQ4 (32,037 bp), pAQ5 (38,515 bp), pAQ6 (124,029 bp), and pAQ7 (186,451 bp).
Genome Annotation3299 predicted ORFs have been annotated for the genome by Manatee annotation system (TIGR), in which 2827 ORFs, two sets of rRNA genes cluster and 42 tRNA genes have been annotated onto the chromosome. All the ORFs have been reviewed manually.
Molecule’s Copy numberQuantitative Real-Time PCR has been used to determine the copy numbers of the chromosome and the plasmids on a cellular basis. The results show that pAQ6 and pAQ7 are present at the same copy number as the chromosomal DNA molecule. The estimated copy number for each molecule per cell in exponential phase is chromosome:pAQ1:pAQ3:pAQ4:pAQ5:pAQ6:pAQ7 = 7:40:31:17:11:7:6 at OD550 nm = 0.7 and about half this number of molecules per cell at OD550 nm = 3.0
Photosystem I, structure, function and biogenesisIn cyanobacteria and plants, the Photosystem I (PS I) reaction center is a membrane-bound, multisubunit complex, which functions as the light-driven plastocyanin (or cytochrome c6)/ferredoxin (or flavodoxin) oxidoreductase (see Figure 1). Collaborating with Dr. John Golbeck’s group (link), we are focusing our studies on the structural and functional relationships of this biological light-energy converter, on the biogenesis and assembly of the PS I complexes in thylakoid membranes, and on the physical and biochemical properties of PS I cofactors in PS I-mediated electron transfer processes.
Cyanobacteria have served as model systems in elucidating structure and function relationships for the PS I reaction center. Two cyanobacterial strains are used in our studies: Synechococccus sp. PCC 7002 and Synechocystis sp. PCC 6803. The PS I complex of cyanobacteria is structurally and functionally equivalent to the PS I core complex of higher plants. However, the cyanobacterial strains used in our lab are naturally transformable, which makes them suitable for many genetic manipulations. Their capacity for photoheterotrophic growth in presence of an external carbon source (either glycerol or glucose, respectively) allows these bacteria to live in the absence of most photosynthetic genes. The greater stability of trimeric PS I complexes, especially from Synechococcus sp. PCC 7002, genomic sequencing and the improved PS I X-ray structure (see Figure 2) provide other compelling reasons for using these cyanobacterial strains as model systems for our studies on PS I.
The biogenesis of photosynthetic complexes in cyanobacteria and higher plant chloroplasts is a complex process that includes apoprotein translation, protein folding and insertion into thylakoid membranes, chlorophyll a and carotenoid binding, iron-sulfur cluster assembly, and ordered association of the individual subunits to form multisubunit complexes. Our research has primarily focused on regulation and biogenesis of PS I in the thylakoids, especially efforts to identify the cofactor and pigment-binding polypeptides required for maturation of complexes. Comparative genomic analyses and gene-targeted mutagenesis have identified several genes (e.g., rubA in Figure 3) involved in the biogenesis and regulation of photosystems in cynobacteria.
Fe/S cluster biogenesisFe-S clusters serve as cofactors in many proteins that have essential physiological functions. The biosynthesis of Fe/S clusters is a multistep process that requires multiple gene products to mediate assembly of these complex metal factors. In cyanobacteria, genes for two major systems of Fe/S cluster biogenesis (named SUF and ISC) have been identified in the genomes of several sequenced cyanobacteria. In collaboration with Dr. John Golbeck’s group, we are studying the functions of genes involved in the regulation and assembly of Fe-S clusters in cyanobacteria by functional genomics. Our research has focused on the following questions:
Through reverse genetics, we have shown that the SUF system, composed of SufB, C, D, S and E, serves as the essential machinery for Fe/S cluster biogenesis in cyanobacteria. Results from mutagenesis studies demonstrate that SufR functions as a transcriptional repressor of the suf regulon in response to redox and oxidative stress, and Nfu, but not SufA or IscA, is the essential Fe/S scaffold protein in cyanobacteria. Very recently, we have successfully demonstrated Nfu-dependent Fe/S cluster transfer to apo-PsaC in a reaction that rapidly reconstitutes Photosystem I function. The physiological competence and gene expression profiles of sufA and iscA mutant strains suggest that SufA and IscA may play regulatory roles in iron homeostasis, redox poise, and the capacity for Fe/S cluster biogenesis in cyanobacterial cells. Figure 4 shows a working model that summarizes our present knowledge of Fe/S biogenesis and regulation in cyanobacteria.
Phycobiliprotein biogenesisPhycobilisomes are macromolecular light harvesting structures used by cyanobacteria to harvest light for photosynthesis (see Figure 1). Although the production of these structures is a major expense for the cells, the investment pays off by allowing the cells to exploit light environments in which they would otherwise not be able to grow. When producing phycobilisomes, cyanobacteria carefully monitor environmental variables such as light wavelength, intensity, and nutrient status in order to assemble phycobilisomes that are finely tuned to utilize available resources most efficiently. Research into the complex process of phycobilisome assembly has shown that there are many steps at which such regulation can occur. Recently our lab, in collaboration with Dr. Wendy Schluchter’s lab, have continued this line of investigation by exploring the mechanisms of attachment of linear tetrapyrrole chromophores to the phycobiliproteins. Phycobilisomes are composed of two classes of proteins: the pigmented phycobiliproteins and the mostly non-pigmented linker proteins. The linker proteins have structural roles in phycobilisome assembly and provide a scaffold for assembly of the structure as well as locational information that places the colored proteins in their appropriate positions. They also fine-tune the energy transfer properties of some of the chromophores of the phycobiliproteins. Phycobiliproteins are the major light-harvesting proteins in cyanobacteria, red algae, cryptomonads and glaucophytes. These proteins owe their brilliant colors and visible light-harvesting capabilities to specific linear tetrapyrrole chromophores covalently bound to specific binding sites on the protein. One or more linear tetrapyrrole chromophores, known as phycobilins, are bound to the phycobiliprotein subunits by cysteinyl thioether linkages. Chromophores are attached to these cysteine residues by a class of proteins known as phycobilin lyases. Our lab has recently discovered new lyases that function to attach phycocyanobilin chromophores (see Figure 2) to the beta subunits of the phycocyanin and the alpha and beta subunits of allophycocyanin (see Figure 3). Along with the previously identified CpeEF heterodimer, which encodes a phycocyanobilin lyase specific for the phycocyanin alpha subunit, our lab has now identified, and has mutants defective in, (See Figure 4) the complete set of lyases required for phycocyanin and allophycocyanin assembly in Synechococcus sp. PCC 7002.
Many cyanobacteria also utilize the phycobiliprotein known as phycoerythrin to enhance their absorption profile in the green regions of the visible light spectrum. Phycoerythrin binds the chromophore phycoerythrobilin (see Figure 2) giving cells with this type of phycobiliprotein a reddish coloring. We have chosen Synechococcus sp. PCC 7335 as our model organism for our investigation of the phycoerythrin lyases (see Figure 5). Unlike Synechococcus sp. PCC 7002, this cyanobacterium is capable of producing phycoerythrin in response to the wavelength of light in its environment. Through a process known as complementary chromatic adaptation, this organism dramatically changes its absorption profile in response to red or green light in its environment. It becomes red when grown in green light and green when grown in red light. Although they remain largely uncharacterized, it is known that several phycoerythrin-specific lyases are utilized by this organism to ensure the proper attachment of phycoerythroblin to phycoerythrin during growth in green light. As part of our continued research into phycobiliprotein biogenesis we are working to determine the full set of lyases involved in phycoerythrin chromophorylation in this organisim.
All phycobiliprotein beta subunits (e.g., CpcB, ApcB, and ApcF) are modified by methylation of the gamma-nitrogen of Asn72 (see Figure 3). We have recently identified the gene, cpcM, encoding the phycobiliprotein Asn72 methyltransferase. Mutants lacking CpcM are more sensitive to high light intensity than wild-type cells and are unable to perform state transitions. Carotenoid BiosynthesisCyanobacterial carotenoids are tetraterpenoid (C-40) compounds with poly-ene chromophores. Carotenoids are powerful antioxidants that play essential roles in photoprotection, phototrophic light capture, conversion, and sensing. Mutants lacking the ability to synthesize carotenoids are not viable in the presence of even modest light intensities in the presence of oxygen. Although cyanobacteria only produce a few types of chlorophylls, including Chl a, Chl b, Chl d and a few derivatives, a rich diversity of carotenoids has been isolated and described. In spite of the known structural diversity of these compounds, the biosynthesis of most of these compounds have remained partially defined or completely unknown. There is still no cyanobacterium for which the entire carotenoid biosynthetic pathway has been fully described. We have recently sought to correct this deficiency by defining the biosynthetic pathway for carotenoids in Synechococcus sp. PCC 7002. As shown in Figure 1, Synechococcus sp. PCC 7002 produces seven carotenoids that accumulate to significant amounts during standard exponential growth: b-carotene, zeaxanthin, cryptoxanthin, echinenone, hydroxy-echinenone, myxoxanthophyll, and a newly discovered aromatic carotenoid, synechoxanthin. The branching pathway that leads to the production of these compounds is quite complex, but all of the intermediates shown have been detected and characterized. The enzymes responsible for two conversions, the 2’-hydroxylating enzyme in the myxoxanthophyll pathway and the anticipated dehydrogenase(s) in the synechoxanthin pathways, remain to be identified.
Synechoxanthin, c,c-caroten-18,18’-dioic acid, is the first aromatic carotenoid to be documented in cyanobacteria. The structure of the purified compound (Figure 2) has been determined by NMR in collaboration with Dr. Juliette Lecomte. In stationary phase, this extremely polar carotenoid accumulates in large amounts in the cell wall and the external growth medium (Figure 3). The absorption spectrum under these conditions suggests that synechoxanthin should be highly effective as a sunscreen agent to protect cells from harmful UV-A illumination. In future studies we will characterize the physiology of mutants lacking specific carotenoids and we will determine the transcriptional regulation of the genes encoding the enzymes of this complex pathway.
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