Transfer and replication of RSF1010-derived plasmids in several cyanobacteria of the generaSynechocystis andSynechococcus

RSF1010-derived plasmids are most efficiently transferred by conjugation to the unicellular cyanobacteriaSynechocystis strains sp. PCC6803 and PCC6714 andSynechococcus strains sp. PCC7942 and PCC6301, where they replicate autonomously, even though they contain no cyanobacterial DNA. These results are especially important in the case of the facultative heterotrophic strainSynechocystis PCC6714, which is not transformable [Mol Gen Genet 204:185, 1986]     

Transformation and gene replacement in the facultatively chemoheterotrophic,unicellular cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC6714 by electroporation

The unicellular cyanobacterium Synechocystis sp. PCC6714 can grow not only under photoautotrophic conditions, but also under chemoheterotrophic conditions if glucose is added to the medium. This makes it useful for the study of many aspects of bioenergetic mechanisms. In contrast to its closely related strain Synechocystis sp. PCC6803, which cannot grow chemoheterotrophically, Synechocystis PCC6714 is not naturally transformable. To enable gene transfer in this strain, we established a method for the introduction of self-replicating IncQ plasmids and for gene replacement using electroporation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Effects of fatty acid activation on photosynthetic production of fatty acid-based biofuels in Synechocystis sp. PCC6803

Background

Direct conversion of solar energy and carbon dioxide to drop in fuel molecules in a single biological system can be achieved from fatty acid-based biofuels such as fatty alcohols and alkanes. These molecules have similar properties to fossil fuels but can be produced by photosynthetic cyanobacteria.

Results

Synechocystis sp. PCC6803 mutant strains containing either overexpression or deletion of the slr1609 gene, which encodes an acyl-ACP synthetase (AAS), have been constructed. The complete segregation and deletion in all mutant strains was confirmed by PCR analysis. Blocking fatty acid activation by deleting slr1609 gene in wild-type Synechocystis sp. PCC6803 led to a doubling of the amount of free fatty acids and a decrease of alkane production by up to 90 percent. Overexpression of slr1609 gene in the wild-type Synechocystis sp. PCC6803 had no effect on the production of either free fatty acids or alkanes. Overexpression or deletion of slr1609 gene in the Synechocystis sp. PCC6803 mutant strain with the capability of making fatty alcohols by genetically introducing fatty acyl-CoA reductase respectively enhanced or reduced fatty alcohol production by 60 percent.

Conclusions

Fatty acid activation functionalized by the slr1609 gene is metabolically crucial for biosynthesis of fatty acid derivatives in Synechocystis sp. PCC6803. It is necessary but not sufficient for efficient production of alkanes. Fatty alcohol production can be significantly improved by the overexpression of slr1609 gene.     

Computational protein structure modeling and analysis of UV-B stress protein in Synechocystis PCC 6803

This study focuses on Ultra Violet stress (UVS) gene product which is a UV stress induced protein from cyanobacteria, Synechocystis PCC 6803. Three dimensional structural modeling of target UVS protein was carried out by homology modeling method. 3F2I pdb from Nostoc sp. PCC 7120 was selected as a suitable template protein structure. Ultimately, the detection of active binding regions was carried out for characterization of functional sites in modeled UV-B stress protein. The top five probable ligand binding sites were predicted and the common binding residues between target and template protein was analyzed. It has been validated for the first time that modeled UVS protein structure from Synechocystis PCC 6803 was structurally and functionally similar to well characterized UVS protein of another cyanobacterial species, Nostoc sp PCC 7120 because of having same structural motif and fold with similar protein topology and function. Investigations revealed that UVS protein from Synechocystis sp. might play significant role during ultraviolet resistance. Thus, it could be a potential biological source for remediation for UV induced stress.     

Genome sequences published outside of Standards in Genomic Sciences, October �C November 2011

The purpose of this table is to provide the community with a citable record of publications of ongoing genome sequencing projects that have led to a publication in the scientific literature. While our goal is to make the list complete, there is no guarantee that we may have omitted one or more publications appearing in this time frame. Readers and authors who wish to have publications added to subsequent versions of this list are invited to provide the bibliographic data for such references to the SIGS editorial office.

• Phylum Crenarchaeota
• Thermoproteus tenax, strain Kra1, DSM 2078^T sequence accession {"type":"entrez-nucleotide","attrs":{"text":"FN869859","term_id":"350274033"}}FN869859 [1]
• Phylum Euryarchaeota
• Haloarcula hispanica CGMCC 1.2049, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002921","term_id":"343781660"}}CP002921 (chromosome I), {"type":"entrez-nucleotide","attrs":{"text":"CP002922","term_id":"343784702"}}CP002922 (chromosome II), and {"type":"entrez-nucleotide","attrs":{"text":"CP002923","term_id":"343785159"}}CP002923 (plasmid pHH400) [2]
• Methanococcus maripaludis, strain X1 (unculturable) sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002913","term_id":"339903450"}}CP002913 [3]
• Phylum Proteobacteria
• Acinetobacter baumannii strain 1656-2, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP001921","term_id":"322506180"}}CP001921 [4]
• Arcobacter butzleri strain ED-1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AP012047","term_id":"345468266"}}AP012047, {"type":"entrez-nucleotide","attrs":{"text":"AP012048","term_id":"345470425"}}AP012048, and {"type":"entrez-nucleotide","attrs":{"text":"AP012049","term_id":"345473271"}}AP012049 [5]
• Brucella suis strain 1330, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002997","term_id":"343381878"}}CP002997 and {"type":"entrez-nucleotide","attrs":{"text":"CP002998","term_id":"343384001"}}CP002998 [6]
• Campylobacter fetus subsp. venerealis NCTC 10354, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFGH01000000","term_id":"342326654"}}AFGH01000000 [7]
• “Chromobacterium sp.” strain C-61, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CAEE01000001","term_id":"344197715"}}CAEE01000001 to {"type":"entrez-nucleotide","attrs":{"text":"CAEE01001118","term_id":"344196598"}}CAEE01001118 [8]
• Cronobacter sakazakii strain E899, sequence accession {"type":"entrez-protein","attrs":{"text":"AFMO00000000","term_id":"333956885"}}AFMO00000000 [9]
• “Desulfovibrio sp.” strain A2, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGFG01000000","term_id":"347520603"}}AGFG01000000 [10]
• “Erythrobacter sp.” strain NAP1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"NZ_AAMW00000000","term_id":"85710750"}}NZ_AAMW00000000 [11]
• Escherichia coli strain XH140A, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFVX01000000","term_id":"342364931"}}AFVX01000000 [12]
• Escherichia coli strain XH001, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFYG01000000","term_id":"344195679"}}AFYG01000000 [13]
• Haemophilus haemolyticus strain {"type":"entrez-nucleotide","attrs":{"text":"M19107","term_id":"1059793989"}}M19107, sequence accession {"type":"entrez-protein","attrs":{"text":"AFQN00000000","term_id":"341956667"}}AFQN00000000 [14]
• Haemophilus haemolyticus strain {"type":"entrez-nucleotide","attrs":{"text":"M19501","term_id":"147422"}}M19501, sequence accession {"type":"entrez-protein","attrs":{"text":"AFQO00000000","term_id":"341951174"}}AFQO00000000 [14]
• Haemophilus haemolyticus strain {"type":"entrez-nucleotide","attrs":{"text":"M21127","term_id":"1015602694"}}M21127, sequence accession {"type":"entrez-protein","attrs":{"text":"AFQP00000000","term_id":"341951709"}}AFQP00000000 [14]
• Haemophilus haemolyticus strain {"type":"entrez-nucleotide","attrs":{"text":"M21621","term_id":"189206"}}M21621, sequence accession {"type":"entrez-protein","attrs":{"text":"AFQQ00000000","term_id":"341955662"}}AFQQ00000000 [14]
• Haemophilus haemolyticus strain {"type":"entrez-nucleotide","attrs":{"text":"M21639","term_id":"1036032350"}}M21639, sequence accession {"type":"entrez-protein","attrs":{"text":"AFQR00000000","term_id":"341956942"}}AFQR00000000 [14]
• Idiomarina sp.” strain A28L, sequence accession {"type":"entrez-nucleotide-range","attrs":{"text":"AFPO01000001 to AFPO01000028","start_term":"AFPO01000001","end_term":"AFPO01000028","start_term_id":"336283061","end_term_id":"336280888"}}AFPO01000001 to AFPO01000028 [15]
• Ketogulonicigenium vulgare” strain WSH-001, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002018","term_id":"343461404"}}CP002018 (chromosome), {"type":"entrez-nucleotide","attrs":{"text":"CP002019","term_id":"343464001"}}CP002019 (plasmid pKVU_100), and {"type":"entrez-nucleotide","attrs":{"text":"CP002020","term_id":"343464245"}}CP002020 (plasmid pKVU_200) [16]
• Methylobacter tundripaludum strain SV96, sequence accession {"type":"entrez-protein","attrs":{"text":"AEGW00000000","term_id":"344263587"}}AEGW00000000 [17]
• Pseudogulbenkiania sp.” strain NH8B, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AP012224","term_id":"345641016"}}AP012224 [18]
• Pseudomonas aeruginosa NCGM1179, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"DF126593","term_id":"346055306"}}DF126593 through {"type":"entrez-nucleotide","attrs":{"text":"DF126613","term_id":"346061544"}}DF126613 [19]
• Pseudomonas putida strain B001, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CAED01000001","term_id":"344196578"}}CAED01000001 to {"type":"entrez-nucleotide","attrs":{"text":"CAEE01000262","term_id":"344197454"}}CAEE01000262 [20]
• Pseudomonas putida strain B6-2, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGCS01000000","term_id":"1026254431"}}AGCS01000000 [21]
• Pseudomonas stutzeri CGMCC 1.1803, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002881","term_id":"338799449"}}CP002881 [22]
• Ralstonia solanacearum phylotype IB, strain Y45, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFWL01000000","term_id":"343176764"}}AFWL01000000 [23]
• Rheinheimera sp.” strain A13L, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFHI01000001","term_id":"335881665"}}AFHI01000001 through {"type":"entrez-nucleotide","attrs":{"text":"AFHI01000072","term_id":"335877953"}}AFHI01000072 [24]
• Sphingobium yanoikuyae strain XLDN2-5, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFXE01000000","term_id":"345099327"}}AFXE01000000 [25]
• Vibrio cholerae strain Amazonia, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFSV01000000","term_id":"341584524"}}AFSV01000000 [26]
• Phylum Firmicutes
• Bacillus coagulans strain XZL4, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFWM01000000","term_id":"343381451"}}AFWM01000000 [27]
• Bacillus megaterium strain WSH-002, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003017","term_id":"345441867"}}CP003017 (chromosome), plasmids {"type":"entrez-nucleotide","attrs":{"text":"CP003018","term_id":"345447048"}}CP003018 (plasmid pBME_100), {"type":"entrez-nucleotide","attrs":{"text":"CP003019","term_id":"345447118"}}CP003019 (plasmid pBME_200), and {"type":"entrez-nucleotide","attrs":{"text":"CP003020","term_id":"345447130"}}CP003020 (plasmid pBME_300) [28]
• Bacillus pumilus strain S-1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGBY00000000","term_id":"346722060"}}AGBY00000000 [29]
• “Desulfosporosinus sp.” strain OT, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGAF01000000","term_id":"344330753"}}AGAF01000000 [30]
• Lentibacillus jeotgali strain Grbi, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGAV01000000","term_id":"345115115"}}AGAV01000000 [31]
• Leuconostoc carnosum KCTC 3525, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"BACM01000000","term_id":"338736686"}}BACM01000000 [32]
• Listeria ivanovii subsp. ivanovii strain PAM 55, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"FR687253","term_id":"346980581"}}FR687253 [33]
• Paenibacillus riograndensis strain SBR5, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGBD01000000","term_id":"345128217"}}AGBD01000000 [34]
• Sporolactobacillus inulinus strain CASD, sequence accession {"type":"entrez-protein","attrs":{"text":"AFVQ00000000","term_id":"823094357"}}AFVQ00000000 [35]
• Streptococcus pseudopneumoniae strain IS7493, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002925","term_id":"341932553"}}CP002925 and {"type":"entrez-nucleotide","attrs":{"text":"CP002926","term_id":"341934784"}}CP002926 [36]
• Streptococcus salivarius strain 57.I, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002888","term_id":"339291081"}}CP002888 and {"type":"entrez-nucleotide","attrs":{"text":"CP002889","term_id":"339293023"}}CP002889 [37]
• Streptococcus salivarius strain M18, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGBV01000000","term_id":"345528121"}}AGBV01000000 [38]
• Streptococcus suis SS12, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002640","term_id":"353733193"}}CP002640 [39]
• Streptococcus suis D9, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002641","term_id":"353735273"}}CP002641 [39]
• Streptococcus suis D12, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002644","term_id":"353737348"}}CP002644 [39]
• Streptococcus suis ST1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002651","term_id":"353739427"}}CP002651 [39]
• Weissella thailandensis strain fsh4-2, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"HE575133","term_id":"341819811"}}HE575133 through {"type":"entrez-nucleotide","attrs":{"text":"HE575182","term_id":"341821297"}}HE575182 [40]
• Phylum Tenericutes
• Mycoplasma anatis strain 1340, sequence accession {"type":"entrez-protein","attrs":{"text":"AFVJ00000000","term_id":"341579585"}}AFVJ00000000 [41]
• Mycoplasma capricolum subsp. capripneumoniae strain M1601, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AENG01000000","term_id":"1067112011"}}AENG01000000 [42]
• Mycoplasma putrefaciens Type strain KS1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003021","term_id":"343956693"}}CP003021 [43]
• Corynebacterium pseudotuberculosis strain PAT10, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP002924","term_id":"341823823"}}CP002924 [44]
• Phylum Actinobacteria
• Bifidobacterium animalis subsp. lactis strain BLC1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP003039","term_id":"447219159"}}CP003039 [45]
• Bifidobacterium breve strain DPC 6330, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFXX01000000","term_id":"347344070"}}AFXX01000000 [46]
• Brachybacterium squillarum strain M-6-3, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGBX01000000","term_id":"346317973"}}AGBX01000000 [47]
• “Citricoccus sp.” strain CH26A, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFXQ01000000","term_id":"344198218"}}AFXQ01000000 [48]
• Corynebacterium glutamicum strain S9114, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFYA01000000","term_id":"344046353"}}AFYA01000000 [49]
• Dietzia alimentaria strain 72, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGFF01000000","term_id":"347366987"}}AGFF01000000 [50]
• Mycobacterium colombiense CECT 3035, sequence accession {"type":"entrez-protein","attrs":{"text":"AFVW00000000","term_id":"400333858"}}AFVW00000000 [51]
• Mycobacterium tuberculosis NCGM2209, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"DF126614","term_id":"358229918"}}DF126614 and {"type":"entrez-nucleotide","attrs":{"text":"DF126615","term_id":"358233644"}}DF126615 [52]
• Rhodococcus erythropolis strain XP, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGCF01000000","term_id":"345896954"}}AGCF01000000 [53]
• Serinicoccus profundi MCCC 1A05965^T, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFYF00000000","term_id":"345107103"}}AFYF00000000 [54]
• Phylum Spirochaetes
• Leptospira interrogans, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP001221","term_id":"353456277"}}CP001221 (CI), {"type":"entrez-nucleotide","attrs":{"text":"CP001222","term_id":"353459699"}}CP001222 (CII) [55]
• Phylum Bacteroidetes
• Bacteroides faecis Type strain MAJ27^T, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AGDG01000000","term_id":"347014739"}}AGDG01000000 [56]
• Bizionia argentinensis, Type strain JUB59^T sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFXZ01000000","term_id":"344049519"}}AFXZ01000000 [57]
• Flavobacterium branchiophilum strain FL-15, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"FQ859183","term_id":"345528129"}}FQ859183 [58]
• “Flavobacteriaceae” strain S85, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"AFPK00000000","term_id":"339778866"}}AFPK00000000 [59]
• Phylum Thermotogae
• “Thermotoga sp.” strain RQ2, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"CP000969","term_id":"170175310"}}CP000969 [60]

Non-Bacterial genomes

• Aspergillus kawachii IFO 4308, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"DF126447","term_id":"358365263"}}DF126447 through {"type":"entrez-nucleotide","attrs":{"text":"DF126592","term_id":"358376882"}}DF126592, BACL01000001 through BACL01001641, {"type":"entrez-nucleotide","attrs":{"text":"AP012272","term_id":"354548779"}}AP012272 [61]
• Cajanus cajan pigeonpea, sequence accession PRJNA72815 [62]
• Coxsackievirus A22, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JN542510","term_id":"354682107"}}JN542510 [63]
• Gordonia phage GRU1, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JF923797","term_id":"356471393"}}JF923797 [64]
• Gordonia phage GTE5, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JF923796","term_id":"356471300"}}JF923796 [64]
• Heterocephalus glaber naked mole rat, sequence accession {"type":"entrez-protein","attrs":{"text":"AFSB00000000","term_id":"351716052"}}AFSB00000000, {"type":"entrez-nucleotide","attrs":{"text":"AFSB01000000","term_id":"351581530"}}AFSB01000000 [65]
• Human Adenovirus Prototype 17, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"HQ910407","term_id":"324105303"}}HQ910407 [66]
• Macaca mulatta lasiota rhesus macaque, sequence accession {"type":"entrez-protein","attrs":{"text":"AEHL00000000","term_id":"355786683"}}AEHL00000000 [67]
• Macaca mulatta mulatta rhesus macaque, sequence accession {"type":"entrez-protein","attrs":{"text":"AEHK00000000","term_id":"355710505"}}AEHK00000000 [67]
• Porcine epidemic diarrhea virus, sequence accession {"type":"entrez-nucleotide","attrs":{"text":"JN547228","term_id":"351630105"}}JN547228 [68]

     

Degradation of Phycobilisomes in Synechocystis sp. PCC6803: EVIDENCE FOR ESSENTIAL FORMATION OF AN NblA1/NblA2 HETERODIMER AND ITS CODEGRADATION BY A Clp PROTEASE COMPLEX

When cyanobacteria acclimate to nitrogen deficiency, they degrade their large (3–5-MDa), light-harvesting complexes, the phycobilisomes. This massive, yet specific, intracellular degradation of the pigmented phycobiliproteins causes a color change of cyanobacterial cultures from blue-green to yellow-green, a process referred to as chlorosis or bleaching. Phycobilisome degradation is induced by expression of the nblA gene, which encodes a protein of ∼7 kDa. NblA most likely acts as an adaptor protein that guides a Clp protease to the phycobiliproteins, thereby initiating the degradation process. Most cyanobacteria and red algae possess just one nblA-homologous gene. As an exception, the widely used “model organism” Synechocystis sp. PCC6803 expresses two such genes, nblA1₆₈₀₃ and nblA2₆₈₀₃, both of whose products are required for phycobilisome degradation. Here, we demonstrate that the two NblA proteins heterodimerize in vitro and in vivo using pull-down assays and a Förster energy-transfer approach, respectively. We further show that the NblA proteins form a ternary complex with ClpC (the HSP100 chaperone partner of Clp proteases) and phycobiliproteins in vitro. This complex is susceptible to ATP-dependent degradation by a Clp protease, a finding that supports a proposed mechanism of the degradation process. Expression of the single nblA gene encoded by the genome of the N₂-fixing, filamentous cyanobacterium Nostoc sp. PCC7120 in the nblA1/nblA2 mutant of Synechocystis sp. PCC6803 induced phycobilisome degradation, suggesting that the function of the NblA heterodimer of Synechocystis sp. PCC6803 is combined in the homodimeric protein of Nostoc sp. PCC7120.     

Genomic Structure of the Cyanobacterium Synechocystis sp. PCC 6803 Strain GT-S

Synechocystis sp. PCC 6803 is the most popular cyanobacterial strain, serving as a standard in the research fields of photosynthesis, stress response, metabolism and so on. A glucose-tolerant (GT) derivative of this strain was used for genome sequencing at Kazusa DNA Research Institute in 1996, which established a hallmark in the study of cyanobacteria. However, apparent differences in sequences deviating from the database have been noticed among different strain stocks. For this reason, we analysed the genomic sequence of another GT strain (GT-S) by 454 and partial Sanger sequencing. We found 22 putative single nucleotide polymorphisms (SNPs) in comparison to the published sequence of the Kazusa strain. However, Sanger sequencing of 36 direct PCR products of the Kazusa strains stored in small aliquots resulted in their identity with the GT-S sequence at 21 of the 22 sites, excluding the possibility of their being SNPs. In addition, we were able to combine five split open reading frames present in the database sequence, and to remove the C-terminus of an ORF. Aside from these, two of the Insertion Sequence elements were not present in the GT-S strain. We have thus become able to provide an accurate genomic sequence of Synechocystis sp. PCC 6803 for future studies on this important cyanobacterial strain.     

Molecular genetics of the cyanobacteriumSynechocystis sp. PCC 6803: Principles and possible biotechnology applications

The cyanobacteriumSynechocystis sp. PCC 6803 is readily amenable to targeted mutagenesis: Foreign DNA is taken up spontaneously, and after uptake DNA can be integrated into the organism's genome by homologous recombination. Using appropriate DNA constructs for transformation, specific genes in the organism can be interrupted, deleted, or replaced by modified gene copies. The organism can grow under a number of different conditions, ranging from photoautotrophic to fully heterotrophic modes, making genetic modifications that alter fundamental processes such as photosynthesis and/or respiration feasible. For example, deletion of photosystem I leads to an obligate (photo)heterotrophic strain in which photosystem II-generated electrons appear to be consumed by respiratory processes, whereas deletion of photosystem II leads to an obligate (photo)heterotrophic strain in which cyclic electron flow around photosystem I appears to remain active. A major advantage ofSynechocystis sp. PCC 6803 is that its entire genome has been sequenced (by S. Tabata and co-workers), opening many avenues to address basic and applied research problems. For example, genes can be introduced, modified or deleted, and hypotheses regarding the function of an open reading frame can be tested by deletion of this open reading frame. Methods to modify genes are numerous. In addition to site-directed mutagenesis, novel molecular genetic approaches including targeted random mutagenesis, combinatorial mutagenesis and introduction of hybrid genes have come of age and have proven to be very powerful tools in protein engineering. These approaches have been utilized primarily in this strain to study photosynthesis, but applications of this technology, including pathway engineering, alterations of substrate specificity of enzymes and introduction of tolerance to a variety of stresses, are equally feasible in relation to more applied aims. For optimal utilization of the potential of theSynechocystis sp. PCC 6803 system, however, an increased emphasis toward understanding the biochemistry and molecular physiology of cyanobacteria will also be critically important.     

Increase in the rate of photosynthetic linear electron transport in cyanobacteruim <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 lacking phycobilisomes

Compensating changes in the pigment apparatus of photosynthesis that resulted from a complete loss of phycobilisomes (PBS) were investigated in the cells of a PAL mutant of cyanobacterium Synechocystis sp. PCC 6803. The ratio PBS/chlorophyll calculated on the basis of the intensity of bands in the action spectra of photosynthetic activity of two photosystems in the wild strain was 1: 70 for PSII and 1: 300 for PSI. Taking into consideration the number of chlorophyll molecules per reaction center in each photosystem, these ratios could be interpreted as association of PBS with dimers of PSII and trimers of PSI as well as greater dependence of PSII as compared with PSI on light absorption by PBS. The ratio PSI/PSII determined by photochemical cross-section of the reactions of two photosystems was 3.5: 1.0 for wild strain of Synechocystis sp. PCC 6803 and 0.7: 1.0 for the PAL mutant. A fivefold increase in the relative content of PSII in pigment apparatus corresponds to a 5-fold increase in the intensity of bands at 685 and 695 nm as related to the band of PSI at 726 nm recorded in low-temperature fluorescence spectrum of the PAL mutant. Inhibition of PSII with diuron resulted in a pronounced stimulation of chlorophyll fluorescence in the PAL mutant as compared to the wild strain of Synechocystis sp. PCC 6803; these data suggested an activation of electron transfer between PSII and PSI in the mutant cells. Thus, the lack of PBS in the mutant strain of Synechocystis sp. PCC 6803 was compensated for by the higher relative content of PSII in the pigment apparatus of photosynthesis and by a rise in the rate of linear electron transport.     

The atp1 and atp2 operons of the cyanobacterium Synechocystis sp. PCC 6803

The two operons atp1 and atp2, encoding the subunits of the F_OF₁ ATP-synthase, have been cloned and sequenced from the cyanobacterium Synechocystis sp. PCC 6803. The organization of the different genes in the operons have been found to resemble that of the cyanobacteria Synechococcus sp. PCC 6301 and Anabaena sp. PCC 7120. The Synechocystis F_OF₁ ATP-synthase has nine subunits. A tenth open reading frame with unknown function was detected at the 5 end of atp1, coding for a putative gene product similar to uncI in Escherichia coli.A promoter structure was inferred for the Synechocystis atp operons and compared to other known promoters of cyanobacteria. Even though the operon structure of atp1 and atp2 in Synechocystis resembles the corresponding operons of Synechococcus, the amino acid sequences of individual gene products show marked differences. Genetic distances between cyanobacterial genes and genes for ATP-synthase subunits from other species have been calculated and compiled into evolutionary trees.     

Growth arrest of Synechocystis sp. PCC6803 by superoxide generated from heterologously expressed Rhodobacter sphaeroides chlorophyllide a reductase

The photosynthetic growth of Synechocystis sp. PCC6803 ceased upon expression of Rhodobacter sphaeroides chlorophyllide a reductase (COR). However, an increase in cytosolic superoxide dismutase level in the recombinant Synechocystis sp. PCC6803 completely reversed the growth cessation. This demonstrates that COR generates superoxide in Synechocystis sp. PCC6803. Considering the dissolved oxygen (DO) level suitable for COR, the intracellular DO of this oxygenic photosynthetic cell appears to be low enough to support COR-mediated superoxide generation. The growth arrest of Synechocystis sp. PCC6803 by COR may give an insight into the evolutionary path from bacteriochlorophyll a biosynthetic pathway to chlorophyll a, which bypasses COR reaction.     

Characterization of a two-component signal transduction system involved in the induction of alkaline phosphatase under phosphate-limiting conditions in Synechocystis sp. PCC 6803

The gene products of sll0337 and slr0081 in Synechocystis sp. PCC 6803 have been identified as the homologues of the Escherichia coli phosphate-sensing histidine kinase PhoR and response regulator PhoB, respectively. Interruption of sll0337, the gene encoding the histidine protein kinase, by a spectinomycin-resistance cassette blocked the induction of alkaline phosphatase activity under phosphate-limiting conditions. A similar result was obtained when slr0081, the gene encoding the response regulator, was interrupted with a cassette conferring resistance to kanamycin. In addition, the phosphate-specific transport system was not up-regulated in our mutants when phosphate was limiting. Unlike other genes for bacterial phosphate-sensing two-component systems, sll0337 and slr0081 are not present in the same operon. Although there are three assignments for putative alkaline phosphatase genes in the Synechocystis sp. PCC 6803 genome, only sll0654 expression was detected by northern analysis under phosphate limitation. This gene codes for a 149 kDa protein that is homologous to the cyanobacterial alkaline phosphatase reported in Synechococcus sp. PCC 7942 [Ray, J.M., Bhaya, D., Block, M.A. and Grossman, A.R. (1991) J. Bact. 173: 4297–4309]. An alignment identified a conserved 177 amino acid domain that was found at the N-terminus of the protein encoded by sll0654 but at the C-terminus of the protein in Synechococcus sp. PCC 7942.