利用同源重组构建蓝藻集胞藻6803ndhO基因突变株及其分子鉴定

蓝藻NADPH脱氢酶(NDH-1)是一种重要的光合膜蛋白复合体,参与CO2吸收、围绕光系统I的循环电子传递和细胞呼吸。迄今为止,人们在蓝藻细胞中已鉴定出15种NDH-1复合体亚基(NdhA-NdhO)。然而,人们对NdhO亚基的研究尚不够,至今未见有反向遗传学等方面的研究。在通过构建同源重组载体、自然转化和多次继代筛选后,对转化子进行了PCR和蛋白免疫印迹鉴定。结果表明,卡那霉素基因已成功地插入到ndhO基因的保守区域,并完全破坏了ndhO基因的蛋白表达,从而获得了ndhO基因缺失的突变株,为进一步研究NdhO亚基对NDH-1复合体的稳定性和生理功能等奠定了实验基础。     

Mutants of Cyanobacterium Synechocystis sp. PCC 6803 with Insertional Inactivation of the sodB Gene Encoding Fe-Superoxide Dismutase

The sodB gene encoding the only superoxide dismutase (Fe-SOD) in cells of the cyanobacterium Synechocystis sp. PCC 6803 was inactivated with gentamycin resistance aacC1 marker insertions located in the direct or reverse direction in the sodB gene. The corresponding sodB12 and sodB22 mutants are characterized by the complete absence of superoxide dismutase activity and the loss of viability upon standard photoautotrophic cultivation. Mutant cells can grow under conditions of a decreased illumination intensity and upon addition of NaHCO₃ with catalase or bovine serum albumin in the growth medium. The sodB22 mutant is auxotrophic for leucine due to the polar effect of insertion into the sodB gene on the downstream leuB gene controlling leucine biosynthesis. These data suggest that Fe-SOD is very important for providing resistance of Synechocystis 6803 cells to oxidative stress and thatsodB and leuB genes are organized into a single operon.     

蓝细菌6803agp基因的分子克隆和缺失突变株的构建

PCR扩增了蓝细菌集胞藻6803(Synechocystis sp.PCC6803)的agp基因（编码ADP－葡萄糖焦磷酸羧化酶）,进一步以pUC118为载体将其克隆到大肠杆菌中,构建了pUCA质粒。通过DNA体外重组,以红霉素抗性基因部分取代agp基因片段,构建了既含agp基因上游及下游序列、又携带选择性标记－红霉素抗性的pUCAE质粒。该质粒转化野生型集胞藻6803细胞,获得了能在含红霉素的培养基上正常生长的agp基因缺失突变株。对该突变株基因组DNA进行PCR扩增,验邝了其基因结构的正确性。突变株细胞生长速度较野生型细胞快,胞内的叶绿素含量比野生型细胞高,表明该突变株具有较高的光合效率。在突变株中未检测到糖原的存在,进一步从生理水平上验证了突变株构建的正确性。     

Molecular Cloning and Construction of agp Gene Deletion-mutant in Cyanobacterium Synechocystis sp. PCC 6803

Nine compounds were isolated from Elsholtzia blanda (Benth.) Benth. Their　structures were identified with spectral and chemical methods as follows: 5,6-dihydro-6-styry-2-pyrone (1), friedelin (2), 4-hydroxy-3-methoxystyrene (3), 5,2′-dimethoxy-6,7-methylene dioxyflavanone (4), 5-hydroxy-7-methoxy-6-O-［α- L -rhamnopyranosyl(1→2)-β- D -fucopyranosyl］ flavone glycoside (5), 5,5′-dihydroxy-7-acetoxyl-6,8,3″,3″-tetramethylpyran　(3′,4′)　flavone (6), 5,5′-dihydroxy-7-(α-methyl) butyroxyl-6,8,3″,3″-tetramethylpyran (3′,4′) flavone (7), 5,5′-dihydroxy-6,7-methylenedioxy-8,3″,3″-trimethylpyran　(3′,4′)　flavone (8), glucosyringic acid (9). Among them, 6, 7 and 8 are new compounds, named as sifanghaoine Ⅰ,Ⅱ and Ⅲ, respectively.     

Functional Characterization of the slr1944 Gene of Cyanobacterium Synechocystis sp. PCC 6803

The properties of Slr1944 protein encoded by the slr1944 gene and participating in the metabolism of lipophilic compounds in a cyanobacterium Synechocystis were under study. Located in the periplasm, this protein comprises a conserved pentapeptide G-X-S-X-G characteristic of lipases, acetylcholinesterases, and thioesterases. An attempt to delete the gene from the cyanobacterial genome failed; this fact presumes an essential function of Slr1944 protein under the optimum growth conditions. Expression of the slr1944 gene in Escherichia coli cells demonstrated a high affinity of the product for lipophilic compounds. An enhanced slr1944 expression deprived Synechocystis cells of the ability to restore the activity of the photosynthetic electron-transport chain following photoinactivation. The authors believe that Slr1944 participates in the biogenesis of the lipophilic components of photosynthetic complexes.     

r{beta}Carotene Hydroxylase Gene from the Cyanobacterium Synechocystis sp. PCC6803

The ORF sll1468 of Synechocystis sp. PCC6803 was identifiedas a gene for rß-carotene hydroxylase by functionalcomplementation in a rß-carotene-producing Escherichiacoll. The gene product of ORF sll11468 added hydroxyl groupsto the rß-ionone rings of rß-carotene (rß,rß-carotene)to form zeaxanthin (rß,rß-carotene-3,3'-diol).This newly identified rß-carotene hydroxylase doesnot show overall amino acid sequence similarity to the knownrß-carotene hydroxylases. However, it showed significantsequence similarity to rß-carotene ketolases of marinebacteria and a green alga. (Received November 29, 1997; Accepted March 6, 1998)     

Ethylene Regulates the Physiology of the Cyanobacterium Synechocystis sp. PCC 6803 via an Ethylene Receptor

Ethylene is a plant hormone that plays a crucial role in the growth and development of plants. The ethylene receptors in plants are well studied, and it is generally assumed that they are found only in plants. In a search of sequenced genomes, we found that many bacterial species contain putative ethylene receptors. Plants acquired many proteins from cyanobacteria as a result of the endosymbiotic event that led to chloroplasts. We provide data that the cyanobacterium Synechocystis (Synechocystis sp. PCC 6803) has a functional receptor for ethylene, Synechocystis Ethylene Response1 (SynEtr1). We first show that SynEtr1 directly binds ethylene. Second, we demonstrate that application of ethylene to Synechocystis cells or disruption of the SynEtr1 gene affects several processes, including phototaxis, type IV pilus biosynthesis, photosystem II levels, biofilm formation, and spontaneous cell sedimentation. Our data suggest a model where SynEtr1 inhibits downstream signaling and ethylene inhibits SynEtr1. This is similar to the inverse-agonist model of ethylene receptor signaling proposed for plants and suggests a conservation of structure and function that possibly originated over 1 billion years ago. Prior research showed that SynEtr1 also contains a light-responsive phytochrome-like domain. Thus, SynEtr1 is a bifunctional receptor that mediates responses to both light and ethylene. To our knowledge, this is the first demonstration of a functional ethylene receptor in a nonplant species and suggests that that the perception of ethylene is more widespread than previously thought.Ethylene is a gaseous hormone that influences the growth and development of plants (Abeles et al., 1992). The signal transduction pathway for ethylene has been studied predominantly in the flowering plant Arabidopsis (Arabidopsis thaliana), but research on plant species from more ancient lineages suggests that ethylene signaling probably evolved in plants prior to the colonization of land (Rensing et al., 2008; Banks et al., 2011; Gallie, 2015; Ju et al., 2015).In plants, the perception of ethylene is mediated by a family of receptors that contain a conserved N-terminal transmembrane ethylene-binding domain consisting of three transmembrane α-helices with seven conserved amino acids required for the binding of ethylene (Schaller and Bleecker, 1995; Wang et al., 2006). Several of these amino acids are believed to coordinate a copper cofactor required for ethylene binding (Rodríguez et al., 1999). These receptors have homology to bacterial two-component receptors that function via His autophosphorylation, followed by transfer of this phosphate to an Asp residue on a downstream response regulator protein (Chang et al., 1993). Plants acquired many proteins from cyanobacteria as a result of an endosymbiotic event approximately 1.5 billion years ago that led to chloroplasts (Yoon et al., 2004). Because of the endosymbiotic gene transfer that occurred, it has been proposed that components of several two-component-like receptors in plants, such as ethylene receptors and phytochromes, were acquired from the cyanobacterium that gave rise to the chloroplasts of plants (Kehoe and Grossman, 1996; Martin et al., 2002; Mount and Chang, 2002; Timmis et al., 2004; Schaller et al., 2011).Phytochrome-like receptors (Vierstra and Zhang, 2011), but not ethylene receptors, have been characterized in nonplant species. Some cyanobacterial species have saturable ethylene-binding sites and contain genes predicted to encode proteins with ethylene-binding domains (Rodríguez et al., 1999; Wang et al., 2006), but the distribution and function of ethylene receptors in bacteria are unknown. In a search of sequenced genomes, we found that genes encoding putative ethylene receptors are found in diverse bacterial species. One of these genes, slr1212, is in the model cyanobacterium Synechocystis (Synechocystis sp. PCC 6803). We previously showed that disruption of this gene eliminates ethylene-binding activity in Synechocystis, leading to the speculation that it encodes an ethylene-binding protein (Rodríguez et al., 1999). This gene, called Synechocystis Ethylene Response1 (SynEtr1), as originally designated by Ulijasz et al. (2009) because of its putative role as an ethylene receptor, also has been called Positive Phototaxis A (Narikawa et al., 2011) and UV Intensity Response Sensor (Song et al., 2011), because of its role in light signaling. Despite these observations, there has been no research published that demonstrates that SynEtr1 directly binds ethylene or functions as an ethylene receptor.We focused on SynEtr1 to determine whether it is a functional ethylene receptor. Expression of the N-terminal portion of SynEtr1 in P. pastoris led to the generation of ethylene-binding sites, demonstrating that this region of the protein directly binds ethylene. Treatment of Synechocystis with ethylene or disruption of SynEtr1 caused measurable changes in physiology, including faster movement toward light, slower cell sedimentation, enhanced biofilm production, a larger number of type IV pili, and higher levels of PSII. Additionally, SynEtr1-deficient Synechocystis cells transformed with a mutant SynEtr1 that cannot bind ethylene do not respond to ethylene. Our research demonstrates that SynEtr1 is an ethylene receptor and, in the context of prior research (Ulijasz et al., 2009; Narikawa et al., 2011; Song et al., 2011), likely functions as a dual input receptor for both light and ethylene. To our knowledge, this is the first report of a functional ethylene receptor in a cyanobacterium, making it the first ethylene receptor characterized in a nonplant species.     

The Insertional Inactivation of Genes Encoding Eukaryotic-Type Serine/Threonine Protein Kinases in the Cyanobacterium Synechocystis sp. PCC 6803

Synechocystis sp. PCC 6803 mutants, in which one of the eukaryotic-type serine/threonine protein kinase genes pknD, pknE, pknG, and pknH was inactivated, were obtained by insertion mutagenesis. None of these mutants differed phenotypically from the wild-type strain, indicating that the pknD, pknE, pknG, and pknHgenes are not of crucial importance for the photoautotrophically grown cyanobacterium. The mutant with the inactivatedpknE gene was resistant to L-methionine-D,L-sulfoximine and especially to methylamine. The resistance was due neither to the impaired transport of these compounds nor to the inhibition of the production of toxic -glutamylmethylamide from methylamine. The data presented suggest that resistance to methylamine may be associated with alterations in the regulation of the glutamine synthetase system and that the PknE protein kinase may be involved in the regulation of nitrogen metabolism in the cyanobacterium studied.     

Complete Genome Structure of the Unicellular Cyanobacterium Synechocystis sp. PCC6803

Cyanobacteria are photoautotrophic organisms capable of oxygen-producingphotosynthesis similar to that in eukaryotic algae and plants,and because of this, they have been used as model organismsfor the study of the mechanism and regulation of oxygen-producingphotosynthesis. To understand the entire genetic system in cyanobacteria,the nucleotide sequence of the entire genome of the unicellularcyanobacterium Synechocystis sp. PCC6803 has been determined.The total length of the circular genome is 3,573,470 bp, witha GC content of 47.7%. A total of 3,168 potential protein codinggenes were assigned. Of these, 145 (4.6%) were identical toreported genes, and 1,259 (39.6%) and 342 (10.8%) showed similarityto reported and hypothetical genes, respectively. The remaining1,422 (45.0%) showed no apparent similarity to any genes registeredin the databases. Classification of the genes by their biologicalfunction and comparison of the gene complement with those ofother organisms have revealed a variety of features of the geneticinformation characteristic of a photoautotrophic organism. Thesequence data, as well as other information on the Synechocystisgenome, is presented in CyanoBase on WWW [http://www.kazusa.or.jp/cyano/]. (Received July 24, 1997; Accepted September 17, 1997)     

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.     

Effect of Gravity Changes on the Cyanobacterium Synechocystis sp. PCC 6803

The impact of hypergravity and simulated weightlessness were studied to check whether cyanobacteria perceive changes of gravity as stress. Hypergravity generated by a low-speed centrifuge increased slightly the overall activity of dehydrogenases, but the increase was the same for 90 g and 180 g. The protein pattern did not show qualitative alterations during hypergravity treatment up to 180 g. Cells of Synechocystis PCC 6803 subjected to common stressors like salt, heat, and light clearly accumulated at least four general stress proteins (25, 31, 34, and 63 kDa, respectively). Three of these proteins could also be detected after hypergravity, but in such small amounts that their occurrence could only be taken as a weak indication of stress. Low-molecular-weight stress metabolites were not synthesized in response to hypergravity, indicating that this gravity change was unable to activate the osmotic signal transduction chain. Gravity-dependent alterations were observed only during simulated weightlessness (generated by a fast-rotating clinostat). The glutamate/glutamine ratio was significantly shifted toward a higher glutamine portion. Altogether, the results may indicate that moderate changes of gravity were hardly, if ever, sensed as stress by cyanobacteria. Received: 20 May 1997 / Accepted: 25 June 1997     

Genomic Responses to Arsenic in the Cyanobacterium Synechocystis sp. PCC 6803

Arsenic is a ubiquitous contaminant and a toxic metalloid which presents two main redox states in nature: arsenite [As^III] and arsenate [As^V]. Arsenic resistance in Synechocystis sp. strain PCC 6803 is mediated by the arsBHC operon and two additional arsenate reductases encoded by the arsI1 and arsI2 genes. Here we describe the genome-wide responses to the presence of arsenate and arsenite in wild type and mutants in the arsenic resistance system. Both forms of arsenic produced similar responses in the wild type strain, including induction of several stress related genes and repression of energy generation processes. These responses were transient in the wild type strain but maintained in time in an arsB mutant strain, which lacks the arsenite transporter. In contrast, the responses observed in a strain lacking all arsenate reductases were somewhat different and included lower induction of genes involved in metal homeostasis and Fe-S cluster biogenesis, suggesting that these two processes are targeted by arsenite in the wild type strain. Finally, analysis of the arsR mutant strain revealed that ArsR seems to only control 5 genes in the genome. Furthermore, the arsR mutant strain exhibited hypersentivity to nickel, copper and cadmium and this phenotype was suppressed by mutation in arsB but not in arsC gene suggesting that overexpression of arsB is detrimental in the presence of these metals in the media.     

Cloning and Inactivation of a Gene Essential to Inorganic Carbon Transport of Synechocystis PCC6803

A clone (HP-1) which transforms the high CO₂-requiring mutant (RKb) of Synechocystis PCC6803 defective in inorganic carbon transport to the wild-type (WT) phenotype was isolated from a WT genomic library. The clone contained a 5.4 kilobase-pair DNA insert. Complementation tests with subclones derived from HP-1 allowed the mutation in RKb to be located within 141 base-pair nucleotides. Sequencing of nucleotides in this region revealed an open reading frame encoding a hydrophobic protein consists of 80 amino acids. A defined mutant (M9) constructed by inactivating this putative inorganic carbon transport gene, designated ictA, was unable to transport CO₂ and HCO₃⁻ into the intracellular inorganic carbon pool. Cloning and sequence analysis of the respective RKb gene revealed a base substitution which generates a stop codon in the middle of ictA.