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.     

Sll1330 controls the expression of glycolytic genes in Synechocystis sp. PCC 6803

In the complete annotated genome sequences of cyanobacterium Synechocystis sp. PCC 6803, one can find many putative genes for two-component response regulators that include a helix-turn-helix DNA-binding domain. The mRNA level of one of the putative genes, sll1330, was increased by glucose, especially in the presence of light. We successfully disrupted the sll1330 gene by targeted mutagenesis with a spectinomycin resistance cassette. Deltasll1330 could not grow well under light-activated heterotrophic growth conditions. Analyses of the expression of glycolytic genes revealed that the mRNA levels of five glycolytic genes, that is, glk (sll0593), pfkA (sll1196), fbaA (sll0018), gpmB (slr1124), and pk (sll0587), were decreased, and were regulated by Sll1330 under light and glucose-supplemented conditions. The Synechocystis sp. PCC 6803 genome each encodes two isozymes for these five glycolytic genes, suggesting that each of the two isozymes is regulated by Sll1330 at the mRNA level.     

Polymerase chain reaction-based mutageneses identify key transporters belonging to multigene families involved in Na+ and pH homeostasis of Synechocystis sp. PCC 6803

Primary ion pumps and antiporters exist as multigene families in the Synechocystis sp. PCC 6803 genome and show very strong homologies to those found in higher plants. The gene knock-outs of five putative Na+/H+ antiporters (slr1727, sll0273, sll0689, slr1595 and slr0415) and seven cation ATPases (sll1614, sll1920, slr0671-72, slr0822, slr1507-08-09, slr1728- 29 and slr1950) in the model cyanobacterium (http://www.kazusa.or.jp/cyano/cyano.html) were performed in this study relying on homologous recombination with mutagenenic fragments constructed using a fusion polymerase chain reaction (PCR) approach. The impacts of these gene knock-outs were evaluated in terms of Na+ and pH, and light-induced acidification and alkalization that are asso-ciated with inorganic carbon uptake. Two of the five putative antiporter mutants exhibit a characteristic interplay between the pH and Na+ dependence of growth, but only one of the antiporters appears to be necessary for high NaCl tolerance. On the other hand, the mutation of one of the two copper-trafficking ATPases produces a cell line that shows acute NaCl sensitivity. Additionally, disruptions of a putative Ca2+-ATPase and a gene cluster encoding a putative Na+-ATPase subunit also cause high NaCl sensitivity. The findings and possible mechanisms are discussed in relation to the potential roles of these transporters in Synechocystis sp. PCC 6803.     

Slr1122影响Hik12磷酸化并参与调节Synechocystis sp.PCC 6803的色素合成

Synechocystis sp.PCC 6803是一种良好的研究光合作用的模式生物,其中slr1122编码一个250个氨基酸的未知蛋白。据报道Slr1122可能与杂合传感激酶（hybrid sensory kinase）Sll1672（Hik12）相互作用,本研究通过复合物实验证实了Slr1122与Sll1672确实存在相互作用。利用32P标记证明,在加入Slr1122后Hik12的磷酸化受到了明显的影响,推测其可能参与该双组分系统的调控。通过同源双交换,用卡那霉素抗性基因替换slr1122,将slr1122从Synechocystis sp.PCC 6803中敲除,构建了slr1122的缺失体Δslr1122。研究发现在Δslr1122中,编码PSⅡ中核心蛋白D1亚基的slr1181（psbAI）的转录水平明显降低,使PSⅡ光合作用受到影响,导致Δslr1122的生长速率低于野生型（WT）。同时slr1122的缺失使得蓝细菌对光的敏感性增强,在弱光条件下,Δslr1122对光能的利用效率高于WT,其生长速率也较WT高,但与此相反,Δslr1122对强光的耐受力及生长速率则不及WT。Δslr1122体内的藻胆蛋白含量与色素含量均降低,尤其是类胡萝卜素,RT-PCR的结果也显示合成类胡萝卜素过程中的5个关键酶转录水平均下降。这可能是Δslr1122对氧化胁迫变得敏感的原因之一。总之,Slr1122影响杂合传感激酶Hik12磷酸化并参与调节Synechocystis sp.PCC 6803的光合色素合成。     

Chorismate Pyruvate-Lyase and 4-Hydroxy-3-solanesylbenzoate Decarboxylase Are Required for Plastoquinone Biosynthesis in the Cyanobacterium Synechocystis sp. PCC6803

Plastoquinone is a redox active lipid that serves as electron transporter in the bifunctional photosynthetic-respiratory transport chain of cyanobacteria. To examine the role of genes potentially involved in cyanobacterial plastoquinone biosynthesis, we have focused on three Synechocystis sp. PCC 6803 genes likely encoding a chorismate pyruvate-lyase (sll1797) and two 4-hydroxy-3-solanesylbenzoate decarboxylases (slr1099 and sll0936). The functions of the encoded proteins were investigated by complementation experiments with Escherichia coli mutants, by the in vitro enzyme assays with the recombinant proteins, and by the development of Synechocystis sp. single-gene knock-out mutants. Our results demonstrate that sll1797 encodes a chorismate pyruvate-lyase. In the respective knock-out mutant, plastoquinone was hardly detectable, and the mutant required 4-hydroxybenzoate for growth underlining the importance of chorismate pyruvate-lyase to initiate plastoquinone biosynthesis in cyanobacteria. The recombinant Slr1099 protein displayed decarboxylase activity and catalyzed in vitro the decarboxylation of 4-hydroxy-3-prenylbenzoate with different prenyl side chain lengths. In contrast to Slr1099, the recombinant Sll0936 protein did not show decarboxylase activity regardless of the conditions used. Inactivation of the sll0936 gene in Synechocystis sp., however, caused a drastic reduction in the plastoquinone content to levels very similar to those determined in the slr1099 knock-out mutant. This proves that not only slr1099 but also sll0936 is required for plastoquinone synthesis in the cyanobacterium. In summary, our data demonstrate that cyanobacteria produce plastoquinone exclusively via a pathway that is in the first reaction steps almost identical to ubiquinone biosynthesis in E. coli with conversion of chorismate to 4-hydroxybenzoate, which is then prenylated and decarboxylated.     

Nucleotide sequence, transcriptional analysis, and expression of genes encoded within the form I CO2 fixation operon of Rhodobacter sphaeroides

In Rhodobacter sphaeroides, many of the structural genes encoding enzymes of the Calvin cycle are duplicated and grouped within two separate clusters. In this study, the nucleotide sequence of a 5627-base pair region of DNA that contains the form I Calvin cycle gene cluster has been determined. The five open reading frames are arranged in the order, fbpA prkA cfxA rbcL rbcS and are tightly linked and oriented in the same direction. The results of insertional mutagenesis studies suggest the genes are organized within an operon. Consistent with this proposal, the cfxA gene has been tentatively identified as a gene encoding the Calvin cycle enzyme, aldolase. Measurement of the activities of various Calvin cycle enzymes in the insertion mutants showed that inactivation of genes within one CO2 fixation cluster affected expression of genes within the second cluster, revealing a complex regulatory network.     

The Effect of Various Stresses on the Expression of Genes Encoding the Secreted Proteins of the Cyanobacterium Synechocystis sp. PCC 6803

The effects of various stresses (osmotic, salt, low-temperature, high-temperature, and high-light stress) on the amount of mRNA of eight genes encoding the secreted proteins of Synechocystis sp. PCC 6803 were studied. Osmotic stress (0.5 M sorbitol) reduced the amount of all mRNAs, with the exception of slr0924. Supposedly, this gene encodes Tic22, a polypeptide involved in the formation of the transport system for proteins crossing the internal thylakoid membrane on the way to the lumen. Salt stress (0.5 M NaCl) inhibited the expression of all genes for secreted proteins almost completely. Low temperature (20°C) did not affect the expression of the sll1891 gene of an unknown function and the slr0924 gene. The high temperature (44°C) suppressed the expression of all genes tested. A detailed study of the expression of the sll1694 (pilA1) gene, which encodes the main structural protein of cyanobacterial pili, pilin PilA1, demonstrated that virtually all stresses suppressed its expression. Thus, various stresses were shown to suppress the expression of most genes encoding Synechocystis secreted proteins.     

Dissecting the Photoprotective Mechanism Encoded by the flv4‐2 Operon: a Distinct Contribution of Sll0218 in Photosystem II Stabilization

In Synechocystis sp. PCC 6803, the flv4‐2 operon encodes the flavodiiron proteins Flv2 and Flv4 together with a small protein, Sll0218, providing photoprotection for Photosystem II (PSII). Here, the distinct roles of Flv2/Flv4 and Sll0218 were addressed, using a number of flv4‐2 operon mutants. In the ?sll0218 mutant, the presence of Flv2/Flv4 rescued PSII functionality as compared with ?sll0218‐flv2, where neither Sll0218 nor the Flv2/Flv4 heterodimer are expressed. Nevertheless, both the ?sll0218 and ?sll0218‐flv2 mutants demonstrated deficiency in accumulation of PSII proteins suggesting a role for Sll0218 in PSII stabilization, which was further supported by photoinhibition experiments. Moreover, the accumulation of PSII assembly intermediates occurred in Sll0218‐lacking mutants. The YFP‐tagged Sll0218 protein localized in a few spots per cell at the external side of the thylakoid membrane, and biochemical membrane fractionation revealed clear enrichment of Sll0218 in the PratA‐defined membranes, where the early biogenesis steps of PSII occur. Further, the characteristic antenna uncoupling feature of the ?flv4‐2 operon mutants is shown to be related to PSII destabilization in the absence of Sll0218. It is concluded that the Flv2/Flv4 heterodimer supports PSII functionality, while the Sll0218 protein assists PSII assembly and stabilization, including optimization of light harvesting.     

Identification of a glucokinase that generates a major glucose phosphorylation activity in the cyanobacterium Synechocystis sp. PCC 6803

In silico analysis of genome of the cyanobacterium Synechocystis sp. PCC 6803 identified two genes, slr0329 and sll0593, that might participate in glucose (Glc) phosphorylation (www.kazusa.or.jp/cyano). In order to determine the functions of these two genes, we generated deletion mutants, and analyzed their phenotypes and enzymatic activities. In the presence of 10 mM Glc, wild-type (WT) and slr0329 defective strain (M1) grew fast with increased respiratory activity and NADPH production, whereas the sll0593 deletion mutant (M2) failed to show any of the Glc responses. WT and M1 were not significantly different in their glucokinase activity, but M2 had 90% less activity. Therefore, we propose that Sll0593 plays a major role in the phosphorylation of glucose in Synechocystis cells.     

Mutational analysis of genes involved in pilus structure, motility and transformation competency in the unicellular motile cyanobacterium Synechocystis sp. PCC 6803

The relevance of pilus-related genes to motility, pilus structure on the cell surface and competency of natural transformation was studied by gene disruption analysis in the unicellular motile cyanobacterium SYNECHOCYSTIS: sp. PCC 6803. The genes disrupted in this study were chosen as related to the pil genes for biogenesis of the type IV pili in a Gram-negative bacterium PSEUDOMONAS: aeruginosa. It was found that motility of SYNECHOCYSTIS: cells was lost in the mutants of slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 together with a simultaneous loss of the thick pili on the cell surface. Competency of the natural transformation was lost in the mutants listed above and slr0197-disruptant. The gene slr0197 was previously predicted as a competence gene by a search with sequence-independent DNA-binding structure [Yura et al. (1999) DNA Res. 6: 75]. It was suggested that both DNA uptake for natural transformation and motility are mediated by a specific type IV-like pilus structure, while a putative DNA-binding protein encoded by slr0197 is additionally required for the DNA uptake. Based on the homology with the pil genes in P: aeruginosa, slr0063, slr1274, slr1275, slr1276, slr1277 and sll1694 were designated pilB1, pilM, pilN, pilO, pilQ and pilA1, respectively. The gene slr0197 was designated comA.