Degradation of PsbO by the Deg Protease HhoA Is Thioredoxin Dependent

The widely distributed members of the Deg/HtrA protease family play an important role in the proteolysis of misfolded and damaged proteins. Here we show that the Deg protease rHhoA is able to degrade PsbO, the extrinsic protein of the Photosystem II (PSII) oxygen-evolving complex in Synechocystis sp. PCC 6803 and in spinach. PsbO is known to be stable in its oxidized form, but after reduction by thioredoxin it became a substrate for recombinant HhoA (rHhoA). rHhoA cleaved reduced eukaryotic (specifically, spinach) PsbO at defined sites and created distinct PsbO fragments that were not further degraded. As for the corresponding prokaryotic substrate (reduced PsbO of Synechocystis sp. PCC 6803), no PsbO fragments were observed. Assembly to PSII protected PsbO from degradation. For Synechocystis sp. PCC 6803, our results show that HhoA, HhoB, and HtrA are localized in the periplasma and/or at the thylakoid membrane. In agreement with the idea that PsbO could be a physiological substrate for Deg proteases, part of the cellular fraction of the three Deg proteases of Synechocystis sp. PCC 6803 (HhoA, HhoB, and HtrA) was detected in the PSII-enriched membrane fraction.     

The family of Deg proteases in cyanobacteria and chloroplasts of higher plants

The family of Deg proteases is present in nearly all organisms from bacteria to higher plants. This family consists of ATP-independent serine endopeptidases with a catalytic domain of trypsin type and up to three PDZ domains, involved in protein–protein interactions. Sixteen deg genes (originally named deg P1–16) were found in Arabidopsis thaliana , and the chloroplast location was predicted or experimentally proven for seven proteins. The cyanobacterium Synechocystis sp. PCC6803 contains three Deg homologues, HtrA (DegP), HhoA (DegQ) and HhoB (DegS), but their number can vary between one and six in other photosynthetic Prokaryota. Interestingly, all of these proteases are evolutionarily more closely related within one species than proteases with the same names present in other organisms. This means that Deg proteases from A. thaliana are not necessarily the closest relatives of cyanobacterial DegP. Therefore, we propose to change the misleading original name 'DegP' to 'Deg' for A. thaliana enzymes. Here, we summarize the expression, location and functions of Deg proteases from cyanobacteria and chloroplasts of higher plants, with special emphasis on their role in the photosystem II (PSII) repair cycle under light stress conditions.     

The serine protease HhoA from Synechocystis sp. strain PCC 6803: substrate specificity and formation of a hexameric complex are regulated by the PDZ domain

Enzymes of the ATP-independent Deg serine endopeptidase family are very flexible with regard to their substrate specificity. Some family members cleave only one substrate, while others act as general proteases on unfolded substrates. The proteolytic activity of Deg proteases is regulated by PDZ protein interaction domains. Here we characterized the HhoA protease from Synechocystis sp. strain PCC 6803 in vitro using several recombinant protein constructs. The proteolytic activity of HhoA was found to increase with temperature and basic pH and was stimulated by the addition of Mg(2+) or Ca(2+). We found that the single PDZ domain of HhoA played a critical role in regulating protease activity and in the assembly of a hexameric complex. Deletion of the PDZ domain strongly reduced proteolysis of a sterically challenging resorufin-labeled casein substrate, but unlabeled beta-casein was still degraded. Reconstitution of the purified HhoA with total membrane proteins isolated from Synechocystis sp. wild-type strain PCC 6803 and a DeltahhoA mutant resulted in specific degradation of selected proteins at elevated temperatures. We concluded that a single PDZ domain of HhoA plays a critical role in defining the protease activity and oligomerization state, combining the functions that are attributed to two PDZ domains in the homologous DegP protease from Escherichia coli. Based on this first enzymatic study of a Deg protease from cyanobacteria, we propose a general role for HhoA in the quality control of extracytoplasmic proteins, including membrane proteins, in Synechocystis sp. strain PCC 6803.     

Isolation of outer membrane of Synechocystis sp. PCC 6803 and its proteomic characterization

In this report, we describe a newly developed method for isolating outer membranes from Synechocystis sp. PCC 6803 cells. The purity of the outer membrane fraction was verified by immunoblot analysis using antibodies against membrane-specific marker proteins. We investigated the protein composition of the outer membrane using two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry followed by database identification. Forty-nine proteins were identified corresponding to 29 different gene products. All of the identified proteins have a putative N-terminal signal peptide. About 40% of the proteins identified represent hypothetical proteins with unknown function. Among the proteins identified are a Toc75 homologue, a protein that was initially found in the outer envelope of chloroplasts in pea, as well as TolC, putative porins, and a pilus protein. Other proteins identified include ABC transporters and GumB, which has a suggested function in carbohydrate export. A number of proteases such as HtrA were also found in the outer membrane of Synechocystis sp. PCC 6803.     

The deg proteases protect Synechocystis sp. PCC 6803 during heat and light stresses but are not essential for removal of damaged D1 protein during the photosystem two repair cycle

Members of the DegP/HtrA (or Deg) family of proteases are found widely in nature and play an important role in the proteolysis of misfolded and damaged proteins. As yet, their physiological role in oxygenic photosynthetic organisms is unclear, although it has been widely speculated that they participate in the degradation of the photodamaged D1 subunit in the photosystem two complex (PSII) repair cycle, which is needed to maintain PSII activity in both cyanobacteria and chloroplasts. We have examined the role of the three Deg proteases found in the cyanobacterium Synechocystis sp. PCC 6803 through analysis of double and triple insertion mutants. We have discovered that these proteases show overlap in function and are involved in a number of key physiological responses ranging from protection against light and heat stresses to phototaxis. In previous work, we concluded that the Deg proteases played either a direct or an indirect role in PSII repair in a glucose-tolerant version of Synechocystis 6803 (Silva, P., Choi, Y. J., Hassan, H. A., and Nixon, P. J. (2002) Philos. Trans. R. Soc. Lond. B Biol. Sci. 357, 1461-1467). In this work, we have now been able to demonstrate unambiguously, using a triple deg mutant created in the wild type strain of Synechocystis 6803, that the Deg proteases are not obligatory for PSII repair and D1 degradation. We therefore conclude that although the Deg proteases are needed for photoprotection of Synechocystis sp. PCC 6803, they do not play an essential role in D1 turnover and PSII repair in vivo.     

Involvement of the HtrA family of proteases in the protection of the cyanobacterium Synechocystis PCC 6803 from light stress and in the repair of photosystem II

Photosystem II (PSII) is prone to irreversible light-induced damage, with the D1 polypeptide a major target. Repair processes operate in the cell to replace a damaged D1 subunit within the complex with a newly synthesized copy. As yet, the molecular details of PSII repair are relatively obscure despite the critical importance of this process for maintaining PSII activity and cell viability. We are using the cyanobacterium Synechocystis sp. PCC 6803 to identify the various proteases and chaperones involved in D1 turnover in vivo. Two families of proteases are being studied: the FtsH family (four members) of Zn(2+)-activated nucleotide-dependent proteases; and the HtrA (or DegP) family (three members) of serine-type proteases. In this paper, we report the results of our studies on a triple mutant in which all three copies of the htrA gene family have been inactivated. Growth of the mutant on agar plates was inhibited at high light intensities, especially in the presence of glucose. Oxygen evolution measurements indicated that, under conditions of high light, the rate of synthesis of functional PSII was less in the mutant than in the wild-type. Immunoblotting experiments conducted on cells blocked in protein synthesis further indicated that degradation of D1 was slowed in the mutant. Overall, our observations indicate that the HtrA family of proteases are involved in the resistance of Synechocystis 6803 to light stress and play a part, either directly or indirectly, in the repair of PSII in vivo.     

Purification and characterization of class-I and class-II fructose-1,6-bisphosphate aldolases from the cyanobacterium Synechocystis sp. PCC 6803

The whole genome sequence database for Synechocystis sp. PCC 6803 has revealed the presence of genes encoding class-I (CI) and class-II (CII) fructose-1,6-bisphosphate aldolases (FBAs) in this organism. Two types of FBA from Synechocystis sp. PCC 6803 were separated by chromatography on phenyl-Sepharose. The activity of the enzyme in the major peak was inhibited by the presence of 25 mM EDTA; however, the activity in the minor peak was not. Therefore, the FBA in the former fractions was designated as CII-FBA, and in the latter designated as CI-FBA. CI-FBA was functionally redundant in Synechocystis sp. PCC 6803, while no disruptant for the gene encoding CII-FBA was obtained under photoautotrophic conditions. The kinetic parameters of CI- and CII-FBAs purified from Synechocystis sp. PCC 6803 in the cleavage reaction of FBP were generally similar, except in their reactivity for SBP. The SBP/FBP activity ratio of the CII-FBA was two times higher than that of the CI-FBA.     

The rnb gene of Synechocystis PCC6803 encodes a RNA hydrolase displaying RNase II and not RNase R enzymatic properties

Cyanobacteria are photosynthetic prokaryotic organisms that share characteristics with bacteria and chloroplasts regarding mRNA degradation. Synechocystis sp. PCC6803 is a model organism for cyanobacteria, but not much is known about the mechanism of RNA degradation. Only one member of the RNase II-family is present in the genome of Synechocystis sp PCC6803. This protein was shown to be essential for its viability, which indicates that it may have a crucial role in the metabolism of Synechocystis RNA. The aim of this work was to characterize the activity of the RNase II/R homologue present in Synechocystis sp. PCC6803. The results showed that as expected, it displayed hydrolytic activity and released nucleoside monophosphates. When compared to two E. coli counterparts, the activity assays showed that the Synechocystis protein displays RNase II, and not RNase R characteristics. This is the first reported case where when only one member of the RNase II/R family exists it displays RNase II and not RNase R characteristics.     

The family of Deg/HtrA proteases: from Escherichia coli to Arabidopsis

In the genomic era, an increasing number of protease genes have been identified in various organisms. During the last few years, many of these proteases have been characterized using biochemical as well as molecular biological techniques. However, neither the precise location nor the physiological substrates of these enzymes has been identified in many cases, including the Deg/HtrA proteases, a family of serine-type ATP-independent proteases. This family has become especially interesting for many researchers following the determination of the crystal structures of an Escherichia coli and a human Deg/HtrA protease. A breakthrough in photosynthesis research has revealed that a Deg/HtrA protease of Arabidopsis thaliana is involved in the degradation of the D1 protein of photosystem II following photoinhibition. In this review, the available data on Deg/HtrAs of different organisms are compared with those from the photoautotroph cyanobacterium Synechocystis sp. PCC 6803 and the plant Arabidopsis thaliana .     

集胞藻PCC6803铜离子诱导表达平台的构建

在集胞藻PCC6803中,基因敲除是研究基因功能的最直接有效的方法,但是对于某些生存必需的基因则无法通过这种方法获得突变株。为研究集胞藻PCC6803中此类基因的功能,在其基因组中构建了一个petE基因启动子（PpetE）控制的铜离子诱导表达的平台。将集胞藻PpetE装配在lacZ报告基因的上游,通过同源双交换整合到这种蓝藻的基因组中。通过调节培养基中铜离子的浓度发现,lacZ的表达能够人为控制。特别是当铜离子浓度在6－400nmoL／L范围时,LacZ活力随铜离子浓度增加呈S型增长关系。利用这个铜离子诱导表达平台,可以控制某些必需基因的表达：提供铜离子维持细胞生存;而撤去铜离子时则关闭基因的表达,可以观察其对生命活动的影响。     

Isolation and functional characterization of Ca2+/H+ antiporters from cyanobacteria

Genome sequences of cyanobacteria, Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120, and Thermosynechococcus elongatus BP-1 revealed the presence of a single Ca2+/H+ antiporter in these organisms. Here, we isolated the putative Ca2+/H+ antiporter gene from Synechocystis sp. PCC 6803 (synCAX) as well as a homologous gene from a halotolerant cyanobacterium Aphanothece halophytica (apCAX). In contrast to plant vacuolar CAXs, the full-length apCAX and synCAX genes complemented the Ca2+-sensitive phenotype of an Escherichia coli mutant. ApCAX and SynCAX proteins catalyzed specifically the Ca2+/H+ exchange reaction at alkaline pH. Immunological analysis suggested their localization in plasma membranes. The Synechocystis sp. PCC 6803 cells disrupted of synCAX exhibited lower Ca2+ efflux activity and a salt-sensitive phenotype. Overexpression of ApCAX and SynCAX enhanced the salt tolerance of Synechococcus sp. PCC 7942 cells. Mutagenesis analyses indicate the importance of two conserved acidic amino acid residues, Glu-74 and Glu-324, in the transmembrane segments for the exchange activity. These results clearly indicate that cyanobacteria contain a Ca2+/H+ antiporter in their plasma membranes, which plays an important role for salt tolerance.     

ggpS基因过表达对集胞藻PCC 6803甘油葡萄糖苷和甘油合成的影响

为了研究甘油葡萄糖苷磷酸合成酶(GgpS)在集胞藻PCC 803甘油葡萄糖苷和甘油合成中的作用,本研究在前期获得高产甘油葡萄糖苷藻株的基础上分别过量表达来自于集胞藻PCC 6803自身和聚球藻PCC7002的甘油葡萄糖苷磷酸合成酶基因ggpS,并测定了在不同浓度NaCl胁迫时突变藻株的甘油葡萄糖苷和甘油积累量。结果发现获得的突变株甘油葡萄糖苷合成没有提高,但是甘油合成显著增强。此外,当培养基NaCl浓度从600 mmol/L提高到900 mmol/L时,集胞藻PCC 6803自身ggpS过表达藻株的甘油合成进一步提高75%。这些结果显示了GgpS在将碳代谢流导入集胞藻甘油合成途径中的作用。研究成果也为进一步通过基因工程改造提高集胞藻甘油葡萄糖苷和甘油合成效率奠定了基础。     

Comparison of tryptophan fluorescence lifetimes in cyanobacterial photosystem I frozen in the light and in the dark

Photosynthesis Research - The dependence on temperature of tryptophan fluorescence lifetime in trimeric photosystem I (PSI) complexes from cyanobacteria Synechocystis sp. PCC 6803 during the...     

Characterization of cyanobacterial carotenoid ketolase CrtW and hydroxylase CrtR by complementation analysis in Escherichia coli

The pathway from beta-carotene to astaxanthin is a crucial step in the synthesis of astaxanthin, a red antioxidative ketocarotenoid that confers beneficial effects on human health. Two enzymes, a beta-carotene ketolase (carotenoid 4,4'-oxygenase) and a beta-carotene hydroxylase (carotenoid 3,3'-hydroxylase), are involved in this pathway. Cyanobacteria are known to utilize the carotenoid ketolase CrtW and/or CrtO, and the carotenoid hydroxylase CrtR. Here, we compared the catalytic functions of CrtW ketolases, which originated from Gloeobacter violaceus PCC 7421, Anabaena (also known as Nostoc) sp. PCC 7120 and Nostoc punctiforme PCC 73102, and CrtR from Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and Anabaena variabilis ATCC 29413 by complementation analysis using recombinant Escherichia coli cells that synthesized various carotenoid substrates. The results demonstrated that the CrtW proteins derived from Anabaena sp. PCC 7120 as well as N. punctiforme PCC 73102 (CrtW148) can convert not only beta-carotene but also zeaxanthin into their 4,4'-ketolated products, canthaxanthin and astaxanthin, respectively. In contrast, the Anabaena CrtR enzymes were very poor in accepting either beta-carotene or canthaxanthin as substrates. By comparison, the Synechocystis sp. PCC 6803 CrtR converted beta-carotene into zeaxanthin efficiently. We could assign the catalytic functions of the gene products involved in ketocarotenoid biosynthetic pathways in Synechocystis sp. PCC 6803, Anabaena sp. PCC 7120 and N. punctiforme PCC 73102, based on the present and previous findings. This explains why these cyanobacteria cannot produce astaxanthin and why only Synechocystis sp. PCC 6803 can produce zeaxanthin.     

Enhanced poly-beta-hydroxybutyrate accumulation in a unicellular cyanobacterium, Synechocystis sp. PCC 6803

AIM: To stimulate poly-beta-hydroxybutyrate (PHB) accumulation in Synechocystis sp. PCC 6803 by manipulating culture conditions. METHODS AND RESULTS: Stationary phase cultures of Synechocystis sp. PCC 6803 were subjected to N- and P-deficiency, chemoheterotrophy and limitations of gas-exchange. Enhanced PHB accumulation was observed under all the above conditions. However, interaction of P-deficiency with gas-exchange limitation (GEL) in the presence of exogenous carbon boosted PHB accumulation maximally. CONCLUSIONS: Combined effects of P-deficiency and GEL boosted PHB accumulation up to 38% (w/w) of dry cell weight (dcw) in Synechocystis sp. PCC 6803 in the presence of fructose and acetate. This value is about eightfold higher as compared with the accumulation under photoautotrophic growth condition. SIGNIFICANCE AND IMPORTANCE OF THE STUDY: These results showed a good potential of Synechocystis sp. PCC 6803 in accumulating poly-beta-hydroxybutyrate, an appropriate raw material for biodegradable and biocompatible plastic. Poly-beta-hydroxybutyrate could be an important material for plastic and pharmaceutical industries.     

细胞周期蛋白依赖性激酶抑制剂Roscovitine对3种蓝藻生长和活性的影响

为了明确蓝藻中丝氨酸/苏氨酸激酶的功能是否与调控细胞的生长分裂相关,以丝状鱼腥藻7120、单细胞集胞藻6803和聚球藻7002为对象,利用OD750光吸收测定和MTT方法研究了不同浓度丝氨酸苏氨酸激酶抑制剂roscovitine对其生长和脱氢酶活性的影响。结果表明:4 h roscovitine处理后对鱼腥藻7120和集胞藻6803生长量影响不大,对聚球藻7002的生长有促进作用。4 h roscovitine的处理对鱼腥藻7120有浓度依赖的显著抑制活性,对集胞藻6803的活性无影响,但是却促进聚球藻7002的活性。药物作用4 d后,7120的生长和活性均显著降低,并有浓度效应;6803的生长量较对照减少,但活性变化不明显;聚球藻7002的生长和活性均未受影响。显微观察结果显示,roscovitine对3种细胞形态没有影响,但药物作用4 d后的7120藻丝体较短。结果表明丝氨酸/苏氨酸抑制剂roscovitine影响丝状藻7120的生长和活性。     

Effect of increased PHA synthase activity on polyhydroxyalkanoates biosynthesis in Synechocystis sp. PCC6803

Polyhydroxyalkanoate (PHA) synthase activity in Synechocystis sp. PCC6803 was increased two-fold by introducing the PHA biosynthetic genes of Ralstonia eutropha. The resulting recombinant Synechocystis sp. PCC6803 strain was subjected to conditions that favor PHA accumulation and the effects of various carbon sources were studied. In addition, the fine structure of both wild-type and recombinant Synechocystis sp. PCC6803 was examined using freeze-fracture electron microscopy technique. The PHA granules in the recombinant Synechocystis sp. PCC6803 were localised near the thylakoid membranes. Maximum amount of PHA accumulation was obtained in the presence of acetate, where the number of granules in the recombinant cells ranged from 4 to 6 and their sizes were in the range of 70-240 nm. In comparison to wild-type Synechocystis sp. PCC6803, recombinant cells with increased PHA synthase activity showed only a marginal increase in PHA content suggesting that PHA synthase is not the rate limiting enzyme of PHA biosynthesis in Synechocystis sp. PCC6803.