Bifunctional enzyme FBPase/SBPase is essential for photoautotrophic growth in cyanobacterium Synechocystis sp PCC 6803

From a random insertion mutant library of Synechocystis sp. PCC 6803, a mutant defective in photoautotrophic growth was obtained. The interrupted gene was identified to be slr2094 (rbpl), which encodes the fructose-1,6-biphosphatase (FBPase)/sedoheptulose-1,7-biphosphatase (SBPase) bifunctional enzyme (F-I). Two other independently constructed slr2094 mutants showed an identical phenotype. The FBPase activity was found to be virtually lacking in an slr2094 mutant, which was sensitive to light under mixotrophic growth conditions. These results indicate that slr2094 is the only active FBPase-encoding gene in this cyanobacterium. Inactivation of photosystem II by interrupting psbB in slr2094 mutant alleviated the sensitiveness to light. This report provides the direct genetic evidence for the essential role of F-I in the photosynthesis of Synechocystis sp. PCC 6803. (c) 2007 National Natural Science Foundation of China and Chinese Academy of Sciences. Published by Elsevier Limited and Science in China Press. All rights reserved.

alpha-tocopherol is essential for acquired chill-light tolerance in the cyanobacterium Synechocystis sp strain PCC 6803

Unlike Escherichia coli, the cyanobacterium Synechocystis sp. strain PCC 6803 is insensitive to chill (5 degrees C) in the dark but rapidly losses viability when exposed to chill in the light (100 mu mol photons m(-2) s(-1)). Preconditioning at a low temperature (15 degrees C) greatly enhances the chill-light tolerance of Synechocystis sp. strain PCC 6803. This phenomenon is called acquired chill-light tolerance (ACLT). Preconditioned wild-type cells maintained a substantially higher level of alpha-tocopherol after exposure to chill-light stress. Mutants unable to synthesize alpha-tocopherol, such as slr1736, slr1737, slr0089, and slr0090 mutants, almost completely lost ACLT. When exposed to chill without light, these mutants showed no or a slight difference from the wild type. When complemented, the slr0089 mutant regained its ACLT. Copper-regulated expression of slr0090 from P-petE controlled the level of et-tocopherol and ACLT. We conclude that alpha-tocopherol is essential for ACLT of Synechocystis sp. strain PCC 6803. The role of a-tocopherol in ACLT may be based largely on a nonantioxidant activity that is not possessed by other tocopherols or pathway intermediates.

A TPR-family membrane protein gene is required for light-activated heterotrophic growth of the cyanobacterium Synechocystis sp PCC 6803

The unicellular cyanobacterium Synechocystis sp. PCC6803 can grow heterotrophically in complete darkness, given that a brief period of illumination is supplemented every day (light-activated heterotrophic growth, LAHG), or under very weak ( < 0.5 mumol m(-2) s(-1)) but continuous light. By random insertion of the genome with an antibiotic resistance cassette, mutants defective in LAHG were generated. In two identical mutants, sll0886, a tetratricopeptide repeat (TPR)-family membrane protein gene, was disrupted. Targeted insertion of sll0886 and three downstream genes showed that the phenotype was not due to a polar effect. The sll0886 mutant shows normal photoheterotrophic growth when the light intensity is at 2.5 mumol m(-2) s(-1) or above, but no growth at 0.5 mumol m(-2) s(-1). Homologs to sll0886 are also present in cyanobacteria that are not known of LAHG. sll0886 and homologs may be involved in controlling different physiological processes that respond to light of low fluence. (C) 2003 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.

硫代硫酸钠及葡萄糖对蓝细菌Synechocystis sP.PCC6803脂及光合器组成的影响

本文通过对蓝细菌Synechocystis sp. PCC 6803在添加葡萄糖、Na2S203的BG-11培养基中的生长特性、脂类及脂肪酸组成、细胞低温荧光、色素组成进行分析测定，总结出如下规律： 当蓝细菌Synechocystis sp. PCC 6803在添加有葡萄糖的BG-11培养基中培养时细胞出现了一种新的糖脂（记为糖脂-x），在添加果糖、麦芽糖、乳糖等其它碳源的培养基中生长的细胞中也检测到糖脂-x糖脂-x的出现经推测是与活性氧相作用的产物，当在含糖的培养基中加入活性氧猝灭剂Na2S203时能有效地抑制糖脂-x的出现。糖脂一x的出现伴随着其它脂、尤其是双半乳糖甘油二酯(DGDG)的含量下降，这可能与细胞营养代谢类型的转变相适应。糖脂-x的出现使细胞适应异养生长条件，这时藻胆体(PBS)，光系统II(PSII)，光系统I(PSD降解，叶绿素消失。 糖脂-x经1H-NMR波谱术检测证实为甘油糖脂，经气质联谱分析其脂肪酸组成中含大量的枝链脂肪酸，12-甲基十四碳酸、12-甲基十五碳酸、12-甲基十六碳酸以及两种稀有的含氮脂肪酸。这些脂肪酸在添加高浓度葡萄糖的培养基中生长的．Synechocystis sp. PCC 6803中的单半乳糖甘油二酯(MGDG)也能检测到。ESI-MS以及P-SI-MS测定结果表明糖脂．x含一分子的脂酰基侧链以及两分子的己糖，半乳糖与葡萄糖。 对．Synechocystis sp. PCC 6803生长在不同浓度的葡萄糖与Na2S203培养基中脂类组成与脂肪酸组成进行比较，发现Na2S203能有效地增加膜脂中硫代异鼠李糖二酰基甘油(SQDG)和磷脂酰甘油(PG)的百分含量，培养基中同时添加葡萄糖时能抵消Na2S203的这一效应。此外，Na2S203能显著增加单半乳糖甘油二酯(MGDG)、双半乳糖甘油二酯(DGDG)中十六碳酸(C16:0)的百分含量，这一效应也能为葡萄糖恢复。Na2S203不能显著地改变SQDG中C16:0的百分含量，加入葡萄糖时能降低C16:0的百分含量。这些结果说明Na2S203可能充当一种还原剂使膜脂处于一种低的不饱和状态，同时加入葡萄糖时能降低Na2S203的还原力。此外，Na2S203还可作为SQDG合成中的硫供体。 用HPLC测定．Synechocystis sp. PCC 6803在添加不同浓度的Na2S203，葡萄糖的BG-11培养基中生长时的叶绿素与类胡萝卜素浓度，结果表明葡萄糖表现出对叶绿素与类胡萝卜素水平的抑制效应，Na2S203在低浓度时表现出对叶绿素与类胡萝卜素水平的促进效应，但在高浓度时表现出抑制效应。因此适当浓度的Na2S203的加入有利于维持蓝细菌在培养基中添加葡萄糖的生长条件下的低水平自由基，能使葡萄糖表现出促进细胞生长的特性。 通过测定Synechocystis sp. PCC 6803生长曲线中葡萄糖、Na2S203的浓度效应，结果表明葡萄糖在低浓度（例如5 mmoI.L-l）时表现出促进细胞的生长，在相对高的浓度表现出抑制细胞生长的效应。在培养基中同时加入Na2S203时可恢复葡萄糖对细胞的生长的促进效应。单独加入Na2S203表现出对细胞生长的抑制效应。这说明葡萄糖、Na2S203对细胞的生长存在着正的协同效应。

Cloning and characterization of the gene for UDPGlc dehydrogenase from the cyanobacterium, Microcystis aeruginosa FACHB 905

Using degenerate primers based on conserved regions of the UDP-glucose dehydrogenase (UDPGDH) gene, an initial 476-bp DNA fragment was amplified from the water-bloom forming cyanobacterium, Microcystis aeruginosa FACHB 905. TAIL-PCR and ligation-mediated PCR were used to amplify the flanking regions to isolate an about 2.5-kb genomic DNA fragment. Sequence analysis revealed an ORF encoding a putative 462 amino acid protein, designated Mud for Microcystis UDPGDH. The Mud amino acid sequence is closely related to UDPGDH sequences from cyanobacterium Synechocystis PCC6803 (73% identity, 81% similarity), and bacterium Bacillus subtilis (51% identity and 67% similarity). The cloned mud gene was expressed in Escherichia coli using the pGEX-4T-1 fusion expression vector system to generate a GST-Mud fusion protein that exhibited UDPGDH activity. The cytosolic fraction of M aeruginosa FACHB 905 was subjected to Western analysis with an anti-Mud antibody, which revealed a single band of approximately 49 kD, consistent with the deduced molecular mass of the enzyme. The Mud protein could thus be characterized as a UDP-glucose dehydrogenase, which was a key enzyme for polysaccharide synthesis and has, for the first time, been studied in algae.

DnaJ-like protein gene sll1384 is involved in phototaxis in Synechocystis sp PCC 6803

In Synechocystis sp. PCC 6803, gene sll1384 encodes a protein with a DnaJ domain at its N-terminal portion and a TPR domain at the C-terminal portion. An sll1384 mutant shows no difference from the wild type in adaptation to different temperatures, but almost completely loses its capability of phototactic movement. After complementation with sll1384, the mutant regains the phototaxis. As shown with electron microscopy, on the cell surface, mutant cells have pili that appear to be the same as that of the wild type. Also, the transformation efficiency remains unchanged in the mutant. It is postulated that Sll1384 regulates phototaxis of Synechocystis through protein-protein interaction. It is the first DnaJ-like protein gene identified in a cyanobacterium for a role in phototaxis.

Isolation of a novel cyanophage infectious to the filamentous cyanobacterium Planktothrix agardhii (Cyanophyceae) from Lake Donghu, China

A previously unknown cyanophage, PaV-LD (Planktothrix agardhii Virus isolated from Lake Donghu), which causes lysis of the bloom-forming filamentous cyanobacterium P. agardhii, was isolated from Lake Donghu, Wuhan, China. PaV-LD only lysed P. agardhii strains isolated from Lake Donghu and not those isolated from other lakes. The PaV-LD particle has an icosahedral, non-tailed structure, ca. 70 to 85 nm (mean +/- SD = 76 +/- 6 nm) in diameter. PaV-LD was stable at freezing temperature, but lost its infectivity at temperatures >50 degrees C. Lysis of host cells was delayed about 3 d after the PaV-LD treatment with chloroform, and the virus was inactivated by exposure to low pH (<= 4). The latent period and burst size of the PaV-LD were estimated to be 48 to 72 h and about 340 infectious units per cell, respectively. The regrowth cultures of surviving host filaments were not lysed by the PaV-LD suspension. To our knowledge, this is the first isolation and cultivation of a virus infectious to the filamentous bloom-forming cyanobacterium Planktothrix from a freshwater lake.

Cytochemical changes in the developmental process of Nostoc sphaeroides (cyanobacterium)

There are several apparent developmental stages in the life cycle of Nostoc sphaeroides Kutzing, an edible cyanobacterium found mainly in paddy fields in central China. The cytochemical changes in developmental stages such as hormogonia, aseriate stage, filamentous stage and colony in N. sphaeroides were examined using fluorescent staining and colorimetric methods. The staining of acidic and sulfated polysaccharides increased with development when hormogonia were used as the starting point. Acidic polysaccharides (AP) were most abundant at the aseriate stage and then decreased, while the sulfated polysaccharides (SP) were highest at the colony stage. Quantitatively, along the developmental process from hormogonia to colony, total carbohydrates first increased, then became stable, and then reached their highest level at the colony stage, while reducing sugars were highest at the hormogonia stage and then decreased sharply once development began. SP were not detectable in the hot water soluble polysaccharides (HWSP), and hormogonia had the lowest content of AP, while old colonies had the highest. The AP content of the aseriate stage, filamentous stage and young colony stage were very similar. The evolutionary relationships reflected in the developmental stages of N. sphaeroides are discussed.

Microcystin-RR induces physiological stress and cell death in the cyanobacterium Aphanizomenon sp DC01 isolated from Lake Dianchi, China

The phytoplankton community in Lake Dianchi (Yunnan Province, Southwestern China) is dominated in April by a bloom of Aphanizomenon, that disappears Suddenly and is displaced by a Microcystis bloom in May. The reasons for the rapid bloom disappearance phenomenon and the temporal variability in the composition of phytoplankton assemblages are poorly understood. Cell growth, ultrastructure and physiological changes were examined in cultures of Aphanizomenon sp. DC01 isolated from Lake Dianchi exposed to different closes of rnicrocystin-RR (MC-RR) produced by the Microcystis bloom. MC-RR concentrations above 100 mu g L-1 markedly inhibited the pigment (chlorophyll-a, phycocyanin) synthesis and caused an increase of soluble carbohydrate and protein contents and nitrate reductase activity of toxin-treated blue-green algae. A drastic. reduction in photochemical efficiency of PSII (Fv/Fm) was also found. Morphological examinationn showed that the Aphanizomenon filaments disintegrated and file cells lysed gradually after 48 h Of toxin exposure. Transmission electron microscopy revealed that cellular inclusions of stressed cells almost leaked out completely and the cell membranes were grossly damaged. These findings demonstrate the allelopathic activity of Microcystis aeruginosa inducing physiological stress and cell death of Aphanizomenon sp. DC01 Although the active concentrations of microcystin were rather high, we propose that microcystin may function as allelopathic Substance due to inhomogeneous toxin concentrations close to Microcystis cells. Hence, it may play a role in species Succession of Aphanizomenon and Microcystis in Lake Dianchi.

Different tolerances and responses to low temperature and darkness between waterbloom forming cyanobacterium Microcystis and a green alga Scenedesmus

The dynamics of planktonic cyanobacteria in eutrophicated freshwaters play an important role in formation of annual summer blooms, yet overwintering mechanisms of these water bloom forming cyanobacteria remain unknown. The responses to darkness and low temperature of three strains (unicellular Microcystis aeruginosa FACHB-905, colonial M. aeruginosa FACHB-938, and a green alga Scenedesmus quadricauda FACHB-45) were investigated in the present study. After a 30-day incubation under darkness and low temperature, cell morphology, cell numbers, chlorophyll a, photosynthetic activity (ETRmax and I-k), and malodialdehyde (MDA) content exhibited significant changes in Scenedesmus. In contrast, Microcystis aeruginosa cells did not change markedly in morphology, chlorophyll a, photosynthetic activity, and MDA content. The stress caused by low temperature and darkness resulted in an increase of the antioxidative enzyme-catalase (CAT) in all three strains. When the three strains re-grew under routine cultivated condition subjected to darkness and low temperature, specific growth rate of Scenedesmus was lower than that of Microcystis. Flow cytometry (FCM) examination indicated that two distinct types of metabolic response to darkness and low temperature existed in the three strains. The results from the present study reveal that the cyanobacterium Microcystis, especially colonial Microcystis, has greater endurance and adaptation ability to the stress of darkness and low temperature than the green alga Scenedesmus.

Solar UV radiation drives CO2 fixation in marine phytoplankton: A double-edged sword

Photosynthesis by phytoplankton cells in aquatic environments contributes to more than 40% of the global primary production (Behrenfeld et al., 2006). Within the euphotic zone (down to 1% of surface photosynthetically active radiation [PAR]), cells are exposed not only to PAR (400-700 nm) but also to UV radiation (UVR; 280-400 nm) that can penetrate to considerable depths (Hargreaves, 2003). In contrast to PAR, which is energizing to photosynthesis, UVR is usually regarded as a stressor (Hader, 2003) and suggested to affect CO2-concentrating mechanisms in phytoplankton (Beardall et al., 2002). Solar UVR is known to reduce photosynthetic rates (Steemann Nielsen, 1964; Helbling et al., 2003), and damage cellular components such as D1 proteins (Sass et al., 1997) and DNA molecules (Buma et al., 2003). It can also decrease the growth (Villafane et al., 2003) and alter the rate of nutrient uptake (Fauchot et al., 2000) and the fatty acid composition (Goes et al., 1994) of phytoplankton. Recently, it has been found that natural levels of UVR can alter the morphology of the cyanobacterium Arthrospira (Spirulina) platensis (Wu et al., 2005b). On the other hand, positive effects of UVR, especially of UV- A (315-400 nm), have also been reported. UV- A enhances carbon fixation of phytoplankton under reduced (Nilawati et al., 1997; Barbieri et al., 2002) or fast-fluctuating (Helbling et al., 2003) solar irradiance and allows photorepair of UV- B-induced DNA damage (Buma et al., 2003). Furthermore, the presence of UV-A resulted in higher biomass production of A. platensis as compared to that under PAR alone (Wu et al., 2005a). Energy of UVR absorbed by the diatom Pseudo-nitzschia multiseries was found to cause fluorescence (Orellana et al., 2004). In addition, fluorescent pigments in corals and their algal symbiont are known to absorb UVR and play positive roles for the symbiotic photosynthesis and photoprotection (Schlichter et al., 1986; Salih et al., 2000). However, despite the positive effects that solar UVR may have on aquatic photosynthetic organisms, there is no direct evidence to what extent and howUVR per se is utilized by phytoplankton. In addition, estimations of aquatic biological production have been carried out in incubations considering only PAR (i. e. using UV-opaque vials made of glass or polycarbonate; Donk et al., 2001) without UVR being considered (Hein and Sand-Jensen, 1997; Schippers and Lurling, 2004). Here, we have found that UVR can act as an additional source of energy for photosynthesis in tropical marine phytoplankton, though it occasionally causes photoinhibition at high PAR levels. While UVR is usually thought of as damaging, our results indicate that UVR can enhance primary production of phytoplankton. Therefore, oceanic carbon fixation estimates may be underestimated by a large percentage if UVR is not taken into account.

Identification of a gene, ccr-1 (sll1242), required for chill-light tolerance and growth at 15 degrees C in Synechocystis sp PCC 6803

Synechocystis sp. PCC 6803 exposed to chill (5 degrees C)-light (100 mu mol photons m(-2) s(-1)) stress loses its ability to reinitiate growth. From a random insertion mutant library of Synechocystis sp. PCC 6803, a sll1242 mutant showing increased sensitivity to chill plus light was isolated. Mutant reconstruction and complementation with the wild-type gene confirmed the role of sll1242 in maintaining chill-light tolerance. At 15 degrees C, the autotrophic and mixotrophic growth of the mutant were both inhibited, paralleled by decreased photosynthetic activity. The expression of sll1242 was upregulated in Synechocystis sp. PCC 6803 after transfer from 30 to 15 degrees C at a photosynthetic photon flux density of 30 mu mol photons m(-2) S-1. sll1242, named ccr (cyanobacterial cold resistance gene)- 1, may be required for cold acclimation of cyanobacteria in light.

The functional divergence of two glgP homologues in Synechocystis sp PCC 6803

Glycogen phosphorylase (GlgP, EC 2.4.1.1) catalyzes the cleavage of glycogen into glucose-1-phosphate (Glc-1-P), the first step in glycogen catabolism. Two glgP homologues are found in the genome of Synechocystis sp. PCC 6803, a unicellular cyanobacterium: sll1356 and slr1367. We report on the different functions of these glgP homologues. sll1356, rather than slr1367, is essential for growth at high temperatures. On the other hand, when CO2-fixation and the supply of glucose are both limited, slr1367 is the key factor in glycogen metabolism. In cells growing autotrophically, sll1356 plays a more important role in glycogen digestion than slr1367. This functional divergence is also supported by a phylogenetic analysis of glgP homologues in cyanobacteria.