Fine-structural and chemical analyses on inner and outer sheath of the cyanobacteriumGloeothece sp. PCC 6909

Cells of the unicellular cyanobacteriumGloeothece sp. PCC 6909 are surrounded by an inner (enclosing 1–2 cells) and an outer (enclosing cell groups) sheath. Using conventional Epon-embedding in combination with ruthenium-red staining, the inner and outer sheaths appeared similar and displayed multiple bands of electron-dense subunits. However, embedding in Nanoplast resin to avoid shrinkage led to the detection of two distinct zones (inner and outer zone) each with several distinct layers. The zone delimited by the electron-dense thick inner sheath layer, and the zone enclosed by the thin electron-dense outer sheath layer, are composed of a homogeneous material of little electron-contrast. Whereas the outer zone appears to be of even contrast, the inner zone is characterized by a distinct electron-transparent layer. Element distribution analysis revealed that the electron-transparent layer contained relatively large amounts of sulfur, carbon, and oxygen but only little nitrogen.Inner and outer sheath fractions were isolated by differential mechanical cell breakage and centrifugation. The outer sheath fraction was less hydrated than the inner one. The two fractions differed little in their contents of uronic acids, carbohydrate and protein, although the outer sheath fraction contained less sulfate. A soluble polysaccharide with a chemical composition similar to that of inner and outer sheath fractions was also obtained from the culture supernatant.     

2-O-methyl-d-xylose containing sheath in the cyanobacterium Gloeothece sp. PCC 6501

The sheath of the unicellular cyanobacterium Gloeothece sp. PCC 6501 was isolated from cell homogenates by differential and sucrose gradient centrifugation, followed by lysozyme treatment and hot sodium dodecyl sulfate extraction. The sheath contains a major fraction of carbohydrate consisting of galactose, glucose, mannose, rhamnose, 2-O-methyl-d-xylose, xylose, glucuronic and galacturonic acids, but only traces of fatty acids and phosphate. A protein content of about 2% (of fraction dry weight) could not be removed by the detergent treatment.Abbreviation SDS sodium dodecyl sulfate     

Oxygen and the light–dark cycle of nitrogenase activity in two unicellular cyanobacteria

Cyanobacteria capable of fixing dinitrogen exhibit various strategies to protect nitrogenase from inactivation by oxygen. The marine Crocosphaera watsonii WH8501 and the terrestrial Gloeothece sp. PCC6909 are unicellular diazotrophic cyanobacteria that are capable of aerobic nitrogen fixation. These cyanobacteria separate the incompatible processes of oxygenic photosynthesis and nitrogen fixation temporally, confining the latter to the dark. Although these cyanobacteria thrive in fully aerobic environments and can be cultivated diazotrophically under aerobic conditions, the effect of oxygen is not precisely known due to methodological limitations. Here we report the characteristics of nitrogenase activity with respect to well‐defined levels of oxygen to which the organisms are exposed, using an online and near real‐time acetylene reduction assay combined with sensitive laser‐based photoacoustic ethylene detection. The cultures were grown under an alternating 12–12 h light–dark cycle and acetylene reduction was recorded continuously. Acetylene reduction was assayed at 20%, 15%, 10%, 7.5%, 5% and 0% oxygen and at photon flux densities of 30 and 76 μmol m^?2 s^?1 provided at the same light–dark cycle as during cultivation. Nitrogenase activity was predominantly but not exclusively confined to the dark. At 0% oxygen nitrogenase activity in Gloeothece sp. was not detected during the dark and was shifted completely to the light period, while C. watsonii did not exhibit nitrogenase activity at all. Oxygen concentrations of 15% and higher did not support nitrogenase activity in either of the two cyanobacteria. The highest nitrogenase activities were at 5–7.5% oxygen. The highest nitrogenase activities in C. watsonii and Gloeothece sp. were observed at 29°C. At 31°C and above, nitrogenase activity was not detected in C. watsonii while the same was the case at 41°C and above in Gloeothece sp. The differences in the behaviour of nitrogenase activity in these cyanobacteria are discussed with respect to their presumed physiological strategies to protect nitrogenase from oxygen inactivation and to the environment in which they thrive.     

Sheath and outer membrane components from the cyanobacterium Fischerella sp. PCC 7414

A purified sheath fraction and an outer membrane fraction were obtained from the cyanobacterium Fischerella sp. PCC 7414. The sheath had a fine structure with osmiophilic fibers running in parallel to the cell surface in two distinct layers. The sheath fraction contained mainly neutral sugars (Glc, Man, Gal, Xyl, Fuc, 2-O-methylhexose), GlcN, uronic acids, and minor components such as amino acids, sulfate, phosphate, and fatty acids. The protein moiety was removable from the sheath fraction by treatment with boiling sodium dodecyl sulfate. The presence of three different 3-hydroxy fatty acids (3-OH-14:0, 3-OH-16:0, 3-OH-18:0) in addition to GlcN indicated the presence of lipopolysaccharide in the outer membrane. One major (M_r 50,000) and two minor (M_r 54,000 and 65,000) proteins were detected as constituents of the outer membrane.Abbreviations A₂pm diaminopimelic acid - GLC gas-liquid chromatography - GlcN glucosamine - Ino inositol - MurN muramic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecyl sulfate     

Nucleotide sequence and homology comparison of two genes of the sulfate transport operon from the cyanobacterium Synechocystis sp. PCC 6803

The genome of Synechocystis sp. PCC 6803 contains an operon with homology to the sulfate permease of other prokaryotes. We used antibodies raised against cytoplasmic membrane protein to find three genes with strong homology to sbpA, orf81 and cysT genes of the cyanobacterium Synechococcus sp. PCC 7942, Escherichia coli, Salmonella typhymurium and Marchantia polymorpha. It is likely that the permease genes are expressed and the proteins are inserted into the cytoplasmic membrane.     

迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白在蓝藻中的表达

在蓝藻中表达迟缓爱德华氏菌Eta1-L-Gapdh融合蛋白。提取迟缓爱德华氏菌基因组DNA为模板,用PCR技术分别扩增两个已知具有较强免疫原性的基因eta1和gapdh,再采用重叠延伸PCR将这两个基因融合,获得目的融合基因eta1-L-gapdh。将目的基因连接到表达载体pRL489的两个Bam H I酶切位点之间构建表达载体,用质粒提取、PCR、酶切、测序等手段对表达载体进行验证。验证正确的表达载体通过三亲接合转化野生鱼腥藻PCC7120,用新霉素抗性筛选出转基因藻落,通过质粒提取和PCR验证转基因藻。用RT-PCR和Western-blot分别从转录水平和翻译水平对转基因藻中融合基因的表达进行了检测。结果表明,含目的基因的表达载体构建成功,目的基因在蓝藻中转录并表达蛋白,该蛋白在蓝藻中的表达量为2.46%。     

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.     

The IdiA protein of Synechococcus sp. PCC 7942 functions in protecting the acceptor side of Photosystem II under oxidative stress

Synechococcus sp. strains PCC 7942 and PCC 6301 contain a 35 kDa protein called IdiA (Iron deficiency induced protein A) that is expressed in elevated amounts under Fe deficiency and to a smaller extent also under Mn deficiency. Absence of this protein was shown to mainly damage Photosystem II. To decide whether IdiA has a function in optimizing and/or protecting preferentially either the donor or acceptor side reaction of Photosystem II, a comparative analysis was performed of Synechococcus sp. PCC 7942 wild-type, the IdiA-free mutant, the previously constructed PsbO-free Synechococcus PCC 7942 mutant and a newly constructed Synechococcus PCC 7942 double mutant lacking both PsbO and IdiA. Measurements of the chlorophyll fluorescence and determinations of Photosystem II activity using a variety of electron acceptors gave evidence that IdiA has its main function in protecting the acceptor side of Photosystem II. Especially, the use of dichlorobenzoquinone, preferentially accepting electrons from Q_A, gave a decreased O₂ evolving activity in the IdiA-free mutant. Investigations of the influence of hydrogen peroxide treatment on cells revealed that this treatment caused a significantly higher damage of Photosystem II in the IdiA-free mutant than in wild-type. These results suggest that although the IdiA protein is not absolutely required for Photosystem II activity in Synechococcus PCC 7942, it does play an important role in protecting the acceptor side against oxidative damage. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Slr0924 protein of Synechocystis sp. strain PCC 6803 resembles a subunit of the chloroplast protein import complex and is mainly localized in the thylakoid lumen

An isolated 25 kDa protein of Synechocystis sp. PCC 6803 was N-terminally sequenced and assigned to a protein encoded by the ORF slr0924. This ORF shows a certain degree of sequence similarity to a subunit from the protein Translocon at the Inner envelope of pea Chloroplasts (Tic22). The deduced amino acid sequence of Slr0924 has a N-terminal extension, that contains two possible translational start points and two possible cleavage sites for leader peptidases. Immunostaining with an antibody raised to the over-produced protein revealed two cross-reacting forms, which probably correspond to a larger intermediate and the mature protein. Immunogold labelling of thin sections showed that the protein is located mainly in the thylakoid region. This result was verified by thylakoid membrane fractionation indicating that Slr0924 is a lumenal protein. The slr0924 gene product is essential for the viability of Synechocystis sp. PCC 6803 as shown by interposon mutagenesis. The merodiploid strain showed reduced photosynthetic activity compared to the wild-type. Furthermore, growth of the merodiploid strain was found to be completely inhibited after cultivation with glucose. Accordingly, the amount of the slr0924 gene product was regulated by glucose and light intensities in wild-type cells. The potential function of the protein in Synechocystis sp. PCC 6803 will be discussed.     

Organization of the nif genes in cyanobacteria in symbiotic association with Azolla and Anthoceros

The sizes of endonuclease digestion fragments of DNA from cyanobacteria in symbiotic association with Azolla caroliniana or Anthoceros punctatus, or in free-living culture, were compared by Southern hybridization using cloned nitrogenase (nif) genes from Anabaena sp. PCC 7120 as probes. The restriction fragment pattern produced by cyanobacteria isolated from A. caroliniana by culture through symbiotic association with Anthoceros differed from that of the major symbiotic cyanobacterium freshly separated from A. caroliniana. The results indicate that minor cyanobacterial symbionts occur in association with Azolla and that the dominant symbiont was not cultured in the free-living state. Both the absence of hybridization to an xisA gene probe and the mapping of restriction fragments indicated a contiguous nifHDK organization in all cells of the symbiont in association with Azolla. On the other hand, in the cultured isolate from Azolla and in Nostoc sp. 7801, the nifD and nifK genes are nominally separated by an interval of unknown length, compatible with the interruption of the nifHDK operon by a DNA element as observed in Anabaena sp. PCC 7120. In the above cultured strains, restriction fragments consistent with a contiguous nifHDK operon were also present at varying hybridization intensities, especially in Nostoc sp. 7801 grown in association with Anthoceros, presumably due to gene rearrangement in a fraction of the cells.Non-standard abbreviations bp base pairs - kb kilobase pairs - kd kilodaltons     

Construction of insertion mutants of Synechocystis sp. PCC 6803: evidence for an essential function of subunit IV of the cytochrome b6/f complex

The gene encoding subunit IV of the cytochrome b₆/f complex (petD) has been isolated from a genomic library of the unicellular cyanobacterium Synechocystis sp. PCC 6803. The coding region consists of 480 nucleotides and can code for a polypeptide with a molecular weight of 17.5 kDa. The deduced amino acid sequence shows high identity with the corresponding sequences of both the photoautotrophic prokaryote Nostos sp. PCC 7906 as well as of lower and higher photoautotrophic eukaryotes (e.g. Chlorella protothecoides, Nicotiana tabacum). Transformation of Synechocystis sp. PCC 6803 with a plasmid containing the cloned petD gene in which the coding sequence is interrupted by the aminoglycoside 3-phosphotransferase gene (aph) from Tn903 resulted in the formation of km resistant transformants. The molecular analysis of independent transformants revealed that all clones were merodiploid containing both uninterrupted wild-type as well as interrupted mutant petD copies. Approaches to segregate these two genomes were unsuccessful implying an essential function of the petD gene product in Synechocystis sp. PCC 6803.Abbreviations aph aminoglycoside 3-phosphotransferase - cpDNA chloroplast DNA - km kanamycin - PSI photosystem I - PSII photosystem II     

The structural nif genes of the cyanobacteria Gloeothece sp. and Calothrix sp. share homology with those of Anabaena sp., but the Gloeothece genes have a different arrangement.

Probes carrying the Anabaena sp. strain PCC 7120 nitrogenase reductase (nifH) and nitrogenase (nifK and nifD) genes were hybridized to Southern blots of DNA from the unicellular, aerobic nitrogen-fixing cyanobacterium Gloeothece sp. strain PCC 6909 and from the filamentous cyanobacterium Calothrix sp. strain PCC 7601. These data suggest that the Gloeothece sp. nif structural proteins must be similar to those of other diazotrophs and that the ability for aerobic nitrogen fixation does not reside in the nif protein complex. We also found that the nif structural genes of Gloeothece sp. are clustered, whereas those of Calothrix sp. are arranged more like those of Anabaena sp.     

MORPHOLOGICAL AND PHYLOGENETIC STUDIES ON UNICELLULAR DIAZOTROPHIC CYANOBACTERIA (CYANOPHYTES) ISOLATED FROM THE COASTAL WATERS AROUND SINGAPORE1

Six unicellular diazotrophic cyanobacteria were isolated from the coast around Singapore. The isolates grew under both light:dark (L:D) cycles and continuous illumination (CL) in media without combined nitrogen and exhibited an ability to fix nitrogen (as measured by acetylene reduction) under aerobic conditions. The cells of all isolates were surrounded by a thick fibrous outer wall layer, and they divided by transverse binary fission. The arrangement of photosynthetic thylakoids was of the dispersed type. Three isolates were identified as form‐genus Gloeothece as cells were divided in a single plane, and the other three isolates were identified as form‐genus Gloeocapsa as cells were divided in multiple planes. Phylogenetic analyses based on the DNA sequences of the genes encoding 16S rRNA and dinitrogenase reductase (nifH) revealed the following: (i) Our six isolates formed a monophyletic cluster. (ii) The monophyletic cluster was subdivided into two phylogenetic groups, which taxonomically corresponded with the form‐genera Gloeothece and Gloeocapsa. However, (iii) a diazotrophic strain of form‐genus Gloeothece, Gloeothece membranacea (Rabenh.) Bornet PCC6501, was not closely related to our isolates, and (iv) some, but not all, diazotrophic unicellular strains of form‐genus Cyanothece were observed to be in a close relationship with our isolates.     

Cell wall and sheath constituents of the cyanobacterium Gloeobacter violaceus

Sheaths isolated from Gloeobacter violaceus were found to be composed of a major polysaccharide moiety (glucose, galactose, rhamnose, mannose, arabinose), a protein moiety, and negatively charged components (glucuronic acids, phosphate, sulfate). Outer membrane polypeptide patterns were dominated by two major peptidoglycan-associated proteins (M_r 62,000 and 53,000). Lipopolysaccharide constituents were glucosamine, 3-hydroxy fatty acids (3-OH-14:0, anteiso-3-OH-15:0, 3-OH-16:0, 3-OH-18:0), carbohydrates, and phosphate. A1-type peptidoglycan and non-peptidoglycan components (mannosamine, glucose, mannose, and glucosamine) indicated the presence of a peptidoglycan-polysaccharide complex in the cell walls of Gloeobacter violaceus.Abbreviations A₂pm diaminopimelic acid - ATCC American Type Culture Collection - CE cell envelope - CM cytoplasmic membrane - CW cell wall - dOcla 3-deoxy-d-manno-2-octulosonic acid - GalN galactosamine - GlcN glucosamine - GlcUA glucuronic acid - HF hydrofluoric acid - LPS lipopolysaccharide - ManN mannosamine - M relative molecular mass - MurN muramic acid - MurN-6-P muramic acid-6-phosphate - OMe O-methyl - PAGE polyacrylamide gel electrophoresis - PCC Pasteur Culture Collection - SDS sodium dodecyl sulfate - SH sheath     

sll1981, an acetolactate synthase homologue of Synechocystis sp. PCC6803, functions as l-myo-inositol 1-phosphate synthase

l-myo-inositol 1-phosphate synthase (EC 5.5.1.4; MIPS) catalyzes the first rate limiting conversion of d-glucose 6-phosphate to l-myo-inositol 1-phosphate in the inositol biosynthetic pathway. In an earlier communication we have reported two forms of MIPS in Synechocystis sp. PCC6803 (Chatterjee et al. in Planta 218:989–998, 2004). One of the forms with a ~50 kDa subunit has been found to be coded by an as yet unassigned ORF, sll1722. In the present study we have purified the second isoform of MIPS as a ~65 kDa protein from the crude extract of Synechocystis sp. PCC6803 to apparent homogeneity and biochemically characterized. MALDI-TOF analysis of the 65 kDa protein led to its identification as acetolactate synthase large subunit (EC 2.2.1.6; ALS), the putatively assigned ORF sll1981 of Synechocystis sp. PCC6803. The PCR amplified ~1.6 kb product of sll1981 was found to functionally complement the yeast inositol auxotroph, FY250 and could be expressed as an immunoreactive ~65 kDa MIPS protein in the natural inositol auxotroph, Schizosaccharomyces pombe. In vitro MIPS activity and cross reactivity against MIPS antibody of purified recombinant sll1981 further consolidated its identity as the second probable MIPS gene in Synechocystis sp. PCC6803. Sequence comparison along with available crystal structure analysis of the yeast MIPS reveals conservation of several amino acids in sll1981 essential for substrate and co-factor binding. Comparison with other prokaryotic and eukaryotic MIPS sequences and phylogenetic analysis, however, revealed that like sll1722, sll1981 is quite divergent from others. It is probable that sll1981 may code for a bifunctional enzyme protein having conserved domains for both MIPS and acetolactate synthase (ALS) activities.Anirban Chatterjee and Krishnarup Ghosh Dastidar contributed equally.     

Modification of the N-Terminal Nucleotide Sequence of Mature hGM-CSF Results in High Expression in the Foreign Host, Anabaena sp. Strain PCC 7120

Cloning and high foreign expression of the human granulocyte-macrophage colony stimulating factor (hGM-CSF) gene were achieved in Anabaena sp. strain PCC 7120 cells. To promote high expression of hGM-CSF in cyanobacterial cells, PCR primers were designed to modify the N-terminal cDNA sequence of mature hGM-CSF, including a GC rich region and some discriminating against codons according to the degeneracy codon rules, selecting for prokaryotic usage codons. The PCR product encoding the modified hGM-CSF was inserted downstream of the promoter, PpsbA of the shuttle vector pRL439, then ligated with pDC-08 to generate the shuttle expression plasmid, pDC-GM1. The resulting shuttle expression plasmid was transferred into the filamentous, heterocyst-forming cyanobacterium, Anabaena sp. strain PCC 7120 using the tri-parental conjugation transfer method. The results of PCR amplification of wild type and transgenic cells indicated that the hGM-CSF gene was successfully cloned into Anabaena sp. strain PCC 7120 cells. Western blot analysis showed that the protein expression of modified hGM-CSF in transgenic cells harboring pDC-GM1 was 136% higher than that of non-modified hGM-CSF in transgenic cells harboring pDC-GM0. Additionally, there were similar rate of growth and content of Chl a as compared to controls, suggesting that foreign hGM-CSF did not impair the photosynthetic activity of host cells. Taken together, the results indicate that modification of the N-terminal nucleotide sequence of mature hGM-CSF results in high expression in the transgenic cells.     

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.     

Sulfonylurea-resistant mutants and natural tolerance of cyanobacteria

The herbicide sulfometuron methyl (SM) inhibited the growth of the cyanobacterium Synechococcus sp. PCC7942, but not of Synechocystis sp. PCC6714. The inhibitory effect was alleviated by the simultaneous addition of valine, leucine and isoleucine. SM resistant mutants were isolated from Synechococcus 7942, two types of which were further analysed. In these mutants, SM3/20 and SM2/32, the activity of acetolactate synthase (ALS) — a key enzyme in the biosynthesis of branched-chain amino acids —appeared 2600- and 300-fold, respectively, more resistant to SM than that of their wild type. Strain SM2/32 also exhibited a low level of ALS activity. Although the growth of the latter mutant was extremely inhibited by valine, the sensitivity of its ALS activity to feed-back inhibition by the amino acid was unaltered. At high concentrations valine inhibited growth of the wild type strains and of the mutant SM3/20. Isoleucine alleviated the valine-induced growth inhibition. Unlike that of Synechococcus 7942, the ALS activity of Synechocystis was found to tolerate high concentrations (100-fold) of the herbicide. The study confirms that the SM mutations are correlated with a cyanobacterial ilv gene.Abbreviations ALS acetolactate synthase; ile, isoleucine - leu leucine - NTG N-methyl-N-nitro-N-nitrosoguanidine - SM sulfometuron methyl - SM^r sulfometuron methyl resistant - val valine     

Modulation of tolerance to Cr(VI) and Cr(VI) reduction by sulfate ion in a <Emphasis Type="Italic">Candida</Emphasis> yeast strain isolated from tannery wastewater

The main aim of this study was to investigate the influence of the sulfate ion on the tolerance to Cr(VI) and the Cr(VI) reduction in a yeast strain isolated from tannery wastewater and identified as Candida sp. FGSFEP by the D1/D2 domain sequence of the 26S rRNA gene. The Candida sp. FGSFEP strain was grown in culture media with sulfate concentrations ranging from 0 to 23.92 mM, in absence and presence of Cr(VI) [1.7 and 3.3 mM]. In absence of Cr(VI), the yeast specific growth rate was practically the same in every sulfate concentration tested, which suggests that sulfate had no stimulating or inhibiting effect on the yeast cell growth. In contrast, at the two initial Cr(VI) concentrations assayed, the specific growth rate of Candida sp. FGSFEP rose when sulfate concentration increased. Likewise, the greater efficiencies and volumetric rates of Cr(VI) reduction exhibited by Candida sp. FGSFEP were obtained at high sulfate concentrations. Yeast was capable of reducing 100% of 1.7 mM Cr(VI) and 84% of 3.3 mM Cr(VI), with rates of 0.98 and 0.44 mg Cr(VI)/L h, with 10 and 23.92 mM sulfate concentrations, respectively. These results indicate that sulfate plays an important role in the tolerance to Cr(VI) and Cr(VI) reduction in Candida sp. FGSFEP. These findings may have significant implications in the biological treatment of Cr(VI)-laden wastewaters.