Evidence for the production of a proteinaceous hemolytic exotoxin by wild-type strain of <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 (Cyanobacteria)

A proteinaceous hemolysin produced by a wild-type strain of Synechocystis sp. PCC 6803 was purified from cell-free culture supernatants by successive column chromatography on DEAE-Sepharose Fast Flow and Sephacryl S-300 High Resolution. The molecular mass of the hemolysin, determined by SDS-PAGE, was approximately 81 kDa. The hemolysin was heat labile and showed potent hemolytic activity against rabbit and sheep erythrocytes. The hemolysin started to be secreted during the exponential growth phase and accumulated maximally at the stationary phase. The production of hemolysin varied with the amount of calcium present in modified BG-11 culture medium. Hemolysin production decreased in calcium-free medium, whereas it increased in medium containing 0.48 mM calcium. In contrast, the potency of hemolysin, as shown by hemolysis assay, was enhanced by deprivation of calcium (EDTA treatment) but decreased in the presence of calcium. Our results show that calcium stimulated production and secretion of hemolysin, but inhibited hemolytic potency.     

Light-dependent expression of superoxide dismutase from cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC 6803

The oxygenic phototrophic cyanobacterium Synechocystis sp. strain PCC 6803 inevitably evolves superoxide during photosynthesis. Synechocystis 6803 contains only one type of superoxide dismutase, designated as SodB; therefore, this protein plays an important role in preventing oxidative damages caused by light. Because there was no direct evidence that SodB in Synechocystis 6803 could be regulated by light, the relationship between SodB and light was investigated in the present study. The activity of SodB from the cells grown in continuous light culture was about 3.5-fold higher than that from the cells cultivated in continuous dark. Illumination maximally activated SodB within 12 h. The level of sodB mRNA increased 12-fold by light, and that of SodB protein proportionally. Therefore, the expression and activity of SodB from Synechocystis 6803 were dependent on the light.     

The response regulator RpaB binds the high light regulatory 1 sequence upstream of the high-light-inducible <Emphasis Type="Italic">hliB</Emphasis> gene from the cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> PCC 6803

Cyanobacteria, like other photosynthetic organisms, respond to the potentially damaging effects of high-intensity light by regulating the expression of a variety of stress-responsive genes through regulatory mechanisms that remain poorly understood. The high light regulatory 1 (HLR1) sequence can be found upstream of many genes regulated by high-light (HL) stress in cyanobacteria. In this study, we identify the factor that binds the HLR1 upstream of the HL-inducible hliB gene in the cyanobacterium Synechocystis PCC 6803 as the RpaB (Slr0947) response regulator.     

On the functional significance of the polypeptide PsbY for photosynthetic water oxidation in the cyanobacterium<Emphasis Type="Italic"> Synechocystis</Emphasis> sp. strain PCC 6803

Recent investigations have revealed that the cyanobacterial photosystem II complex contains more than 26 polypeptides. The functions of most of the low-molecular-mass polypeptides, including PsbY, have remained elusive. Here we present a comparative characterization of the wild-type Synechocystis sp. strain PCC 6803 and a PsbY-free mutant derived from it. The results show that growth of the PsbY-free mutant was comparable to that of the wild-type when cells were cultivated in complete BG11 medium or under initial manganese or chloride limitation, and when illuminated at 20 or 200 E m^–2 s^–1. However, while growth rates of both the wild-type and the PsbY-free mutant were reduced when cells were cultivated in BG11 medium in the absence of calcium, the reduction was significantly greater in the case of the PsbY-free mutant. This differential effect on growth of the mutant relative to the wild-type in CaCl₂ deficient medium was detected when the cells were illuminated with high-intensity light (200 E m^–2 s^–1) but not when light levels were lower (20 E m^–2 s^–1). The differential effect on growth was associated with lower O₂ evolving activity in the mutant compared to wild-type cells. The mutant was also found to be more sensitive to photoinhibition, and showed an altered pattern of fluorescence emission at 77 K. In addition, mass spectrometric analysis revealed that PsbY-free cells cultivated in CaCl₂ sufficient medium (in which no growth reduction was observed) had a significantly higher O₂ evolution from hydrogen peroxide and a lower O₂ evolution from water under flash light illumination than wild-type cells. These results imply that photosystem II is slightly impaired in the PsbY-free mutant, and that the mutant is less capable of coping with low levels of Ca²⁺ than the wild-type.Communicated by R. G. Herrmann     

Functional identification of sll1383 from<Emphasis Type="Italic"> Synechocystis</Emphasis> sp PCC 6803 as L-<Emphasis Type="Italic">myo</Emphasis>-inositol 1-phosphate phosphatase (EC 3.1.3.25): molecular cloning,expression and characterization

The genome sequence of the cyanobacterium Synechocystis sp. PCC6803 revealed four Open reading frame (ORF) encoding putative inositol monophosphatase or inositol monophosphatase-like proteins. One of the ORFs, sll1383, is ∼870 base pair long and has been assigned as a probable myo-inositol 1 (or 4) monophosphatase (IMPase; EC 3.1.3.25). IMPase is the second enzyme in the inositol biosynthesis pathway and catalyses the conversion of L-myo-inositol 1-phosphate to free myo-inositol. The present work describes the functional assignment of ORF sll1383 as myo-inositol 1-phosphate phosphatase (IMPase) through molecular cloning, bacterial overexpression, purification and biochemical characterization of the gene product. Affinity (K _m) of the recombinant protein for the substrate DL-myo-inositol 1-phosphate was found to be much higher (0.0034 ± 0.0003 mM) compared to IMPase(s) from other sources but in comparison V _max (∼0.033 μmol Pi/min/mg protein) was low. Li⁺ was found to be an inhibitor (IC₅₀ 6.0 mM) of this enzyme, other monovalent metal ions (e.g. Na⁺, K⁺ NH₄⁺) having no significant effect on the enzyme activity. Like other IMPase(s), the activity of this enzyme was found to be totally Mg²⁺ dependent, which can be substituted partially by Mn²⁺. However, unlike other IMPase(s), the enzyme is optimally active at ∼42°C. To the best of our knowledge, sll1383 encoded IMPase has the highest substrate affinity and specificity amongst the known examples from other prokaryotic sources. A possible application of this recombinant protein in the enzymatic coupled assay of L-myo-inositol 1-phosphate synthase (MIPS) is discussed.     

Effects of Dark Adaptation on Light-Induced Electron Transport through Photosystem I in the Cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

Kinetics of the redox reactions in the reaction center (P700) of photosystem I (PSI) of the cyanobacterium Synechocystis sp. PCC 6803 have been studied by EPR spectroscopy. The redox kinetics were recorded based on accumulation of the EPRI signal when the final signal was the sum of individual signals produced in response to illumination of the cells. After prolonged (more than 3 sec) dark intervals between illuminations, the kinetic curve of the EPR signal from P700+ was multiphasic. After a sharp increase in the signal amplitude at the beginning of illumination (phase I), the amplitude rapidly (for 0.1-0.2 sec) decreased (phase II). Then the signal amplitude gradually increased (phase III) until the steady rate of electron transfer was established. With short-term (1 sec) dark intervals between the flashes and also in the presence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU), the kinetics of the light-induced increase in the EPR signal from P700+ were monophasic. Inhibition with iodoacetamide of electron transport on the acceptor side of PSI under anaerobic conditions or an increase in the amount of respiration substrates on addition of glucose into a suspension of DCMU-treated wild-type cells increased the level of P700 reduction in phase III. The findings suggest that the kinetic curve of the EPR signal from P700+ is determined by both the electron entrance onto P700+ on the donor side of PSI and activity of electron acceptors of PSI.     

N-terminus deletion affecting the preparation of soluble cyanobacterial glutaredoxin in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Glutaredoxin has been implicated in maintenance of a normal cellular thiol/disufide ratio and the regeneration of oxidatively damaged proteins. In order to obtain more information about these important regulatory proteins in cyanobacteria, we have previously cloned and expressed the first cyanobacterial glutaredoxin gene ssr2061 in Escherichia coli. In this work, the second glutaredoxin gene slr1562 was studied. About 90% of Grx2061 coded by ssr2061 was produced in a soluble form while 90% of Grx1562 coded by slr1562 was found in inclusion bodies. To improve the production of soluble Grx1562, we constructed two mutants: Grx1562NC with cysteines in conserved site substituted by serines, and Grx1562M with N-terminus hydrophobic region deletion. Only the latter mutant was successfully expressed in soluble form with increased glutaredoxin activity and showed less sensitivity in oxidative stress. Spectroscopic analysis shows that the structure of Grx1562M with less hydrophobic nature could give more opportunity for protein solubility and could improve the substrate catalytic efficiency. These results suggest that hydrophobic N-terminus determines the insolubility of Grx1562 and may provide another strategy for increasing expression level of soluble heterologous proteins in E. coli. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 3, pp. 383–391.     

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.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-<Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transformation of<Emphasis Type="Italic"> Helminthosporium turcicum</Emphasis>, the maize leaf-blight fungus

Agrobacterium tumefaciens has the ability to transfer its T-DNA to plants, yeast, filamentous fungi, and human cells and integrate it into their genome. Conidia of the maize pathogen Helminthosporium turcicum were transformed to hygromycin B resistance by a Agrobacterium-tumefaciens-mediated transformation system using a binary plasmid vector containing the hygromycin B phosphotransferase (hph) and the enhanced green fluorescent protein (EGFP) genes controlled by the gpd promoter from Agaricus bisporus and the CaMV 35S terminator. Agrobacterium-tumefaciens-mediated transformation yielded stable transformants capable of growing on increased concentrations of hygromycin B. The presence of hph in the transformants was confirmed by PCR, and integration of the T-DNA at random sites in the genome was demonstrated by Southern blot analysis. Agrobacterium-tumefaciens-mediated transformation of Helminthosporium turcicum provides an opportunity for advancing studies of the molecular genetics of the fungus and of the molecular basis of its pathogenicity on maize.     

Analyses of<Emphasis Type="Italic"> cis</Emphasis> -acting elements that affect the transposition of<Emphasis Type="Italic"> Mos1 mariner</Emphasis> transposons in vivo

The left (5) inverted terminal repeat (ITR) of the Mos1 mariner transposable element was altered by site-directed mutagenesis so that it exactly matched the nucleotide sequence of the right (3) ITR. The effects on the transposition frequency resulting from the use of two 3 ITRs, as well as those caused by the deletion of internal portions of the Mos1 element, were evaluated using plasmid-based transposition assays in Escherichia coli and Aedes aegypti. Donor constructs that utilized two 3 ITRs transposed with greater frequency in E. coli than did donor constructs with the wild-type ITR configuration. The lack of all but 10 bp of the internal sequence of Mos1 did not significantly affect the transposition frequency of a wild-type ITR donor. However, the lack of these internal sequences in a donor construct that utilized two 3 ITRs resulted in a further increase in transposition frequency. Conversely, the use of a donor construct with two 3 ITRs did not result in a significant increase in transposition in Ae. aegypti. Furthermore, deletion of a large portion of the internal Mos1 sequence resulted in the loss of transposition activity in the mosquito. The results of this study indicate the possible presence of a negative regulator of transposition located within the internal sequence, and suggest that the putative negative regulatory element may act to inhibit binding of the transposase to the left ITR. The results also indicate that host factors which are absent in E. coli, influence Mos1 transposition in Ae. aegypti.Communicated by G. P. Georgiev     

Behaviour of the hyphae of<Emphasis Type="Italic"> Laccaria laccata</Emphasis> in the presence of<Emphasis Type="Italic"> Trichoderma harzianum</Emphasis> in vitro

The growth rate and the behaviour of Laccaria laccata and Trichoderma harzianum hyphae in co-culture and in the rhizosphere of 3-month-old Pinus sylvestris seedlings grown in vitro were investigated. In the interaction zone, hyphae of L. laccata became more pigmented and formed short branches growing towards the hyphae of the saprobic fungus, coiled around them and penetrated sporadically. Vacuolated hyphae of T. harzianum showed protoplasm granulation and breaks in walls followed by release of protoplasts. In the rhizosphere, the mantle hyphae of L. laccata showed a tendency to surround conidia of T. harzianum. No obvious penetration of the conidial walls by the hyphae of the mycorrhizal fungus was observed by scanning electron microscopy. Instead, in rare cases, the hyphae of L. laccata showed marked wrinkles, and a partial degradation of a mucilaginous material covering the mantle appeared to occur.     

Antibacterial activity of<Emphasis Type="Italic">Ulva lactuca</Emphasis> against methicillin-resistant<Emphasis Type="Italic">Staphylococcus aureus</Emphasis> (MRSA)

Thein vitro antimicrobial activity of the marine green algaeUlva lactuca was examined against gram-positive bacteria, gram-negative bacteria, and a fungus. The ethyl-ether extract of algae exhibited a broad-spectrum of antibacterial activity. but not antifungal activity againstCandida albicans. In particular, theU. lactuca extract showed strong activity aganst the bacterium methicillin-resistantStaphylococcus aureus (MRSA). This result confirms the potential use of seaweed extracts as a source of antibacterial compounds or as a health-promoting food for aquaculture.     

Structural and transcriptional analysis of<Emphasis Type="Italic"> S</Emphasis>-locus F-box genes in<Emphasis Type="Italic"> Antirrhinum</Emphasis>

A class of ribonucleases termed S-RNases, which control the pistil expression of self-incompatibility, represents the only known functional products encoded by the S locus in species from the Solanaceae, Scrophulariaceae and Rosaceae. Previously, we identified a pollen-specific F-box gene, AhSLF (S locus F-box)-S₂, very similar to S₂-RNase in Antirrhinum, a member of the Scrophulariaceae. In addition, AhSLF-S₂ also detected the presence of its homologous DNA fragments. To identify these fragments, we constructed two genomic DNA libraries from Antirrhinum self-incompatible lines carrying alleles S₁S₅ and S₂S₄, respectively, using a transformation-competent artificial chromosome (TAC) vector. With AhSLF-S₂-specific primers, TAC clones containing both AhSLF-S₂ and its homologs were subsequently identified (S₂TAC, S₅TACa, S₄TAC, and S₁TACa). DNA blot hybridization, sequencing and segregation analyses revealed that they are organized as single allelic copies (AhSLF-S₂, -S₁, -S₄ and -S₅) tightly linked to the S-RNases. Furthermore, clusters of F-box genes similar to AhSLF-S₂ were identified. In total, three F-box genes (AhSLF-S₂, -S₂A and -S₂C) in S₂TAC (51 kb), three (AhSLF-S₄, -S₄A and -S₄D) in S₄TAC (75 kb), two (AhSLF-S₅ and -S₅A) in S₅TACa (55 kb), and two (AhSLF-S₁ and -S₁E) in S₁TACa (71 kb), respectively, were identified. Paralogous copies of these genes show 38–54% identity, with allelic copies sharing 90% amino acid identity. Among these genes, three (AhSLF-S₂C, -S₄D and -S₁E) were specifically expressed in pollen, similar to AhSLF-S₂, implying that they likely play important roles in pollen, whereas three AhSLF-SA alleles showed no detectable expression. In addition, several types of retroelements and transposons were identified in the sequenced regions, revealing some detailed information on the structural diversity of the S locus region. Taken together, these results indicate that both single allelic and tandemly duplicated genes are associated with the S locus in Antirrhinum. The implications of these findings in evolution and possible roles of allelic AhSLF-S genes in the self-incompatible reaction are discussed in species like Antirrhinum.Sequence data from this article have been deposited with the EMBL/GenBank databases under accession numbers AJ300474, AJ515534, AJ515536 and AJ515535     

A homozygous <Emphasis Type="Italic">recA</Emphasis> mutant of <Emphasis Type="Italic">Synechocystis</Emphasis> PCC6803: construction strategy and characteristics eliciting a novel RecA independent UVC resistance in dark

We report here the construction of a homozygous recA460::cam insertion mutant of Synechocystis sp. PCC 6803 that may be useful for plant molecular genetics by providing a plant like host free of interference from homologous recombination. The homozygous recA460::cam mutant is highly sensitive to UVC under both photoreactivating and nonphotoreactivating conditions compared to the wild type (WT). The liquid culture of the mutant growing in ~800 lx accumulates nonviable cells to the tune of 86% as estimated by colony counts on plates incubated at the same temperature and light intensity. The generation time of recA mutant in standard light intensity (2,500 lx) increases to 50 h compared to 28 h in lower light intensity (~800 lx) that was used for selection, thus explaining the earlier failures to obtain a homozygous recA mutant. The WT, in contrast, grows at faster rate (23 h generation time) in standard light intensity compared to that at ~800 lx (26 h). The Synechocystis RecA protein supports homologous recombination during conjugation in recA ^— mutant of Escherichia coli, but not the SOS response as measured by UV sensitivity. It is suggested that using this homozygous recA460::cam mutant, investigations can now be extended to dissect the network of DNA repair pathways involved in housekeeping activities that may be more active in cyanobacteria than in heterotrophs. Using this mutant for the first time we provide a genetic evidence of a mechanism independent of RecA that causes enhanced UVC resistance on light to dark transition.     

The <Emphasis Type="Italic">Caenorhabditis</Emphasis><Emphasis Type="Italic">briggsae</Emphasis> genome contains active <Emphasis Type="Italic">CbmaT1</Emphasis> and <Emphasis Type="Italic">Tcb1</Emphasis> transposons

The maT clade of transposons is a group of transposable elements intermediate in sequence and predicted protein structure to mariner and Tc transposons, with a distribution thus far limited to a few invertebrate species. We present evidence, based on searches of publicly available databases, that the nematode Caenorhabditis briggsae has several maT-like transposons, which we have designated as CbmaT elements, dispersed throughout its genome. We also describe two additional transposon sequences that probably share their evolutionary history with the CbmaT transposons. One resembles a fold back variant of a CbmaT element, with long (380-bp) inverted terminal repeats (ITRs) that show a high degree (71%) of identity to CbmaT1. The other, which shares only the 26-bp ITR sequences with one of the CbmaT variants, is present in eight nearly identical copies, but does not have a transposase gene and may therefore be cross mobilised by a CbmaT transposase. Using PCR-based mobility assays, we show that CbmaT1 transposons are capable of excising from the C. briggsae genome. CbmaT1 excised approximately 500 times less frequently than Tcb1 in the reference strain AF16, but both CbmaT1 and Tcb1 excised at extremely high frequencies in the HK105 strain. The HK105 strain also exhibited a high frequency of spontaneous induction of unc-22 mutants, suggesting that it may be a mutator strain of C. briggsae.     

Expression patterns of <Emphasis Type="Italic">engrailed</Emphasis> and <Emphasis Type="Italic">dpp</Emphasis> in the gastropod <Emphasis Type="Italic">Lymnaea stagnalis</Emphasis>

We isolated the full-length cDNAs of engrailed and dpp-BMP2/4 orthologues from the pond snail Lymnaea stagnalis and examined their expression patterns during development by the whole mount in situ hybridization. At the gastrula and trochophore stages, engrailed is expressed in the peripheral ectoderm of the presumptive and invaginating shell gland, corroborating its role in the shell formation that is widely conserved among molluscs. At the same stages, dpp-BMP2/4 is expressed in the right-hand side ectoderm of the shell gland and in the invaginating stomodaeum. Unlike in the gastropod Patella vulgata, our results suggested that dpp-BMP2/4 has a role in the shell formation, rather than in the regional specification and that it could be involved in the specification pathway of the left–right asymmetry of the developing shell in L. stagnalis.