Influence of ultraviolet-C on structure and function of <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942 photolyase

In this work, an over-expressed cyclobutane pyrimidine dimer (CPD) photolyase of Synechococcus sp. PCC 7942 was used to investigate UV-C (ultraviolet irradiation of C-region) influence on photoreactivation. In vivo photoreactivation experiments indicated that the survival rate decreased from 100 to 2.6% when the UV-C flux was increased from 1.1 to 68.5 μW/cm². It seemed that the photolyase was easily inactivated at UV-C intensities ≥25.5 μW/cm². Spectrometric analysis indicated that tertiary structure of the photolyase changed evidently when the UV-C fluxes were ≥25.5 μW/cm², while the secondary structure was almost unchanged even at 170 μW/cm². Band shift assay indicated that catalytic activity of the photolyase was impaired at fluxes ≥25.5 μW/cm², but no significant influence on DNA-binding activity was observed. These results suggest that photoreactivation is efficient at UV-C fluxes ≤25.5 μW/cm², but would be impaired by intense UV-C irradiation due to structure changes of the photolyase. Published in Russian in Biokhimiya, 2007, Vol. 72, No. 5, pp. 668–673.     

Electron self-exchange and self-amplified posttranslational modification in the hemoglobins from <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 and <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002

Many heme proteins undergo covalent attachment of the heme group to a protein side chain. Such posttranslational modifications alter the thermodynamic and chemical properties of the holoprotein. Their importance in biological processes makes them attractive targets for mechanistic studies. We have proposed a reductively driven mechanism for the covalent heme attachment in the monomeric hemoglobins produced by the cyanobacteria Synechococcus sp. PCC 7002 and Synechocystis sp. PCC 6803 (GlbN) (Nothnagel et al. in J Biol Inorg Chem 16:539–552, 2011). These GlbNs coordinate the heme iron with two axial histidines, a feature that distinguishes them from most hemoglobins and conditions their redox properties. Here, we uncovered evidence for an electron exchange chain reaction leading to complete heme modification upon substoichiometric reduction of GlbN prepared in the ferric state. The GlbN electron self-exchange rate constants measured by NMR spectroscopy were on the order of 10²–10³ M⁻¹ s⁻¹ and were consistent with the proposed autocatalytic process. NMR data on ferrous and ferric Synechococcus GlbN in solution indicated little dependence of the structure on the redox state of the iron or cross-link status of the heme group. This allowed the determination of lower bounds to the cross-exchange rate constants according to Marcus theory. The observations illustrate the ability of bishistidine hemoglobins to undergo facile interprotein electron transfer and the chemical relevance of such transfer for covalent heme attachment.     

Alleviation of NaCl toxicity in the cyanobacterium <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7942 by exogenous calcium supplementation

Salinity (NaCl) is one of the major problems associated with irrigated agricultural lands, especially rice fields. Being the common inhabitants of rice fields, cyanobacteria frequently experience high concentration of NaCl which in turn causes cellular damage. Therefore, mitigation of NaCl stress in cyanobacteria, plant growth-promoting microorganisms, is of utmost importance. The present study was designed to investigate the role of calcium in the alleviation of NaCl stress-induced cellular in Synechococcus sp. PCC 7942. The cyanobacterium was subjected to sub-lethal concentration of NaCl (800 mM) with and without the supplementation of calcium (1 mM CaCl₂) for 8 days. The results showed a drastic reduction in growth due to excess NaCl, but supplementation of CaCl₂ reduced the salt stress damage and partially restored growth. Application of calcium increased pigment contents, photosynthetic efficiency, antioxidative enzyme activity, osmolyte contents and reduced the intracellular sodium ion concentration, MDA content, electrolyte leakage and free oxygen radical generation. Furthermore, proteins involved in photosynthesis, respiration, ATP synthesis and protein synthesis along with two hypothetical proteins were also observed to be upregulated in the cyanobacterium in presence of calcium. Furthermore, proteins related to oxidative stress defence, nitrogen metabolism, carbohydrate metabolism, fatty acid metabolism and secondary metabolism were found to be upregulated by several fold. Therefore, our study suggests that calcium suppresses salt toxicity in Synechococcus sp. PCC 7942 by restricting the entry of Na⁺ into the cell, increasing osmolyte production and upregulating defence-related proteins.     

Chemical reactivity of <Emphasis Type="Italic">Synechococcus</Emphasis> sp. PCC 7002 and <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803 hemoglobins: covalent heme attachment and bishistidine coordination

In the absence of an exogenous ligand, the hemoglobins from the cyanobacteria Synechocystis sp. PCC 6803 and Synechococcus sp. PCC 7002 coordinate the heme group with two axial histidines (His46 and His70). These globins also form a covalent linkage between the heme 2-vinyl substituent and His117. The in vitro mechanism of heme attachment to His117 was examined with a combination of site-directed mutagenesis, NMR spectroscopy, and optical spectroscopy. The results supported an electrophilic addition with vinyl protonation being the rate-determining step. Replacement of His117 with a cysteine demonstrated that the reaction could occur with an alternative nucleophile. His46 (distal histidine) was implicated in the specificity of the reaction for the 2-vinyl group as well as protection of the protein from oxidative damage caused by exposure to exogenous H₂O₂.     

Isolation of C-phycocyanin from <Emphasis Type="Italic">Synechococcus</Emphasis> sp., (<Emphasis Type="Italic">Anacystis nidulans</Emphasis> BD1)

We report a procedure for obtaining fairly pure phycocyanin from a local isolate of the cyanobacterium Synechococcus sp (Anacystis nidulans BD1). Cells were incubated with 1 mg∙mL⁻¹ of lysozyme at 37°C for 16 h with shaking. The cell-free extract was treated with activated charcoal and chitosan. The purity (A _620/280) of phycocyanin obtained after lysozyme treatment was up to 2.18, which could be improved to 4.72 after incubation with activated charcoal and chitosan. The yield of phycocyanin was 80–100 mg∙g⁻¹ dry weight of cells. The method reported here is a single-step and efficient procedure and has the potential to be adopted for large-scale production of phycocyanin.     

Enhanced biomass and oxidative stress tolerance of <Emphasis Type="Italic">Synechococcus elongatus</Emphasis> PCC 7942 overexpressing the <Emphasis Type="Italic">DHAR</Emphasis> gene from <Emphasis Type="Italic">Brassica juncea</Emphasis>

Objectives

To improve the oxidative stress tolerance, biomass yield, and ascorbate/dehydroascorbate (AsA/DHA) ratio of Synechococcus elongatus PCC 7942 in the presence of H₂O₂, by heterologous expression of the dehydroascorbate reductase (DHAR) gene from Brassica juncea (BrDHAR).

Results

Under H₂O₂ stress, overexpression of BrDHAR in the transgenic strain (BrD) of S. elongatus greatly increased the AsA/DHA ratio. As part of the AsA recycling system, the oxidative stress response induced by reactive oxygen species was enhanced, and intracellular H₂O₂ level decreased. In addition, under H₂O₂ stress conditions, the BrD strain displayed increased growth rate and biomass, as well as higher chlorophyll content and deeper pigmentation than did wild-type and control strains.

Conclusion

By maintaining the AsA pool and redox homeostasis, the heterologous expression of BrDHAR increased S. elongatus tolerance to H₂O₂ stress, improving the biomass yield under these conditions. The results suggest that the BrD strain of S. elongatus, with its ability to attenuate the deleterious effects of ROS caused by environmental stressors, could be a promising platform for the generation of biofuels and other valuable bioproducts.

     

<Emphasis Type="Italic">Kretzschmaria varians</Emphasis> sp. nov., <Emphasis Type="Italic">Xylaria coremiifera</Emphasis> sp. nov. and <Emphasis Type="Italic">Xylaria umbonata</Emphasis> sp. nov. from Costa Rica

Kretzschmaria varians, a species apparently related to K. micropus, is described as new. It is distinguished primarily by having asci with 2 to 8 ascospores with inconstant germination slit length and remains of synnemata on stromata and surrounding substrate. Xylaria coremiifera, described here as new, bears small fragile coremia on pulvinate stromata and the surrounding substrate. Asci often have fewer than 8 ascospores, most frequently 4. Xylaria umbonata, described here as new, produces perithecia around a central umbo that appears to be the remains of a synnema. Ascospores have long spiralling germination slits.     

Molecular and biochemical characterization of <Emphasis Type="Italic">Ba</Emphasis>-EGA,a cellulase secreted by <Emphasis Type="Italic">Bacillus</Emphasis> sp. AC-1 from <Emphasis Type="Italic">Ampullaria crosseans</Emphasis>

A novel gene (Ba-ega) of Bacillus sp. AC-1, encoding an endoglucanase (Ba-EGA), was cloned and expressed in Escherichia coli. Ba-ega, containing a 1,980-bp open reading frame (ORF), encoded a protein of 659 amino acids and had a molecular mass of 74.87 kDa. Ba-EGA was a modular enzyme composed of a family-9 glycosyl hydrolase catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). To investigate the functions of the CBM3 and CM9, a number of truncated derivatives of Ba-EGA were constructed, and all were active. The catalytic module (rCM9) alone was less stable at high temperature than the recombinant Ba-EGA (rBa-EGA). The temperature stability for the complex of rCM9 and rCBM3 was still lower than rBa-EGA, but higher than rCM9 alone. These observations indicated the existence of a non-covalent interaction between CM9 and CBM3 that might strengthen the stability of CM9. However, this interaction is not strong enough to mimic the protective effect of the CBM in the wild-type enzyme.     

Molecular and biochemical characterization of squalene synthase from <Emphasis Type="Italic">Siraitia grosvenorii</Emphasis>

Objectives

To clone and characterize the squalene synthase from Siraitia grosvenorii (SgSQS).

Results

The gene encoding SgSQS was cloned. SgSQS has 417 amino acid residues with an pI of 7.3. There are 32 phosphorylation sites in its sequence: S⁴⁸ as well as S¹⁹⁶ play important roles in regulation of enzyme activity. The enzyme is a monomeric protein with a cave-like active center formed by α helixes and has two transmembrane domains at its C-terminus. SgSQS mRNA expression in stem and root were about twice as much as that in leaf and peel. Full-length SgSQS with measurable catalytic activity was expressed in Escherichia coli. SgSQS activity was optimal at 37 °C and pH 7.5 respectively.

Conclusion

SgSQS gene was cloned, and the molecular structure and biochemical function of SgSQS were characterized.

     

Molecular Detection of <Emphasis Type="Italic">Streptococcus pyogenes</Emphasis> and <Emphasis Type="Italic">Streptococcus dysgalactiae</Emphasis> subsp. <Emphasis Type="Italic">equisimilis</Emphasis>

We developed molecular diagnostic assays for the detection of Streptococcus pyogenes (GAS) and Streptococcus dysgalactiae subsp. equisimilis (SDSE), two streptococcal pathogens known to cause both pharyngitis and more invasive forms of disease in humans. Two real-time PCR assays coupled with an internal control were designed to be performed in parallel. One assay utilizes a gene target specific to GAS, and the other utilizes a gene target common to the two species. Both assays showed 2–3 orders of magnitude improved analytical sensitivity when compared to a commercially available rapid antigen test. In addition, when compared to standard culture in an analysis of 96 throat swabs, the real-time PCR assays resulted in clinical sensitivity and specificity of 91.7 and 100%, respectively. As capital equipment costs for real-time PCR can be prohibitive in smaller laboratories, the real-time PCR assays were converted to a low-density microarray format designed to function with an inexpensive photopolymerization-based non-enzymatic signal amplification (NESA™) method. S. pyogenes was successfully detected on the low-density microarray in less than 4 h from sample extraction through detection.     

<Emphasis Type="Italic">In silico</Emphasis> characterization and Molecular modeling of double-strand break repair protein <Emphasis Type="Italic">MRE11</Emphasis> from <Emphasis Type="Italic">Phoenix dactylifera</Emphasis> v deglet nour

Background

DNA double-strand breaks (DSBs) are highly cytotoxic and mutagenic. MRE11 plays an essential role in repairing DNA by cleaving broken ends through its 3′ to 5′ exonuclease and single-stranded DNA endonuclease activities.

Methods

The present study aimed to in silico characterization and molecular modeling of MRE11 from Phoenix dactylifera L cv deglet nour (DnMRE11) by various bioinformatic approaches. To identify DnMRE11 cDNA, assembled contigs from our cDNA libraries were analysed using the Blast2GO2.8 program.

Results

The DnMRE11 protein length was 726 amino acids. The results of HUMMER show that DnMRE11 is formed by three domains: the N-terminal core domain containing the nuclease and capping domains, the C-terminal half containing the DNA binding and coiled coil region. The structure of DnMRE11 is predicted using the Swiss-Model server, which contains the nuclease and capping domains. The obtained model was verified with the structure validation programs such as ProSA and QMEAN servers for reliability. Ligand binding studies using COACH indicated the interaction of DnMRE11 protein with two Mn²⁺ ions and dAMP. The ConSurf server predicted that residues of the active site and Nbs binding site have high conservation scores between plant species.

Conclusions

A model structure of DnMRE11 was constructed and validated with various bioinformatics programs which suggested the predicted model to be satisfactory. Further validation studies were conducted by COACH analysis for active site ligand prediction, and revealed the presence of six ligands binding sites and two ligands (2 Mn²⁺ and dAMP).

     

<Emphasis Type="Italic">Ty</Emphasis>1<Emphasis Type="Italic">/copia</Emphasis>- and <Emphasis Type="Italic">Ty</Emphasis>3<Emphasis Type="Italic">/gypsy</Emphasis>-like DNA sequences in <Emphasis Type="Italic">Helianthus </Emphasis>species

Two repeated DNA sequences isolated from a partial genomic DNA library of Helianthus annuus, p HaS13 and p HaS211, were shown to represent portions of the int gene of a Ty3 /gypsy retroelement and of the RNase-Hgene of a Ty1 /copia retroelement, respectively. Southern blotting patterns obtained by hybridizing the two probes to BglII- or DraI-digested genomic DNA from different Helianthus species showed p HaS13 and p HaS211 were parts of dispersed repeats at least 8 and 7 kb in length, respectively, that were conserved in all species studied. Comparable hybridization patterns were obtained in all species with p HaS13. By contrast, the patterns obtained by hybridizing p HaS211 clearly differentiated annual species from perennials. The frequencies of p HaS13- and p HaS211-related sequences in different species were 4.3x10(4)-1.3x10(5) copies and 9.9x10(2)-8.1x10(3) copies per picogram of DNA, respectively. The frequency of p HaS13-related sequences varied widely within annual species, while no significant difference was observed among perennial species. Conversely, the frequency variation of p HaS211-related sequences was as large within annual species as within perennials. Sequences of both families were found to be dispersed along the length of all chromosomes in all species studied. However, Ty3 /gypsy-like sequences were localized preferentially at the centromeric regions, whereas Ty1/ copia-like sequences were less represented or absent around the centromeres and plentiful at the chromosome ends. These findings suggest that the two sequence families played a role in Helianthusgenome evolution and species divergence, evolved independently in the same genomic backgrounds and in annual or perennial species, and acquired different possible functions in the host genomes.     

Mercury Tolerance of Thermophilic <Emphasis Type="Italic">Bacillus</Emphasis> sp. and <Emphasis Type="Italic">Ureibacillus</Emphasis> sp

Although resistance of microorganisms to Hg(II) salts has been widely investigated and resistant strains have been reported from many eubacterial genera, there are few reports of mercuric ion resistance in extremophilic microorganisms. Moderately thermophilic mercury resistant bacteria were selected by growth at 62 °C on Luria agar containing HgCl₂. Sequence analysis of 16S rRNA genes of two isolates showed the closest matches to be with Bacillus pallidus and Ureibacillus thermosphaericus. Minimum inhibitory concentration (MIC) values for HgCl₂ were 80 μg/ml and 30 μg/ml for these isolates, respectively, compared to 10 μg/ml for B. pallidus H12 DSM3670, a mercury-sensitive control. The best-characterised mercury-resistant Bacillus strain, B. cereus RC607, had an MIC of 60 μg/ml. The new isolates had negligible mercuric reductase activity but removed Hg from the medium by the formation of a black precipitate, identified as HgS by X-ray powder diffraction analysis. No volatile H₂S was detected in the headspace of cultures in the absence or presence of Hg²⁺, and it is suggested that a new mechanism of Hg tolerance, based on the production of non-volatile thiol species, may have potential for decontamination of solutions containing Hg²⁺ without production of toxic volatile H₂S.     

Taxonomic characterization of <Emphasis Type="Italic">Haloferax</Emphasis> sp. ("<Emphasis Type="Italic">H. alicantei</Emphasis>") strain Aa 2.2: description of <Emphasis Type="Italic">Haloferax lucentensis</Emphasis> sp. nov.

An extremely halophilic archaeon, previously named as Haloferax sp. strain Aa 2.2 or "Haloferax alicantei" that has been extensively used for genetic studies with halobacteria, was taxonomically characterized by using phenotypic tests (including morphological, physiological, biochemical and nutritional features), DNA-DNA hybridization and 16S rRNA sequence phylogenetic analysis. This organism was isolated in 1986 by Torreblanca et al. from a pond of a Spanish saltern located in Alicante. The cells were pleomorphic, Gram negative and grew optimally at 25% NaCl. The polar lipid composition was similar to that of species of the genus Haloferax. The DNA G+C content of this strain was 64.5 mol%. Phylogenetic analysis based on 16S rRNA sequence comparison confirmed that this archaeon is a member of the genus Haloferax and was most closely related to Haloferax volcanii. DNA-DNA hybridization between strain Aa 2.2 and the type strain of all named species of the genus Haloferax revealed low levels of relatedness (25-2%), supporting the placement of this organism in a new species. On the basis of the phenotypic characteristics, molecular data and phylogenetic analysis we propose to name strain Aa 2.2 as a new species, Haloferax lucentensis sp. nov. The type strain is Aa 2.2 (=JCM 9276=NCIMB 13854=CIP 107410=DSM 14919=CECT 5871=CCM 7023).     

Transcription of the extended <Emphasis Type="Italic">hyp</Emphasis>-operon in <Emphasis Type="Italic">Nostoc</Emphasis>sp. strain PCC 7120

Background  

The maturation of hydrogenases into active enzymes is a complex process and e.g. a correctly assembled active site requires the involvement of at least seven proteins, encoded by hypABCDEF and a hydrogenase specific protease, encoded either by hupW or hoxW. The N₂-fixing cyanobacterium Nostoc sp. strain PCC 7120 may contain both an uptake and a bidirectional hydrogenase. The present study addresses the presence and expression of hyp-genes in Nostoc sp. strain PCC 7120.