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.     

<Emphasis Type="Italic">In vitro</Emphasis> DNA-binding properties of a manganese response regulator (ManR) from <Emphasis Type="Italic">Anabaena</Emphasis> sp.

ManR of Anabaena sp. PCC 7120 is a manganese response regulator. Two ManR molecules bind to the specific DNA sequences at the same time, which was demonstrated by our previous results. From size exclusion chromatography, ManR exits as monomer in solution. Therefore, cooperative interactions of ManR–ManR play a role in DNA binding of the ManR, suggesting that ManR molecules bind co-operatively to DNA. When serial deletions of N-terminal of the ManR were also carried out the mutant proteins, ManRC111, ManRC130 and ManRC158, had completely lost the in DNA binding activity. Mutants ManRC 196, ManRC206, ManRC221 and ManRC230, however, could specifically bind to DNA, indicating that the amino acid residues between Val₁₆ and Ile₇₈ of the N-terminal of ManR are necessary for the DNA binding activity of C-terminal domain.Revisions requested 20 Ocotober 2004/15 November 2004; Revisions received 10 November/13 December 2004     

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.     

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.     

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.     

Modulation of biocidal activity of <Emphasis Type="Italic">Calothrix</Emphasis> sp. and <Emphasis Type="Italic">Anabaena</Emphasis> sp. by environmental factors

An investigation was undertaken to evaluate a set of cyanobacterial strains in terms of production of biocidal compounds exhibiting allelochemical and fungicidal properties. Two cyanobacterial strains — Anabaena sp. and Calothrix sp. were selected for further investigation, on the basis of their larger inhibition zones on the lawn of Synechocystis and Synechococcus sp. and two phytopathogenic fungi — Rhizoctonia bataticola and Pythium debaryanum. The diameter of the inhibition zone was largest when extracellular filtrates of the two cultures incubated at high light intensity (90–100 μmol photons m⁻² s⁻¹) and temperature (40 ± 2 °C) or grown in medium containing two-folds higher P (1.4 mg/L, as compared to 0.7 mg/L in BG 11 medium) were taken. A pH of 8 was the most optimal for both strains, in terms of growth and biocidal activity. Partial purification of ethyl acetate extract using TLC, followed by GLC revealed a single peak. This study highlights the importance of environmental factors in aggravating or reducing the toxic effects of these harmful cyanobacteria and their potential as a biocontrol agent.     

Secondary structure estimation of recombinant <Emphasis Type="Italic">psbH</Emphasis>, encoding a photosynthetic membrane protein of cyanobacterium <Emphasis Type="Italic">Synechocystis</Emphasis> sp. PCC 6803

The PsbH protein of cyanobacterium Synechocystis sp. PCC 6803 was expressed as a fusion protein with glutathione-S transferase (GST) in E. coli grown on a mineral medium enriched in ¹⁵N isotope. After enzymatic cleavage of the fusion protein, the ¹H-¹⁵N-HSQC spectrum of PsbH protein in presence of the detergent β-D-octyl-glucopyranoside (OG) was recorded on a Bruker DRX 500 MHz NMR spectrometer equipped with a 5 mm TXI cryoprobe to enhance the sensitivity and resolution. Non-labelled protein was used for secondary structure estimation by deconvolution from circular dichroism (CD) spectra. Experimental results were compared with our results from a structural model of PsbH using a restraint-based comparative modelling approach combined with molecular dynamics and energetic modelling. We found that PsbH shows 34–38% α-helical structure (Thr36-Ser60), a maximum of around 15% of β-sheet, and 12–19% of β-turn.     

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.     

Paired cloning vectors for complementation of mutations in the cyanobacterium<Emphasis Type="Italic"> Anabaena</Emphasis> sp. strain PCC 7120

The clones generated in a sequencing project represent a resource for subsequent analysis of the organism whose genome has been sequenced. We describe an interrelated group of cloning vectors that either integrate into the genome or replicate, and that enhance the utility, for developmental and other studies, of the clones used to determine the genomic sequence of the cyanobacterium, Anabaena sp. strain PCC 7120. One integrating vector is a mobilizable BAC vector that was used both to generate bridging clones and to complement transposon mutations. Upon addition of a cassette that permits mobilization and selection, pUC-based sequencing clones can also integrate into the genome and thereupon complement transposon mutations. The replicating vectors are based on cyanobacterial plasmid pDU1, whose sequence we report, and on broad-host-range plasmid RSF1010. The RSF1010- and pDU1-based vectors provide the opportunity to express different genes from either cell-type-specific or -generalist promoters, simultaneously from different plasmids in the same cyanobacterial cells. We show that pDU1 ORF4 and its upstream region play an essential role in the replication and copy number of pDU1, and that ORFs alr2887 and alr3546 (hetF _A ) of Anabaena sp. are required specifically for fixation of dinitrogen under oxic conditions.     

Extracellular phycoerythrin-like protein released by freshwater cyanobacteria <Emphasis Type="Italic">Oscillatoria</Emphasis> and <Emphasis Type="Italic">Scytonema</Emphasis> sp.

During growth of the freshwater cyanobacteria, Oscillatoria sp. BTCC/A0004, and Scytonema sp. TISTR 8208, a pink pigment is released into the growth medium. The pigment from each source had a molecular weight of approximately 250 kDa and had adsorption maxima at 560 and 620 nm. These results suggest that pink pigment is a phycoerythrin-like protein. It inhibited the growth of green algae, Chlorella fusca and Chlamydomonas reinhardtii, but not other cyanobacteria or true bacteria. The concentration at which growth inhibition 50% occurred was 0.5, 6 and more than 10 mg ml⁻¹, respectively.     

Effect of temperature on the life history of <Emphasis Type="Italic">Eretmocerus</Emphasis> sp. nr. <Emphasis Type="Italic">furuhashii</Emphasis>, a parasitoid of <Emphasis Type="Italic">Bemisia tabaci</Emphasis>

Eretmocerus sp. nr. furuhashii (Hymenoptera: Aphelinidae) is an indigenous parasitoid of Bemisia tabaci (Gennadius)(Hemiptera: Aleyrodidae) from southern China; the effects of constant temperatures on the life history of E. sp. nr. furuhashii were examined in the laboratory. The developmental period ranged from 39.2 days at 20°C to 12.40 days at 32°C. A total of 263.4 degree-days were required to complete development with a lower developmental threshold temperature of 11.1°C. Of the eggs produced, 59.3% completed development at 20°C with completion increasing to 71.5% at 26°C. Adult female longevity was 10.8 days at 20°C and 5.2 days at 32°C while the mean daily offspring reproduced per female was highest at 29°C with 5.9 offspring. Adult oviposition peaked three days after emergence at 26, 29 and 32°C, and four days post-emergence at 20°C and 23°C. The total numbers of offspring produced per female ranged from 25.7 individuals at 32°C to 41.1 individuals at 20°C. The sex ratio had a female bias and ranged from 0.72 at 17°C to 0.51 at 35°C. The intrinsic rate of increase was 0.1727 at 29°C followed with 0.1606 at 32°C. Results indicated that E. sp. nr. furuhashii reaches its maximum biological potential at temperatures ranging from 26°C to 32°C.     

Four novel <Emphasis Type="Italic">Candida</Emphasis> species in the <Emphasis Type="Italic">Candida albicans</Emphasis>/<Emphasis Type="Italic">Lodderomyces elongisporus</Emphasis> clade isolated from the gut of flower beetles

Flower-visiting beetles belonging to three species of Cetoniidae were collected on three mountains near Beijing, China, and yeasts were isolated from the gut of the insects collected. Based on the 26S rDNA D1/D2 domain and internal transcribed spacer (ITS) region sequence analysis and phenotypic characterization, four novel anamorphic yeast species located in the Candida albicans/Lodderomyces elongisporus clade were identified from 18 of the strains isolated. The new species and type strains are designated as Candida blackwellae AS 2.3639^T (=CBS 10843^T), Candida jiufengensis AS 2.3688^T (=CBS 10846^T), Candida oxycetoniae AS 2.3656^T (=CBS 10844^T), and Candida pseudojiufengensis AS 2.3693^T (=CBS 10847^T). C. blackwellae sp. nov. was basal to the branch formed by C. albicans and C. dubliniensis with moderately strong bootstrap support. The closest relative of C. oxycetoniae was L. elongisporus. C. jiufengensis sp. nov. and C. pseudojiufengensis sp. nov. were closely related with each other and formed a branch in a subclade represented by C. parapsilosis and L. elongisporus.     

Mineralization of chlorpyrifos by co-culture of <Emphasis Type="Italic">Serratia</Emphasis> and <Emphasis Type="Italic">Trichosporon</Emphasis> spp.

A bacterial strain (Serratia sp.) that could transform chlorpyrifos to 3,5,6-trichloro-2-pyridinol (TCP) and a TCP-mineralizing fungal strain (Trichosporon sp.) were isolated from activated sludge by enrichment culture technique. The fungus could also degrade 50 mg chlorpyrifos l(-1) within 7 days. Co-cultures completely mineralized 50 mg chlorpyrifos l(-1) within 18 h at 30 degrees C and pH 8 using a total inocula of 0.15 g biomass l(-1).     

Allelopathic impact of artificially applied coumarin on <Emphasis Type="Italic">Fusarium oxysporum</Emphasis> f.sp. <Emphasis Type="Italic">niveum</Emphasis>

Watermelon production is threatened by fusarium wilt caused by Fusarium oxysporum f.sp. niveum (FON) in continuous cultivation system. Some elements, mainly allelochemicals, released from living roots or decayed plants might be associated with the disease. The purpose of this work was to evaluate the possible impact of coumarin, one kind of watermelon allelochemical, on FON. Furthermore, possible new mechanisms might be investigated during the ecological interactions of plant-microbe. Results showed that coumarin strongly inhibited growth of FON leading to a decrease in its biomass, dry weight of mycelia of FON in a liquid culture. The dry weight was decreased by 62.9% compared with control. The hyphal growth of FON on plates was stopped at high (>400 mg l⁻¹) concentrations of coumarin. At 320 mg l⁻¹, sporulation and enzyme activities of FON were also severely suppressed by coumarin. The yield of conidia, and the activities of proteinase, cellulase, and amylase were reduced by 98.9%, 79.7%, 29.8% and 15.9% respectively. However, conidial germination and mycotoxin (MT) production of FON were greatly stimulated, being increased by 55.7% and 14.9 fold at 320 mg l⁻¹ respectively. We conclude that coumarin acted as an allelochemical substance to inhibit growth and pathogenic enzyme activities of FON but to stimulate mycotoxin production and conidial germination. It was suggested that coumarin acted as a signal transduction element bridging plant and pathogen in the process of plant-microbe interactions.     

Characterization of <Emphasis Type="Italic">twist</Emphasis> and <Emphasis Type="Italic">snail</Emphasis> gene expression during mesoderm and nervous system development in the polychaete annelid <Emphasis Type="Italic">Capitella</Emphasis> sp. I

To investigate the evolutionary history of mesoderm in the bilaterian lineage, we are studying mesoderm development in the polychaete annelid, Capitella sp. I, a representative lophotrochozoan. In this study, we focus on the Twist and Snail families as candidate mesodermal patterning genes and report the isolation and in situ expression patterns of two twist homologs (CapI-twt1 and CapI-twt2) and two snail homologs (CapI-sna1 and CapI-sna2) in Capitella sp. I. CapI-twt1 is expressed in a subset of mesoderm derivatives during larval development, while CapI-twt2 shows more general mesoderm expression at the same stages. Neither twist gene is detected before the completion of gastrulation. The two snail genes have very distinct expression patterns. At cleavage and early gastrula stages, CapI-sna1 is broadly expressed in precursors of all three germ layers and becomes restricted to cells around the closing blastopore during late gastrulation; CapI-sna2 expression is not detected at these stages. After gastrulation, both snail genes are expressed in the developing central nervous system (CNS) at stages when neural precursor cells are internalized, and CapI-sna1 is also expressed laterally within the segmental mesoderm. Based on the expression patterns in this study, we suggest a putative function for Capitella sp. I twist genes in mesoderm differentiation and for snail genes in regulating CNS development and general cell migration during gastrulation. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

<Emphasis Type="Italic">Streptomyces</Emphasis><Emphasis Type="Italic">mayteni</Emphasis> sp. nov., a novel actinomycete isolated from a Chinese medicinal plant

An actinomycete strain, designated YIM 60475^T, was isolated from the roots of Maytenus austroyunnanensis and was characterized by using a polyphasic approach. The strain was determined to belong to the genus Streptomyces, based on its phenotypic and phylogenetic characteristics. The strain produced spiral spore chains on aerial mycelium. The cell wall contained ll-diaminopimelic acid. Whole-cell hydrolysates contained galactose, glucose, and xylose. The phospholipid was type II. The DNA G+C content of the type strain was 73.3 mol%. DNA–DNA hybridization and comparison of physiological and chemical characteristics suggested that strain YIM 60475^T is a new Streptomyces species, for which the name Streptomyces mayteni sp. nov. is proposed. The type strain is YIM 60475^T (=CCTCC AA 207005^T = KCTC 19383^T). Hua-Hong Chen and Sheng Qin contributed equally to this work.     

Sorbitol regulates energy transfer from allophycocyanin to the terminal emitter within phycobilisomes in <Emphasis Type="Italic">Synechocystis</Emphasis> sp

The effects of sorbitol on energy transfer of phycobilisomes (PBSs) in vivo were investigated in a chlN deletion mutant of Synechocystis sp. PCC 6803. When the mutant was grown in the dark, it contained intact and functional PBSs but essentially no chlorophyll or photosystems. Therefore, the structural and functional changes of the mutant PBSs in vivo can be detected by measurement of low temperature (77 K) and room temperature fluorescence emission spectra. Our results, for the first time, demonstrate that sorbitol decreases the energy transfer from allophycocyanin to the terminal emitter, indicating the site for osmotic regulation of excitation transfer in PBSs.     

<Emphasis Type="Italic">Sarcocystis</Emphasis> sp. in red foxes (<Emphasis Type="Italic">Vulpes vulpes</Emphasis>) from northern Croatia

The prevalence of Sarcocystis sp. was assessed in foxes from northern Croatia (city of Varadin). The intestine of 50 (29 male and 21 female) foxes aged 1–5 years, killed during the rabies control campaign, were examined. Sarcocystis sp. was identified in 62% of the fox intestinal samples examined. There was no sex difference in the rate of invasion. However, the rate of invasion differed significantly between the animals aged 1 year (47%), and those aged 2 (71%) and 3–5 years (71%).