Interaction of <Emphasis Type="Italic">Bdellovibrio bacteriovorus</Emphasis> with bacteria <Emphasis Type="Italic">Campylobacter jejuni</Emphasis> and <Emphasis Type="Italic">Helicobacter pylori</Emphasis>

Interaction of Bdellovibrio bacteriovorus 100NCJB with bacteria Campylobacter jejuni (strains 1, 2, 3, 4, and 5) and Helicobacter pylori, strain TX30a, was confirmed. The results indicate that lytic activity of bdellovibrios both in liquid media and cells attached to a surface was observed. The potential use of the antimicrobial activity of predatory bacteria for environmental bioprotection and public health is discussed.     

Isolation of <Emphasis Type="Italic">madA</Emphasis> homologs in <Emphasis Type="Italic">Pilobolus crystallinus</Emphasis>

Pilobolus crystallinus shows unique photoresponses at various growing stages. cDNAs for putative photoreceptors were cloned from this fungus. Three genes named pcmada1, pcmada2, and pcmada3 were identified from the PCR fragments, and amplified with degenerated primers for the LOV domain, which is conserved in many blue-light receptors. Deduced amino acid sequences for PCMADA1, PCMADA2, and PCMADA3 had one light-oxygen-voltage (LOV)-sensing and two PER-ARNT-SIM (PAS) domains. A zinc finger DNA-binding motif was conserved in the C-terminals of PCMADA1 and PCMADA3. However, PCMADA2 lacked the zinc finger motif. Expression of pcmada1 was suppressed by blue light whereas that of pcmada3 was promoted by blue-light irradiation.     

Retrotransposon <Emphasis Type="Italic">gtwin</Emphasis>: Structural analysis and distribution in <Emphasis Type="Italic">Drosophila</Emphasis> strains

A search for noncanonical variants of the gypsy retrotransposon ( MDG4 ) in the genome of the Drosophila melanogaster strain G32 led to the cloning of four copies of the poorly studied 7411-bp gtwin element. Sequence analysis showed that gtwin belongs to a family of endogeneous retroviruses, which are widespread in the Drosophila genome and have recently been termed insect erantiviruses. The gtwin retrotransposon is evolutionarily closest to MDG4, as evident from a good alignment of their nucleotide sequences including ORF2 (the pol gene) and ORF3 (the env gene), as well as the amino acid sequences of their protein products. These regions showed more than 75% homology. The distribution of gtwin was studied in several strains of the genus Drosophila. While strain G32 contained more than 20 copies of the element, ten other D. melanogaster strains carried gtwin in two to six copies per genome. The gtwin element was not detected in D. Hydei or D. Virilis. Comparison of the cloned gtwin sequences with the gtwin sequence available from the D. melanogaster genome database showed that the two variants of the mobile element differ by the presence or absence of a stop codon in the central region of ORF3. Its absence from the gtwin copies cloned from the strain G32 may indicate an association between the functional state of ORF3 and amplification of the element.Translated from Genetika, Vol. 41, No. 1, 2005, pp. 23–29.Original Russian Text Copyright © 2005 by Kotnova, Karpova, Feoktistova, Lyubomirskaya, Kim, Ilyin.     

<Emphasis Type="Italic">PVAS3</Emphasis>, a class-II ubiquitous asparagine synthetase from the common bean (<Emphasis Type="Italic">Phaseolus vulgaris)</Emphasis>

A gene encoding a putative asparagine synthetase (AS; EC 6.3.5.4) has been isolated from common bean (Phaseolus vulgaris). A 2.4 kb cDNA clone of this gene (PVAS3) encodes a protein of 570 amino acids with a predicted molecular mass of 64,678 Da, an isoelectric point of 6.45, and a net charge of −5.9 at pH 7.0. The PVAS3 protein sequence conserves all the amino acid residues that are essential for glutamine-dependent AS, and PVAS3 complemented an E. coli asparagine auxotroph, that demonstrates that it encodes a glutamine-dependent AS. PVAS3 displayed significant similarity to other AS. It showed the highest similarity to soybean SAS3 (92.9% identity), rice AS (73.7% identity), Arabidopsis ASN2 (73.2%) and sunflower HAS2 (72.9%). A phylogenetic analysis revealed that PVAS3 belongs to class-II asparagine synthetases. Expression analysis by real-time RT-PCR revealed that PVAS3 is expressed ubiquitously and is not repressed by light.     

Response to different oxidants of <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis><Emphasis Type="Italic">ure2Δ</Emphasis> mutant

Growth of Saccharomyces cerevisiae ure2Δ mutant strain was investigated in the presence of diverse oxidant compounds. The inability of the strain to grow on a medium supplemented with H₂O₂ was confirmed and a relationship between diminishing levels of glutathione (GSH) and peroxide sensitivity was established. Data for the lack of significant effect of URE2 disruption on the cellular growth in the presence of paraquat and menadione were obtained. The possible role of Ure2p in acquiring sensitivity to oxidative stress by means of its regulatory role in the GATA signal transduction pathway was discussed. It was suggested that the susceptibility of ure2Δ mutant to the exogenous hydrogen peroxide can result from increased GSH degradation due to the deregulated localization of the γ-glutamyl transpeptidase activating factors Gln3/Gat1. The important role of Ure2p in in vivo glutathione-mediated reactive oxygen species (ROS) scavenging was shown by measuring the activity of antioxidant enzymes glutathione peroxidase, superoxide dismutase (SOD) and catalase in an URE2 disrupted strain. A time-dependent increase in SOD and catalase activity was observed. More importantly, it was shown that the ure2 mutation could cause significant disturbance in cellular oxidant balance and increased ROS level.     

Proteolysis of <Emphasis Type="Italic">α</Emphasis>-amylase from <Emphasis Type="Italic">Saccharomycopsis fibuligera</Emphasis>: characterization of digestion products

α-Amylase from Saccharomycopsis fibuligera R-64 was successfully purified by butyl Toyopearl hydrophobic interaction chromatography, followed by Sephadex G-25 size exclusion and DEAE Toyopearl anion exchange chromatography. The enzyme has a molecular mass of 54 kDa, as judged by SDS PAGE analysis. Upon tryptic digestion, two major fragments with relative molecular masses of 39 kDa and 10 kDa, which resemble the A/B and C-terminal domains in the homologous Taka-amylase, were obtained and were successfully separated with the Sephadex G-50 size exclusion column. The 39-kDa fragment demonstrated a similar amylolytic activity to that of the undigested enzyme. However, it was found that the K _m value of the 39-kDa fragment was about two-times higher than that of the undigested enzyme. Moreover, thermostability studies showed a lower half-life time for the 39-kDa fragment. These findings suggest that the 39-kDa fragment is the catalytic domain, while the 10-kDa fragment is the C-terminal one, which plays a role in thermostability and starch binding. Although the undigested enzyme is able to act on raw starches at room temperature, with maize starches as the best substrate, neither the undigested enzyme nor the fragments adsorb the tested raw starches. These results propose Saccharomycopsis fibuligera α-amylase as a raw starch-digesting but not adsorbing amylase, with a similar domain organization to that of Taka-amylase A.     

Chromosomes are eliminated in the symmetric fusion between <Emphasis Type="Italic">Arabidopsis </Emphasis><Emphasis Type="Italic">thaliana</Emphasis> L. and <Emphasis Type="Italic">Bupleurum scorzonerifolium</Emphasis> Willd.

In order to investigate chromosome elimination in symmetric somatic hybridization between Bupleurum scorzonerifolium and Arabidopsis thaliana, protoplasts were isolated from suspension cultures of both A. thaliana and B. scorzonerifolium parents. Biparental protoplasts were mixed at a rate of 1.5:1 and fused with PEG-method. After protoplast fusion, the products were cultured in the P5 liquid medium for microcallus formation. Single cell lines formed from microcalli after subculturing on the MB1 (Xia and Chen, Plant Sci 120:197–203, 1996) solid medium. The putative somatic hybrid cell lines were identified by cytological and molecular analysis. Of the 132 somatic cell lines generated, 16 were identified as somatic hybrids, with the phenotypes resembled B. scorzonerifolium parent. These hybrids showed a complete set of B. scorzonerifolium chromosome and 0–2 small chromosome(s) of A. thaliana. A few of them showed nuclear and cytoplasmic SSR fragments of A. thaliana. These hybrid cell lines could differentiate to green spots, buds/leaves through complementation of regeneration ability. The chromosomes elimination of A. thaliana was discussed. Wang Minqin and Zhao Junsheng contributed equally to the work.     

Influence of <Emphasis Type="Italic">Daphnia</Emphasis> infochemicals on functional traits of <Emphasis Type="Italic">Microcystis</Emphasis> strains (Cyanobacteria)

We conducted a laboratory experiment to investigate the influence of Daphnia infochemicals on growth rate, microcystin production, colony formation and cell size of eight Microcystis strains isolated from two lakes. The strains were characterized genetically by their 16S-23S rDNA ITS sequence. The experiment was composed of four treatments: (1) a control using filtered WC medium, (2) addition of Scenedesmus obliquus culture medium filtrate, (3) addition of Daphnia magna culture medium filtrate and (4) addition of sodium octyl sulphate, a commercially available Daphnia infochemical. Our results showed that sympatric strains differed strongly for the measured functional traits, while no correlations between traits were found. Between-strain differences in growth rate, microcystin production, colony formation and cell size were generally larger than the differences in phenotypes observed between treatments. Despite this, several strains reacted to the infochemicals by changing functional trait values. Daphnia culture medium filtrate and, to a lesser extent, sodium octyl sulphate had a negative influence on the growth rate of half of the strains and stimulated microcystin production in one strain, but the latter effect was not Daphnia-specific as Scenedesmus culture medium filtrate had the same effect. Daphnia culture medium filtrate also induced colony formation in one strain. Our data suggest that Daphnia infochemicals generally have a weak influence on growth rate, microcystin production and colony formation of Microcystis strains as compared to the inter-strain variability, while existing inducible effects are highly strain-specific.     

Study on the scavenging and anti-<Emphasis Type="Italic">Staphylococcus</Emphasis><Emphasis Type="Italic">aureus</Emphasis> activities of the extracts,fractions and subfractions of two <Emphasis Type="Italic">Volvariella volvacea</Emphasis> strains

Scavenging and anti-Staphylococcus aureus activities of extracts, fractions and subfractions from the in vitro mycelium of two strains of the edible mushroom Volvariella volvacea were determined. Chloroform subfractions of the ATCC62890 strain showed the highest inhibition percentage of the DPPH radical (over 80%) after 180 min. When chloroform and hexane subfractions of the R83 strain were combined they showed moderate (inhibition zone of 8.99 ± 0.78 mm) to high (inhibition zone of 13.06 ± 0.41 mm) activity against the Gram-positive bacteria Staphylococcus aureus, which are 74.4 and 51.2% of tetracycline (inhibition zone of 17.55 ± 0.11 mm). Partitioning suggested that the substances in the chloroform and hexane fractions of the R83 strain act synergistically to give the antimicrobial activity, while separating the substances of the ATCC62890 subfractions reduced their activity. In general, the R83 strain seems to be a source of antimicrobial substances, while the ATCC62890 strain appears to be an alternative source of antioxidants.     

Identification of <Emphasis Type="Italic">DUF538</Emphasis> cDNA clone from <Emphasis Type="Italic">Celosia cristata</Emphasis> expressed sequences of nonstressed and stressed leaves

DUF538 domain-containing protein family consists of several plant proteins of unknown functions. This protein family has already been discovered by genome annotation tools and cloned as an inducible gene product under various environmental stress conditions. For the first time, we presented a full length DUF538 cDNA (encoding 170 amino acid residues) clone, which was randomly isolated from Celosia cristata leaf cDNA library constructed under normal growth conditions and consistently amplified from leaf cDNA populations prepared from nonstressed and drought-stressed leaves. We predicted that a DUF538 gene product can be a putative candidate for common stress-related protein (regulatory factor) in the plant system. The nucleotide and deduced amino acid sequences of the isolated clone have been submitted to EMBL data bases under accession no. AJ535713.     

Characterization of the nematocidal toxocyst in <Emphasis Type="Italic">Pleurotus</Emphasis> subgen. <Emphasis Type="Italic">Coremiopleurotus</Emphasis>

Toxocysts of the genus Pleurotus are blastoconidia-like ovoid structures surrounded by a liquid droplet containing a toxin that paralyzes nematodes. This study investigated toxocyst development using a strain S396 of Pleurotus cystidiosus subsp. abalonus (subgen. Coremiopleurotus). The surface of the liquid droplet was found to be an elastic envelope. When a nematode touches the toxocyst, the envelope adheres to the worm and bursts. Toxocysts are induced simultaneously with coremia formation in the absence of nematodes and developed only from aerial hyphae in which nuclear division had ceased. In the early stage of toxocyst development, liquid springs repeatedly from the tip of the sterigma-like stipe before ovoid (blastoconidium-like structure) formation. A certain substance in the liquid might polymerize to form the envelope while the ovoid simultaneously budded in the droplet. The nucleus tends to locate near the toxocyst, especially in early stage of toxocyst development. Each dikaryotic cell predominantly formed one or two toxocyst(s) while each monokaryotic cell predominantly formed one. In rare cases, a nucleus existed in the toxocyst, suggesting the possibility that the toxocyst is a vestigial blastoconidium.     

Comparative analysis of <Emphasis Type="Italic">iol</Emphasis> clusters in <Emphasis Type="Italic">Lactobacillus casei</Emphasis> strains

The present study was designed to expand genetic knowledge of myo -inositol (MI) metabolism in Lactobacillus casei. Twenty-four L. casei isolates of dairy origin were tested for the presence of iol cluster. PCR screening revealed eight strains encoded functions involved in MI utilization, of which one strain was able to use MI as carbon source. To gain a deeper understanding of the function of iol genes, four of the eight observed iol clusters were subjected to the full sequencing procedure. The results showed that the iol cluster was not a common feature among dairy L. casei strains. In addition, the four iol clusters were highly similar to one another in terms of sequence similarity and operon architecture. However, abundant polymorphisms that comprised a majority of synonymous mutations were detected throughout the full sequences. Three of them distributed among iolB, iolC, and iolT genes were found in linkage to MI-negative phenotype. Compared with other bacterial iol clusters, the iol cluster of L. casei showed a high similarity with that of Bacillus subtilis.     

Medium optimization for the feather-degradation by <Emphasis Type="Italic">Streptomyces fradiae Var</Emphasis><Emphasis Type="Italic">S-221</Emphasis> using the response surface methodology

In order to accelerate biodegradation of feather into more amino acids, the fermentation medium of feather-biodegrading Streptomyces fradiae Var S-221 was optimized in this paper. In the first optimization step, the effects of feather powder, beet molasses, (NH₄)₂SO₄ and KH₂PO₄ on amino acids formation were evaluated by using full factorial design. The results showed that feather powder and (NH₄)₂SO₄ had significant and positive effects on feather-biodegradation into amino acids. Then, the method of the steepest ascent was used to access the optimal region of the two significant factors. In the third step, the concentration of feather powder and (NH₄)₂SO₄ were further optimized with central composite design and response surface analysis. As a result, the composition of the optimal medium for S. fradiae Var S-221 fermentation were as follows (g/100 ml): feather powder, 19.504; beet molasses, 4.0; (NH₄)₂SO₄, 1.467; KH₂PO₄, 0.3; MgSO₄, 0.15; FeSO₄, 0.001; ZnSO₄, 0.0001; and MnSO₄, 0.0001. Using this optimal fermentation medium, the amino acids concentration was increased from 4.61 to 6.13 g/100 ml.