Development of a real-time TaqMan assay to detect <Emphasis Type="Italic">mendocina</Emphasis> sublineage <Emphasis Type="Italic">Pseudomonas</Emphasis> species in contaminated metalworking fluids

A TaqMan quantitative real-time polymerase chain reaction (qPCR) assay was developed for the detection and enumeration of three Pseudomonas species belonging to the mendocina sublineage (P. oleovorans, P. pseudoalcaligenes, and P. oleovorans subsp. lubricantis) found in contaminated metalworking fluids (MWFs). These microbes are the primary colonizers and serve as indicator organisms of biodegradation of used MWFs. Molecular techniques such as qPCR are preferred for the detection of these microbes since they grow poorly on typical growth media such as R2A agar and Pseudomonas isolation agar (PIA). Traditional culturing techniques not only underestimate the actual distribution of these bacteria but are also time-consuming. The primer–probe pair developed from gyrase B (gyrB) sequences of the targeted bacteria was highly sensitive and specific for the three species. qPCR was performed with both whole cell and genomic DNA to confirm the specificity and sensitivity of the assay. The sensitivity of the assay was 10¹ colony forming units (CFU)/ml for whole cell and 13.7 fg with genomic DNA. The primer–probe pair was successful in determining concentrations from used MWF samples, indicating levels between 2.9 × 10³ and 3.9 × 10⁶ CFU/ml. In contrast, the total count of Pseudomonas sp. recovered on PIA was in the range of <1.0 × 10¹ to 1.4 × 10⁵ CFU/ml for the same samples. Based on these results from the qPCR assay, the designed TaqMan primer–probe pair can be efficiently used for rapid (within 2 h) determination of the distribution of these species of Pseudomonas in contaminated MWFs.     

Activity-density of <Emphasis Type="Italic">Pardosa cribata</Emphasis> in Spanish citrus orchards and its predatory capacity on <Emphasis Type="Italic">Ceratitis capitata</Emphasis> and <Emphasis Type="Italic">Myzus persicae</Emphasis>

The wolf spider Pardosa cribata Simon is the most abundant ground-dwelling spider inhabiting citrus orchards in eastern Spain. However, little is known about its activity-density and its predatory role in the citrus agrosystem. Here we report on the activity-density of P. cribata monitored by pitfall traps, and on its capacity to prey on two citrus pests that appear both in the citrus canopy and the ground cover, Ceratitis capitata (Wiedemman) and Myzus persicae (Sulzer), respectively. Pardosa cribata was present in citrus orchards throughout the year, with a peak in spring and a higher peak in summer. Pardosa cribata preyed on adults and third-instar larvae but not on pupae of C. capitata. A type II functional response was obtained for teneral-like adults, with an estimated attack rate (a′) of 0.771 ± 0.213 days⁻¹ and a handling time (T _h) of 0.051 ± 0.013 days. Pardosa cribata also preyed efficiently on M. persicae, giving a type II functional response with an estimated attack rate and handling time of 2.833 ± 0.578 days⁻¹ and 0.031 ± 0.001 days, respectively. The data reported here indicate that this wolf spider could play an important role in regulating both these pests, and therefore might contribute to developing conservation biological control strategies for citrus pests. Handling Editor: Arne Jenssen.     

<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.     

Isolation and characterization of a novel 2-<Emphasis Type="Italic">sec</Emphasis>-butylphenol-degrading bacterium <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. strain MS-1

A novel bacterium capable of utilizing 2-sec-butylphenol as the sole carbon and energy source, Pseudomonas sp. strain MS-1, was isolated from freshwater sediment. Within 30 h, strain MS-1 completely degraded 1.5 mM 2-sec-butylphenol in basal salt medium, with concomitant cell growth. A pathway for the metabolism of 2-sec-butylphenol by strain MS-1 was proposed on the basis of the identification of 3 internal metabolites—3-sec-butylcatechol, 2-hydroxy-6-oxo-7-methylnona-2,4-dienoic acid, and 2-methylbutyric acid—by gas chromatography-mass spectrometry analysis. Strain MS-1 degraded 2-sec-butylphenol through 3-sec-butylcatechol along a meta-cleavage pathway. Degradation experiments with various alkylphenols showed that the degradability of alkylphenols by strain MS-1 depended strongly on the position (ortho ≫ meta = para) of the alkyl substitute, and that strain MS-1 could degrade 2-alkylphenols with various sized and branched alkyl chain (o-cresol, 2-ethylphenol, 2-n-propylphenol, 2-isopropylphenol, 2-sec-butylphenol, and 2-tert-butylphenol), as well as a dialkylphenol (namely, 6-tert-butyl-m-cresol).     

Functional characterization of <Emphasis Type="Italic">Arabidopsis thaliana</Emphasis> transthyretin-like protein

Background  

Arabidopsis thaliana transthyretin-like (TTL) protein is a potential substrate in the brassinosteroid signalling cascade, having a role that moderates plant growth. Moreover, sequence homology revealed two sequence domains similar to 2-oxo-4-hydroxy-4-carboxy-5-ureidoimidazoline (OHCU) decarboxylase (N-terminal domain) and 5-hydroxyisourate (5-HIU) hydrolase (C-terminal domain). TTL is a member of the transthyretin-related protein family (TRP), which comprises a number of proteins with sequence homology to transthyretin (TTR) and the characteristic C-terminal sequence motif Tyr-Arg-Gly-Ser. TRPs are single domain proteins that form tetrameric structures with 5-HIU hydrolase activity. Experimental evidence is fundamental for knowing if TTL is a tetrameric protein, formed by the association of the 5-HIU hydrolase domains and, in this case, if the structural arrangement allows for OHCU decarboxylase activity. This work reports about the biochemical and functional characterization of TTL.     

<Emphasis Type="Italic">Pseudomonas</Emphasis> bacteria and phosphorous fertilization,affecting wheat (<Emphasis Type="Italic">Triticum</Emphasis><Emphasis Type="Italic">aestivum</Emphasis> L.) yield and P uptake under greenhouse and field conditions

We have recently indicated the plant growth promoting activities of Pseudomonas sp. as well as their alleviating effects on some soil stressors such as salinity. This is because in recent years, biological fertilizers have received special attention by scientists in sustainable agriculture. Accordingly, it is pertinent to specify the beneficiary level of such soil bacteria on plant growth including phosphorous (P) uptake. Hence, the objectives were to determine: (1) the plant growth promoting effects of the tested Pseudomonas sp., and (2) its combined effects with different P fertilization rates on the nutrient uptake (N, P, and K) and yield of wheat (Triticum aestivum L.) under greenhouse and field conditions. The experiments were factorially arranged on the basis of a completely randomized block design with three replicates and were conducted at the Research Farm of Agriculture and Natural Resources Research Center of Khorasan, Mashhad, Iran. P was fertilized at three levels including 0, 25 and 50 kg/ha P₂O₅. Pseudomonas sp. including Pseudomonas fluorescens 153, P. fluorescens 169, P. putida 4, and P. putida 108 were tested. Activities such as production of ACC deaminase and IAA-like products, as well as P solubilization were among the most important activities of the tested Pseudomonas sp. Such bacterial effects greatly enhanced wheat growth and yield under greenhouse and field conditions. The results also showed that the effects of Pseudomonas sp. on wheat nutrient uptake and the effects of bacteria as well as P fertilization on wheat yield were significant. P. putida 108 was the most effective strain enhancing wheat P uptake and grain yield under greenhouse (96 and 58%) and field (80 and 37%) conditions, respectively. Hence, although Pseudomonas sp. could be a suitable replacement for high P fertilization, however, the optimum wheat yield resulted when the bioinoculants are combined with 50% (25 kg/ha P₂O₅) P fertilization. This finding has great agricultural and environmental implications.     

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.     

Structural characteristics of the chloroplast <Emphasis Type="Italic">rpS16</Emphasis> intron in <Emphasis Type="Italic">Allium sativum</Emphasis> and related <Emphasis Type="Italic">Allium</Emphasis> species

The intron sequence of chloroplast rpS16 and the secondary structure of its pre-mRNA were characterized for the first time in 26 Allium sativum accessions of different ecologo-geographical origins and seven related Allium species. The boundaries and main stem-loop consensus sequences were identified for all six domains of the intron. Polymorphism was estimated for the total intron and its regions. The structural regions of the rpS16 intron proved to be heterogeneous for mutation rate and spectrum. Mutations were most abundant in domains II and IV, and transition predominated in domains I, III, V, and VI. In addition to structural elements and motifs typical for group IIB introns, several Allium-specific micro- and macrostructural mutations were revealed. A 290-bp deletion involving domains III and IV and part of domain V was observed in A. altaicum, A. fistulosum, and A. schoenoprasum. Several indels and nucleotide substitutions were found to cause a deviation of the pre-mRNA secondary structure from the consensus model of group II introns.     

Direct gene transfer study and transgenic plant regeneration after electroporation into mesophyll protoplasts of <Emphasis Type="Italic">Pelargonium</Emphasis> <Emphasis Type="Italic">×</Emphasis> <Emphasis Type="Italic">hortorum</Emphasis>, ‘Panaché Sud’

Direct genetic transformation of mesophyll protoplasts was studied in Pelargonium × hortorum. Calcein and green-fluorescent protein (GFP) gene were used to set up the process. Electroporation (three electric pulses from a 33-μF capacitor in a 250-V cm⁻¹ electric field) was more efficient than PEG 6000 for membrane permeation, protoplast survival and cell division. Transient expression of GFP was detected in 33–36% of electroporated protoplasts after 2 days and further in colonies. A protoplast suspension conductivity of >1,500 μS cm⁻¹ allowed high colony formation and plant regeneration. Stable transformation was obtained using the plasmid FAJ3000 containing uidA and nptII genes. When selection (50 mg l⁻¹ kanamycin) was achieved 6 weeks after electroporation, regenerated shoots were able to grow and root on 100 mg l⁻¹ kanamycin. The maximum transformation efficiency was 4.5%, based on the number of colonies producing kanamycin-resistant rooted plants or 0.7% based on the number of cultured protoplasts. Polymerase chain reaction (PCR) analysis on in vitro micropropagated plants showed that 18 clones out of 20 contained the nptII gene, while the uidA gene was absent. These results were confirmed after PCR analyses of five glasshouse-acclimatized clones.     

Utilization of dibenzothiophene as sulfur source by <Emphasis Type="Italic">Microbacterium</Emphasis> sp. <Emphasis Type="Italic">NISOC-06</Emphasis>

Oil-polluted soils were sampled from National Iranian South Oil Company (NISOC) for isolation and screening of C–S and not C–C targeted Dibenzothiophene (DBT) degrading microorganisms. Microbacterium sp. NISOC-06, a C–S targeted DBT degrading bacterium, was selected and its desulfurization ability was studied in aqueous phase and water-gasoline biphasic systems. The 16srRNA gene was amplified using universal eubacteria-specific primers, PCR product was sequenced and the sequence of nearly 1,500 bp 16srDNA was studied. Based on Gas Chromatography results Microbacterium sp. NISOC-06 utilized 94.8% of 1 mM DBT during the 2 weeks of incubation. UV Spectrophotometry and biomass production measurements showed that the Microbacterium sp. NISOC-06 was not able to utilize DBT as a carbon source. There was no accumulation of phenolic compounds as Gibb’s assay showed. Biomass production in a biphasic system for which DBT-enriched gasoline was used as the sulfur source indicated the capability of Microbacterium sp. NISOC-06 to desulfurize gasoline.     

Molecular cloning and characterization of a novel glyoxalase I gene <Emphasis Type="Italic">TaGly I</Emphasis> in wheat (<Emphasis Type="Italic">Triticum aestivum</Emphasis> L.)

Methylglyoxal is a kind of poisonous metabolite that can react with RNA, DNA and protein, which generally results in a number of side advert effects to cell. Glyoxalase I is a member of glyoxalase system that can detoxify methylglyoxal. An EST encoding a glyoxalase I was isolated from a SSH (suppression subtractive hybridization)-cDNA library of wheat spike inoculated by Fusarium graminearum. The corresponding full length gene, named TaGly I, was cloned, sequenced and characterized. Its genomic sequence consists of 2,719 bp, including seven exons and six introns, and its coding sequence is 929 bp with an open reading frame encoding 291 amino acids. Sequence alignment showed that there were two glyoxalase I domains in the deduced protein sequence. By using specific primers, TaGly I was mapped to chromosome 7D of wheat via a set of durum wheat ‘Langdon’ D-genome disomic-substitution lines. The result of Real-time quantitative polymerase chain reaction demonstrated that TaGly I was induced by the inoculation of Fusarium graminearum in wheat spikes. Additionally, it was also induced by high concentration of NaCl and ZnCl₂. When TaGly I was overexpressed in tobacco leaves via Agrobacterium tumefaciens infection, the transgenic tobacco showed stronger tolerance to ZnCl₂ stress relative to transgenic control with GFP. The above facts indicated that TaGly I might play a role in response to diverse stresses in plants.     

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.     

A <Emphasis Type="Italic">sirA</Emphasis>-like gene, <Emphasis Type="Italic">sirA2</Emphasis>, is essential for 3-succinoyl-pyridine metabolism in the newly isolated nicotine-degrading <Emphasis Type="Italic">Pseudomonas</Emphasis> sp. HZN6 strain

A novel nicotine-degrading Pseudomonas sp. strain, HZN6, was isolated from a pesticide-wastewater treatment facility in Hangzhou. The strain could grow on nicotine as its sole source of carbon, nitrogen, and energy. The strain’s main intermediate metabolites were determined to be pseudooxynicotine, 3-succinoyl-pyridine (SP), and 6-hydroxy-3-succinoyl-pyridine (HSP). A Tn5 transposon mutant was generated in which the degradation pathway was blocked at the SP. A 4,583-bp DNA fragment flanking the transposon insertion site was obtained through self-formed adaptor PCR and analyzed. The mutant gene orfC displays 89% deduced amino acid sequence identity with the sirA-like gene (sirA2, a sulfurtransferase homologue gene) of Pseudomonas stutzeri A1501. The orfC-disrupted strain lost the ability to degrade SP, and the complementation strains with the orfC from the Pseudomonas sp. HZN6 and the sirA2 (PP_1233) from Pseudomonas putida KT2440 recovered the degradation ability. Though the orfC-disrupted strain also lost the xanthine dehydrogenase activity, the effects of tungsten on the degradation of SP and hypoxanthine revealed that the hydroxylation of SP to HSP was not a xanthine dehydrogenase type. These results demonstrated that the orfC gene was essential for the SP metabolism involved in the nicotine metabolic pathway in the Pseudomonas sp. HZN6 strain. This study might advance the understanding of the nicotine metabolic mechanism in Pseudomonas.     

Synthesis of orthogonally protected (<Emphasis Type="Italic">3R</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)- and (<Emphasis Type="Italic">3S</Emphasis>,<Emphasis Type="Italic">4S</Emphasis>)-4,5-diamino-3-hydroxypentanoic acids

Summary.  The paper describes two methods of the synthesis of ethyl (3R,4S)- and (3S,4S)-4-[(benzyloxycarbonyl)amino]-5-[(tert-butyloxycarbonyl)amino]-3-hydroxypentanoates, useful for the syntheses of edeine analogs. Differently N-protected (S)-2,3-diaminopropanoic acid was used as a substrate in both procedures. The absolute configuration of newly generated asymmetric carbon atoms C-3 in β-hydroxy-γ,δ-diamino products was assigned by means of ¹H NMR spectroscopy after their transformation into corresponding piperidin-2-ones. Received May 24, 2002 Accepted October 10, 2002 Published online December 18, 2002 Acknowledgment The authors are indebted to the Faculty of Chemistry, Technical University of Gdańsk for financial support. Authors' address: Zbigniew Czajgucki, M. Sc., Department of Pharmaceutical Technology and Biochemistry, Faculty of Chemistry, Technical University of Gdańsk, 11/12 Narutowicza St., 80-952 Gdańsk, Poland, Fax +48 58 347 11 44, E-mail: zmczaj@wp.pl     

<Emphasis Type="Italic">RAD18</Emphasis> gene product of yeast <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis> controls mutagenesis induced by hydrogen peroxide

Within eukaryotes, tolerance to DNA damage is determined primarily by the repair pathway controlled by the members of the RAD6 epistasis group. Genetic studies on a yeast Saccharomyces cerevisiae model showed that the initial stage of postreplication repair (PRR), i.e., initiation of replication through DNA damage, is controlled by Rad6–Rad18 ubiquitin-conjugating enzyme complex. Mutants of these genes are highly sensitive to various genotoxic agents and reduce the level of induced mutagenesis. In this case, the efficiency of mutagenesis suppression depends on the type of damage. In this study we showed that DNA damage induced by hydrogen peroxide at the same mutagen doses causes significantly more mutations and lethal events in the rad18 mutant cells compared to control wild-type cells.     

Frequencies of the <Emphasis Type="Italic">DRB1</Emphasis>, <Emphasis Type="Italic">DQA1</Emphasis>, <Emphasis Type="Italic">DQB1</Emphasis> and <Emphasis Type="Italic">TNFA</Emphasis> alleles in immigrant population of west Siberia

Based on population analysis of the DRB1, DQA1, DQB1 and TNFA allele frequency distribution patterns, regional features of immunogenetic structure of the population of West Siberia were investigated. Statistically significant linkage disequilibrium within the HLA class II region, as well as between the TNFA and DRB1, DQA1, and DQB1 was demonstrated. Population frequency distribution patterns of two- and multilocus haplotypes were examined.     

Quorum sensing regulation in <Emphasis Type="Italic">Pseudomonas</Emphasis>

Expression of many bacterial genes is regulated in a cell density-dependent manner via small signal molecules known as autoinducers; this type of regulation is termed quorum sensing (QS). The QS systems that employ N-acyl-homoserine lactones (HSLs) are best un derstood in Gram-negative bacteria. QS regulates expression of various genes, including the genes responsible for the production of virulence factors, synthesis of exoenzymes and antibiotics, antagonistic properties of bacteria, etc. The QS systems of the genus Pseudomonas are linked to other global regulatory networks of the cell, and their functions are controlled by numerous additional regulatory factors. Such regulators and the QS systems together form an intricate multifactorial cascade regulatory network. The review considers the QS systems of several Pseudomonas species, their interaction with other regulatory systems, and their roles in the regulation of cell processes.     

Overexpression of <Emphasis Type="Italic">odc</Emphasis> (ornithine decarboxylase) in <Emphasis Type="Italic">Datura innoxia</Emphasis> enhances the yield of scopolamine

Scopolamine is widely used for its anticholinergic properties. Because of higher physiological activity and less side effects the world demand of scopolamine is estimated to be ten times greater than other anticholinergic agents, hyoscyamine and atropine. Since natural production is limited, alternatives are required to boost the production. We report the introduction of mouse odc gene of polyamine biosynthesis pathway which is also the primary pathway of tropane alkaloids in Datura innoxia. Polyamines, mainly putrescine, serve as the common metabolite for tropane alkaloids and nicotine. We have overexpressed odc gene to modulate the metabolic flux downstream and eventually achieved higher accumulation of scopolamine in transgenic plants. Among six independent transformed lines one line (O10) produced scopolamine (0.258 μg/g dry weight) almost six times higher than that produced by control plants (0.042 μg/g DW). To our knowledge, this is the first report of odc overexpression in D. innoxia leading to higher scopolamine yield.