Production of <Emphasis Type="Italic">N</Emphasis><Superscript><Emphasis Type="Italic">α</Emphasis></Superscript>-acetylated thymosin α1 in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Background  

Thymosin α1 (Tα1), a 28-amino acid N ^α -acetylated peptide, has a powerful general immunostimulating activity. Although biosynthesis is an attractive means of large-scale manufacture, to date, Tα1 can only be chemosynthesized because of two obstacles to its biosynthesis: the difficulties in expressing small peptides and obtaining N ^α -acetylation. In this study, we describe a novel production process for N ^α -acetylated Tα1 in Escherichia coli.     

Practical considerations for investigation of protein conformational dynamics by <Superscript>15</Superscript>N <Emphasis Type="Italic">R</Emphasis><Subscript>1ρ</Subscript> relaxation dispersion

It is becoming increasingly apparent that proteins are not static entities and that their function often critically depends on accurate sampling of multiple conformational states in aqueous solution. Accordingly, the development of methods to study conformational states in proteins beyond their ground-state structure (“excited states”) has crucial biophysical importance. Here we investigate experimental schemes for optimally probing chemical exchange processes in proteins on the micro- to millisecond timescale by ¹⁵N R _1ρ relaxation dispersion. The schemes use selective Hartmann–Hahn cross-polarization (CP) transfer for excitation, and derive peak integrals from 1D NMR spectra (Korzhnev et al. in J Am Chem Soc 127:713–721, 2005; Hansen et al. in J Am Chem Soc 131:3818–3819, 2009). Simulation and experiment collectively show that in such CP-based schemes care has to be taken to achieve accurate suppression of undesired off-resonance coherences, when using weak spin-lock fields. This then (i) ensures that relaxation dispersion profiles in the absence of chemical exchange are flat, and (ii) facilitates extraction of relaxation dispersion profiles in crowded regions of the spectrum. Further improvement in the quality of the experimental data is achieved by recording the free-induction decays in an interleaved manner and including a heating-compensation element. The reported considerations will particularly benefit the use of CP-based R _1ρ relaxation dispersion to analyze conformational exchange processes in larger proteins, where resonance line overlap becomes the main limiting factor.     

High-throughput genotyping-by-sequencing facilitates molecular tagging of a novel rust resistance gene, <Emphasis Type="Italic">R</Emphasis><Subscript><Emphasis Type="Italic">15</Emphasis></Subscript>, in sunflower (<Emphasis Type="Italic">Helianthus annuus</Emphasis> L.)

Key message

A novel rust resistance gene, R ₁₅ , derived from the cultivated sunflower HA-R8 was assigned to linkage group 8 of the sunflower genome using a genotyping-by-sequencing approach. SNP markers closely linked to R ₁₅ were identified, facilitating marker-assisted selection of resistance genes.

Abstract

The rust virulence gene is co-evolving with the resistance gene in sunflower, leading to the emergence of new physiologic pathotypes. This presents a continuous threat to the sunflower crop necessitating the development of resistant sunflower hybrids providing a more efficient, durable, and environmentally friendly host plant resistance. The inbred line HA-R8 carries a gene conferring resistance to all known races of the rust pathogen in North America and can be used as a broad-spectrum resistance resource. Based on phenotypic assessments of 140 F₂ individuals derived from a cross of HA 89 with HA-R8, rust resistance in the population was found to be conferred by a single dominant gene (R ₁₅ ) originating from HA-R8. Genotypic analysis with the currently available SSR markers failed to find any association between rust resistance and any markers. Therefore, we used genotyping-by-sequencing (GBS) analysis to achieve better genomic coverage. The GBS data showed that R ₁₅ was located at the top end of linkage group (LG) 8. Saturation with 71 previously mapped SNP markers selected within this region further showed that it was located in a resistance gene cluster on LG8, and mapped to a 1.0-cM region between three co-segregating SNP makers SFW01920, SFW00128, and SFW05824 as well as the NSA_008457 SNP marker. These closely linked markers will facilitate marker-assisted selection and breeding in sunflower.

     

Pistil-function breakdown in a new <Emphasis Type="Italic">S</Emphasis>-allele of European pear, <Emphasis Type="Italic">S</Emphasis><Subscript><Emphasis Type="Italic">21</Emphasis></Subscript>°, confers self-compatibility

European pear exhibits RNase-based gametophytic self-incompatibility controlled by the polymorphic S-locus. S-allele diversity of cultivars has been extensively investigated; however, no mutant alleles conferring self-compatibility have been reported. In this study, two European pear cultivars, ‘Abugo’ and ‘Ceremeño’, were classified as self-compatible after fruit/seed setting and pollen tube growth examination. S-genotyping through S-PCR and sequencing identified a new S-RNase allele in the two cultivars, with identical deduced amino acid sequence as S ₂₁ , but differing at the nucleotide level. Test-pollinations and analysis of descendants suggested that the new allele is a self-compatible pistil-mutated variant of S ₂₁ , so it was named S ₂₁ °. S-genotypes assigned to ‘Abugo’ and ‘Ceremeño’ were S ₁₀ S ₂₁ ° and S ₂₁ °S ₂₅ respectively, of which S ₂₅ is a new functional S-allele of European pear. Reciprocal crosses between cultivars bearing S ₂₁ and S ₂₁ ° indicated that both alleles exhibit the same pollen function; however, cultivars bearing S ₂₁ ° had impaired pistil-S function as they failed to reject either S ₂₁ or S ₂₁ ° pollen. RT-PCR analysis showed absence of S ₂₁ °-RNase gene expression in styles of ‘Abugo’ and ‘Ceremeño’, suggesting a possible origin for S ₂₁ ° pistil dysfunction. Two polymorphisms found within the S-RNase genomic region (a retrotransposon insertion within the intron of S ₂₁ ° and indels at the 3′UTR) might explain the different pattern of expression between S ₂₁ and S ₂₁ °. Evaluation of cultivars with unknown S-genotype identified another cultivar ‘Azucar Verde’ bearing S ₂₁ °, and pollen tube growth examination confirmed self-compatibility for this cultivar as well. This is the first report of a mutated S-allele conferring self-compatibility in European pear.     

<Emphasis Type="Italic">Bacillus anthracis</Emphasis>, <Emphasis Type="Italic">Francisella tularensis</Emphasis> and <Emphasis Type="Italic">Yersinia pestis</Emphasis>. The most important bacterial warfare agents — <Emphasis Type="Italic">review</Emphasis>

There are three most important bacterial causative agents of serious infections that could be misused for warfare purposes: Bacillus anthracis (the causative agent of anthrax) is the most frequently mentioned one; however, Fracisella tularensis (causing tularemia) and Yersinia pestis (the causative agent of plague) are further bacterial agents enlisted by Centers for Disease Control and Prevention into the category A of potential biological weapons. This review intends to summarize basic information about these bacterial agents. Military aspects of their pathogenesis and the detection techniques suitable for field use are discussed.     

Functional expression of the lipase gene Lip2 of <Emphasis Type="Italic">Pleurotus</Emphasis> <Emphasis Type="Italic">sapidus</Emphasis> in <Emphasis Type="Italic">Escherichia</Emphasis> <Emphasis Type="Italic">coli</Emphasis>

The lipase Lip2 of the edible basidiomycete, Pleurotus sapidus, is an extracellular enzyme capable of hydrolysing xanthophyll esters with high efficiency. The gene encoding Lip2 was expressed in Escherichia coli TOP10 using the gene III signal sequence to accumulate proteins in the periplasmatic space. The heterologous expression under control of the araBAD promoter led to the high level production of recombinant protein, mainly as inclusion bodies, but partially in a soluble and active form. A fusion with a C-terminal His tag was used for purification and immunochemical detection of the target protein. This is the first example of a heterologous expression and periplasmatic accumulation of a catalytically active lipase from a basidiomycete fungus.     

Brood cell size of <Emphasis Type="Italic">Apis</Emphasis> <Emphasis Type="Italic">mellifera</Emphasis> modifies the reproductive behavior of <Emphasis Type="Italic">Varroa</Emphasis> <Emphasis Type="Italic">destructor</Emphasis>

We undertook a field study to determine whether comb cell size affects the reproductive behavior of Varroa destructor under natural conditions. We examined the effect of brood cell width on the reproductive behavior of V. destructor in honey bee colonies, under natural conditions. Drone and worker brood combs were sampled from 11 colonies of Apis mellifera. A Pearson correlation test and a Tukey test were used to determine whether mite reproduction rate varied with brood cell width. Generalized additive model analysis showed that infestation rate increased positively and linearly with the width of worker and drone cells. The reproduction rate for viable mother mites was 0.96 viable female descendants per original invading female. No significant correlation was observed between brood cell width and number of offspring of V. destructor. Infertile mother mites were more frequent in narrower brood cells.     

Rapid induction of <Emphasis Type="Italic">Agrobacterium</Emphasis> <Emphasis Type="Italic">tumefaciens</Emphasis>-mediated transgenic roots directly from adventitious roots in <Emphasis Type="Italic">Panax</Emphasis> <Emphasis Type="Italic">ginseng</Emphasis>

Root segments from seedlings of Panax ginseng produced adventitious roots directly when cultured on 1/2 MS solid medium lacking NH₄NO₃ and containing 3.0 mg l⁻¹ IBA. Using this adventitious root formation, we developed rapid and efficient transgenic root formation directly from adventitious root segments in P. ginseng. Root segments were co-cultivated with Agrobacterium tumefaciens (GV3101) caring β-glucuronidase (GUS) gene. Putative transgenic adventitious roots were formed directly from root segments on medium with 400 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin. Kanamycin resistant adventitious roots were selected and proliferated as individual lines by subculturing on medium with 300 mg l⁻¹ cefotaxime and 50 mg l⁻¹ kanamycin at two weeks subculture interval. Frequency of transient and stable expression of GUS gene was enhanced by acetosyringon (50 mg l⁻¹) treatment. Integration of transgene into the plants was confirmed by the X-gluc reaction, PCR and Southern analysis. Production of transgenic plants was achieved via somatic embryogenesis from the embryogenic callus derived from independent lines of adventitious roots. The protocol for rapid induction of transgenic adventitious roots directly from adventitious roots can be applied for a new Agrobacterium tumefaciens-mediated genetic transformation protocol in P. ginseng.     

<Superscript>15</Superscript>N–{<Superscript>1</Superscript>H} NOE experiment at high magnetic field strengths

The heteronuclear ¹⁵N–{¹H} NOE values are typically determined by taking the ratio of ¹⁵N signal intensities recorded in the presence and absence of ¹H saturation prior to evolution of ¹⁵N magnetization. Since the intensity ratio of two independent experiments is taken, complete recovery of ¹⁵N magnetization during the scan repetition delay is critical to obtain reliable NOE values. Because it may not be practical to wait for the complete recovery of magnetization at high magnetic fields, Solomon equations may be used to correct measured NOE values. Here, based on experiments and simulations, we show that since the cross-correlation between ¹H–¹⁵N dipole and ¹⁵N chemical shift anisotropy becomes significant at high fields for small or deuterated proteins, measured NOE values can not be accurately corrected based on the Solomon equations. We also discuss ranges of rotational correlation times and proton spin-flip rate, in which the NOE values can be corrected by the equations.     

Microsecond motions probed by near-rotary-resonance <Emphasis Type="Italic">R</Emphasis><Subscript>1ρ</Subscript><Superscript>15</Superscript>N MAS NMR experiments: the model case of protein overall-rocking in crystals

F_oF₁-ATP synthase catalyzes ATP hydrolysis/synthesis coupled with a transmembrane H⁺ translocation in membranes. The F_o c-subunit ring plays a major role in this reaction. We have developed an assignment strategy for solid-state ¹³C NMR (ssNMR) signals of the F_o c-subunit ring of thermophilic Bacillus PS3 (TF_o c-ring, 72 residues), carrying one of the basic folds of membrane proteins. In a ssNMR spectrum of uniformly ¹³C-labeled sample, the signal overlap has been a major bottleneck because most amino acid residues are hydrophobic. To overcome signal overlapping, we developed a method designated as COmplementary Sequential assignment with MInimum Labeling Ensemble (COSMILE). According to this method, we generated three kinds of reverse-labeled samples to suppress signal overlapping. To assign the carbon signals sequentially, two-dimensional C^α(i+1)–C′C^α(i) correlation and dipolar assisted rotational resonance (DARR) experiments were performed under magic-angle sample spinning. On the basis of inter- and intra-residue ¹³C–¹³C chemical shift correlations, 97% of C^α, 97% of C^β and 92% of C′ signals were assigned directly from the spectra. Secondary structure analysis predicted a hairpin fold of two helices with a central loop. The effects of saturated and unsaturated phosphatidylcholines on TF_o c-ring structure were examined. The DARR spectra at 15 ms mixing time are essentially similar to each other in saturated and unsaturated lipid membranes, suggesting that TF_o c-rings have similar structures under the different environments. The spectrum of the sample in saturated lipid membranes showed better resolution and structural stability in the gel state. The C-terminal helix was suggested to locate in the outer layer of the c-ring.     

Delimiting a rice wide-compatibility gene <Emphasis Type="Italic">S</Emphasis><Stack><Subscript>5</Subscript><Superscript><Emphasis Type="Italic">n</Emphasis></Superscript></Stack> to a 50 kb region

Wide-compatibility (WC) is one of the most important traits in rice, which can overcome the fertility barrier in the indica/japonica hybrids, and hence to make it possible to utilize the higher yield potential of inter-subspecific hybrids. The S ₅ ⁿ gene located on chromosome 6 has been previously reported to be responsible for the wide-compatibility in rice. Here we report the precise location of the S ₅ ⁿ gene. In the first-pass mapping, the S ₅ ⁿ gene was restricted within a 200 kb region by using a population of 242 isogenic lines in combination with high-density markers developed in the S ₅ region. In the fine mapping, the S ₅ region was further saturated with newly developed markers and more isogenic lines (549 in total) were investigated. Eventually, the S ₅ ⁿ gene was mapped within a 50 kb region delimited by the left marker J13 and the right marker J17. One BAC clone screened from the BAC library of the WC rice variety 02428 covered the whole S ₅ region. Sequence analysis of the 50 kb region revealed two candidate genes, coding an aspartyl protease and a hypothetical protein. This result would greatly accelerate both cloning and marker-assisted selection of this important S ₅ ⁿ gene. Qing Ji and Jufei Lu have contributed equally to this paper.     

Optimization of <Emphasis Type="Italic">R</Emphasis>-(+)-α-terpineol production by the biotransformation of <Emphasis Type="Italic">R</Emphasis>-(+)-limonene

R-(+)-limonene is an abundant and non-expensive by-product of the citrus industry and is, therefore, a suitable starting material for the production of natural flavor and fragrance compounds. The biotransformation of R-(+)-limonene to R-(+)-alpha-terpineol by Fusarium oxysporum 152b has already been reported, although the influence of the main process parameters on the production has not yet been evaluated. In this paper, a Plackett-Burman screening design was used to define the effects of the medium composition (glucose, peptone, yeast extract, malt extract and pH), the presence of a co-substrate (biosurfactant), the cultivation conditions (temperature, agitation), the substrate concentration and the inoculum/culture medium ratio on the absolute amount of R-(+)-alpha-terpineol resulting from this biotransformation. The process conditions were further optimized applying response surface methodology (RSM). The volatiles were extracted using a SPME device and were subsequently quantified by GC-FID and identified by GC-MS. The best results were obtained using 0.5% (v/m) R-(+)-limonene in pure distilled water as the culture medium with an inoculum/culture medium ratio of 0.25 (m/m) and 72 h cultivation at 26 degrees C/240 rpm. Under these conditions the concentration of R-(+)-alpha-terpineol in the culture medium reached 2.4 g L(-1), a production almost six times greater than in earlier trials. The presence of a biosurfactant (0-500 mg L(-1)) did not significantly increase the yield.     

A new minimum fluorescence parameter,as generated using pulse frequency modulation,compared with pulse amplitude modulation:<Emphasis Type="Italic"> F</Emphasis><Subscript>α</Subscript> versus<Emphasis Type="Italic"> F</Emphasis><Subscript>o</Subscript>

The minimum fluorescence parameter (F (alpha)), generated using the new pulse frequency modulation (PFM) technology, was compared with the minimum fluorescence parameter (F (o)), generated by pulse amplitude modulation (PAM), in response to a reversible low-oxygen stress in 'Honeycrisp'trade mark (HC) apples (Malus domestica) and an irreversible osmotic stress induced by water loss in two grape (Vitis spp.) cultivars ('L'Acadie' (LAc) and 'Thompson Seedless' (TS)). The minimum fluorescence values produced by both fluorometer types in response to a reversible low-oxygen stress in apples were indistinguishable: both F (o) and F (alpha) increased when O(2) levels were lowered below the anaerobic compensation point (ACP); when gas levels returned to normoxia both parameters dipped below, then returned to, the original fluorescence baseline. The two parameters also responded similarly to the irreversible osmotic stress in grapes: in both cultivars, F (alpha) and F (o) first decreased before reaching an inflection point at approximately 20% mass loss and then increased towards a second inflection point. However, the two parameters were not analogous under the irreversible osmotic stress; most notably, the relative F (alpha) values appeared to be lower than F (o) during the later stages of dehydration. This was likely due to the influence of the F (m) parameter and an overestimation of F (alpha) when measuring the fluorescence from healthy and responsive chloroplasts as found in grapes experiencing minimal water loss, but not in grapes undergoing moderate to severe dehydration. An examination of the data during a typical PFM scan reveals this fluorometer system may yield new fluorescence information with interesting biological applications.     

<Emphasis Type="Italic">Carex</Emphasis> × <Emphasis Type="Italic">involuta</Emphasis> and <Emphasis Type="Italic">Carex juncella</Emphasis> in the flora of Slovakia

The paper brings information on an isolated occurrence and morphological characters of Carex × involuta and C. juncella populations in the Vel’ká Fatra Mts. Their presence has been known neither from the territory of Slovakia nor from the whole Western Carpathians till now.     

Polymorphism of <Emphasis Type="Italic">SFBB</Emphasis><Superscript>−γ</Superscript> and its use for <Emphasis Type="Italic">S</Emphasis> genotyping in Japanese pear (<Emphasis Type="Italic">Pyrus pyrifolia</Emphasis>)

Japanese pear (Pyrus pyrifolia) exhibits the S-RNase-based gametophytic self-incompatibility where the pollen-part determinant, pollen S, had long remained elusive. Recent identification of S locus F-box brothers (SFBB) in Japanese pear and apple suggested that the multiple F-box genes are the pollen S candidates as they exhibited pollen specific expression, S haplotype-specific polymorphisms and linkage to the S locus. In Japanese pear, three SFBBs were identified from a single S haplotype, and they were more homologous to other haplotype genes of the same group (i.e., α-, β- and γ-groups). In this study, we isolated new seven PpSFBB ^−γ genes from different S genotypes of Japanese pear. These genes showed S haplotype-specific polymorphisms, however, sequence similarities among them were very high. Based on the sequence polymorphisms of the PpSFBB ^−γ genes, we developed a CAPS/dCAPS system for S genotyping of the Japanese pear cultivars. This new S genotyping system was found to not only be able to discriminate the S ¹–S ⁹, but also be suitable for identification of the mutant S ^4sm haplotype for the breeding of self-compatible cultivars, and detection of new S haplotypes such as S ^k.     

Molecular characterization of <Emphasis Type="Italic">Vibrio cholerae</Emphasis> Δ<Emphasis Type="Italic">relA</Emphasis> Δ<Emphasis Type="Italic">spoT</Emphasis> double mutants

In Escherichia coli cellular levels of pppGpp and ppGpp, collectively called (p)ppGpp, are maintained by the products of two genes, relA and spoT. Like E. coli, Vibrio cholerae also possesses relA and spoT genes. Here we show that similar to E. coli, V. cholerae ΔrelA cells can accumulate (p)ppGpp upon carbon starvation but not under amino acid starved condition. Although like in E. coli, the spoT gene function was found to be essential in V. cholerae relA ⁺ background, but unlike E. coli, several V. cholerae ΔrelA ΔspoT mutants constructed in this study accumulated (p)ppGpp under glucose starvation. The results suggest a cryptic source of (p)ppGpp synthesis in V. cholerae, which is induced upon glucose starvation. Again, unlike E. coli ΔrelA ΔspoT mutant (ppGpp⁰ strain), the V. cholerae ΔrelA ΔspoT mutants showed certain unusual phenotypes, which are (a) resistance towards 3-amino-1,2,4-triazole (AT); (b) growth in nutrient poor M9 minimal medium; (c) ability to stringently regulate cellular rRNA accumulation under glucose starvation and (d) initial growth defect in nutrient rich medium. Since these phenotypes of ΔrelA ΔspoT mutants could be reverted back to ΔrelA phenotypes by providing SpoT in trans, it appears that the spoT gene function is crucial in V. cholerae. Part of this work was presented at the International Symposium on Chemical Biology, Kolkata, India, 7–9 March 2007.     

Carbohydrate and Ethane Release with <Emphasis Type="Italic">Erwinia carotovora</Emphasis> Subspecies <Emphasis Type="Italic">betavasculorum</Emphasis><Emphasis Type="Italic">–</Emphasis>Induced Necrosis

Erwinia carotovora subspecies betavasculorum, also known as E. betavasculorum and Pectobacterium betavasculorum, is a soil bacterium that has the capacity to cause root rot necrosis of sugarbeets. The qualitatively different pathogenicity exhibited by the virulent E. carotovora strain and two avirulent strains, a Citrobacter sp. and an Enterobacter cloacae, was examined using digital analysis of photographic evidence of necrosis as well as for carbohydrate, ethane, and ethylene release compared with uninoculated potato tuber slices. Visual scoring of necrosis was superior to digital analysis of photographs. The release of carbohydrates and ethane from potato tuber slices inoculated with the soft rot necrosis-causing Erwinia was significantly greater than that of potato tuber slices that had not been inoculated or that had been inoculated with the nonpathogenic E. cloacae and Citrobacter sp. strains. Interestingly, ethylene production from potato slices left uninoculated or inoculated with the nonpathogenic Citrobacter strain was 5- to 10-fold higher than with potato slices inoculated with the pathogenic Erwinia strain. These findings suggest that (1) carbohydrate release might be a useful measure of the degree of pathogenesis, or relative virulence; and that (2) bacterial suppression of ethylene formation may be a critical step in root rot disease formation.     

Expression and purification of <Emphasis Type="Italic">Zantedeschia aethiopica</Emphasis> agglutinin in <Emphasis Type="Italic">Escherichia </Emphasis><Emphasis Type="Italic">coli</Emphasis>

Recombinant Zantedeschia aethiopica agglutinin (ZAA) was expressed in Escherichia coli as N-terminal His-tagged fusion. After induction with isopropylthio-β-d-galactoside (IPTG), the recombinant ZAA was purified by metal-affinity chromatography. The purified ZAA protein was applied in anti-fungal assay and the result showed that recombinant ZAA had anti-fungal activity towards leaf mold (Fulvia fulva), one of the most serious phytopathogenic fungi causing significant yield loss of crops. This study suggests that ZAA could be an effective candidate in genetic engineering of plants for the control of leaf mold.