<Emphasis Type="Italic">Muscadinia rotundifolia</Emphasis> ‘Noble’ defense response to <Emphasis Type="Italic">Plasmopara viticola</Emphasis> inoculation by inducing phytohormone-mediated stilbene accumulation

Downy mildew (DM), one of the most devastating grape diseases worldwide, is caused by the biotrophic oomycete Plasmopara viticola (Pv). In general, grapevine responds to Pv infection with the accumulation of phytoalexins as part of the innate immune system, and diverse phytoalexins are induced on grapevines with different DM-resistance levels in response to Pv invasion. However, the regulation of phytoalexin biosynthesis during grapevine against Pv is still unclear. Herein, we detected stilbenes by UPLC-ESI-MS/MS and found that resveratrol was accumulated to higher level and earlier in the DM-immune Muscadinia rotundifolia ‘Noble’ than that in the DM-susceptible Vitis vinifera ‘Thompson Seedless’ after Pv inoculation. Additionally, a considerable amount of pterostilbene and ε-viniferin was found in ‘Noble’, while a little was detected in ‘Thompson Seedless’. Resveratrol was glycosylated into piceid both in ‘Noble’ and ‘Thompson Seedless’ after Pv inoculation. The qPCR analysis of gene expression indicated that the resveratrol-synthesis gene (STS) was induced by Pv inoculation earlier in ‘Noble’ than that in ‘Thompson Seedless’, while the pterostilbene-synthesis gene (ROMT) was induced in ‘Noble’ but not in ‘Thompson Seedless’ at all. The piceid-synthesis gene (GT) was generally up-regulated in both cultivars. Sequence analysis of STS, ROMT, and GT promoters revealed that they contained cis-regulatory elements responsive to phytohormones and pathogens. Following Pv inoculation, the level of SA, MeJA, and ABA was found to be consistently higher in ‘Noble’ than those in ‘Thompson Seedless’. The results of exogenous hormone elicitation further demonstrated that the accumulation of stilbenes was regulated by phytohormones. The earlier and higher accumulation of phytohormones and consequent induction of stilbene synthesis may play an important role in grapevine defense against downy mildew disease.     

Conjugal transfer using the bacteriophage ϕC31 <Emphasis Type="Italic">att</Emphasis>/<Emphasis Type="Italic">int</Emphasis> system and properties of the <Emphasis Type="Italic">attB</Emphasis> site in <Emphasis Type="Italic">Streptomyces ambofaciens</Emphasis>

To facilitate molecular genetic studies of Streptomyces ambofaciens that produces spiramycin, a commercially important macrolide antibiotic used in human medicine against Gram-positive pathogenic bacteria, the conditions for the conjugal transfer of DNA from E. coli to S. ambofaciens were established using a bacteriophage ϕC31 att/int system. The transconjugation efficiency of S. ambofaciens varied with the medium used; the highest frequency was obtained on AS-1 medium containing 10 mM MgCl₂ without heat treatment of the spores. In addition, by cloning and sequencing the attB site, we identified that S. ambofaciens contains a single attB site within an ORF coding for a pirin homolog, and its attB site sequence shows 100% nt identity to the sequence of S. coelicolor and S. lividans, which have the highest efficiency in transconjugation using the ϕC31 att/int system.     

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.     

Regeneration of transformed verbena (<Emphasis Type="Italic">Verbena </Emphasis>× <Emphasis Type="Italic">hybrida</Emphasis>) by <Emphasis Type="Italic">Agrobacterium tumefaciens</Emphasis>

Verbena (Verbena x hybrida), an important floricultural species, was successfully regenerated from stem segments on Murashige and Skoog's basal medium supplemented with thidiazuron and indole-3-acetic acid. A transformation system was developed using cvs. Temari Scarlet, Temari Sakura, Tapien Rose and TP-P2. Agrobacterium tumefaciens strain Agl0 harboring the sGFP gene was infected into stem segments. Transformation efficiency was improved by evaluating and manipulating the age of the plant material, the concentration of kanamycin in the medium during selection, and the length of the culture period in the dark. After 2-3 months of culture on the selection medium, GFP-positive shoots were obtained in all four of the cultivars tested. These shoots were successfully acclimated and set flowers within 2-3 months in a greenhouse. GFP was expressed in all of the organs including the floral parts. Stable genomic transformation was confirmed by Southern blot analysis. No morphological differences were observed between the transformed plants and their host plants.     

AFLP markers closely linked to a major gene essential for nucellar embryony (apomixis<Emphasis Type="Italic">)</Emphasis> in <Emphasis Type="Italic">Citrus maxima</Emphasis> × <Emphasis Type="Italic">Poncirus trifoliata</Emphasis>

Some citrus varieties express a form of apomixis termed nucellar embryony in which the adventive embryos develop from nucellus tissue surrounding the embryo sac. This trait results in many seeds containing multiple embryos (polyembryony). Inheritance of the frequency of polyembryony was studied in 88 progeny from a cross of Citrus maxima (monoembryonic) × Poncirus trifoliata (polyembryonic). The frequency of polyembryonic seed produced by each progeny was determined by scoring 100–500 seeds for the number of seedlings to emerge from each seed. Two groups of eight individuals from each extreme of the population were chosen for bulked segregant analysis with amplified fragment length polymorphism markers amplified with 256 primer combinations. Candidate markers identified in the bulks as linked to the trait were tested on the 32 individuals used to create the bulks and then on the remaining plants in the population. Five candidate markers tightly linked to polyembryony in P. trifoliata were identified. Specific marker alleles were present in nearly all progeny that produced polyembryonic seed, and alternate alleles were present in nearly all progeny that produced only monoembryonic seed. The region defined by these markers very likely contains a gene that is essential for the production of polyembryonic seeds by apomixis, but also shows segregation distortion. The proportion of polyembryonic seeds varied widely among the hybrid progeny, probably due to other genes. Scoring 119 progeny of a P. trifoliata selfed population for the closely linked markers and the proportion of polyembryonic seeds confirmed close linkage between these markers and polyembryony.     

Characteristics of β-lactamases and their genes (<Emphasis Type="Italic">blaA</Emphasis> and <Emphasis Type="Italic">blaB</Emphasis>) in <Emphasis Type="Italic">Yersinia intermedia</Emphasis> and <Emphasis Type="Italic">Y. frederiksenii</Emphasis>

Background  

The presence of β-lactamases in Y. enterocolitica has been reported to vary with serovars, biovars and geographical origin of the isolates. An understanding of the β-lactamases in other related species is important for an overall perception of antibiotic resistance in yersiniae. The objective of this work was to study the characteristics of β-lactamases and their genes in strains of Y. intermedia and Y. frederiksenii, isolated from clinical and non-clinical sources in India.     

Cryopreservation of cell suspension cultures of <Emphasis Type="Italic">Taxus</Emphasis> × <Emphasis Type="Italic">media</Emphasis> and <Emphasis Type="Italic">Taxus floridana</Emphasis>

Different lines of cell suspension cultures of Taxus × media Rehd. and Taxus floridana Nutt. were cryopreserved with a two-step freezing method using a simple and inexpensive freezing container instead of a programmable freezer. Four to seven days old suspension cell cultures were precultured in growth medium supplemented with 0.5 M mannitol for 2 d. The medium was then replaced with cryoprotectant solution (1 M sucrose, 0.5 M glycerol and 0.5 M dimethylsulfoxide) and the cells incubated on ice for 1 h. Before being plunged into liquid nitrogen, cells were frozen with a cooling rate of approximately −1 °C per min to −80 °C. The highest post-thaw cell viability was 90 %. The recovery was line dependent. The cryopreservation procedure did not alter the nuclear DNA content of the cell lines. The results indicate that cryopreservation of Taxus cell suspension cultures using inexpensive freezing container is possible.     

Sequence Analysis of the <Emphasis Type="Italic">Lactoferrin</Emphasis> Gene and Variation of <Emphasis Type="Italic">g</Emphasis>.<Emphasis Type="Italic">7605C</Emphasis>→<Emphasis Type="Italic">T</Emphasis> in 10 Chinese Indigenous Goat Breeds

Much attention has been focused on the study of lactoferrin at the protein or nucleotide level in mice, humans, and cattle, but little is known about it in goats. The goat LF gene from 5' UTR to exon 17 was amplified, and the variation of g.7605C→T in 10 Chinese indigenous goat breeds was analyzed. Among the three ruminant species (cattle, sheep, and goats), the intron-exon distribution pattern was similar, and all the exons had the same length, but the length of introns varied greatly due to insertions or deletions. The frequency of allele T at g.7605C→T (50.12%) was a little higher than that of allele C (49.88%), and the genotype distribution differed greatly between goat populations. The g.7605C→T site showed higher genetic diversity in goat populations. The genetic differentiation was 0.0783, and gene flow was 2.9433 among the 10 Chinese indigenous goat populations.     

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.     

Overexpression of an optimized <Emphasis Type="Italic">Aspergillus sulphureus β</Emphasis>-mannanase gene in <Emphasis Type="Italic">Pichia pastoris</Emphasis>

β-Mannanase (EC 3.2.1.78) is a key enzyme to hydrolyze the β-mannosidic linkages in mannan and heteromannan. The expression of a wild type β-mannanase (manWT) of Aspergillus sulphureus in Pichia pastoris is not high enough for its application in feed supplement. To earn a high expression level, the manWT gene was firstly optimized to manM according to the code bias of P. pastoris, which was then inserted into pPICzαA and transformed into P. pastoris strain X-33. In the induction by methanol, β-mannanase was expressed in high level with 32% increase in comparison with the manWT gene expressed in P. pastoris in shaken flask. In a 10-L fermenter, the manM was expressed in 9-fold higher level than that in shaken flask, which yielded the enzyme activity of 1100 U/mL. This is the first study on codon bias effect on the β-mannanase gene expression level, which helps to achieve high β-mannanase yield and enzymatic activity in P. pastoris.     

Novel peroxidases of <Emphasis Type="Italic">Marasmius scorodonius</Emphasis> degrade <Emphasis Type="Italic">β</Emphasis>-carotene

Two extracellular enzymes (MsP1 and MsP2) capable of efficient β-carotene degradation were purified from culture supernatants of the basidiomycete Marasmius scorodonius (garlic mushroom). Under native conditions, the enzymes exhibited molecular masses of ~150 and ~120 kDa, respectively. SDS-PAGE and mass spectrometric data suggested a composition of two identical subunits for both enzymes. Biochemical characterisation of the purified proteins showed isoelectric points of 3.7 and 3.5, and the presence of heme groups in the active enzymes. Partial amino acid sequences were derived from N-terminal Edman degradation and from mass spectrometric ab initio sequencing of internal peptides. cDNAs of 1,604 to 1,923 bp, containing open reading frames (ORF) of 508 to 513 amino acids, respectively, were cloned from a cDNA library of M. scorodonius. These data suggest glycosylation degrees of ~23% for MsP1 and 8% for MsP2. Databank homology searches revealed sequence homologies of MsP1 and MsP2 to unusual peroxidases of the fungi Thanatephorus cucumeris (DyP) and Termitomyces albuminosus (TAP).     

Does sequence polymorphism of <Emphasis Type="Italic">FLC</Emphasis> paralogues underlie flowering time QTL in <Emphasis Type="Italic">Brassica </Emphasis><Emphasis Type="Italic">oleracea</Emphasis>?

Previous locations of flowering time (FT) QTL in several Brassica species, coupled with Arabidopsis synteny, suggest that orthologues of the genes FLC, FY or CONSTANS might be the candidates. We focused on FLC, and cloned paralogous copies in Brassica oleracea, obtained their genomic DNA sequences, and confirmed their locations relative to those of known FT-QTL by genetical mapping. They varied in total length mainly due to the variable size of the first and last introns. A high level of identity was observed among Brassica FLC genes at the amino acid level but non-synonymous differences were present. Comparative analysis of the promoter and intragenic regions of BoFLC paralogues with Arabidopsis FLC revealed extensive differences in overall structure and organisation but showed high conservation within those segments known to be essential in regulating FLC expression. Four B. oleracea FLC copies (BoFLC1, BoFLC3, BoFLC4 and BoFLC5) were located to their respective linkage groups based on allelic sequence variation in lines from a doubled haploid population. All except BoFLC4 were within the confidence intervals of known FT-QTL. Sequence data indicated that relevant non-synonymous polymorphisms were present between parents A12DHd and GDDH33 for BoFLC genes. However, BoFLC alleles segregated independently of FT in backcrosses while the study provided evidence that BoFLC4 and BoFLC5 contain premature stop codons and so could not contribute to flowering time variation. Therefore, there is strong evidence against any of the 4 BoFLC being FT-QTL candidates in this population.     

Stereoselective Inhibition of Cholesterol Esterase by Enantiomers of <Emphasis Type="Italic">exo</Emphasis>- and <Emphasis Type="Italic">endo</Emphasis>-2-Norbornyl-<Emphasis Type="Italic">N</Emphasis>–<Emphasis Type="Italic">n</Emphasis>-butylcarbamates

Four stereoisomers of 2-norbornyl-N–n-butylcarbamates are characterized as the pseudo substrate inhibitors of cholesterol esterase. Cholesterol esterase shows enantioselective inhibition for enantiomers of exo- and endo-2-norbornyl-N–n-butylcarbamates. For the inhibitions by (R)-(+)- and (S)-(−)-exo-2-norbornyl-N–n-butylcarbamates, the R-enantiomer is 6.8 times more potent than the S-enantiomer. For the inhibitions by (R)-(+)- and (S)-(−)-endo-2-norbornyl-N–n-butyl-carbamates, the S-enantiomer is 4.6 times more potent than the R-enantiomer. The enzyme-inhibitor complex models have been proposed to explain these different enantioselectivities.     

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.     

<Emphasis Type="Italic">Chromobacterium violaceum</Emphasis> and its important metabolites — <Emphasis Type="Italic">review</Emphasis>

C. violaceum appeared as important bacterium in different applications and mainly these aspects are related to the production of violacein. This review discusses the last reports on biosynthetic pathways, production, genetic aspects, biological activities, pathological effects, antipathogenic screening through quorum sensing, environmental effects and the products of C. violaceum with industrial interest. An important discussion is on biological applications in medicine and as industrial products such as textile and in cosmetics.     

<Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated transformation of <Emphasis Type="Italic">Codonopsis lanceolata</Emphasis> using the <Emphasis Type="Italic">γ-TMT</Emphasis> gene

Efficient transformation of leaf disc-derived callus of Codonopsis lanceolata was obtained using Agrobacterium tumefaciens strain LBA4404 harboring a binary vector, pYBI121, that carries the neomycin phosphotransferase (npt II) gene as a selectable marker. The green shoots recovered from agroinfected explants on selection medium (containing 0.1 mg/l α-naphthaleneacetic acid (NAA), 1 mg/l 6-benzylaminopurine (BAP), 100 mg/l kanamycin, and 250 mg/l cefotaxime) were rooted on Murashige and Skoog (MS) medium supplemented with 2 mg/l IBA and 10 mg/l kanamycin. To optimize the transformation conditions, several factors were assessed, including the co-cultivation period, the duration of pre- and post-culture in darkness and light, the kanamycin concentration, and the Agrobacterium densities. We produced transgenic Codonopsis lanceolata overexpressing γ-tocopherol methyltransferase (γ-TMT) by this protocol. Moreover, the α-tocopherol content of the plants was enhanced by the overexpression of this gene. Bimal Kumar Ghimire and Eun Soo Seong contributed equally to this work.     

Carbon isotope fractionation during <Emphasis Type="Italic">cis</Emphasis>–<Emphasis Type="Italic">trans</Emphasis> isomerization of unsaturated fatty acids in <Emphasis Type="Italic">Pseudomonas putida</Emphasis>

The molecular mechanism of the unique cis to trans isomerization of unsaturated fatty acids in the solvent-tolerant bacterium Pseudomonas putida S12 was studied. For this purpose, the carbon isotope fractionation of the cis–trans isomerase was estimated. In resting cell experiments, addition of 3-nitrotoluene for activation of the cis–trans isomerase resulted in the conversion of the cis-unsaturated fatty acids into the corresponding trans isomers. For the conversion of C16:1 cis to its corresponding trans isomer, a significant fractionation was measured. The intensity of this fractionation strongly depended on the rate of cis–trans isomerization and the added concentration of 3-nitrotoluene, respectively. The presence of a significant fractionation provides additional indication for a transition from the sp² carbon linkage of the cis-double bond to an intermediate sp³ within an enzyme–substrate complex. The sp² linkage is reconstituted after rotation to the trans configuration has occurred. As cytochrome c plays a major role in the catabolism of Cti polypeptide, these findings favour a mechanism for the enzyme in which electrophilic iron (Fe³⁺), provided by a heme domain, removes an electron of the cis double bond thereby transferring the sp² linkage into sp³.