Expression of<Emphasis Type="Italic"> Sulfolobus solfataricus</Emphasis> α-glucosidase in<Emphasis Type="Italic"> Lactococcus lactis</Emphasis>

The industrial potential to use extreme thermophilic microorganisms and their enzymes lies in applications in which the temperature cannot be adjusted (cooled) at will. The production of enzymes from wild-type thermophiles is very low, therefore, for industrial applications, it is necessary to use recombinant microorganisms. In this paper, the cloning of a heat-stable -glucosidase from Sulfolobus solfataricus using lactic acid bacteria as expression system is reported. The extremophilic -glucosidase was cloned in Lactococcus lactis and correctly folded despite being expressed at a lower temperature. The recombinant cells were assayed for enzyme residual activity at 75 °C in order to analyze the direct use of whole cells as biocatalysts. Maximum activity corresponded to 40 U/l in static cultures. The protein yield was further improved by optimizing fermentation and reached 600 U/l in batch mode. Microfiltration led to an even higher enzyme production of 850 U/l as a result of increased biomass. The overall production of -glucosidase using the engineered L. lactis strain in microfiltration fermentation is 1,000-fold higher than obtained using the wild-type.     

Metabolic flux distribution for γ-linolenic acid synthetic pathways in<Emphasis Type="Italic">Spirulina platensis</Emphasis>

Spirulina produces γ-linolenic acid (GLA), an important pharmaceutical substance, in a relatively low level compared with fungi and plants, prompting more research to improve its GLA yield. In this study, metabolic flux analysis was applied to determine the cellular metabolic flux distributions in the GLA synthetic pathways of twoSpirulina strains, wild type BP and a high-GLA producing mutant Z19/2. Simplified pathways involving the GLA synthesis ofS. platensis formulated comprise of photosynthesis, gluconeogenesis, the pentose phosphate pathway, the anaplerotic pathway, the tricarboxylic cycle, the GLA synthesis pathway, and the biomass synthesis pathway. A stoichiometric model reflecting these pathways contains 17 intermediates and 22 reactions. Three fluxes—the bicarbonate (C-source) uptake rate, the specific growth rate, and the GLA synthesis rate—were measured and the remaining fluxes were calculated using linear optimization. The calculation showed that the flux through the reaction converting acetyl-CoA into malonyl-CoA in the mutant strain was nearly three times higher than that in the wild-type strain. This finding implies that this reaction is rate controlling. This suggestion was supported by experiments, in which the stimulating factors for this reaction (NADPH and MgCl₂) were added into the culture medium, resulting in an increased GLA-synthesis rate in the wild type strain.     

The <Emphasis Type="Italic">Endo-β-Mannanase</Emphasis> gene families in <Emphasis Type="Italic">Arabidopsis</Emphasis>, rice,and poplar

Mannans are widespread hemicellulosic polysaccharides in plant cell walls. Hydrolysis of the internal β-1,4-d-mannopyranosyl linkage in the backbone of mannans is catalyzed by endo-β-mannanase. Plant endo-β-mannanase has been well studied for its function in seed germination. Its involvement in other plant biological processes, however, remains poorly characterized or elusive. The completed genome sequences of Arabidopsis (Arabidopsis thaliana), rice (Oryza sativa), and poplar (Populus trichocarpa) provide an opportunity to conduct comparative genomic analysis of endo-β-mannanase genes in these three species. In silico sequence analysis led to the identification of eight, nine and 11 endo-β-mannanase genes in the genomes of Arabidopsis, rice, and poplar, respectively. Sequence comparisons revealed the conserved amino acids and motifs that are critical for the active site of endo-β-mannanases. Intron/exon structure analysis in conjunction with phylogenetic analysis implied that both intron gain and intron loss has played roles in the evolution of endo-β-mannanase genes. The phylogenetic analysis that included the endo-β-mannanases from plants and other organisms implied that plant endo-β-mannanases have an ancient evolutionary origin. Comprehensive expression analysis of all Arabidopsis and rice endo-β-mannanase genes showed divergent expression patterns of individual genes, suggesting that the enzymes encoded by these genes, while carrying out the same biochemical reaction, are involved in diverse biological processes.     

Molecular population genetics of the<Emphasis Type="Italic">β-esterase</Emphasis> gene cluster of<Emphasis Type="Italic">Drosophila melanogaster</Emphasis>

We have investigated nucleotide polymorphism at theβ-esterase gene cluster including theEst-6 gene andψEst-6 putative pseudogene in four samples ofDrosophila melanogaster derived from natural populations of southern Africa (Zimbabwe), Europe (Spain), North America (USA: California), and South America (Venezuela). A complex haplotype structure is revealed in bothEst-6 andψEst-6. Total nucleotide diversity is twice inψEst-6 as inEst-6; diversity is higher in the African sample than in the non-African ones. Strong linkage disequilibrium occurs within theβ-esterase gene cluster in non-African samples, but not in the African one. Intragenic gene conversion events are detected withinEst-6 and, to a much greater extent, withinyEst-6; intergenic gene conversion events are rare. Tests of neutrality with recombination are significant for theβ-esterase gene cluster in the non-African samples but not significant in the African one. We suggest that the demographic history (bottleneck and admixture of genetically differentiated populations) is the major factor shaping the pattern of nucleotide polymorphism in theb-esterase gene cluster. However there are some ’footprints’ of directional and balancing selection shaping specific distribution of nucleotide polymorphism within the cluster. Intergenic epistatic selection betweenEst-6 andψEst-6 may play an important role in the evolution of theβ-esterase gene cluster preserving the putative pseudogene from degenerative destruction and reflecting possible functional interaction between the functional gene and the putative pseudogene.Est-6 andyEst-6 may represent an indivisible intergenic complex (‘intergene’) in which each single component (Est-6 orψEst-6) cannot separately carry out the full functional role.     

Screening interspecific hybrids of<Emphasis Type="Italic"> Populus</Emphasis> (<Emphasis Type="Italic">P. ciliata</Emphasis> ×<Emphasis Type="Italic"> maximowiczii</Emphasis>) using AFLP markers

Hybrids of Populus ciliata × maximowiczii are very vigorous and outperform both the parents in growth performance and yield. Genetic evaluation of 24 of these interspecific hybrids along with the two mother trees (Populus ciliata), and five male-parent (Populus maximowiczii) genotypes was carried out using the AFLP marker assay. Eight AFLP primer combinations detected 428 markers, of which 280 (66%) were polymorphic. Genetic relationships within the samples were evaluated by generating the similarity matrix based on Jaccards coefficient. The phenetic dendrograms, as well as the PCO plots, separated the hybrids and the two parent species into three distinct clusters. The hybrids grouped closer to the P. ciliata (female parent) cluster as compared to the P. maximowiczii (male parent) cluster. The hybrid cluster contained internal groupings, which correlated to some extent with growth performance. The four best performing hybrids (42m1, 65m1, 23m2, Cm2-5-20/91) formed a distinct sub-cluster. Data from a single primer combination was sufficient for distinguishing the hybrids from the parents and assigning paternity. The hybrids showed 22 markers that were absent in P. ciliata but were monomorphically present in all the hybrids, suggesting outcrossing and common paternity. Further, these 22 markers were found in all the P. maximowiczii genotypes confirming it as the male parent. These male-specific markers can be converted to SCAR markers and used for rapid screening of the P.ciliata × maximowiczii hybrids. The primer combination E-AAC × M-CAA was identified as most suitable for ascertaining true hybridity. AFLP proves to be a useful tool for screening of P. ciliata × maximowiczii hybrids at the early stages of development.Communicated by H.F. Liskens     

Transient expression of<Emphasis Type="Italic"> Arabidopsis thaliana</Emphasis> ascorbate peroxidase 3 in<Emphasis Type="Italic"> Nicotiana benthamiana</Emphasis> plants infected with recombinant potato virus X

We have explored the transient over-expression of Arabidopsis thaliana ascorbate peroxidase 3 (APX3) in Nicotiana benthamiana using a viral vector based on the potato virus X (PVX). Plants infected with a PVX:APX3 hybrid had a similar progression of viral particles compared to control plants infected with a PVX:GFP hybrid, indicating that infection was not affected by the over-expression of heterologous APX3. Our results also showed that in PVX:APX3-infected plants, the hybrid virus directed a high level of APX3 expression and the recombinant protein was functional, as inferred from the higher APX activity compared to mock and PVX:GFP hybrid-infected plants. The PVX recombinant expression system used is a simple and quick method for transient expression of heterologous APXs, which are expected to suffer specific processing in plant cells.     

Identification of aryl-phospho-β-<Emphasis Type="SmallCaps">d</Emphasis>-glucosidases in<Emphasis Type="Italic"> Bacillus subtilis</Emphasis>

Four aryl-phospho--d-glucosidases were identified in Bacillus subtilis by using 4-methylumbelliferyl-phospho--d-glucopyranoside as a substrate. Two of these enzymes are the products of the bglA and bglH genes, previously suggested to encode aryl-phospho--d-glucosidases, while the other enzymes are encoded by the yckE and ydhP genes. Together, these four genes account for >99.9% of the glucosidase activity in B. subtilis on aryl-phospho--d-glucosides. yckE was expressed at a low and constant level during growth, sporulation, and spore germination, and was not induced by aryl--d-glucosides. ydhP was also not induced by aryl--d-glucosides. However, while ydhP was expressed at only a very low level in exponential-phase cells and germinating spores, this gene was expressed at a higher levels upon entry into the stationary phase of growth. Strains lacking yckE or ydhP exhibited no defects in growth, sporulation, or spore germination or in growth on aryl--d-glucosides. However, a strain lacking bglA, bglH and yckE grew poorly if at all on aryl--d-glucosides as the sole carbon source.Abbreviations MU 4-Methylumbelliferone - MUG 4-Methylumbelliferyl--d-glucopyranoside - MUGal 4-Methylumbelliferyl--d-galactopyranoside - MUG-P 4-Methylumbelliferyl--d-glucopyranoside-6-phosphate     

How promising is <Emphasis Type="Italic">Lasioseius floridensis</Emphasis> as a control agent of <Emphasis Type="Italic">Polyphagotarsonemus</Emphasis><Emphasis Type="Italic">latus</Emphasis>?

The blattisociid mite Lasioseius floridensis Berlese was found associated with the broad mite, Polyphagotarsonemus latus (Banks), on gerbera leaves in Mogi das Cruzes, State of Sao Paulo, Brazil. Blattisociid mites are not common on aerial plant parts, except under high air humidity levels. Some Lasioseius species have been mentioned as effective control agents of rice pest mites, but nothing is known about the biology of L. floridensis. The objective of this study was to evaluate whether the observed co-occurrence of L. floridensis and P. latus was just occasional or whether the latter could be important as food source for the former, assumed by laboratory evaluation of the ability of the predator to maintain itself, reproduce and develop on that prey. Biological parameters of L. floridensis were compared when exposed to P. latus and to other items as food. The study showed that mating is a pre-requisite for L. floridensis to oviposit and that oviposition rate was much higher on the soil nematode Rhabditella axei (Cobbold) (Rhabditidae) than on P. latus. Ovipositon on the acarid mite Tyrophagus putrescentiae (Schrank) was about the same as on P. latus, but it was nearly zero when the predator was fed the fungi Aspergillus flavus Link or Penicillium sp., or cattail (Typha sp.) pollen. Survivorship was higher in the presence of pollen and lower in the presence of A. flavus or Penicillium sp. than in the absence of those types of food. Life table parameters indicated that the predator performed much better on R. axei than on P. latus. To evaluate the potential effect of L. floridensis as predator of P. latus, complementary studies are warranted to determine the frequency of migration of L. floridensis to aerial plant parts, when predation on P. latus could occur.     

The W303 genetic background affects the<Emphasis Type="Italic">isw2</Emphasis>Δ mutant phenotype in<Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

We performed detailed phenotypic analysis of the isw2 delta strains of the W303 genetic background and compared its results with those obtained previously in BY-derived genetic background. Shmoolike morphology was observed in the isw2 delta strain of alpha-mating type of the BY strains, but not in its W303-derived counterpart. On the other hand, derepression of a-specific genes in the isw2 delta (MAT alpha) strain was observed in both genetic backgrounds, although to a different extent. Unlike in BY-derived strain hyperactivation of the Ras2/cAMP pathway reduced invasiveness of the isw2 delta strain (MAT alpha) of the W303 background. Sensitivity to Calcofluor White indicating a cell wall-integrity defect was significantly increased in the isw2 delta strains of the W303 background in contrast to BY-derived strains. Our data indicate that the effects of the isw2 deletion strongly depend on the background in which the deletion, is made.     

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.     

Production of γ-aminobutyric acid by <Emphasis Type="Italic">Streptococcus salivarius</Emphasis> subsp. <Emphasis Type="Italic">thermophilus</Emphasis> Y2 under submerged fermentation

Summary. γ-Aminobutyric acid (GABA), a major inhibitory neurotransmitter in the central nervous system, has several well-known physiological functions and has been applied to the production of many drugs and functional foods. The technology of GABA production via submerged fermentation by Streptococcus salivarius subsp. thermophilus Y2 was investigated in this paper. It indicated that the GABA production was related to the biochemical characteristics of glutamate decarboxylase (GAD) of S. salivarius subsp. thermophilus Y2. After 24 h of fermentation at 37 °C, which is the suitable culture conditions for GAD-production, then the culture condition were adjusted to the optimal temperature (40 °C) and pH (4.5) for the GAD reaction activity in biotransformation of cells and pyridoxal 5′-phosphate (0.02 mmol/l) were added to the broth at the 48 h, the GABA production was increased up to 1.76-fold, reaching 7984.75 ± 293.33 mg/l. The strain shows great potential use as a starter for GABA-containing yoghurt, cheese and other functional fermented food productions. Authors’ address: Zhao-Xin Lu, College of Food Science and Technology, Nanjing Agricultural University, Nanjing 210095, P.R. China     

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.     

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.     

Overexpression of <Emphasis Type="Italic">ppc</Emphasis> or deletion of <Emphasis Type="Italic">mdh</Emphasis> for improving production of γ-aminobutyric acid in recombinant <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

l-Glutamate decarboxylase (GAD) transforms l-glutamate into γ-aminobutyric acid (GABA). Corynebacterium glutamicum that expresses exogenous GAD gene(s) can synthesize GABA from its own produced l-glutamate. To enhance GABA production in recombinant C. glutamicum strain SH, metabolic engineering strategies were used to improve the supply of the GABA precursor, l-glutamate. Five new strains were constructed here. First, the ppc gene was coexpressed with two GAD genes (gadB1 and gadB2). Then, the mdh gene was deleted in C. glutamicum SH. Next, gadB1-gadB2 and gadB1-gadB2-ppc co-expression plasmids were transformed into C. glutamicum strains SH and Δmdh, resulting in four recombinant GAD strains SE1, SE2, SDE1, and SDE2, respectively. Finally, the mdh gene was overexpressed in mdh-deleted SDE1, generating the mdh-complemented GAD strain SDE3. After fermenting for 72 h, GABA production increased to 26.3?±?3.4, 24.8?±?0.7, and 25.5?±?3.3 g/L in ppc-overexpressed SE2, mdh-deleted SDE1, and mdh-deleted ppc-overexpressed SDE2, respectively, which was higher than that in the control GAD strain SE1 (22.7?±?0.5 g/L). While in the mdh-complemented SDE3, GABA production decreased to 20.0?±?0.6 g/L. This study demonstrates that the recombinant strains SE2, SDE1, and SDE2 can be used as candidates for GABA production.     

Efficient bioconversion of <Emphasis Type="SmallCaps">l</Emphasis>-glutamate to γ-aminobutyric acid by <Emphasis Type="Italic">Lactobacillus brevis</Emphasis> resting cells

This work investigated the efficient bioconversion process of l-glutamate to GABA by Lactobacillus brevis TCCC 13007 resting cells. The optimal bioconversion system was composed of 50 g/L 48 h cultivated wet resting cells, 0.1 mM pyridoxal phosphate in glutamate-containing 0.6 M citrate buffer (pH 4.5) and performed at 45 °C and 180 rpm. By 10 h bioconversion at the ratio of 80 g/L l-glutamic acid to 240 g/L monosodium glutamate, the final titer of GABA reached 201.18 g/L at the molar bioconversion ratio of 99.4 %. This process presents a potential for industrial and commercial applications and also offers a promising feasibility of continuous GABA production coupled with fermentation. Besides, the built kinetics model revealed that the optimum operating conditions were 45 °C and pH 4.5, and the bioconversion kinetics at low ranges of substrate concentration (0 < S < 80 g/L) was assumed to follow the classical Michaelis–Menten equation.     

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.     

Inappropriate annotation of a key defence marker in<Emphasis Type="Italic"> Arabidopsis</Emphasis>: will the real<Emphasis Type="Italic"> PR-1</Emphasis> please stand up?

PR-1 has been extensively used as a marker for salicylic acid (SA)-mediated defence and systemic and local acquired resistance. The Arabidopsis Genome Project annotates At2g19990 as PR-1. This gene is also identified as PR-1 in two full genome Arabidopsis microarrays, and TAIR cites approximately 60 articles to describe its patterns of expression. However, most of these citations are incorrect; the probes used were not At2g19990, but a homologous gene At2g14610, which is annotated as PR-1-like. Because of the potential for confusion, we analyzed the expression of both genes in Arabidopsis thaliana (L.) Heynh. At2g14610 (PR-1-like) showed the archetypal patterns of SA-responsive expression: mRNA levels increased following SA-treatment, inoculation with an avirulent (but not a virulent) strain of Pseudomonas syringae, and in wild-type (but not NahG) Arabidopsis infected with cauliflower mosaic virus (CaMV). In cpr5 mutants it was expressed constitutively. In contrast, expression of At2g19990 (annotated as PR-1) was detectable in neither SA-treated Col-0 nor in cpr5. Infection by virulent and avirulent isolates of P. syringae up-regulated expression, but to a similar level, and infection by CaMV induced a modest increase in expression in both the wild type and NahG. At2g19990, although pathogen responsive, does not show the SA-dependent patterns of expression expected from a member of the PR-1 regulon, and its annotation as PR-1 is inappropriate. The annotations should identify At2g14610 as the authentic PR-1.