In planta transformation of <Emphasis Type="Italic">Notocactus scopa</Emphasis> cv. <Emphasis Type="Italic">Soonjung</Emphasis> by <Emphasis Type="Italic">Agrobacterium </Emphasis><Emphasis Type="Italic">tumefaciens</Emphasis>

Notocactus scopa cv. Soonjung was subjected to in planta Agrobacterium tumefaciens-mediated transformation with vacuum infiltration, pin-pricking, and a combination of the two methods. The pin-pricking combined with vacuum infiltration (20-30 cmHg for 15 min) resulted in a transformation efficiency of 67-100%, and the expression of the uidA and nptII genes was detected in transformed cactus. The established in planta transformation technique generated a transgenic cactus with higher transformation efficiency, shortened selection process, and stable gene expression via asexual reproduction. All of the results showed that the in planta transformation method utilized in the current study provided an efficient and time-saving procedure for the delivery of genes into the cactus genome, and that this technique can be applied to other asexually reproducing succulent plant species.     

High-level overproduction of <Emphasis Type="Italic">Thermus</Emphasis> enzymes in <Emphasis Type="Italic">Streptomyces lividans</Emphasis>

Biotechnology needs to explore the capacity of different organisms to overproduce proteins of interest at low cost. In this paper, we show that Streptomyces lividans is a suitable host for the expression of Thermus thermophilus genes and report the overproduction of the corresponding proteins. This capacity was corroborated after cloning the genes corresponding to an alkaline phosphatase (a periplasmic enzyme in T. thermophilus) and that corresponding to a beta-glycosidase (an intracellular enzyme) in Escherichia coli and in S. lividans. Comparison of the production in both hosts revealed that the expression of active protein achieved in S. lividans was much higher than in E. coli, especially in the case of the periplasmic enzyme. In fact, the native signal peptide of the T. thermophilus phosphatase was functional in S. lividans, being processed at the same peptide bond in both organisms, allowing the overproduction and secretion of this protein to the S. lividans culture supernatant. As in E. coli, the thermostability of the expressed proteins allowed a huge purification factor upon thermal denaturation and precipitation of the host proteins. We conclude that S. lividans is a very efficient and industry-friendly host for the expression of thermophilic proteins from Thermus spp.     

High-efficiency <Emphasis Type="Italic">Agrobacterium rhizogenes</Emphasis>-mediated transformation of heat inducible sHSP18.2-GUS in <Emphasis Type="Italic">Nicotiana tabacum</Emphasis>

The chimerical gene, Arabidopsis thaliana sHSP18.2 promoter fused to E. coli gusA gene, was Agrobacterium rhizogenes-mediated transformed into Nicotiana tabacum as a heat-regulatable model, and the thermo-inducible expression of GUS activity in N. tabacum transgenic hairy roots was profiled. An activation of A. rhizogenes with acetosyringone (AS) before cocultured with tobacco's leaf disc strongly promoted transgenic hairy roots formation. Transgenic hairy roots formation efficiency of A. rhizogenes precultured with 200 μM AS supplementation was 3.1-fold and 7.5-fold, respectively, compared to the formation efficiency obtained with and without AS supplementation in coculture. Transgenic hairy roots transformed with different AS concentration exhibited a similar pattern of thermo-inducibility after 10 min to 3 h heat treatments detected by GUS expression. The peak of expressed GUS specific activity, 399,530 pmol MUG per mg total protein per min, of the transgenic hairy roots was observed at 48 h after 3 h of 42°C heat treatment, and the expressed GUS specific activity was 7–26 times more than that reported in A. thaliana, tobacco BY-2 cells and Nicotiana plumbaginifolia. Interference caused by AS supplementation on the growth of transgenic hairy roots, time-course of GUS expression and its expression level were not observed.     

Inactivation of barotolerant strains of <Emphasis Type="Italic">Listeria monocytogenes</Emphasis> and <Emphasis Type="Italic">Escherichia coli</Emphasis> O157:H7 by ultra high pressure and <Emphasis Type="Italic">tert</Emphasis>-butylhydroquinone combination

Antimicrobial efficacy of ultra-high-pressure (UHP) can be enhanced by application of additional hurdles. The objective of this study was to systematically assess the enhancement in pressure lethality by TBHQ treatment, against barotolerant strains of Escherichia coli O157:H7 and Listeria monocytogenes. Two L. monocytogenes Scott A and the barotolerant OSY-328 strain, and two E. coli O157:H7 strains, EDL-933 and its barotolerant mutant, OSY-ASM, were tested. Cell suspensions containing TBHQ (50 ppm, dissolved in dimethyl sulfoxide) were pressurized at 200 to 500 MPa (23+/-2 degrees C) for 1 min, plated on tryptose agar and enumerated the survivors. The TBHQ-UHP combination resulted in synergistic inactivation of both pathogens, with different degrees of lethality among strains. The pressure lethality threshold, for the combination treatment, was lower for E. coli O157:H7 (> or = 200 MPa) than for L. monocytogenes (> 300 MPa). E. coli O157:H7 strains were extremely sensitive to the TBHQ-UHP treatment, compared to Listeria strains. Interestingly, a control treatment involving DMSO-UHP combination consistently resulted in higher inactivation than that achieved by UHP alone, against all strains tested. However, sensitization of the pathogens to UHP by the additives (TBHQ in DMSO) was prominently greater for UHP than DMSO. Differences in sensitivities to the treatment between these two pathogens may be attributed to discrepancies in cellular structure or physiological functions.     

The Novel Use of a Combination of Sonication and Vacuum Infiltration in <Emphasis Type="Italic">Agrobacterium</Emphasis>-mediated Transformation of Kidney Bean (<Emphasis Type="Italic">Phaseolus vulgaris</Emphasis> L.) with <Emphasis Type="Italic">lea</Emphasis> Gene

An efficient gene transfer system without tissue culture steps was developed for kidney bean by using sonication and vacuum infiltration assisted, Agrobacterium-mediated transformation. Transgenic kidney bean with a group 3 lea (late embryogenesis abundant) protein gene from Brassica napus was produced through this approach. Among 18 combinations of transformation methods, Agrobacterium-mediated transformation combined with 5 min sonication and 5 min vacuum infiltration turned to be optimal, resulting in the highest transformation efficiency. Transgenic kidney bean plants demonstrated enhanced growth ability under salt and water deficit stress conditions. The increased tolerance was also reflected by delayed development of damage symptoms caused by drought stress. Transgenic lines with high level of lea gene expression showed higher stress tolerance than lines with lower expression level. Stress tolerance of transgenic kidney bean correlated much better with lea gene expression levels than with gene integration results. There is no prior report on the production of transgenic kidney bean using both ultrasonic and vacuum infiltration assisted, Agrobacterium-mediated transformation.     

Sucrose and overexpression of trehalose biosynthetic genes (<Emphasis Type="Italic">otsBA</Emphasis>) increase desiccation tolerance of recombinant <Emphasis Type="Italic">Escherichia coli</Emphasis>

Survival after desiccation was highest for recombinant strains of E. coli engineered to produce ethanol (KO11 and LY163) and lactate (TG106) when sucrose was provided as the fermentable sugar. Desiccation tolerance was lower with glucose and xylose. Further improvements in desiccation tolerance with sucrose were obtained by combining this with increased expression of otsBA genes encoding trehalose biosynthesis, removal of products from metabolism by resuspending in fresh medium, and harvesting cells prior to the end of log phase. With sucrose and otsBA expression, survivals of 20%–80% were readily achieved. Fermentation tests with EM2L, a derivative of LY163 expressing ostBA, demonstrated that ethanol production from seed fermentations begun with desiccated cells is equivalent to that of an undesiccated control.     

Cyclohexanone-induced stress metabolism of <Emphasis Type="Italic">Escherichia coli</Emphasis> and <Emphasis Type="Italic">Corynebacterium glutamicum</Emphasis>

Solvent stress occurs during whole-cell biocatalysis of organic chemicals. Organic substrates and/or products may accumulate in the cellular membranes of whole cells, causing structural destabilization of the membranes, which leads to disturbances in cellular carbon and energy metabolism. Here, we investigate the effect of cyclohexanone on carbon metabolism in Escherichia coli BL21 and Corynebacterium glutamicum ATCC13032. Adding cyclohexanone to the culture medium (i.e., glucose mineral medium) resulted in a decreased specific growth rate and increased cellular maintenance energy in both strains of bacteria. Notably, carbon metabolism, which is mainly involved to increase cellular maintenance energy, was very different between the bacteria. Carbon flux into the acetic acid fermentation pathway was dominantly enhanced in E. coli, whereas the TCA cycle appeared to be activated in C. glutamicum. In fact, carbon flux into the TCA cycle in E. coli appeared to be reduced with increasing amounts of cyclohexanone in the culture medium. Metabolic engineering of E. coli cells to maintain or improve TCA cycle activity and, presumably, that of the electron transport chain, which are involved in regeneration of cofactors (e.g., NAD(P)H and ATP) and formation of toxic metabolites (e.g., acetic acid), may be useful in increasing solvent tolerance and biotransformation of organic chemicals (e.g., cyclohexanone).     

Modulation of cell proliferation during palatogenesis by the interplay between <Emphasis Type="Italic">Tbx3</Emphasis> and <Emphasis Type="Italic">Bmp4</Emphasis>

During secondary palate development, two shelves are elevated to a horizontal position above the tongue through a process involving many cellular mechanisms, including proliferation. In particular, the expression patterns of Tbx3 and Bmp4, which are colocalized at embryonic day 13.5 (E13.5) and have unique expression patterns in specific regions at E14.5, have been investigated in early mouse palatogenesis. Tbx3 expression is reported to be associated with Bmp4 signaling during the process of organogenesis in other areas, such as limb development. However, the function of Tbx3 and the relationship between Tbx3 and Bmp4 in palate development have not been determined. We have examined the gene expression pattern and cell proliferation in order to understand the mutual interactions and function of Tbx3 and Bmp4. An electroporation method was used to investigate the altered pattern of these genes after their over-expression in organ cultures. NOGGIN protein-soaked beads were also implanted into the cultured palate to determine the function of Bmp4 in palatogenesis. After electroporation and NOGGIN bead implantation, the number of PCNA-positive cells was counted. The results showed that Tbx3 and Bmp4 strongly up- and down-regulated each other in order to control the proliferation of the palatal shelf. Thus, Tbx3 expression is induced by Bmp4 in the mesenchyme of the anterior palatal shelves, whereas mesenchymal expression of Tbx3 down-regulates Bmp4 expression in the mesenchyme of the palate. The harmonization between Tbx3 and Bmp4 therefore controls cell proliferation to regulate secondary palate development. This research was supported by the International Cooperation Research Program of the Ministry of Science & Technology (M6-0302-00-0044).