Brian S. Hooker

Expression of Acidothermus cellulolyticus endoglucanase E1 in transgenic tobacco: biochemical characteristics and physiological effects

The expression of the Acidothermus cellulolyticus endoglucanase E1 gene in transgenic tobacco (Nicotiana tabacum) was examined in this study, where E1 coding sequence was transcribed under the control of a leaf specific Rubisco small subunit promoter (tomato RbcS-3C). Targeting the E1 protein to the chloroplast was established using a chloroplast transit peptide of Rubisco small subunit protein (tomato RbcS-2A) and confirmed by immunocytochemistry. The E1 produced in transgenic tobacco plants was found to be biologically active, and to accumulate in leaves at levels of up to 1.35% of total soluble protein. Optimum temperature and pH for E1 enzyme activity in leaf extracts were 81°C and 5.25, respectively. E1 activity remained constant on a gram fresh leaf weight basis, but dramatically increased on a total leaf soluble protein basis as leaves aged, or when leaf discs were dehydrated. E1 protein in old leaves, or after 5h dehydration, was partially degraded although E1 activity remained constant. Transgenic plants exhibited normal growth and developmental characteristics with photosynthetic rates similar to those of untransformed SR1 tobacco plants. Results from these biochemical and physiological analyses suggest that the chloroplast is a suitable cellular compartment for accumulation of the hydrolytic E1 enzyme.

Optimization of Acidothermus cellulolyticus Endoglucanase (E1) Production in Transgenic Tobacco Plants by Transcriptional, Post-transcription and Post-translational Modification

An attempt was made to obtain a high-level production of intact Acidothermus cellulolyticus endoglucanase (E1) in transgenic tobacco plants. The E1 expression was examined under the control of the constitutive and strong Mac promoter or light-inducible tomato Rubisco small sub-unit (RbcS-3C) promoter with its original or Alfalfa Mosaic Virus (AMV) RNA4 5′-untranslated leader (UTL) and targeted to different sub-cellular compartments via transit peptides. The transit peptides included native E1, endoplasmic reticulum, vacuole, apoplast, and chloroplast. E1 expression and its stability in transgenic plants were determined via E1 activity, protein immunoblotting, and RNA gel-blotting analyses. Effects of sub-cellular compartments on E1 production and its stability were determined in transgenic tobacco plants carrying one of six transgene expression vectors, where the E1 was under the control of Mac promoter, mannopine synthase transcription terminator, and one of the five transit peptides. Transgenic tobacco plants with an apoplastic transit peptide had the highest average E1 activity and protein accumulation, which was about 0.25% of total leaf soluble proteins estimated via E1 specific activity and protein gel blots. Intercellular fluid analyses confirmed that E1 signal peptide functioned properly in tobacco cells to secret E1 protein into the apoplast. By replacing RbcS-3C UTL with AMV RNA4 UTL E1 production was enhanced more than twofold, while it was less effective than the mannopine synthase UTL. It was observed that RbcS-3C promoter was more favorable for E1 expression in transgenic plants than the Mac promoter. E1 activity in dried tobacco seeds stored one year at room temperature was 45% higher than that observed immediately after harvesting, suggesting that E1 protein can be stored at room temperature for a long period. E1 stability in different sub-cellular compartments and the optimal combination of promoter, 5′-UTL, and sub-cellular compartmentation for heterologous protein production in transgenic plants are discussed.

Response of plant tissue culture to a high shear environment

Abstract Plant cell suspension cultures of Nicotiana tabacum were tested in a Couette-type shearing device to determine the effect of shear rate on culture viability, cell lysis, and the accumulation of secondary metabolites (phenolics). The effects of increasing shear and variable culture age were both investigated. Increasing shear caused an increase in cell death, lysis, and phenolics secretion. In testing suspension cultures of various ages, it was found that those cultures in the latter stages of exponential growth and early in the stationary phase were more susceptible to shear damage than cultures in the lag phase, early exponential phase, or later stationary phase. An increase in extracellular phenolics as a response to shear was more apparent with older cultures. The amount of phenolics secreted from the cells was directly related to the intensity of shear. This may have been due to an increase in cell permeabilization with higher shear rates .

Overexpression of multi-heme C-type cytochromes

ABSTRACT-Because they contain covalently attached hemes, c-type cytochromes, especially those with multi-heme, are difficult to over-express. The gram negative bacterium Shewanella oneidensis MR-1 has been successfully used for over-expression of multi-heme c-type cytochromes...

Expression of Trichoderma reesei exo-cellobiohydrolase I in transgenic tobacco leaves and calli

Expression of Trichoderma reesei exo-cellobiohydrolase I (CBHI) gene in transgenic tobacco was under the control of CaMV 35S promoter. In transgenic leaf tissues, CBHI activity up to 66.1 micromol/h/g total protein was observed. In transgenic calli, the highest CBHI activity was 83.6 micromol h/g total protein. Protein immunoblot analysis confirms the presence of CBHI enzyme in both transgenic calli and leaf tissues. CBHI expression levels accounted for about 0.11% and 0.082% of total protein in transgenic leaf tissues and calli, respectively. Furthermore, expression of CBHI gene did not affect normal growth and development of transgenic plants.

Cultivation of plant cells in aqueous two-phase polymer systems

SummarySuspension cultures ofNicotiana tabacum have been successfully grown in aqueous, two-phase systems comprised of polyethylene glycol (PEG) and dextran in a modified LS medium. Aqueous two-phase systems may be advantageous for plant tissue cultivation since cells can be immobilized in one phase while secondary products are collected and withdrawn in the other phase, thus enhancing productivity. Culture growth rate was compared in a variety of two-phase systems, covering a range of both polymer molecular weight and concentration. Systems exhibiting relatively higher phase miscibility yielded increased growth rates as compared to less miscible phase formulations. The highest observed growth rate occurred in 3% PEG 20000/5% crude dextran and approached growth rates and cell densities of cultures grown in standard LS medium.

Transgenic phytoremediation blasts onto the scene.

The EPA National Priority List contains 22 ammunition production and processing sites that are laden with explosive and propellant wastes. With levels of 2,4,6-trinitrotoluene (TNT) contamination as high as 200 g/kg of solids, some of these sites are literally on the verge of exploding. They also present serious exposure risks to humans and wildlife, as many of these contaminants are also strong toxins and mutagens. In this issue, French et al. describe a new option for cleaning up this dangerous mixture: the use of transgenic plants. They engineered plants to express a bacterial enzyme that can completely denitrify TNT and trinitroglycerin (GTN) into harmless compounds.

Effects of several electron donors on tetrachloroethylene dechlorination in anaerobic soil microcosms

Abstract Anaerobic microcosms of subsurface soils from three location were used to investigate the separate effects of several electron donors on tetrachloroethylene (PCE) dechlorination activity. The substrates tested were methanol, lactate, acetate and sucrose. Various levels of sulfate-reducing, acetogenic, fermentative, and methanogenic activity were observed in all sediments. PCE dechlorination was detected in all microcosms, but the amount of dehalogenation varied by several orders of magnitude. Trichloroethylene as the primary dehalogenation product; however, small amounts of cis -1,2-dichloroethylene, 1,1-dichloroethylene, and vinyl chloride were also detected in several microcosms. Lactate-amended microcosms showed large amounts of dehalogenation in two of the three sediments. One of the two sediments which showed positive activity with lactate also had large amounts of dehalogenation with methanol. Amendment with formate, acetate, or sucrose resulted in only slight dehalogenation in the three sediments. Elevated levels of dehalogenation were not consistently associated with any observable anaerobic metabolisms (sulfate reduction, acetogenesis, fermentation, or methanogensis).

Effect of reaction kinetics on predicted concentration profiles during subsurface bioremediation

The lack of a complete mechanistic description of subsurface interactions between contaminants and microbes has limited the ability to predict the effectiveness of subsurface bioremediation processes. Important microbial processes which must be included in such mechanistic descriptions of bioremediation are microbial growth, death and transport, including microbial attachment to and detachment from the soil surfaces. Recent advances have provided a better understanding of the relationships between contaminant destruction rates and such microbial processes. In this manuscript, contaminant profiles predicted using more realistic descriptions are compared with those obtained using less complete, previously published mathematical models. The more comprehensive model described here includes mathematical descriptions for microbial growth and transport. Contaminant profiles predicted using this model are compared with those obtained when the contaminant destruction processes are described by assuming that (1) no reaction occurs, (2) the contaminant reacts instantaneously with any available oxygen, and (3) dual-substrate Monod kinetics without consideration of microbial growth and transport. This work demonstrates that if reaction kinetics are ignored, the size of the plume will be overpredicted at large times and if the reaction is assumed to occur instantaneously, the plume will be underpredicted at small times. Thus, if these processes are not included in the mathematical description of bioremediation, erroneous interpretations of site characterization data collected during this period would be likely.

Evaluation of bacterial detachment rates in porous media

The ability of published biomass detachment rate expressions to describe experimental data obtained from porous media reactors usingPseudomonas aeruginosa grown aerobically on glucose was evaluated. A first-order rate expression on attached biomass concentration best reflected effluent substrate concentration for combined data sets. Detachment rate coefficientkd1 was dependent on initial substrate concentration. Simulation of porous media reactor experiments indicated that responses using higher influent substrate concentrations possessed greater sensitivity to variations inkd1. Simulations of field bioremediation systems suggest the use of accurate biofilm development kinetics is important in the prediction of well bore biofouling.