Trichloroethylene oxidative metabolism in plants: the trichloroethanol pathway.

Trichloroethylene (TCE) is a widespread and persistent environmental contaminant. Recently, plants, poplar trees in particular, have been investigated as a tool to remove TCE from soil and groundwater. The metabolism of TCE in plants is being investigated for two reasons: one, plant uptake and metabolism represent an important aspect of the environmental fate of the contaminant; two, metabolism pattern and metabolite identification will help assess the applicability of phytoremediation. It was previously shown that TCE metabolites in plants are similar to ones that result from cytochrome P450-mediated oxidation in mammals: trichloroethanol, trichloroacetate and dichloroacetate. Our measurements indicate that one of these metabolites, trichloroethanol, is further glycosylated in tobacco and poplar. The glycoside was detected in all tissues (roots, stems and leaves) in comparable levels, and was at least 10 fold more abundant than free trichloroethanol. The glycoside in tobacco was identified as the ss-D-glucoside of trichloroethanol by comparison of the mass spectra and the chromatographic retention time of its acetylation product to that of the synthesized standard. Trichloroethanol and its glucoside did not persist in plant tissue once plants are removed from TCE contaminated water, indicating further metabolism.

Enhanced metabolism of halogenated hydrocarbons in transgenic plants containing mammalian cytochrome P450 2E1.

Chlorinated solvents, especially trichloroethylene (TCE), are the most widespread groundwater contaminants in the United States. Existing methods of pumping and treating are expensive and laborious. Phytoremediation, the use of plants for remediation of soil and groundwater pollution, is less expensive and has low maintenance; however, it requires large land areas and there are a limited number of suitable plants that are known to combine adaptation to a particular environment with efficient metabolism of the contaminant. In this work, we have engineered plants with a profound increase in metabolism of the most common contaminant, TCE, by introducing the mammalian cytochrome P450 2E1. This enzyme oxidizes a wide range of important pollutants, including TCE, ethylene dibromide, carbon tetrachloride, chloroform, and vinyl chloride. The transgenic plants had a dramatic enhancement in metabolism of TCE of up to 640-fold as compared with null vector control plants. The transgenic plants also showed an increased uptake and debromination of ethylene dibromide. Therefore, transgenic plants with this enzyme could be used for more efficient remediation of many sites contaminated with halogenated hydrocarbons.

Discrete regions of the sensor protein virA determine the strain-specific ability of Agrobacterium to agroinfect maize.

The ability of Agrobacterium strains to infect transformation-recalcitrant maize plants has been shown to be determined mainly by the virA locus, implicating vir gene induction as the major factor influencing maize infection. In this report, we further explore the roles of vir induction-associated bacterial factors in maize infection using the technique of agroinfection. The Ti plasmid and virA source are shown to be important in determining the ability of a strain to infect maize, and the monosaccharide binding protein ChvE is absolutely required for maize agroinfection. The linker domain of VirAC58 from an agroinfection-competent strain, C58, is sufficient to convert VirAA6 of a nonagroinfecting strain, A348,to agroinfection competence. The periplasmic domain of VirAC58 is also able to confer a moderate level of agroinfection competence to VirAA6. In addition, the VirAA6 protein from A348 is agroinfection competent when removed from its cognate Ti plasmid background and placed in a pTiC58 background. The presence of a pTiA6-encoded, VirAA6-specific inhibitor is hypothesized and examined.

Mutational analysis of the input domain of the VirA protein of Agrobacterium tumefaciens.

The transmembrane sensor protein VirA activates VirG in response to high levels of acetosyringone (AS). In order to respond to low levels of AS, VirA requires the periplasmic sugar-binding protein ChvE and monosaccharides released from plant wound sites. To better understand how VirA senses these inducers, the C58 virA gene was randomly mutagenized, and 14 mutants defective in vir gene induction and containing mutations which mapped to the input domain of VirA were isolated. Six mutants had single missense mutatiions in three widely separated areas of the periplasmic domain. Eight mutants had mutations in or near an amphipathic helix, TM1, or TM2. Four of the mutations in the periplasmic domain, when introduced into the corresponding A6 virA sequence, caused a specific defect in the vir gene response to glucose. This suggests that most of the periplasmic domain is required for the interaction with, or response to, ChvE. Three of the mutations from outside the periplasmic domain, one from each transmembrane domain and one from the amphiphathic helix, were made in A6 virA. These mutants were defective in the vir gene response to AS. These mutations did not affect the stability or topology of VirA or prevent dimerization; therefore, they may interfere with detection of AS or transmission of the signals to the kinase domain. Characterization of C58 chvE mutants revealed that, unlike A6 VirA, C58 VirA requires ChvE for activation of the vir genes.

On the pitches of the components of a complex tone.

The pitches of the harmonics (numbers 1, 2, 3, 4, 5, 7, 9, and 11) of a complex tone were measured in a matching experiment. The harmonics to be matched were mistuned (8% or less) either positively, or negatively, or not at all. For all mistuned harmonics and all listeners the matching pitches were found to be exaggerations of the mistunings, i.e., the data exhibited pitch shifts with the same sign as the mistunings. This result is shown to be contrary to place models of pitch perception, such as the spectral pitch algorithm of Terhardt, in which pitch shifts are caused by the interaction of excitation patterns for the individual harmonics. An alternative model, in which pitch is determined by neural timing, also fails to account for the data. However, a hybrid model, combining effects of excitation pattern interaction with neural timing, does agree with most of the data.

Construction of Agrobacterium strains by electroporation of genomic DNA and its utility in analysis of chromosomal virulence mutations.

We have extended the technique of electroporation as a genetic tool for manipulating the Agrobacterium tumefaciens chromosome. We used this technique to introduce chromosomal DNA into recipient A. tumefaciens strains by electroporation and constructed isogenic chvE mutants that share the same chromosomal background but differ in their types of pTi (octopine or nopaline). Both nopaline and octopine pTi-carrying chvE mutants were deficient in vir regulon induction and exhibited similar reductions in host range.

The chromosomal virulence gene, chvE, of Agrobacterium tumefaciens is regulated by a LysR family member.

Certain plant phenolic compounds and monosaccharides induce the transcription of virulence (vir) genes of Agrobacterium tumefaciens through the VirA-VirG two-component regulatory system. The product of the chromosomal virulence gene chvE is homologous to galactose-binding protein of Escherichia coli and is required for vir gene induction by sugars. Adjacent to, but divergent in transcription from, chvE is an open reading frame, now termed gbpR (galactose-binding protein regulator), that is homologous to the LysR family of transcriptional regulators. chvE::lacZ expression was induced by L-arabinose, D-galactose, and D-fucose when gbpR was present. In the absence of inducer, GbpR repressed chvE::lacZ expression. In addition, GbpR negatively regulated its own expression.

Humoral immune response to oral microorganisms in periodontitis.

Serum antibody titers from patients with periodontitis were compared with those from periodontally healthy subjects. With the micro-enzyme-linked immunosorbent assay, immunoglobulin G (IgG), IgA, and IgM antibody titers to isolates of Streptococcus sanguis, Actinomyces viscosus, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides gingivalis, Bacteroides melaninogenicus subsp. intermedius, Bacteroides ochraceus, and Fusobacterium nucleation were determined. Antibody titers of the IgG and IgA classes to B. melaninogenicus, B. ochraceus, F. nucleatum, and S. sanguis were found to be significantly higher in the controls than in the patients. No correlations were found with serum IgM titers. These findings indicate that periodonitis may be associated with depressed antibacterial serum antibody titers of the IgG and IgA classes.