Growth of Alcaligenes eutrophus JMP134 on 2,4-dichlorophenoxyacetate requires a 2,4-dichlorophenol hydroxylase encoded by gene tfdB. Catabolism of either 2,4-dichlorophenoxyacetate or 3-chlorobenzoate involves enzymes encoded by the chlorocatechol oxidative operon consisting of tfdCDEF, which converts 3-chloro-and 3,5-dichlorocatechol to maleylacetate and chloromaleylacetate, respectively. Transposon mutagenesis has localized tfdB and t.fdCDEF to EcoRI fragment B of plasmid pJP4 (R. H. Don, A. J. Wieghtman, H.-J. Knackmuss, and K. N. Timmis, J. Bacteriol. 161:85-90, 1985). We present the complete nucleotide sequence of tfdB and tfdCDEF contained within a 7,954-base-pair HindIII-SstI fragment from EcoRI fragment B. Sequence and expression analysis of tfdB in Escherichia coli suggested that 2,4-dichlorophenol hydroxylase consists of a single subunit of 65 kilodaltons. The amino acid sequences of proteins encoded by tfdD and tfdE were found to be 63 and 53% identical to those of functionally similar enzymes encoded by clcB and ckD, respectively, from plasmid pAC27 of Pseudomonas putida. P. putida(pAC27) can utilize 3-chlorocatechol but not dichlorinated catechols. A region of DNA adjacent to clcD in pAC27 was found to be 47% identical in amino acid sequence to tfdiF, a gene important in catabolizing dichlorocatechols. The region in pAC27 does not appear to encode a protein, suggesting that the absence of a functional trans-chlorodienelactone isomerase may prevent P. putida(pAC27) from utilizing 3,5-dichlorocatechol.Bacteria play a crucial role in the dissimilation of environmental pollutants. In general, the catabolism of aromatic compounds is channeled through catechol intermediates (33). Hence, the ability of an organism to form and catabolize catechols or their derivatives can play a major part in the utilization of an aromatic compound. Comparison of the degradative pathways of different aromatics may help in understanding the evolution of growth substrate specificity.Chlorinated aromatics, such as 2,4-dichlorophenoxyacetic acid (2,4D) and 3-chlorobenzoate (3CBA), are catabolized via a modified ortho pathway (11,12,20,28,34,35; for a review, see reference 33). Pseudomonas putida plasmid pAC27, a deletion derivative of pAC25, confers catabolism of 3CBA (5), whereas Alcaligenes eutrophus JMP134 plasmid pJP4 enables degradation of both 2,4D and 3CBA (10, 11). In both cases, 3CBA appears to be degraded to 3-chlorocatechol by enzymes encoded by chromosomal genes. 2,4D is catabolized by A. eutrophus JMP134 to 2,4-dichlorophenol (2,4DCP) by 2,4D monooxygenase. 2,4DCP is then hydroxylated by 2,4DCP hydroxylase to form 3,5-dichlorocatechol (11; Fig. 1).3-Chlorocatechol is oxidized to maleylacetic acid by enzymes encoded by the clcABD operon in pAC27 (13) and by the tfdCDEF operon of pJP4 (11). The maleylacetic acid is then catabolized by chromosomally encoded enzymes (21). Enzymes involved in this transformation are chlorocatechol 1,2-dioxygenase (catechol 1,2-dioxygenase II or pyrocatechase II; EC 1.13.11.1), encoded b...

Electroporation of cells in the presence of DNA is widely used for the introduction of transgenes either stably or transiently into bacterial, fungal, animal, and plant cells. A review of the literature shows that electroporation parameters are often reported in an incomplete or incorrect manner, forcing researchers to rely too much on a purely empirical trial and error approach. The goal of this article is to provide the reader with an understanding of electrical circuits used in electroporation experiments as well as physical and biological aspects of the electroporation process itself. Further, a simple paradigm is provided which unites all electroporation parameters. This article should be particularly useful to those new to the technique.

Cowpea (Vigna unguiculata Walp) embryos mechanically isolated from mature seeds and incubated in the presence of plasmid DNA harboring chimeric gus genes were shown to germinate into seedlings expressing β-glucuronidase activity in a variety of tissues, including the apical meristem. Embryo electroporation in the presence of DNA and protectants such as spermine and Lipofectin(TM) increased both the proportion of embryo-derived seedlings expressing the chimeric gene and the level of gene expression. Microscopic observations of thin sections showed that the blue crystals representing the end product of transgene activity on X-glu were exclusively located inside the treated cells. Histological localization of the blue dye crystals varied with the promoter used to drive the transgene.

Electroporation-mediated gene transfer into intact plant tissues was demonstrated in pea, cowpea, lentil, and soybean plants. Transient expression of a chimeric gus reporter gene was used to monitor the uptake and expression of the introduced DNA in electroporated nodal axillary buds in vivo. The branches that grew out of the nodal meristems were chimeric and expressed the introduced gene up to 20 d after electroporation. Transgenic R1 pea, lentil, and cowpea plants were recovered from seeds originating on these chimeric branches as shown by Southern blot hybridization and GUS expression. Transgenic R2 soybean and lentil plants were also obtained. Segregation ratios in these populations showed a strong bias against transgene presence or expression.

Lecithin and lecithin/cholesterol liposomes formed in aqueous solutions of DNA entrap covalently closed circular, open circular and linear DNA molecules of size up to at least 13 kilobases. The sequestered DNA molecules are efficiently protected against exogenous deoxyribonuclease action although nicking and linearization of circular DNA can be observed. The size of these liposomes ranges from approximately 0.5 to 7.5 mu with an average of 2.5--4 mu. DNA filled liposomes strongly interact with plant protoplasts under conditions inducing protoplast fusion. Results suggest that sequestered plasmid DNA can be transferred to protoplast nuclei.

The Alcaligenes eutrophus JMP134 plasmid pJP4 contains genes necessary for the complete degradation of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoic acid. tfdA encodes 2,4-D monooxygenase, the initial enzyme in the 2,4-D catabolic pathway. The (fdA locus has recently been localized to a region on pJP4 13 kilobases away from a cluster of five genes, #'dB to ifdF, which encode the enzymes responsible for the further degradation of 2,4-D to chloromaleylacetic acid (W. R. Streber, K. N. Timmis, and M. H. Zenk, J. Bacteriol. 169:2950-2955. A second, dissimilar locus on pJP4, fdAII, has been observed which encodes 2,4-D monooxygenase activity. Gas chromatographic analysis of the 2,4-D metabolites of A. eutrophus harboring pJP4 or subclones thereof localized fd4AII to within a 9-kilobase SstI fragment of pJP4 which also carries the genes #fdBCDEF. This fragment was further characterized in Escherichia coli by deletion and subcloning analysis. A region of 2.5 kilobases, adjacent to afdC, enabled E. coli extracts to degrade 2,4-D to 2,4-dichlorophenol. Hybridization under low-stringency conditions was observed between tfdA and ifdAII, signifying that the 2,4-D monooxygenase gene was present as two related copies on pJP4.Halogenated aromatics have become increasingly prevalent in our environment as a result of industrial pollution and herbicide use in agriculture. Several genera of soil bacteria are known to degrade and utilize a variety of these compounds via the presence of partial or complete catabolic pathways encoded by large plasmids. The enzymes responsible for catalyzing the initial steps in the degradative pathways are of particular interest because of their potential use in the development of bacteria and plants capable of degrading and detoxifying xenobiotic compounds (10,17,20).The plasmid pJP4 of Alcaligenes eutrophus JMP134 encodes enzymes for the catabolism of 2,4-dichlorophenoxyacetic acid (2,4-D) and 3-chlorobenzoate (3-CBA) (5, 7). The first enzyme in the 2,4-D catabolic pathway cleaves the ether linkage of 2,4-D to produce glyoxylate and 2,4-dichlorophenol (2,4-DCP). Five genes, tfdB to tfdF, converting 2,4-DCP to chloromaleylacetic acid, are located on a 6.3-kilobase (kb) region of EcoRI fragment B (7) (Fig. 1). A gene encoding 2,4-D monooxygenase, tfdA, has been localized to a region 13 kb distant from the tfdBCDEF genes (19) (Fig. 1).When grown on 3-chlorobenzoate as a sole carbon source, pJP4 can undergo a wide variety of genetic rearrangements (10). This may reflect a capacity of the bacterium to adapt itself to grow more efficiently under different environmental conditions. Such rearrangements include deletions and duplications of specific regions of the plasmid. We have found a second locus which encodes an additional 2,4-D monooxygenase activity in pJP4. We have designated the enzyme 2,4-D monooxygenase II and the gene tfdAII. This locus was found adjacent to the gene encoding chlorocatechol 1,2-dioxygenase, tfdC. The tfdAII locus is clustered with genes tfdBCDEF on plasmid pJP4...

Transient expression and stable integration and expression of transgenes were observed in the tissues and offspring of certain leguminous plants after electroporation of DNA into intact nodal meristems in planta. The method described in this article thus allows the study of transgene expression in tissues differentiating from meristematic cells present in the treated buds. In addition, transgenic plants can be recovered in the offspring of electroporated individuals. Therefore, this technique allows the production of transgenic leguminous plants without the need for in vitro tissue culture, often a major hurdle with this family.

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