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Poplar, a plant species frequently used for phytoremediation of groundwater contaminated with organic solvents, was inoculated with the endophyte Burkholderia cepacia VM1468. This strain, whose natural host is yellow lupine, contains the pTOM-Bu61 plasmid coding for constitutively expressed toluene degradation. Noninoculated plants or plants inoculated with the soil bacterium B. cepacia Bu61(pTOM-Bu61) were used as controls. Inoculation of poplar had a positive effect on plant growth in the presence of toluene and reduced the amount of toluene released via evapotranspiration. These effects were more dramatic for VM1468, the endophytic strain, than for Bu61. Remarkably, none of the strains became established at detectable levels in the endophytic community, but there was horizontal gene transfer of pTOM-Bu61 to different members of the endogenous endophytic community, both in the presence and in the absence of toluene. This work is the first report of in planta horizontal gene transfer among plant-associated endophytic bacteria and demonstrates that such transfer could be used to change natural endophytic microbial communities in order to improve the remediation of environmental insults.

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Two steps in the uptake of DNA by Diplococcus pneumoniae were characterized by analyzing mutants defective in transformation. A strain deficient in the two major deoxyribonucleases of D. pneumoniae takes up DNA normally and converts it to single strands within the cell and oligonucleotide fragments outside the cell. Extracts of this strain contain a residual deoxyribonuclease that produces similar oligonucleotide fragments in vitro. This enzyme is missing in transformationdefective mutants blocked in the second or entry step. Cells of this mutant class bind large amounts of DNA to their surface in a form accessible to external agents.Another class of nontransformable mutants fails to bind DNA at all. Their deoxyribonuclease content is unchanged, and they are apparently blocked in the first or binding step of DNA uptake. The binding step requires a source of energy and prior activation of the cells by competence factor. Entry may be independent of these requirements and may come about by action of the deoxyribonuclease on one strand of DNA with energy for the transport of the intact strand deriving from hydrolysis of the degraded strand. The enzyme may thus act as a DNA translocase.An early step in the genetic transformation of bacteria is the entry of donor DNA into the recipient cell. Prior to entry, DNA appears to be bound to the cell surface in a form sensitive to external agents (1-4). During entry, double-stranded donor DNA is converted to single strands in both Diplococcus pneumoniae (5, 6) and Bacillus subtilii (7,8). The concomitant appearance of donor DNA degradation products in pneumococcal cells suggested the role of a cellular DNase in the entry process (5). Degradation products also appear outside the cells, in proportion to the amount of DNA taken up (3, 9, 10).In a double mutant of D. pneumoniae, which is deficient in the two major DNase activities of the wild type, uptake of DNA and its fate after entry were normal (11). We set out to fractionate and characterize the residual DNase components in this strain, in particular to compare the products of the residual enzymes with the products formed external -to the cell during DNA uptake. We selected mutants of the strain that were more deficient in DNase and, also, mutants that were defective in transformation. Both kinds of mutant were examined with respect to DNase composition, genetic transformability, DNA uptake, and binding of DNA to the outside of the cell. MATERIALS AND METHODSBactrial Strains and Media. Strain designations do. not necessarily indicate genotype. R6endlexo2 and trtlhex4 both are end-i,exo-2 (9, 11). Moendlexo2 and T6trtlhex4 are maltose-negative derivatives. Moendlexo2hex3 is a hex mutant (11). Transformation-defective mutants were obtained in a parental strain after treatment with 1-methyl-3-nitro-1-nitrosoguanidine, and the mutations were transferred by transformation to another strain. A DNase plate assay (11) was used to select noz mutants that failed to form a colorless zone in agar containing DNA and methyl green ...

A chromosomal gene of Streptococcus pneumoniae carrying a spontaneous mutation to sulfonamide resistance was identified. Comparison of its DNA sequence with the wild-type sequence showed that the mutation, sul-d, consisted of an insert of 6 base pairs, a repeat of an adjacent 6-base-pair segment. The gene encoded a 34-kilodalton polypeptide, SulA, which as a dimer or trimer constituted the enzyme dihydropteroate synthase. This was shown by enzyme activity measurements, expression in minicells of Bacillus subtilis, and the amino-terminal sequence of the polypeptide product. Subcloning of the gene in an Eschenichia coli expression vector allowed purification of the enzyme to 80% homogeneity in a single step and at high yield. Although a deleted plasmid, pLS83, produced the mutant dihydropteroate synthase, it did not confer sulfonamide resistance in vivo. It is suggested that the SulA polypeptide is also a component of an enzyme that acts in another step of folate biosynthesis and that this step is inhibited in vivo by either free or conjugated sulfonamides.

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