We demonstrated the genetic transformation of Rhizobium leguminosarum by R68.45 plasmid DNA by freezing and thawing cell suspensions in the presence of R68.45 plasmid DNA and 20 mM MgCl2. Clones resistant to kanamycin and tetracycline were recovered at a frequency of 10(-8) per recipient cell. No colonies that were doubly drug resistant were recovered in parallel control experiments.

We have extended the technique of PCR-directed recombination in Saccharomyces cerevisiae to develop a simple method for plasmid or gene construction in the absence of suitable restriction sites. The DNA to be cloned is PCR-amplified with 30-40 bp of homology to a linearized yeast plasmid. Co-transformation into yeast results in homologous recombination at a position directed by the PCR oligonuc...

A rapid technique for purifying plasmids from yeast Saccharomyces cerevisiae is described that yields high-quality DNA suitable for bacterial transformation, yeast transformation, and direct DNA sequencing. The method requires only small culture volumes and proprietary bacterial plasmid miniprep kits that allow one to simultaneously prepare a large number of samples in a very short period of ti...

Streptococcus pneumoniae is a human pathogen that is naturally transformable. In this study a major component of the homologous recombination pathway, the RexAB exonuclease/helicase, was characterized. rexA and rexB insertional mutants were constructed using mariner mutagenesis and found to have identical phenotypes. Both rexAB mutants displayed poor cell viability, reduced double-strand exonuc...

A recombinant plasmid was constructed by ligation of chromosomal DNA from a sulfanilamide-resistant strain of Bacillus subtilis to the plasmid vector pUB110 which specifies neomycin resistance. Recombinant molecules generated in vitro were introduced into a B. subtilis recipient strain which carried the recE4 mutation, and selection was for neomycin-sulfanilamide-resistant transformants. A sing...

Growing cells of Campylobacter coli and C. jejuni were naturally transformed by naked DNA without the requirement for any special treatment. Transformation frequencies for homologous chromosomal DNA were approximately 10(-3) transformants per recipient cell in C. coli and 10(-4) in C. jejuni. Maximum competence was found in the early log phase of growth. Campylobacters preferentially took up th...