One model of transformation proposed that heat shock destabilizes the cell membrane and allows for plasmid uptake. As lipid composition might also affect the stability of the cell membrane, we hypothesized that a lower growth temperature would alter membrane lipid composition so as to increase transformation efficiency after heat shock. Cells were grown at 20°C and 37°C and treated with calcium...

Lack of a system for genetic manipulation of Chlamydia trachomatis has been a key challenge to advancing understanding the molecular genetic basis of virulence for this bacterial pathogen. We developed a non-viral, dendrimer-enabled system for transformation of this organism and used it to characterize the effects of inserting the common 7.5 kbp chlamydial plasmid into strain L2(25667R), a C. t...

Several attempts to introduce pJH 1, an 80.7-kb R plasmid from Streptococcus faecalis strain JHI and coding for resistance to kanamycin, streptomycin, erythromycin, and tetracycline (Tc), into S. sanguis by transformation have failed. By using pDL3 10, a hybrid plasmid composed of a loto 12-kb DNA segment of pJH1 that includes the Tc resistance determinant, and intact pJH2, a second plasmid har...

The endogenous plasmid pRA2 from Pseudomonas alcaligenes NCIB 9867 was determined to have 32,743 bp with a G+C content of 59.8%. Sequence analysis predicted a total of 29 open reading frames, with approximately half of them contributing towards the functions of plasmid replication, mobilization, and stability. The Pac25I restriction-modification system and two mobile elements, Tn5563 and IS1633...

A stable shuttle vector which replicates in Escherichia coli and Clostridium perfringens was constructed by ligating a 3.6-kilobase (kb) fragment of plasmid pBR322 with C. perfringens plasmid pHB101 (3.1 kb). The marker for this shuttle plasmid originated from the 1.3-kb chloramphenicol resistance gene of plasmid pHR106. The resulting shuttle vector, designated pAK201, is 8 kb in size and codes...

Chromate resistance of Pseudomonas fluorescens LB300, isolated from chromium-contaminated sediment in the upper Hudson River, was found to be plasmid specified. Loss of the plasmid (pLHB1) by spontaneous segregation or mitomycin C curing resulted in a simultaneous loss of chromate resistance. Subsequent transformation of such strains with purified pLHB1 plasmid DNA resulted in a simultaneous re...

An efficient electrotransformation system for Streptococcus suis type 2 is described. It is demonstrated that vectors based on the broad-host-range plasmid pWVO1 replicate in S. suis type 2. Transformation efficiencies of about 10(7) transformants per micrograms of plasmid DNA could be obtained. Derivatives of plasmid pBR322 containing S. suis chromosomal DNA did not replicate but integrated in...

Electrotransformation of several strains of Clostridium thermocellum was achieved using plasmid pIKm1 with selection based on resistance to erythromycin and lincomycin. A custom-built pulse generator was used to apply a square 10-ms pulse to an electrotransformation cuvette consisting of a modified centrifuge tube. Transformation was verified by recovery of the shuttle plasmid pIKm1 from presum...