A Plasmid Which Can Be Transferred Between Escherichia coli and Pasteurella haemolytica by Electroporation and Conjugation

Abstract

Three broad-host-range plasmids (pRK290, pSa4 and pKT230) and one native Pasteurella haemolytica plasmid (pPH33) were used in transformation experiments with P. haemolytica strains T179 (serotype A1), Y216 (serotype A2) and its capsular-deficient variant Y216/NS1. No transformants were detected with either heat-shock or freeze-thaw techniques. However, by electroporation, all P. haemolytica strains were transformed by pPH33 but not by pRK290 or pSa4. The highest frequency obtained was 91 x 10(4) transformants per microgram of pPH33 DNA with P. haemolytica strain Y216/NS1. Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0.3-2.2 x 10(-3) per recipient cell.

Quantification of type VI secretion system activity in macrophages infected with Burkholderia cenocepacia

“…Transformation of E. coli DH5a and E. coli GT115 was done using the calcium chloride method (Cohen et al, 1972). Mobilization of complementing plasmids and mutagenesis plasmids into B. cenocepacia was performed by triparental mating using E. coli DH5a carrying the helper plasmid pRK2013 (Craig et al, 1989;Figurski & Helinski, 1979). DNA amplification by PCR was performed using a PTC-221 DNA engine (MJ Research) with Taq or HotStar HiFidelity DNA polymerases (Qiagen).…”

The suhB gene of Burkholderia cenocepacia is required for protein secretion, biofilm formation, motility and polymyxin B resistance

“…Restriction enzymes, T4 DNA ligase and alkaline phosphatase were obtained from Roche Diagnostics. Conjugations were performed by triparental mating (Craig et al, 1989) with the pRK2013 helper plasmid (Figurski & Helinski, 1979). DNA amplifications by PCR were done with the PTC-0200 or PTC-221 DNA engine (MJ Research) with Taq or HotStar HiFidelity DNA polymerases (Qiagen), and DNA sequencing was performed at the DNA Sequencing Facility, York University (Toronto, Canada).…”

A minor catalase/peroxidase from Burkholderia cenocepacia is required for normal aconitase activity

“…DNA transformations in E. coli were carried out using a calcium chloride protocol, as described elsewhere (Cohen et al, 1972). Transfer of plasmids into B. cepacia complex strains was conducted by tri-parental mating (Craig et al, 1989) using an E. coli helper strain carrying the plasmid pRK2013 (Table 1). DNA sequencing was done in the Mobix Sequencing Facility at the University of McMaster, Hamilton, Ontario, and the DNA Sequencing Facility at York University, Toronto, Ontario.…”