Natural transformation in Campylobacter species

Abstract

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 their own DNA in comparison with Escherichia coli chromosomal DNA, which was taken up very poorly. Three new Campylobacter spp.-to-E. coli shuttle plasmids, which contained additional cloning sites and selectable markers, were constructed from the shuttle vector pILL550A. These plasmid DNAs were taken up by campylobacters much less efficiently than was homologous chromosomal DNA, and transformation into plasmid-free cells was very rare. However, with the use of recipients containing a homologous plasmid, approximately 10(-4) transformants per cell were obtained. The tetM determinant, originally obtained from Streptococcus spp. and not heretofore reported in Campylobacter spp., was isolated from an E. coli plasmid and was introduced, selecting for tetracycline resistance, by natural transformation into C. coli.

Enterochelin acquisition in Campylobacter coli: characterization of components of a binding-protein-dependent transport system

“…T o clone the regions flanking the gene encoding the putative siderophorebinding protein a plasmid rescue method was used. The plasmid pSP120 was introduced into C. coli UA585 by natural transformation (Wang & Taylor, 1990) and tetracycline-resistant transformants were recovered. As the plasmid is not capable of extrachromosomal replication the only way for this antibiotic-resistant phenotype to arise is if the plasmid becomes integrated into the chromosome by a single point crossover driven by homologous recombination at the region of homology.…”

“…Grant & S. F. Park, unpublished data). In this study we have identified the homologue in C. coli UA585, which is naturally competent and amenable to genetic manipulation (Wang & Taylor, 1990). Plasmid rescue was used to clone the DNA sequences flanking the gene encoding this protein from this strain.…”

“…A 513 bp Sau3A fragment, internal to the fragment generated by PCR, was then cloned into the BamHI site of the integrational plasmid pSP105 (Dickinson et al, 1995) and the resulting plasmid was designated pSP120. This plasmid was introduced into C. coli UA585 by natural transformation (Wang & Taylor, 1990). The chromosomal DNA from one tetracycline-resistant transformant, designated CCHl , was analysed by Southern hybridization and the ceuE gene was shown to contain a single integrated copy of pSP120.…”

Identification of Campylobacter jejuni Genes Contributing to Acid Adaptation by Transcriptional Profiling and Genome-Wide Mutagenesis

“…Finally, the DNA was treated with 600 U of T4 DNA ligase (New England Biolabs) for 18 h at 16°C. The repaired transposed DNA was extracted with phenol-chloroform-isoamyl alcohol (25:24:1), pH 6.7, and ethanol precipitated prior to incubation with recipient C. jejuni NCTC 11168 cells and DNA uptake by natural transformation (89). Cells containing EZ-Tn5-Cm were selected by being plated on MH agar plates containing 20 g ml Ϫ1 Cm.…”

Inactivation of the LysR regulator Cj1000 of Campylobacter jejuni affects host colonization and respiration

“…C. jejuni F38011 was transformed with pGEMTcj1000aph by electroporation and the resulting transformants were selected on kanamycin plates and confirmed by PCR. Genomic DNA was extracted and used for natural transformation of C. jejuni NCTC11168 (Wang & Taylor, 1990b). The cj1000 : : aph mutant construction was confirmed by PCR and DNA sequencing.…”