Electrotransformation ofStreptococcus agalactiaewith plasmid DNA

Abstract

A protocol for efficient electrotransformation of Streptococcus agalactiae (group B streptococcus) Lancefield's strain O90R (NTCT 9993) (an unencapsulated derivative of type Ia strain O90) was developed. The Escherichia coli-Streptococcus shuttle vector pDP28 (7.8 kb) carrying the ermB gene for resistance to erythromycin was used as donor DNA. Frozen 'electrocompetent' cells were prepared by repeated washes in 10% glycerol. A 50-microliters aliquot containing about 5 x 10(9) colony forming units of bacteria was subjected to the electric pulse. Optimal conditions for electrotransformation were determined using different media, harvesting cells at different points of the growth curve, and using different field strengths. The dose-response curve for transformation of S. agalactiae with pDP28 showed one-hit kinetics as donor DNA varied between 0.01 and 3 micrograms. The efficiency of electrotransformation for this range of amounts of donor DNA was 1.2 x 10(4) cfu micrograms-1. The transformation frequencies obtained with this electroporation protocol are high enough to allow both subcloning and shotgun cloning of streptococcal DNA in S. agalactiae.

12/111phiA Prophage Domestication Is Associated with Autoaggregation and Increased Ability to Produce Biofilm in Streptococcus agalactiae

“…Genome comparison of 12/111∆relB-metK n • 2 and its complement 12/111∆relB-metK::relB-metK n • 2 (i.e., that restoring the phenotypes) revealed six mutations, three of which in an intergenic region and three synonymous. By contrast, for the pair of remaining mutant and complement (i.e., that not restoring the phenotypes), genome comparisons revealed mutations in intergenic regions, but also, non-synonymous mutations in the complement: one in an ORF encoding a putative Xylulose Kinase, and the second in an ORF encoding a putative Vex1 protein component of an ABC transporter ensuring export of Pep 27 , a peptide involved in growth inhibition, cell death and cell wall autolysis [52,53]. Taking into account the genes affected, it seems plausible that the secondary mutations identified in the genome of the complemented mutants may have hindered or disturbed restoration of phenotype complementation.…”

“…The mutants 12/111∆relB-yafQ, 12/111∆endonuclease-metK, 12/111∆endonuclease and 12/111∆metK were constructed according to the same protocol. The recombinant plasmids were transformed by electroporation into E. coli XL1 for amplification and purification as previously described [26,27] using the Micropulser (Bio-Rad, Hercules, CA, USA) and Ec2 conditions (2.5 kV) with 1 to 2 µg of the appropriate plasmids. The transformants were selected on LB agar with the appropriate antibiotics.…”

The Adc/Lmb System Mediates Zinc Acquisition in Streptococcus agalactiae and Contributes to Bacterial Growth and Survival

“…Electrocompetent E. coli and S. agalactiae cells were produced as previously described (36,37). Both bacterial species were then transformed by electroporation using the Micropulser (Bio-Rad) and the Ec2 conditions (2.5 kV) with 1 to 2 g of appropriate plasmids, respectively.…”

Characterization of a Two-component System in Streptococcus pyogenes Which Is Involved in Regulation of Hyaluronic Acid Production

“…The upstream and downstream fragments were cloned into the MCSI and MCSII sites of pFW6, respectively, and electroporated into streptococci using a method described previously (32). Transformants were selected on Todd Hewitt yeast blood plates with spectinomycin 100 g/ml.…”