Highly efficient protoplast transformation system for Streptococcus faecalis and a new Escherichia coli-S. faecalis shuttle vector

Abstract

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 106 'transformants per ,ug of DNA were consistently obta,ined within 3 days, and cell wall regeneration of protoplasts was virtually 100%. A systematic search for useful vectors showed that the broad-host-range plasmid plP501 could transform S. faecalis at a high frequency (6.3 x 104 transformants per ,ug). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC1W4, we constructed a new E. coli-S. faecalis shuttle vector (pA,M401) having nine unique restriction sites. In a shotgun cloning experiment, we ligated a tetracycline resistance determinant from Streptococcus sanguis chromosomal DNA into pAM401 by direct transformation of S. faecalis, establishing the utility of the protoplast transformation system and of the new shuttle vector.Genetic analyses of Streptococcus faecalis are largely impaired by the lack of an efficient transformation system. While conjugation of plasmids is common in S. faecalis (5, 7), neither transduction nor naturally occurring transformation has been previously described. Since protoplasts of S.faecalis could be reversibly generated by various groups (15,18,23), attempts were undertaken in this laboratory to develop a protoplast transformation system in this species. A recent report by Smith (29) showed that protoplast transformation could be achieved; however, that system worked only at a low frequency and, in our hands, was often difficult to reproduce. The primary objective of the work presented here was to optimize a protoplast transformation system for S. faecalis to a point where its efficiency was high enough that genetic manipulations such as shotgun cloning of chromosomal DNA would be possible.By optimizing various parameters (e.g., growth medium, transformation medium, types of plasmids used, etc.), we have developed a system which reproducibly results in up to 106 regenerated transformants per pug of DNA within 48 h.In addition we constructed a new Escherichia coli-S. faecalis shuttle vector, pAM401, which has nine unique restriction sites. By direct transformation of S. faecalis protoplasts, a chromosomal tetracycline resistance determinant from Streptococcus sanguis was cloned into pAM401, thus demonstrating the general utility of this system. MATERIALS AND METHODSBacterial strains and plasmids. The bacterial strains and plasmids used in this study are listed in Table 1.Media. Unless otherwise noted, the growth medium for E. coli was L broth (26); for S. sanguis, S. faecalis, and Bacillus subtilis, we used brain heart infusion, Todd-Hewitt broth (THB), and Pennassay broth (all from Difco Laboratories), respectively. Other media tested were DM3 (4), nutrient broth no. 2 (Oxoid Ltd.), and methionine assay medium (Difco tetracycline, 10; chloramphenicol, 20; erythromycin, 20; and kanamycin was used at 50 ,ug/ml for E. coli and 500 ,ug/ml for S. faecalis. The concentra...

Pheromone cCF10 and plasmid pCF10‐encoded regulatory molecules act post‐transcriptionally to activate expression of downstream conjugation functions

“…faecalis shuttle vector (Wirth et al, 1986) pINY8101-1 EcoRI C fragment of pCF10 containing lacZ inserted in the Pst I site of prgB, and cloned in the EcoRV site of pWM402 (Chung, 1993) pINY6023 Sal I-XbaI fragment of pCF10 containing the negative control region, cloned in the shuttle vector pDL276 (Ruhfel et al, 1993) pMSP6023D Deletion of prgX and prgQ sequences from pINY6023 pMSP010 pINY8101-1 with a 1 kb BamHI fragment downstream of lacZ deleted pMSP025 prgXQSBlacZ pMSP026 prgXQSBlacZ with frameshift mutation in prgS pMSP028 prgXQBlacZ pMSP029 prgXQB inv lacZ with inverted 5Ј prgB fragment pMSP030 prgXQlacZ pMSP036 pMSP010 with the ribosome-binding site of prgQ abolished pMSP037 pMSP010 with a nonsense mutation at codon 5 of prgQ pMSP038 pMSP010 with a frameshift mutation at codon 11 of prgQ pMSP040 lacZ transcriptional fusion to the 11th codon of prgQ pMSP041 lacZ transcriptional fusion at the XbaI site in prgQ pMSP043 lacZ transcriptional fusion at the HpaI site, just downstream of IRS1 in prgQ pMSP045 lacZ translational fusion to the the first codon of prgS pMSP046 pMSP045 with a frameshift mutation at the 11th codon of prgQ pMSP047 lacZ translational fusion to the the 18th codon of prgS…”

Pheromone‐inducible expression of an aggregation protein in Enterococcus faecalis requires interaction of a plasmid‐encoded RNA with components of the ribosome

“…Genotype/ Description/ Source pWM402 E. coli-E. faecalis shuttle vector (Wirth et al, 1986 EcoRI C and E fragments of pCF10 cloned in the EcoRI site of pWM402 (Christie and Dunny, 1986) pINY4551…”

Genetic organization of the bacterial conjugative transposon Tn916

“…Blood plates contained 4% horse blood in Todd-Hewitt broth (Difco). Media and solutions used in protoplast transformation experiments were as described previously (21). Lambda::TnS stocks were propagated as described elsewhere (22).…”

“…Methods for isolation of plasmid DNA, restriction endonuclease digestion, ligation, agarose gel electrophoresis, nick translation of DNA with [32P]dATP, Southern blot hybridizations, and E. coli transformations were performed as described elsewhere (5,12,14,22). The protocol for protoplast transformation experiments was as described previously (21).…”

“…Using a bacteriophage lambda delivery system, we have recently been successful in generating TnS inserts on plasmid chimeras in E. coli (22). It was also possible to introduce some of the derivatives into S. faecalis via zygotically induced transposition (22) by a recently developed protoplast transformation protocol (21). This has opened the way for analyses of the genetic organization of Tn916; the results of such a study are presented here.…”