Growth inhibition of bacterial variants

Marraro, Robert V.; Pfister, Robert M.; Rheins, Melvin S.

doi:10.1139/m70-109

Cited by 2 publications

(3 citation statements)

References 10 publications

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“…An important observation made here and previously (27,29) is that the growth of S. faecalis protoplasts is inhibited in the presence of nonprotoplasted cells (i.e., rapidly arising colonies not derived from true protoplasts). Our results, based on plating of various dilutions of cells, indicate this inhibiting agent to be a substance excreted by nonprotoplasted cells.…”

Section: Methodssupporting

confidence: 62%

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

Wirth¹,

An²,

Clewell³

1986

J Bacteriol

248

140

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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...

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Section: Methodssupporting

confidence: 62%

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

Wirth¹,

An²,

Clewell³

1986

J Bacteriol

248

140

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“…The regeneration of normal cells from L-phase cells appeared to be slower in OG1-X backgrounds than in OG1-RF backgrounds. As has been reported (22), colonies arising from cells that retain some cell wall or whole cells of S. faecalis inhibited the formation of L-phase colonies nearby, placing an upper limit (dependent on the fraction of protoplasts in the culture) on the amount of a protoplast suspension that could be plated on a drug-free DM3 plate.…”

Section: Generation Of S Faecalis Protoplasts Og1-rf and Fa2-2 Cultmentioning

confidence: 64%

“…The generation and regeneration of protoplasts of S. faecalis were studied in detail by several groups (10,11,14,22). The results vary from strain to strain, but up to 90%o of the S. faecalis protoplasts regenerate on agar containing hypertonic concentrations of salt, sucrose, or a combination of the two (9,11,14).…”

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confidence: 99%

Transformation and fusion of Streptococcus faecalis protoplasts

Smith¹

1985

J Bacteriol

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Nonconjugative plasmids were transferred by protoplast fusion among Streptococcus faecalis strains and from Streptococcus sanguis to S. faecalis. S. faecalis protoplasts were also transformed with several different plasmids, including the Tn917 delivery vehicle pTV1. Transformation was reproducible, but low in frequency (10 6 transformants per viable protoplast). A new shuttle vector (pAM610), able to replicate in Escherichia coli and S. faecalis, was constructed and transformed into S. faecalis protoplasts. pAM610 was mobilized by the conjugative plasmid pAMP1 in matings among S. faecalis strains and from S. sanguis to S. faecalis. Chimeric derivatives of pAM610 were also transformed into S. faecalis. Genetic studies in Streptococcusfaecalis have been hampered by the lack of a reliable transformation system, despite efforts in this laboratory and elsewhere (6, 24). Since the first report (1) of the genetic transformation of Streptomyces sp. protoplasts in the presence of polyethylene glycol (PEG), similar procedures have been successfully applied to protoplasts of Bacillus subtilis (4) and many other species, including Streptococcus lactis (16). Transformation of Streptococcus faecium autoplasts has been reported (P. A. Carson and L. Daneo-Moore, Abstr. Annu. Meet. Am. Soc. Microbiol. 1983, H60, p. 60). Fusion of bacterial protoplasts in the presence of PEG was described originally for B. subtilis (25) and subsequently for many species, including S.

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Growth inhibition of bacterial variants

Cited by 2 publications

References 10 publications

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

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

Transformation and fusion of Streptococcus faecalis protoplasts

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