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PMID: 3005240 Published · ppublish English Journal Article Research Support, Non-U.S. Gov't Research Support, U.S. Gov't, P.H.S.

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

Journal of bacteriology ·Vol. 165 ·No. 3 ·1986-03-00 ·Pages 831-6

Wirth R, An FY, Clewell DB

Abstract

A highly efficient protoplast transformation system for Streptococcus faecalis has been developed by systematically optimizing different parameters. Up to 10(6) transformants per micrograms of DNA were consistently obtained 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 pIP501 could transform S. faecalis at a high frequency (6.3 X 10(4) transformants per microgram). By combining a high-copy-number derivative of pIP501, designated pGB354, with the Escherichia coli vector pACYC184, we constructed a new E. coli-S. faecalis shuttle vector (pAM401) 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.

MeSH Terms

Cloning, Molecular Culture Media DNA Restriction Enzymes Drug Resistance, Microbial Enterococcus faecalis/genetics,ultrastructure Escherichia coli/genetics Genetic Vectors Plasmids Polyethylene Glycols Protoplasts Streptococcus sanguis/drug effects,genetics Tetracycline/pharmacology Transformation, Bacterial

Chemicals

Culture Media Polyethylene Glycols DNA Restriction Enzymes Tetracycline

Authors & Affiliations

3 authors, click to expand affiliations / ORCID

Wirth R

An F Y

Clewell D B

References (24)

24 references, click to expand

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Article Info

Journal

Journal of bacteriology

Abbr.

J Bacteriol

ISSN

0021-9193

DOI

10.1128/jb.165.3.831-836.1986

Published

1986-03-00

Pages

831-6

Language

English

Region

United States

NLM ID

2985120R

PMCID

PMC214503

Subset

Grants

NIAID NIH HHS · AI10318 · United States

NIDCR NIH HHS · DE02731 · United States

NIGMS NIH HHS · GM33956 · United States

External Links

PubMed source DOI: 10.1128/jb.165.3.831-836.1986 PMC Full Text View journal details

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

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