Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis

Christopher J. Kristich; Josephine R. Chandler; Gary M Dunny

doi:10.1016/j.plasmid.2006.08.003

Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis

Christopher J. Kristich, Josephine R. Chandler, Gary M Dunny

Microbiology

Research output: Contribution to journal › Article › peer-review

140 Scopus citations

Abstract

Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and Δeep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.

Original language	English (US)
Pages (from-to)	131-144
Number of pages	14
Journal	Plasmid
Volume	57
Issue number	2
DOIs	https://doi.org/10.1016/j.plasmid.2006.08.003
State	Published - Mar 2007

Bibliographical note

Funding Information:
This work was supported by Grants AI58134 and GM49530 from the NIH to G.M.D. C.J.K. was supported by NRSA fellowship F32-AI56684 from the NIH. J.R.C. is a predoctoral trainee supported by Grant T32DE07288 from the NIH. We thank Tim Leonard for photography and technical advice, and Barry Wanner for bringing the work of Kast and colleagues on E. coli pheS to our attention.

Keywords

Counterselection
Enterococcus faecalis
Epistasis
Markerless exchange
Pheromone biosynthesis
p-chloro-phenylalanine
pheS

Access

10.1016/j.plasmid.2006.08.003

OpenUrl availability

Full text

Cite this

@article{12d238266c7f4b089e24fe55b69276b9,

title = "Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis",

abstract = "Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and Δeep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.",

keywords = "Counterselection, Enterococcus faecalis, Epistasis, Markerless exchange, Pheromone biosynthesis, p-chloro-phenylalanine, pheS",

author = "Kristich, {Christopher J.} and Chandler, {Josephine R.} and Dunny, {Gary M}",

note = "Funding Information: This work was supported by Grants AI58134 and GM49530 from the NIH to G.M.D. C.J.K. was supported by NRSA fellowship F32-AI56684 from the NIH. J.R.C. is a predoctoral trainee supported by Grant T32DE07288 from the NIH. We thank Tim Leonard for photography and technical advice, and Barry Wanner for bringing the work of Kast and colleagues on E. coli pheS to our attention.",

year = "2007",

month = mar,

doi = "10.1016/j.plasmid.2006.08.003",

language = "English (US)",

volume = "57",

pages = "131--144",

journal = "Plasmid",

issn = "0147-619X",

publisher = "Academic Press Inc.",

number = "2",

}

TY - JOUR

T1 - Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis

AU - Kristich, Christopher J.

AU - Chandler, Josephine R.

AU - Dunny, Gary M

N1 - Funding Information: This work was supported by Grants AI58134 and GM49530 from the NIH to G.M.D. C.J.K. was supported by NRSA fellowship F32-AI56684 from the NIH. J.R.C. is a predoctoral trainee supported by Grant T32DE07288 from the NIH. We thank Tim Leonard for photography and technical advice, and Barry Wanner for bringing the work of Kast and colleagues on E. coli pheS to our attention.

PY - 2007/3

Y1 - 2007/3

N2 - Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and Δeep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.

AB - Enterococcus faecalis is a Gram-positive commensal bacterium of the gastrointestinal tract. E. faecalis is also an opportunistic pathogen that frequently exhibits resistance to available antibiotics. Despite the clinical significance of the enterococci, genetic analysis has been restricted by limitations inherent in the available genetic tools. To facilitate genetic manipulation of E. faecalis, we developed a conjugative delivery system for high-frequency introduction of cloned DNA into target strains of E. faecalis and a host-genotype-independent counterselectable marker for use in markerless genetic exchange. We used these tools to construct a collection of E. faecalis mutant strains carrying defined mutations in several genes, including ccfA, eep, gelE, sprE, and an alternative sigma factor (sigH). Furthermore, we combined these mutations in various permutations to create double mutants, triple mutants, and a quadruple mutant of E. faecalis that enabled tests of epistasis to be conducted on the pheromone biosynthesis pathway. Analysis of cCF10 pheromone production by the mutants revealed that both the ccfA2 and Δeep10 mutations are epistatic to mutations in gelE/sprE. To our knowledge, this represents the first example of epistasis analysis applied to a chromosomally encoded biosynthetic pathway in enterococci. Thus, the advanced tools for genetic manipulation of E. faecalis reported here enable efficient and sophisticated genetic analysis of these important pathogens.

KW - Counterselection

KW - Enterococcus faecalis

KW - Epistasis

KW - Markerless exchange

KW - Pheromone biosynthesis

KW - p-chloro-phenylalanine

KW - pheS

UR - http://www.scopus.com/inward/record.url?scp=33846993832&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=33846993832&partnerID=8YFLogxK

U2 - 10.1016/j.plasmid.2006.08.003

DO - 10.1016/j.plasmid.2006.08.003

M3 - Article

C2 - 16996131

AN - SCOPUS:33846993832

SN - 0147-619X

VL - 57

SP - 131

EP - 144

JO - Plasmid

JF - Plasmid

IS - 2

ER -

Development of a host-genotype-independent counterselectable marker and a high-frequency conjugative delivery system and their use in genetic analysis of Enterococcus faecalis

Abstract

Bibliographical note

Keywords

Access

OpenUrl availability

Other files and links

Fingerprint

Cite this