Counterselection system employing mutated pheS for markerless genetic deletion in Bacteroides species

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URI http://shark.lib.kagawa-u.ac.jp/kuir/metadata/28766
Title
Counterselection system employing mutated pheS for markerless genetic deletion in Bacteroides species
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Description

Abstract

Markerless gene deletion is necessary for multiple gene disruptions due to the limited number of antibiotic resistant markers for some bacteria. However, even in transformable strains, obtaining the expected mutation without a marker requires laborious screening of a large number of colonies. Previous studies had success in various bacteria with a counter-selection system where a conditional lethal gene was incorporated into the vector. We examined the efficacy of the mutated pheS gene (pheS*) as a counter-selective marker for gene deletion in Bacteroides. This mutation produces an amino acid substitution (A303G) in the alpha subunit of Bacteroides phenylalanyl tRNA synthetase, which in E. coli alters the specificity of the tRNA synthetase resulting in a conditional lethal mutation due to the incorporation of p-chloro-phenylalanine (p-Cl-Phe) into protein. B. fragilis YCH46 and B. thetaiotaomicron VPI-5482 transformed with a pheS*-harboring shuttle vector were clearly growth-inhibited in the presence of >5 mM p-Cl-Phe in liquid defined minimal media (DMM) and on DMM agar plates. A targeting plasmid was constructed to delete the genetic region for capsular polysaccharide PS2 in B. fragilis or PS1 in B. thetaiotaomicron. After counterselection, p-Cl-Phe-resistant colonies were generated at a frequency of 8.1 × 10-3 for B. fragilis and 1.7 × 10-3 for B. thetaiotaomicron. Of the p-Cl-Phe-resistant colonies, 4.2% and 72% harbored the correct genetic deletion for B. fragilis and B. thetaiotaomicron, respectively. These results indicate that mutated pheS is a useful counter-selective gene to construct markerless genetic deletions in Bacteroides.

KEYWORDS:

Bacteroides; p-chloro-phenylalanine; pheS; tRNA

(医博乙289)

Author
著者 木納 康博
著者(ヨミ) キノウ ヤスヒロ
著者(別表記) Kino Yasuhiro
Publication Title
Anaerobe
Publication Title Alternative
Anaerobe.
Volume
42
Start Page
81
End Page
88
Publisher
Anaerobe Society of the Americas
Elsevier
Published Date
2016-09-14
ISSN
1075-9964
NCID
AA11101129
PMID
27639596
DOI
10.1016/j.anaerobe.2016.09.004
Resource Type
Thesis or Dissertation
Language
eng
Resource URL
https://doi.org/10.1016/j.anaerobe.2016.09.004
Rights
Copyright © 2016 Elsevier Ltd.
この博士論文の本文については、次のエルゼビアの著作権ポリシーの規定により公開しています。「博士論文の場合は、エンバーゴ期間に関係なく機関リポジトリに出版社版を公開することができます。」 The text of this doctoral dissertation is published according to the following Elsevier copyright policy. "In the case of doctoral dissertations, publishers can be published in institutional repositories regardless of the Embargo period."
"This is the Publisher's Version of the following article: Counterselection system employing mutated pheS for markerless genetic deletion in Bacteroides species; Anaerobe; Volume 42 (2016) Pages: 81-88; doi: 10.1016/j.anaerobe.2016.09.004, which has been published in final form at https://doi.org/10.1016/j.anaerobe.2016.09.004 . "
Text Version
ETD
Grant ID
博乙第289号
Grant Date
2019-07-17
Degree Name
博士(医学)
Grantor
香川大学
Set
香川大学
Copyright (C) 2009 Kagawa University All rights reserved.