Development of a genomic site for gene integration and expression in Enterococcus faecalis.
Development of a genomic site for gene integration and expression in Enterococcus faecalis.
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DOI：
10.1016/j.mimet.2012.04.011
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发表时间：
2012-07
影响因子：
2.2
通讯作者：
Garsin, Danielle A.
中科院分区：
文献类型：
--
作者：
DebRoy, Sruti;van der Hoeven, Ransome;Singh, Kavindra V.;Gao, Peng;Harvey, Barrett R.;Murray, Barbara E.;Garsin, Danielle A.
粪肠球菌是一种革兰氏阳性条件致病菌，已成为医院感染的主要致病菌之一。通常情况下，作为胃肠道的居民，抗生素的广泛使用导致粪肠球菌菌株的增加，这些菌株对多种抗生素具有耐药性。这一点，再加上很容易与其他细菌交换抗生素决定因素的能力，使得某些粪肠球菌感染难以用药物治疗。粪肠球菌研究的遗传工具箱近年来有了很大的发展，但缺乏以非破坏性的方式稳定地以单拷贝方式引入基因以补充或表达非本土基因的方法学。在这项研究中，我们确定了粪肠球菌OG1RF基因组中的一个特定位置，该位置可以作为感兴趣基因的表达位置。这个位点在大多数已测序的粪肠球菌基因组中都很保守。还开发了一种载体，通过等位基因交换将基因整合到这个位置。使用这个系统，我们补充了eutV的框内缺失，证明了突变不会引起极性效应。我们还培育了一株稳定表达绿色荧光蛋白的粪肠球菌OG1RF菌株，在体外生长和对线虫和小鼠的致病性方面与亲本菌株相当。这种新方法的另一个主要优势是能够在不需要保持抗生素选择的情况下表达整合的基因，使其成为感染模型和共培养系统中基因功能研究的理想工具。
Enterococcus faecalis, a gram-positive opportunistic pathogen, has become one of the leading causes of nosocomial infections. Normally a resident of the gastrointestinal tract, extensive use of antibiotics has resulted in the rise of E. faecalis strains that are resistant to multiple antibiotics. This, compounded with the ability to easily exchange antibiotic determinants with other bacteria, has made certain E. faecalis infections difficult to treat medically. The genetic toolbox for the study of E. faecalis has expanded greatly in recent years, but has lacked methodology to stably introduce a gene in single copy in a non-disruptive manner for complementation or expression of non-native genes. In this study, we identified a specific site in the genome of E. faecalis OG1RF that can serve as an expression site for a gene of interest. This site is well conserved in most of the sequenced E. faecalis genomes. A vector has also been developed to integrate genes into this site by allelic exchange. Using this system, we complemented an in-frame deletion in eutV, demonstrating that the mutation does not cause polar effects. We also generated an E. faecalis OG1RF strain that stably expresses the green fluorescent protein and is comparable to the parent strain in terms of in vitro growth and pathogenicity in C. elegans and mice. Another major advantage of this new methodology is the ability to express integrated genes without the need for maintaining antibiotic selection, making this an ideal tool for functional studies of genes in infection models and co-culture systems.
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影响因子：
12.3
作者：
Bourgogne, Agathe;Garsin, Danielle A.;Qin, Xiang;Singh, Kavindra V.;Sillanpaa, Jouko;Yerrapragada, Shailaja;Ding, Yan;Dugan-Rocha, Shannon;Buhay, Christian;Shen, Hua;Chen, Guan;Williams, Gabrielle;Muzny, Donna;Maadani, Arash;Fox, Kristina A.;Gioia, Jason;Chen, Lei;Shang, Yue;Arias, Cesar A.;Nallapareddy, Sreedhar R.;Zhao, Meng;Prakash, Vittal P.;Chowdhury, Shahreen;Jiang, Huaiyang;Gibbs, Richard A.;Murray, Barbara E.;Highlander, Sarah K.;Weinstock, George M.
通讯作者：
Weinstock, George M.
影响因子：
3.2
作者：
Baker, Kris Ann;Perego, Marta
通讯作者：
Perego, Marta
影响因子：
3.2
作者：
Palmer, Kelli L.;Carniol, Karen;Gilmore, Michael S.
通讯作者：
Gilmore, Michael S.
影响因子：
6.4
作者：
Singh, KV;Qin, X;Murray, BE
通讯作者：
Murray, BE
影响因子：
4.4
作者：
DUNNY, GM;LEE, LN;LEBLANC, DJ
通讯作者：
LEBLANC, DJ