Alanine racemase mutants of Burkholderia pseudomallei and Burkholderia mallei and use of alanine racemase as a non-antibiotic-based selectable marker.
Alanine racemase mutants of Burkholderia pseudomallei and Burkholderia mallei and use of alanine racemase as a non-antibiotic-based selectable marker.
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DOI：
10.1371/journal.pone.0021523
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发表时间：
2011
期刊：
影响因子：
3.7
通讯作者：
Holmes RK
中科院分区：
文献类型：
--
作者：
Zajdowicz SL;Jones-Carson J;Vazquez-Torres A;Jobling MG;Gill RE;Holmes RK
类鼻疽伯克霍尔德菌和鼻疽伯克霍尔德菌是B类选择性病原体，必须在美国BSL 3控制下进行研究。它们通常对多种抗生素和用于治疗B的抗生素具有耐药性。假鼻疽或B。鼻疽菌感染不能用作相应伯克霍尔德菌属物种的选择性试剂。在这里，我们调查了丙氨酸消旋酶缺陷的突变体的B。类鼻疽和B.鼻疽用于开发基于非抗生素的遗传选择方法和用于减弱毒力。B的基因组。类鼻疽K96243具有两个注释的丙氨酸消旋酶基因（bps 12179和bps 0711），和B。鼻疽ATCC 23344有一个（bma 1575）。这些基因中的每一个编码一种功能酶，可以补充大肠杆菌菌株ALA 1的丙氨酸消旋酶缺陷。在这里，我们表明，B。在bps 12179和bps 0711中具有框内缺失的假鼻疽，或B。在BMA 1575中具有框内缺失的鼻疽菌需要外源D-丙氨酸来生长。将多拷贝质粒上的bpsl 2179引入任一伯克霍尔德氏菌属物种的丙氨酸消旋酶缺陷型变体中消除了对d-丙氨酸的需求。在没有d-丙氨酸的对数生长期，B的丙氨酸消旋酶缺陷突变体的活菌计数。类鼻疽和B.鼻疽在2小时内分别减少约1000倍和10倍，并且在24小时时不存在活细菌。我们构建了几种具有bpsl 2179作为选择性遗传标记的遗传工具，并且我们使用它们而没有任何抗生素选择来构建B的丙氨酸消旋酶缺陷变体中的符合读框的ΔflgK突变体。假鼻疽K96243。在小鼠腹腔巨噬细胞中，野生型B。鼻疽ATCC 23344比野生型B更快地被杀死。假鼻疽K96243。此外，还对B的丙氨酸消旋酶缺陷型突变体进行了研究。与其同基因亲本菌株相比，类鼻疽K96243在鼠腹腔巨噬细胞中的生长和存活表现出衰减。
Burkholderia pseudomallei and Burkholderia mallei are category B select agents and must be studied under BSL3 containment in the United States. They are typically resistant to multiple antibiotics, and the antibiotics used to treat B. pseudomallei or B. mallei infections may not be used as selective agents with the corresponding Burkholderia species. Here, we investigated alanine racemase deficient mutants of B. pseudomallei and B. mallei for development of non-antibiotic-based genetic selection methods and for attenuation of virulence. The genome of B. pseudomallei K96243 has two annotated alanine racemase genes (bpsl2179 and bpss0711), and B. mallei ATCC 23344 has one (bma1575). Each of these genes encodes a functional enzyme that can complement the alanine racemase deficiency of Escherichia coli strain ALA1. Herein, we show that B. pseudomallei with in-frame deletions in both bpsl2179 and bpss0711, or B. mallei with an in-frame deletion in bma1575, requires exogenous d-alanine for growth. Introduction of bpsl2179 on a multicopy plasmid into alanine racemase deficient variants of either Burkholderia species eliminated the requirement for d-alanine. During log phase growth without d-alanine, the viable counts of alanine racemase deficient mutants of B. pseudomallei and B. mallei decreased within 2 hours by about 1000-fold and 10-fold, respectively, and no viable bacteria were present at 24 hours. We constructed several genetic tools with bpsl2179 as a selectable genetic marker, and we used them without any antibiotic selection to construct an in-frame ΔflgK mutant in the alanine racemase deficient variant of B. pseudomallei K96243. In murine peritoneal macrophages, wild type B. mallei ATCC 23344 was killed much more rapidly than wild type B. pseudomallei K96243. In addition, the alanine racemase deficient mutant of B. pseudomallei K96243 exhibited attenuation versus its isogenic parental strain with respect to growth and survival in murine peritoneal macrophages.
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影响因子：
3.5
作者：
CHEREPANOV, PP;WACKERNAGEL, W
通讯作者：
WACKERNAGEL, W
影响因子：
3.2
作者：
DeShazer, D;Brett, PJ;Woods, DE
通讯作者：
Woods, DE
用于选择特工Burkholderia pseudomallei和Burkholderia Mallei的符合遗传操作的等位基因交换系统。
影响因子：
3.5
作者：
Hamad, Mohamad A.;Zajdowicz, Sheryl L.;Holmes, Randall K.;Voskuil, Martin I.
通讯作者：
Voskuil, Martin I.
影响因子：
3.8
作者：
Limmathurotsakul D;Chaowagul W;Chantratita N;Wuthiekanun V;Biaklang M;Tumapa S;White NJ;Day NP;Peacock SJ
通讯作者：
Peacock SJ
DOI：
10.1016/s0304-4165(96)00095-5
发表时间：
1997-03-15
影响因子：
3
作者：
Hamase, K;Homma, H;Imai, K
通讯作者：
Imai, K