Department of Microbiology, University of Colorado School of Medicine, Aurora, Colorado, United States of America.
PLoS One. 2011;6(6):e21523. doi: 10.1371/journal.pone.0021523. Epub 2011 Jun 24.
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.
伯克霍尔德氏菌和鼻疽伯克霍尔德氏菌是 B 类选择剂，在美国必须在 BSL3 控制下进行研究。它们通常对多种抗生素具有抗性，并且用于治疗伯克霍尔德氏菌或鼻疽伯克霍尔德氏菌感染的抗生素可能不能用作相应伯克霍尔德氏菌属的选择剂。在这里，我们研究了伯克霍尔德氏菌和鼻疽伯克霍尔德氏菌的丙氨酸消旋酶缺陷突变体，以开发非抗生素的遗传选择方法和降低毒力。伯克霍尔德氏菌 K96243 的基因组有两个注释的丙氨酸消旋酶基因（bpsl2179 和 bpss0711），鼻疽伯克霍尔德氏菌 ATCC 23344 有一个（bma1575）。这些基因中的每一个都编码一种功能酶，可以补充大肠杆菌菌株 ALA1 的丙氨酸消旋酶缺陷。在此，我们表明，伯克霍尔德氏菌中的 bpsl2179 和 bpss0711 均发生框内缺失，或鼻疽伯克霍尔德氏菌中的 bma1575 发生框内缺失，需要外源性 D-丙氨酸才能生长。将 bpsl2179 导入伯克霍尔德氏菌属的丙氨酸消旋酶缺陷变体中的多拷贝质粒中，消除了对 D-丙氨酸的需求。在没有 D-丙氨酸的对数生长期中，伯克霍尔德氏菌和鼻疽伯克霍尔德氏菌的丙氨酸消旋酶缺陷突变体的活菌数在 2 小时内分别减少了约 1000 倍和 10 倍，24 小时后没有存活的细菌。我们构建了几个带有 bpsl2179 作为可选择遗传标记的遗传工具，并且在没有任何抗生素选择的情况下使用它们，在伯克霍尔德氏菌 K96243 的丙氨酸消旋酶缺陷变体中构建了框内缺失 flgK 的突变体。在鼠腹膜巨噬细胞中，野生型鼻疽伯克霍尔德氏菌 ATCC 23344 的杀伤速度比野生型伯克霍尔德氏菌 K96243 快得多。此外，与同基因亲本菌株相比，伯克霍尔德氏菌 K96243 的丙氨酸消旋酶缺陷突变体在鼠腹膜巨噬细胞中的生长和存活方面表现出衰减。
