Lyell Noreen L, Septer Alecia N, Dunn Anne K, Duckett Drew, Stoudenmire Julie L, Stabb Eric V
Department of Microbiology, University of Georgia, Athens, Georgia, USA.
Department of Microbiology and Plant Biology, University of Oklahoma, Norman, Oklahoma, USA.
Appl Environ Microbiol. 2017 Feb 15;83(5). doi: 10.1128/AEM.02470-16. Print 2017 Mar 1.
Libraries of defined mutants are valuable research tools but necessarily lack gene knockouts that are lethal under the conditions used in library construction. In this study, we augmented a mutant library generated on a rich medium (LBS, which contains [per liter] 10 g of tryptone, 5 g of yeast extract, 20 g of NaCl, and 50 mM Tris [pH 7.5]) by selecting transposon insertion mutants on supplemented LBS and screening for those unable to grow on LBS. We isolated strains with insertions in , (), , several heme biosynthesis genes, and , as well as a mutant disrupted 14 bp upstream of Mutants with insertions in or upstream of were recovered by addition of Mg to LBS, but their cell morphology and motility were affected. The mutant was more strongly affected and formed cells or chains of cells that appeared to wind back on themselves helically. Growth of mutants with insertions in , , or was recovered with -acetylglucosamine (NAG), d-alanine, or d-glutamate, respectively. We hypothesized that NAG, d-alanine, or d-glutamate might be available to in the light organ; however, none of these mutants colonized the host effectively. In contrast, and mutants, which are auxotrophic for δ-aminolevulinate (ALA), colonized at wild-type levels, although mutants later in the heme biosynthetic pathway were severely impaired or unable to colonize. Our findings parallel observations that legume hosts provide symbionts with ALA, but they contrast with virulence phenotypes of mutants in some pathogens. The results further inform our understanding of the symbiotic light organ environment. By supplementing a rich yeast-based medium, we were able to recover mutants with insertions in conditionally essential genes, and further characterization of these mutants provided new insights into this bacterium's symbiotic environment. Most notably, we show evidence that the squid host can provide with enough ALA to support its growth in the light organ, paralleling the finding that legumes provide ALA in symbiotic nodules. Taken together, our results show how a simple method of augmenting already rich media can expand the reach and utility of defined mutant libraries.
特定突变体文库是有价值的研究工具，但必然缺少在文库构建所用条件下致死的基因敲除突变体。在本研究中，我们通过在添加成分的LBS培养基（每升含有10 g胰蛋白胨、5 g酵母提取物、20 g NaCl和50 mM Tris [pH 7.5]）上选择转座子插入突变体并筛选那些在LBS上无法生长的突变体，扩充了在丰富培养基（LBS）上构建的突变体文库。我们分离出了在 、 （）、 、几个血红素生物合成基因以及 中发生插入的菌株，还有一个在 上游14 bp处被破坏的突变体。在 或其上游发生插入的突变体通过向LBS中添加Mg得以恢复，但它们的细胞形态和运动性受到了影响。 突变体受到的影响更强，形成了似乎呈螺旋状自我缠绕的细胞或细胞链。在 、 或 中发生插入的突变体分别通过添加N - 乙酰葡糖胺（NAG）、d - 丙氨酸或d - 谷氨酸得以恢复生长。我们推测NAG、d - 丙氨酸或d - 谷氨酸可能在发光器官中可供 利用；然而，这些突变体均未有效地定殖于宿主。相比之下，对δ - 氨基乙酰丙酸（ALA）营养缺陷的 和 突变体以野生型水平定殖，尽管血红素生物合成途径中靠后的突变体严重受损或无法定殖。我们的发现与豆科宿主为 共生体提供ALA的观察结果相似，但与一些病原体中 突变体的毒力表型形成对比。这些结果进一步加深了我们对共生发光器官环境的理解。通过补充基于酵母的丰富培养基，我们能够获得在条件必需基因中发生插入的 突变体，对这些突变体的进一步表征为这种细菌的共生环境提供了新的见解。最值得注意的是，我们证明了鱿鱼宿主能够为 提供足够多的ALA以支持其在发光器官中的生长，这与豆科植物在共生根瘤中为 提供ALA的发现相似。综上所述，我们的结果表明，一种扩充已丰富培养基的简单方法如何能够扩展特定突变体文库的范围和效用。
