Department of Microbiology, University College Cork, Ireland.
BMC Microbiol. 2010 Feb 11;10:45. doi: 10.1186/1471-2180-10-45.
Photorhabdus are Gram negative entomopathogenic bacteria that also have a mutualistic association with nematodes from the family Heterorhabditis. An essential part of this symbiosis is the ability of the bacterium to colonize the gut of the freeliving form of the nematode called the infective juvenile (IJ). Although the colonization process (also called transmission) has been described phenomonologically very little is known about the underlying molecular mechanisms. Therefore, in this study, we were interested in identifying genes in Photorhabdus that are important for IJ colonization.
In this work we genetically tagged P. luminescens TT01 with gfp and constructed a library containing over 3200 mutants using the suicide vector, pUT-Km2. Using a combination of in vitro symbiosis assays and fluorescent microscopy we screened this library for mutants that were affected in their ability to colonize the IJ i.e. with decreased transmission frequencies. In total 8 mutants were identified with transmission frequencies of <o= 30% compared to wild-type. These mutants were mapped to 6 different genetic loci; the pbgPE operon, galE, galU, proQ, asmA and hdfR. The pbgPE, galE and galU mutants were all predicted to be involved in LPS biosynthesis and, in support of this, we have shown that these mutants are avirulent and sensitive to the cationic antimicriobial peptide, polymyxin B. On the other hand the proQ, asmA and hdfR mutants were not affected in virulence and were either as resistant (proQ) or slightly more sensitive (asmA, hdfR) to polymyxin B than the wild-type (WT).
This is the first report describing the outcome of a comprehensive screen looking for transmission mutants in Photorhabdus. In total 6 genetic loci were identified and we present evidence that all of these loci are involved in the assembly and/or maintenance of LPS and other factors associated with the cell surface. Interestingly several, but not all, of the transmission mutants identified were also avirulent suggesting that there is a significant, but not complete, genetic overlap between pathogenicity and mutualism. Therefore, this study highlights the importance of the cell surface in mediating the symbiotic and pathogenic interactions of Photorhabdus.
Photorhabdus 是革兰氏阴性昆虫病原细菌，与异小杆线虫科的线虫也有共生关系。这种共生关系的一个重要组成部分是细菌能够定植自由生活形式的线虫，即感染性幼虫（IJ）。虽然已经从表型上描述了定植过程（也称为传播），但对于潜在的分子机制知之甚少。因此，在这项研究中，我们对鉴定 Photorhabdus 中对 IJ 定植很重要的基因感兴趣。
在这项工作中，我们用 gfp 标记了 P. luminescens TT01，并使用自杀载体 pUT-Km2 构建了一个包含超过 3200 个突变体的文库。我们结合体外共生实验和荧光显微镜，筛选了这个文库，以寻找在 IJ 定植能力方面受到影响的突变体，即传播频率降低的突变体。总共鉴定出 8 个突变体，其传播频率<o=30%，与野生型相比。这些突变体被映射到 6 个不同的遗传基因座；pbgPE 操纵子、galE、galU、proQ、asmA 和 hdfR。pbgPE、galE 和 galU 突变体均被预测参与 LPS 生物合成，并且我们已经证明这些突变体是无毒性的，并且对阳离子抗菌肽多粘菌素 B 敏感。另一方面，proQ、asmA 和 hdfR 突变体在毒力方面不受影响，并且对多粘菌素 B 的抗性（proQ）或略高于野生型（WT）（asmA、hdfR）。
这是首次报道描述全面筛选 Photorhabdus 中传播突变体的结果。总共鉴定了 6 个遗传基因座，我们提供的证据表明，这些基因座都参与了 LPS 和其他与细胞表面相关的因素的组装和/或维持。有趣的是，鉴定出的一些但不是所有的传播突变体也是无毒性的，这表明致病性和共生性之间存在显著但不完全的遗传重叠。因此，这项研究强调了细胞表面在介导 Photorhabdus 的共生和致病性相互作用中的重要性。
