Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment
Mutational activation of niche-specific genes provides insight into regulatory networks and bacterial function in a complex environment
复制标题
DOI：
10.1073/pnas.0706739104
复制
发表时间：
2007-11-13
影响因子：
11.1
通讯作者：
Rainey, Paul B.
中科院分区：
文献类型：
--
作者：
Giddens, Stephen R.;Jackson, Robert W.;Rainey, Paul B.
植物定植细菌荧光假单胞菌SBW25的基因组含有一组基因，这些基因专门在植物表面表达。这些基因的功能是SBW25生态成功的核心，但他们的研究提出了重大挑战，因为在体外没有表型可区分。在这里，我们描述了一种具有普遍实用价值的遗传策略，该策略将抑制子分析与IVET(SPyVET)相结合，并提供了一种识别利基特定基因的调节因子的方法。这一策略的核心是在植物环境诱导基因座(EIL)和无启动子‘dapB’之间携带操纵子融合的菌株。这些菌株在植物环境中是原养的，但在实验室最低限度的培养基上是营养缺陷型的。通过转座子诱变和在最低限度的基础上筛选原营养体来鉴定调控元件。对27株携带植物eIL的dapB融合的菌株中的每一株都进行了大约106个突变体的筛选，并在大约2,000个假定的调控突变体中确定了转座子的插入点。对调节物进行了功能表征，并用其提供了对EIL表型的洞察。对于一株携带与纤维素编码的wss操纵子融合的菌株，鉴定出五种不同的调节剂，包括二鸟苷环化酶、鞭毛激活剂FleQ和海藻酸盐激活剂AmrZ(AlgZ)。凭借SPyVET策略，进一步的抑制子分析揭示了另外两个调节器，包括激活因子ALGR，并允许确定监管联系。
The genome of the plant-colonizing bacterium Pseudomonas fluorescens SBW25 harbors a subset of genes that are expressed specifically on plant surfaces. The function of these genes is central to the ecological success of SBW25, but their study poses significant challenges because no phenotype is discernable in vitro. Here, we describe a genetic strategy with general utility that combines suppressor analysis with IVET (SPyVET) and provides a means of identifying regulators of niche-specific genes. Central to this strategy are strains carrying operon fusions between plant environment-induced loci (EIL) and promoterless 'dapB. These strains are prototrophic in the plant environment but auxotrophic on laboratory minimal medium. Regulatory elements were identified by transposon mutagenesis and selection for prototrophs on minimal medium. Approximately 106 mutants were screened for each of 27 strains carrying 'dapB fusions to plant EIL and the insertion point for the transposon determined in approximately 2,000 putative regulator mutants. Regulators were functionally characterized and used to provide insight into EIL phenotypes. For one strain carrying a fusion to the cellulose-encoding wss operon, five different regulators were identified including a diguanylate cyclase, the flagella activator, FleQ, and alginate activator, AmrZ (AlgZ). Further rounds of suppressor analysis, possible by virtue of the SPyVET strategy, revealed an additional two regulators including the activator AlgR, and allowed the regulatory connections to be determined.