Consejo Superior de Investigaciones Científicas-EEZ, Department of Environmental Protection, 18008 Granada, Spain.
Environ Microbiol. 2013 Jan;15(1):36-48. doi: 10.1111/j.1462-2920.2012.02732.x. Epub 2012 Mar 28.
We used a combination of in silico and large-scale mutagenesis approaches to expand our current knowledge of the genetic determinants used by Pseudomonas putida KT2440 to attach to surfaces. We first identified in silico orthologues that have been annotated in Pseudomonas aeruginosa as potentially involved in attachment. In this search 67 paired-related genes of P. putida KT2440 and P. aeruginosa were identified as associated to adhesion. To test the potential role of the corresponding gene products in adhesion, 37 knockout mutants of KT2440, available in the Pseudomonas Reference Culture Collection, were analysed with regard to their ability to form biofilms in polystyrene microtitre plates; of these, six mutants were deficient in adhesion. Since mutants in all potential adhesion genes were not available, we generated a genome-wide collection of mutants made of 7684 independent mini-Tn5 insertions and tested them for the formation of biofilm on polystyrene microtitre plates. Eighteen clones that exhibited a reduction of at least twofold in biofilm biomass formation were considered candidate mutants in adhesion determinants. DNA sequencing of the insertion site identified five other new genes involved in adhesion. Phenotypic characterization of the mutants showed that 11 of the inactivated proteins were required for attachment to biotic surfaces too. This combined approach allowed us to identify new proteins with a role in P. putida adhesion, including the global regulator RpoN and GacS, PstS that corresponds to one of the paired-related genes for which a mutant was not available in the mutant collection, and a protein of unknown function (PP1633). The remaining mutants corresponded to functions known or predicted to participate in adhesion based on previous evidence, such as the large adhesion proteins LapA, LapF and flagellar proteins. In silico analysis showed this set of genes to be well conserved in all sequenced P. putida strains, and that at least eight reciprocal genes involved in attachment are shared by P. putida and P. aeruginosa.
我们采用了计算机模拟和大规模诱变方法，扩展了我们目前对 Pseudomonas putida KT2440 用于附着表面的遗传决定因素的认识。我们首先在计算机中识别出已在 Pseudomonas aeruginosa 中注释为可能参与附着的直系同源物。在这个搜索中，我们鉴定了 67 对 Pseudomonas putida KT2440 和 Pseudomonas aeruginosa 的相关基因，这些基因与粘附有关。为了测试相应基因产物在粘附中的潜在作用，我们分析了 Pseudomonas 参考培养物集中可获得的 37 个 KT2440 的敲除突变体，以确定它们在聚苯乙烯微量滴定板中形成生物膜的能力；其中，6 个突变体在粘附方面存在缺陷。由于并非所有潜在的粘附基因的突变体都可用，我们生成了一个由 7684 个独立的 mini-Tn5 插入物组成的全基因组突变体集合，并在聚苯乙烯微量滴定板上测试它们形成生物膜的能力。18 个克隆显示生物膜生物量形成减少至少两倍，被认为是粘附决定因素的候选突变体。插入位点的 DNA 测序确定了另外 5 个参与粘附的新基因。突变体的表型特征表明，11 个失活蛋白也需要附着到生物表面。这种组合方法使我们能够识别出在 P. putida 粘附中起作用的新蛋白，包括全局调节因子 RpoN 和 GacS、PstS，它对应于突变体集合中没有突变体的直系同源物之一，以及一个未知功能的蛋白（PP1633）。其余的突变体对应于以前的证据表明已知或预测参与粘附的功能，例如大粘附蛋白 LapA、LapF 和鞭毛蛋白。计算机模拟分析表明，这组基因在所有测序的 P. putida 菌株中都很好地保守，至少有 8 个参与附着的相互作用基因由 P. putida 和 P. aeruginosa 共享。
