Evaluation and characterization of the predicted diguanylate cyclase-encoding genes in Pseudomonas aeruginosa
Evaluation and characterization of the predicted diguanylate cyclase-encoding genes in Pseudomonas aeruginosa
复制标题
铜绿假单胞菌中预测的二鸟苷酸环化酶编码基因的评估和表征。
DOI：
10.1002/mbo3.975
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发表时间：
2020-02-03
期刊：
影响因子：
3.4
通讯作者：
Ma, Luyan Z.
中科院分区：
文献类型：
--
作者：
Bhasme, Pramod;Wei, Qing;Ma, Luyan Z.
机会致病菌铜绿假单胞菌可引起人类急性和慢性感染。它因形成生物膜而对抗生素产生耐药性而臭名昭著。环二gmp是细菌的第二信使，在生物膜发育过程中起重要作用。铜绿假单胞菌中有40个基因参与c-二gmp的生物合成或降解。这些基因的功能表征是非常耗时的。在这里，我们从P. aeruginosa PAO1中克隆了16个预计编码二胍酸环化酶（DGCs，负责c-di-GMP的生物合成）的基因，并构建了相应的框内缺失突变体。我们分别使用缺失突变体和含有表达16个基因之一的质粒的PAO1菌株来评估细胞内c-di-GMP浓度的测定方法。我们还检测和评估了所有pao1衍生染色剂的功能输出，包括生物膜的形成、胞外多糖的产生、游泳和群体运动。我们的数据表明，使用pCdrA::gfp作为报告因子或LC/MS/MS测量c-di-GMP水平仅表征少数DGC。功能输出结果表明，DGC的过表达比相应的缺失突变体产生更明显的表型，并表明游泳运动测定可以快速估计预测的DGC，以供进一步研究。总体评估表明，16个预测的DGCs中有15个是功能性DGCs，其中6个先前被表征为编码DGCs。总之，我们不仅提供了16个DGC编码基因及其相应的帧内缺失突变的克隆文库，而且为预测DGC的简要表征铺平了道路。
Opportunistic pathogen Pseudomonas aeruginosa can cause acute and chronic infections in humans. It is notorious for its resistance to antibiotics due to the formation of biofilms. Cyclic-di-GMP is a bacterial second messenger that plays important roles during biofilm development. There are 40 genes in P. aeruginosa predicted to participate in c-di-GMP biosynthesis or degradation. It is time-consuming for the functional characterization of these genes. Here, we cloned 16 genes from P. aeruginosa PAO1 that are predicted to encode diguanylate cyclases (DGCs, responsible for c-di-GMP biosynthesis) and constructed their corresponding in-frame deletion mutants. We evaluated the methods to measure the intracellular c-di-GMP concentration by using deletion mutants and PAO1 strains containing a plasmid expressing one of the 16 genes, respectively. Functional outputs of all PAO1-derived stains were also detected and evaluated, including biofilm formation, production of exopolysaccharide, swimming and swarming motilities. Our data showed that measuring the c-di-GMP level only characterized a few DGC by using either pCdrA::gfp as a reporter or LC/MS/MS. Functional output results indicated that overexpression of a DGC gave more pronounced phenotypes than the corresponding deletion mutant and suggested that the swimming motility assay could be a quick way to briefly estimate a predicted DGC for further studies. The overall evaluation suggested 15 out of 16 predicted DGCs were functional DGCs, wherein six were characterized to encode DGCs previously. Altogether, we have provided not only a cloning library of 16 DGC-encoding genes and their corresponding in-frame deletion mutants but also paved ways to briefly characterize a predicted DGC.