Evaluation and characterization of the predicted diguanylate cyclase-encoding genes in Pseudomonas aeruginosa.,MicrobiologyOpen

当前位置： X-MOL 学术 › Microbiologyopen › 论文详情

Our official English website, www.x-mol.net, welcomes your feedback! (Note: you will need to create a separate account there.)

Evaluation and characterization of the predicted diguanylate cyclase-encoding genes in Pseudomonas aeruginosa.
MicrobiologyOpen ( IF 3.9 ) Pub Date : 2020-02-03 , DOI: 10.1002/mbo3.975
Pramod Bhasme _{1,

2} , Qing Wei ₁ , Anming Xu _{1,

2} , Syed Tatheer Alam Naqvi ₁ , Di Wang ₁ , Luyan Z Ma _{1,

2}

Affiliation

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.

中文翻译：

铜绿假单胞菌中预测的双鸟苷酸环化酶编码基因的评估和表征。

机会病原体铜绿假单胞菌可引起人类急性和慢性感染。由于形成生物膜而对抗生素具有抗性，因此臭名昭著。Cyclic-di-GMP是细菌的第二信使，在生物膜发育过程中起着重要作用。铜绿假单胞菌中有40个基因预计会参与c-di-GMP的生物合成或降解。这些基因的功能表征非常耗时。在这里，我们从铜绿假单胞菌中克隆了16个基因预测可编码双鸟苷酸环化酶（DGC，负责c-di-GMP生物合成）的PAO1，并构建其相应的框内缺失突变体。我们评估了通过使用缺失突变体和包含分别表达16个基因之一的质粒的PAO1菌株来测量细胞内c-di-GMP浓度的方法。还检测并评估了所有PAO1衍生染色剂的功能输出，包括生物膜形成，胞外多糖的产生，游泳和成群的运动。我们的数据表明，通过使用pCdrA :: gfp测量c-di-GMP水平仅能表征少量DGC。作为报告者或LC / MS / MS。功能性输出结果表明，与相应的缺失突变体相比，DGC的过表达具有更明显的表型，并且表明游泳运动测定法可能是简要估算预测的DGC进行进一步研究的快速方法。总体评估表明，在16个预测的DGC中，有15个是功能DGC，其中6个以前被表征为编码DGC。总之，我们不仅提供了16个DGC编码基因及其对应的框内缺失突变体的克隆文库，而且还提供了简要表征预测DGC的方法。

更新日期：2020-02-03

点击分享查看原文

点击收藏

公开下载

阅读更多本刊最新论文本刊介绍/投稿指南11