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Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa
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...
Autores principales: | , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
John Wiley and Sons Inc.
2020
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066473/ https://www.ncbi.nlm.nih.gov/pubmed/32012489 http://dx.doi.org/10.1002/mbo3.975 |
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author | Bhasme, Pramod Wei, Qing Xu, Anming Naqvi, Syed Tatheer Alam Wang, Di Ma, Luyan Z. |
author_facet | Bhasme, Pramod Wei, Qing Xu, Anming Naqvi, Syed Tatheer Alam Wang, Di Ma, Luyan Z. |
author_sort | Bhasme, Pramod |
collection | PubMed |
description | 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. |
format | Online Article Text |
id | pubmed-7066473 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2020 |
publisher | John Wiley and Sons Inc. |
record_format | MEDLINE/PubMed |
spelling | pubmed-70664732020-03-18 Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa Bhasme, Pramod Wei, Qing Xu, Anming Naqvi, Syed Tatheer Alam Wang, Di Ma, Luyan Z. Microbiologyopen Original Articles 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. John Wiley and Sons Inc. 2020-02-03 /pmc/articles/PMC7066473/ /pubmed/32012489 http://dx.doi.org/10.1002/mbo3.975 Text en © 2019 The Authors. MicrobiologyOpen published by John Wiley & Sons Ltd. This is an open access article under the terms of the http://creativecommons.org/licenses/by-nc/4.0/ License, which permits use, distribution and reproduction in any medium, provided the original work is properly cited and is not used for commercial purposes. |
spellingShingle | Original Articles Bhasme, Pramod Wei, Qing Xu, Anming Naqvi, Syed Tatheer Alam Wang, Di Ma, Luyan Z. Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa |
title | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa
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title_full | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa
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title_fullStr | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa
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title_full_unstemmed | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa
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title_short | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa
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title_sort | evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in pseudomonas aeruginosa |
topic | Original Articles |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7066473/ https://www.ncbi.nlm.nih.gov/pubmed/32012489 http://dx.doi.org/10.1002/mbo3.975 |
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