Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa
Abstract 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...
| Main Authors: | , , , , , |
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| Format: | Article |
| Language: | English |
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Wiley
2020-03-01
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| Series: | MicrobiologyOpen |
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| Online Access: | https://doi.org/10.1002/mbo3.975 |
| _version_ | 1818176326608093184 |
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| author | Pramod Bhasme Qing Wei Anming Xu Syed Tatheer Alam Naqvi Di Wang Luyan Z. Ma |
| author_facet | Pramod Bhasme Qing Wei Anming Xu Syed Tatheer Alam Naqvi Di Wang Luyan Z. Ma |
| author_sort | Pramod Bhasme |
| collection | DOAJ |
| description | Abstract 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. |
| first_indexed | 2024-12-11T20:14:25Z |
| format | Article |
| id | doaj.art-ee5667d424f24138825f9a4b7b5d5797 |
| institution | Directory Open Access Journal |
| issn | 2045-8827 |
| language | English |
| last_indexed | 2024-12-11T20:14:25Z |
| publishDate | 2020-03-01 |
| publisher | Wiley |
| record_format | Article |
| series | MicrobiologyOpen |
| spelling | doaj.art-ee5667d424f24138825f9a4b7b5d57972022-12-22T00:52:13ZengWileyMicrobiologyOpen2045-88272020-03-0193n/an/a10.1002/mbo3.975Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosaPramod Bhasme0Qing Wei1Anming Xu2Syed Tatheer Alam Naqvi3Di Wang4Luyan Z. Ma5State Key Laboratory of Microbial Resources Institute of Microbiology Chinese Academy of Sciences Beijing ChinaState Key Laboratory of Microbial Resources Institute of Microbiology Chinese Academy of Sciences Beijing ChinaState Key Laboratory of Microbial Resources Institute of Microbiology Chinese Academy of Sciences Beijing ChinaState Key Laboratory of Microbial Resources Institute of Microbiology Chinese Academy of Sciences Beijing ChinaState Key Laboratory of Microbial Resources Institute of Microbiology Chinese Academy of Sciences Beijing ChinaState Key Laboratory of Microbial Resources Institute of Microbiology Chinese Academy of Sciences Beijing ChinaAbstract 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.https://doi.org/10.1002/mbo3.975Cyclic‐di‐GMPdiguanylate cyclasesmotilityPseudomonas aeruginosa |
| spellingShingle | Pramod Bhasme Qing Wei Anming Xu Syed Tatheer Alam Naqvi Di Wang Luyan Z. Ma Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa MicrobiologyOpen Cyclic‐di‐GMP diguanylate cyclases motility Pseudomonas aeruginosa |
| title | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa |
| title_full | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa |
| title_fullStr | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa |
| title_full_unstemmed | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa |
| title_short | Evaluation and characterization of the predicted diguanylate cyclase‐encoding genes in Pseudomonas aeruginosa |
| title_sort | evaluation and characterization of the predicted diguanylate cyclase encoding genes in pseudomonas aeruginosa |
| topic | Cyclic‐di‐GMP diguanylate cyclases motility Pseudomonas aeruginosa |
| url | https://doi.org/10.1002/mbo3.975 |
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