Gene Duplication in Pseudomonas aeruginosa Improves Growth on Adenosine

Toussaint, Jean-Paul; Farrell-Sherman, Anna; Feldman, Tamar P.; Smalley, Nicole E.; Schaefer, Amy L.; Greenberg, E. Peter; Dandekar, Ajai A.

doi:10.1128/jb.00261-17

Cited by 18 publications

(25 citation statements)

References 65 publications

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“…One of these arose by spontaneous gene amplification encompassing gcoAB, a result that further emphasizes the importance of gene amplification. An appreciation for this process continues to grow as evolutionary outcomes are documented in diverse contexts (6,8,42,43). The EASy methodology both accelerates evolution in the laboratory and expands options for phenotypic selection.…”

Section: Discussionmentioning

confidence: 99%

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Tumen-Velasquez

Johnson

Ahmed

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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Experimental evolution is a critical tool in many disciplines, including metabolic engineering and synthetic biology. However, current methods rely on the chance occurrence of a key step that can dramatically accelerate evolution in natural systems, namely increased gene dosage. Our studies sought to induce the targeted amplification of chromosomal segments to facilitate rapid evolution. Since increased gene dosage confers novel phenotypes and genetic redundancy, we developed a method, Evolution by Amplification and Synthetic Biology (EASy), to create tandem arrays of chromosomal regions. In Acinetobacter baylyi, EASy was demonstrated on an important bioenergy problem, the catabolism of lignin-derived aromatic compounds. The initial focus on guaiacol (2-methoxyphenol), a common lignin degradation product, led to the discovery of Amycolatopsis genes (gcoAB) encoding a cytochrome P450 enzyme that converts guaiacol to catechol. However, chromosomal integration of gcoAB in Pseudomonas putida or A. baylyi did not enable guaiacol to be used as the sole carbon source despite catechol being a growth substrate. In ∼1,000 generations, EASy yielded alleles that in single chromosomal copy confer growth on guaiacol. Different variants emerged, including fusions between GcoA and CatA (catechol 1,2-dioxygenase). This study illustrates the power of harnessing chromosomal gene amplification to accelerate the evolution of desirable traits.

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Section: Discussionmentioning

confidence: 99%

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Tumen-Velasquez

Johnson

Ahmed

et al. 2018

Proc. Natl. Acad. Sci. U.S.A.

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“…DNA libraries were prepared by using a Nextera kit (Illumina), and DNA was sequenced with an Illumina MiSeq. Variant analysis was performed as previously described by using Strand NGS version 2.9 software (Strand Life Sciences, Bangalore, India) (27).…”

Section: Methodsmentioning

confidence: 99%

Modulation of Pseudomonas aeruginosa Quorum Sensing by Glutathione

et al. 2019

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Pseudomonas aeruginosa uses quorum sensing (QS) to regulate the production of a battery of secreted products. At least some of these products are shared among the population and serve as public goods. When P. aeruginosa is grown on casein as the sole carbon and energy source, the QS-induced extracellular protease elastase is required for growth. We isolated a P. aeruginosa variant, which showed increased production of QS-induced factors after repeated transfers in casein broth. This variant, P. aeruginosa QS*, had a mutation in the glutathione synthesis gene gshA. We describe several experiments that show a gshA coding variant and glutathione affect the QS response. The P. aeruginosa QS transcription factor LasR has a redox-sensitive cysteine (C79). We report that GshA variant cells with a LasR C79S substitution show a similar QS response to that of wild-type P. aeruginosa. Surprisingly, it is not LasR but the QS transcription factor RhlR that is more active in bacteria containing the variant gshA. Our results demonstrate that QS integrates information about cell density and the cellular redox state via glutathione levels. IMPORTANCE Pseudomonas aeruginosa and other bacteria coordinate group behaviors using a chemical communication system called quorum sensing (QS). The QS system of P. aeruginosa is complex, with several regulators and signals. We show that decreased levels of glutathione lead to increased gene activation in P. aeruginosa, which did not occur in a strain carrying the redox-insensitive variant of a transcription factor. The ability of P. aeruginosa QS transcription factors to integrate information about cell density and cellular redox state shows these transcription factors can fine-tune levels of the gene products they control in response to at least two types of signals or cues.

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“…We used Illumina MiSeq to complete whole-genome sequencing with paired 150-bp reads and StrandNGS version 3.1 (Strand Life Sciences) to align the reads to the published sequence of P. aeruginosa PAO1 (accession no. NC_002516) and to identify genetic polymorphisms including point mutations, deletions, insertions, and inversions (48). Genomic sequences have been deposited at NCBI (BioProject PRJNA514363).…”

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confidence: 99%

Evolution of the Pseudomonas aeruginosa quorum-sensing hierarchy

Kostylev

Kim

Smalley

et al. 2019

Proc. Natl. Acad. Sci. U.S.A.

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The bacterial pathogenPseudomonas aeruginosaactivates expression of many virulence genes in a cell density-dependent manner by using an intricate quorum-sensing (QS) network. QS inP. aeruginosainvolves two acyl-homoserine-lactone circuits, LasI-LasR and RhlI-RhlR. LasI-LasR is required to activate many genes including those coding for RhlI-RhlR.P. aeruginosacauses chronic infections in the lungs of people with cystic fibrosis (CF). In these infections, LasR mutants are common, butrhlR-rhlIexpression has escaped LasR regulation in many CF isolates. To better understand the evolutionary trajectory ofP. aeruginosaQS in chronic infections, we grew LasR mutants of the well-studiedP. aeruginosastrain, PAO1, in conditions that recapitulate an environment where QS signal synthesis by other bacteria might still occur. When QS is required for growth, addition of the RhlI product butyryl-homoserine lactone (C4-HSL), or bacteria that produce C4-HSL, to LasR mutants results in the rapid emergence of a population with a LasR-independent RhlI-RhlR QS system. These evolved populations exhibit subsequent growth without added C4-HSL. The variants that emerge have mutations inmexT, which codes for a transcription factor that controls expression of multiple genes. LasR-MexT mutants have a competitive advantage over both the parent LasR mutant and a LasR-MexT-RhlR mutant. Our findings suggest a plausible evolutionary trajectory for QS inP. aeruginosaCF infections where LasR mutants arise during infection, but because these mutants are surrounded by C4-HSL–producingP. aeruginosa,variants rewired to have a LasR-independent RhlIR system quickly emerge.

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Gene Duplication in Pseudomonas aeruginosa Improves Growth on Adenosine

Cited by 18 publications

References 65 publications

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Accelerating pathway evolution by increasing the gene dosage of chromosomal segments

Modulation of Pseudomonas aeruginosa Quorum Sensing by Glutathione

Evolution of the Pseudomonas aeruginosa quorum-sensing hierarchy

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