RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing
ABSTRACTIn its canonical interpretation, quorum sensing (QS) allows single cells in a bacterial population to synchronize gene expression and hence perform specific tasks collectively once the quorum cell density is reached. However, growing evidence in different bacterial species indicates that con...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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American Society for Microbiology
2023-12-01
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| Series: | mBio |
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| Online Access: | https://journals.asm.org/doi/10.1128/mbio.02039-23 |
| _version_ | 1797378895076392960 |
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| author | Marta Mellini Morgana Letizia Lorenzo Caruso Alessandra Guiducci Carlo Meneghini Stephan Heeb Paul Williams Miguel Cámara Paolo Visca Francesco Imperi Livia Leoni Giordano Rampioni |
| author_facet | Marta Mellini Morgana Letizia Lorenzo Caruso Alessandra Guiducci Carlo Meneghini Stephan Heeb Paul Williams Miguel Cámara Paolo Visca Francesco Imperi Livia Leoni Giordano Rampioni |
| author_sort | Marta Mellini |
| collection | DOAJ |
| description | ABSTRACTIn its canonical interpretation, quorum sensing (QS) allows single cells in a bacterial population to synchronize gene expression and hence perform specific tasks collectively once the quorum cell density is reached. However, growing evidence in different bacterial species indicates that considerable cell-to-cell variation in the QS activation state occurs during growth, often resulting in coexisting subpopulations of cells in which QS is active (quorate cells) or inactive (non-quorate cells). Heterogeneity has been observed in the las QS system of the opportunistic pathogen Pseudomonas aeruginosa. However, the molecular mechanisms underlying this phenomenon have not yet been defined. The las QS system consists of an incoherent feedforward loop in which the LasR transcriptional regulator activates the expression of the lasI synthase gene and rsaL, coding for the lasI transcriptional repressor RsaL. Here, single-cell-level gene expression analyses performed in ad hoc engineered biosensor strains and deletion mutants revealed that direct binding of RsaL to the lasI promoter region increases heterogeneous activation of the las QS system. Experiments performed with a dual-fluorescence reporter system showed that the LasR-dependent expression of lasI and rsaL does not correlate in single cells, indicating that RsaL acts as a brake that stochastically limits the transition of non-quorate cells to the quorate state in a subpopulation of cells expressing high levels of this negative regulator. Interestingly, the rhl QS system that is not controlled by an analogous RsaL protein showed higher homogeneity with respect to the las system.IMPORTANCESingle-cell analyses can reveal that despite experiencing identical physico-chemical conditions, individual bacterial cells within a monoclonal population may exhibit variations in gene expression. Such phenotypic heterogeneity has been described for several aspects of bacterial physiology, including QS activation. This study demonstrates that the transition of non-quorate cells to the quorate state is a graded process that does not occur at a specific cell density and that subpopulations of non-quorate cells also persist at high cell density. Here, we provide a mechanistic explanation for this phenomenon, showing that a negative feedback regulatory loop integrated into the las system has a pivotal role in promoting cell-to-cell variation in the QS activation state and in limiting the transition of non-quorate cells to the quorate state in P. aeruginosa. |
| first_indexed | 2024-03-08T20:14:14Z |
| format | Article |
| id | doaj.art-97b69870ab3c44f5bdb2e0d71b6a23ff |
| institution | Directory Open Access Journal |
| issn | 2150-7511 |
| language | English |
| last_indexed | 2024-03-08T20:14:14Z |
| publishDate | 2023-12-01 |
| publisher | American Society for Microbiology |
| record_format | Article |
| series | mBio |
| spelling | doaj.art-97b69870ab3c44f5bdb2e0d71b6a23ff2023-12-22T19:53:43ZengAmerican Society for MicrobiologymBio2150-75112023-12-0114610.1128/mbio.02039-23RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensingMarta Mellini0Morgana Letizia1Lorenzo Caruso2Alessandra Guiducci3Carlo Meneghini4Stephan Heeb5Paul Williams6Miguel Cámara7Paolo Visca8Francesco Imperi9Livia Leoni10Giordano Rampioni11Department of Science, University Roma Tre, Rome, ItalyDepartment of Science, University Roma Tre, Rome, ItalyDepartment of Science, University Roma Tre, Rome, ItalyDepartment of Science, University Roma Tre, Rome, ItalyDepartment of Science, University Roma Tre, Rome, ItalyNational Biofilms Innovation Centre, Biodiscovery Institute and School of Life Sciences, University of Nottingham, Nottingham, United KingdomNational Biofilms Innovation Centre, Biodiscovery Institute and School of Life Sciences, University of Nottingham, Nottingham, United KingdomNational Biofilms Innovation Centre, Biodiscovery Institute and School of Life Sciences, University of Nottingham, Nottingham, United KingdomDepartment of Science, University Roma Tre, Rome, ItalyDepartment of Science, University Roma Tre, Rome, ItalyDepartment of Science, University Roma Tre, Rome, ItalyDepartment of Science, University Roma Tre, Rome, ItalyABSTRACTIn its canonical interpretation, quorum sensing (QS) allows single cells in a bacterial population to synchronize gene expression and hence perform specific tasks collectively once the quorum cell density is reached. However, growing evidence in different bacterial species indicates that considerable cell-to-cell variation in the QS activation state occurs during growth, often resulting in coexisting subpopulations of cells in which QS is active (quorate cells) or inactive (non-quorate cells). Heterogeneity has been observed in the las QS system of the opportunistic pathogen Pseudomonas aeruginosa. However, the molecular mechanisms underlying this phenomenon have not yet been defined. The las QS system consists of an incoherent feedforward loop in which the LasR transcriptional regulator activates the expression of the lasI synthase gene and rsaL, coding for the lasI transcriptional repressor RsaL. Here, single-cell-level gene expression analyses performed in ad hoc engineered biosensor strains and deletion mutants revealed that direct binding of RsaL to the lasI promoter region increases heterogeneous activation of the las QS system. Experiments performed with a dual-fluorescence reporter system showed that the LasR-dependent expression of lasI and rsaL does not correlate in single cells, indicating that RsaL acts as a brake that stochastically limits the transition of non-quorate cells to the quorate state in a subpopulation of cells expressing high levels of this negative regulator. Interestingly, the rhl QS system that is not controlled by an analogous RsaL protein showed higher homogeneity with respect to the las system.IMPORTANCESingle-cell analyses can reveal that despite experiencing identical physico-chemical conditions, individual bacterial cells within a monoclonal population may exhibit variations in gene expression. Such phenotypic heterogeneity has been described for several aspects of bacterial physiology, including QS activation. This study demonstrates that the transition of non-quorate cells to the quorate state is a graded process that does not occur at a specific cell density and that subpopulations of non-quorate cells also persist at high cell density. Here, we provide a mechanistic explanation for this phenomenon, showing that a negative feedback regulatory loop integrated into the las system has a pivotal role in promoting cell-to-cell variation in the QS activation state and in limiting the transition of non-quorate cells to the quorate state in P. aeruginosa.https://journals.asm.org/doi/10.1128/mbio.02039-23Pseudomonas aeruginosaquorum sensinggene regulationsingle cell analysisheterogeneityLasR |
| spellingShingle | Marta Mellini Morgana Letizia Lorenzo Caruso Alessandra Guiducci Carlo Meneghini Stephan Heeb Paul Williams Miguel Cámara Paolo Visca Francesco Imperi Livia Leoni Giordano Rampioni RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing mBio Pseudomonas aeruginosa quorum sensing gene regulation single cell analysis heterogeneity LasR |
| title | RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing |
| title_full | RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing |
| title_fullStr | RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing |
| title_full_unstemmed | RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing |
| title_short | RsaL-driven negative regulation promotes heterogeneity in Pseudomonas aeruginosa quorum sensing |
| title_sort | rsal driven negative regulation promotes heterogeneity in pseudomonas aeruginosa quorum sensing |
| topic | Pseudomonas aeruginosa quorum sensing gene regulation single cell analysis heterogeneity LasR |
| url | https://journals.asm.org/doi/10.1128/mbio.02039-23 |
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