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Transcriptomic analysis reveals a global alkyl-quinolone-independent regulatory role for PqsE in facilitating the environmental adaptation of Pseudomonas aeruginosa to plant and animal hosts.

Rampioni G, Pustelny C, Fletcher MP, Wright VJ, Bruce M, Rumbaugh KP, Heeb S, Cámara M, Williams P - Environ. Microbiol. (2010)

Bottom Line: To gain insights into the relationship between the AQ stimulon, the PqsE stimulon and the regulatory function of PqsE, we constructed a pqsE inducible mutant (pqsEind) and compared the transcriptomes of the induced and uninduced states with a pqsA mutant.Furthermore, pqsE was required for swarming motility and virulence in plant and animal infection models in the absence of AQs, while mature biofilm development required both pqsA and pqsE.Taken together these data reveal that PqsE is a key regulator within the QS circuitry facilitating the environmental adaptation of P. aeruginosa.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD, UK.

ABSTRACT
The quorum sensing (QS) system of Pseudomonas aeruginosa constitutes a sophisticated genome-wide gene regulatory network employing both N-acylhomoserine lactone and 2-alkyl-4-quinolone (AQ) signal molecules. AQ signalling utilizes 2-heptyl-3-hydroxy-4-quinolone (PQS) and its immediate precursor, 2-heptyl-4-quinolone (HHQ). AQ biosynthesis requires the first four genes of the pqsABCDE operon and while the biochemical function of pqsE is not known, it is required for the production of secondary metabolites such as pyocyanin. To gain insights into the relationship between the AQ stimulon, the PqsE stimulon and the regulatory function of PqsE, we constructed a pqsE inducible mutant (pqsEind) and compared the transcriptomes of the induced and uninduced states with a pqsA mutant. Of 158 genes exhibiting altered expression in the pqsA mutant, 51% were also affected in the pqsE mutant. Following induction of pqsE, 237 genes were differentially expressed compared with the wild-type strain. In the pqsEind strain, pqsA was highly expressed but following induction both pqsA expression and AQ biosynthesis were repressed, revealing a negative autoregulatory role for PqsE. Furthermore, pqsE was required for swarming motility and virulence in plant and animal infection models in the absence of AQs, while mature biofilm development required both pqsA and pqsE. Taken together these data reveal that PqsE is a key regulator within the QS circuitry facilitating the environmental adaptation of P. aeruginosa.

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A. Schematic representation of the pqs locus in P. aeruginosa PAO1 wild type and the IPTG-inducible pqsE strain, pqsEind. The Ω interposon (SmR/SpcR) is from plasmid pHP45Ω: the lacIQ repressor is derived, together with the Ptac promoter, from plasmid pME6032. B. Activity of the PpqsA::lux promoter fusion. The activity of the PpqsA promoter was monitored during growth in PAO1 wild type, pqsEind, rhlR and pqsEind rhlR double mutants. The maximal expression levels reached during the late exponential phase of growth are shown. Where indicated (+), 1 mM IPTG was added to the growth medium. Error bars are calculated from three independent experiments. C. Concentration of HHQ (grey bars) and PQS (white bars) determined by LC-mass spectrometry in PAO1 wild type and the pqsEind mutant. The AQs were extracted from overnight cultures grown in LB broth; where indicated (+), 1 mM IPTG was added to the growth medium. The average of the results from three independent experiments is shown and error bars represent two standard deviations from the mean.
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fig01: A. Schematic representation of the pqs locus in P. aeruginosa PAO1 wild type and the IPTG-inducible pqsE strain, pqsEind. The Ω interposon (SmR/SpcR) is from plasmid pHP45Ω: the lacIQ repressor is derived, together with the Ptac promoter, from plasmid pME6032. B. Activity of the PpqsA::lux promoter fusion. The activity of the PpqsA promoter was monitored during growth in PAO1 wild type, pqsEind, rhlR and pqsEind rhlR double mutants. The maximal expression levels reached during the late exponential phase of growth are shown. Where indicated (+), 1 mM IPTG was added to the growth medium. Error bars are calculated from three independent experiments. C. Concentration of HHQ (grey bars) and PQS (white bars) determined by LC-mass spectrometry in PAO1 wild type and the pqsEind mutant. The AQs were extracted from overnight cultures grown in LB broth; where indicated (+), 1 mM IPTG was added to the growth medium. The average of the results from three independent experiments is shown and error bars represent two standard deviations from the mean.

Mentions: As pqsE influences the production of secondary metabolites such as pyocyanin and the rhamnolipids (Gallagher et al., 2002; Diggle et al., 2007; Farrow et al., 2008), it was not possible to determine whether the differentially regulated genes in the pqsA stimulon were affected as a consequence of a lack of AQs or PqsE. To address this question, we constructed a P. aeruginosa strain (pqsEind) in which the chromosomal pqsE gene was placed under the control of an IPTG-inducible promoter, such that the expression of pqsE is independent from the pqsABCD genes (Fig. 1A). The levels of pqsE expression in pqsEind and wild-type strains were compared by qRT-PCR analysis at an OD600 of 0.5 (early exponential phase) and 1.5 (late exponential phase). In both cases, when grown in the absence of IPTG the pqsEind strain expressed pqsE only at a basal level, while the provision of IPTG resulted in the premature induction and overexpression of pqsE with respect to the parental strain. pqsE transcription increased 9.6-fold at an OD600 0.5, and 18.1-fold at an OD600 1.5 with respect to the wild-type strain at the same OD600 (Appendix S1 and Fig. S2). The growth curve of each of the strains evaluated was identical with or without IPTG (data not shown).


Transcriptomic analysis reveals a global alkyl-quinolone-independent regulatory role for PqsE in facilitating the environmental adaptation of Pseudomonas aeruginosa to plant and animal hosts.

Rampioni G, Pustelny C, Fletcher MP, Wright VJ, Bruce M, Rumbaugh KP, Heeb S, Cámara M, Williams P - Environ. Microbiol. (2010)

A. Schematic representation of the pqs locus in P. aeruginosa PAO1 wild type and the IPTG-inducible pqsE strain, pqsEind. The Ω interposon (SmR/SpcR) is from plasmid pHP45Ω: the lacIQ repressor is derived, together with the Ptac promoter, from plasmid pME6032. B. Activity of the PpqsA::lux promoter fusion. The activity of the PpqsA promoter was monitored during growth in PAO1 wild type, pqsEind, rhlR and pqsEind rhlR double mutants. The maximal expression levels reached during the late exponential phase of growth are shown. Where indicated (+), 1 mM IPTG was added to the growth medium. Error bars are calculated from three independent experiments. C. Concentration of HHQ (grey bars) and PQS (white bars) determined by LC-mass spectrometry in PAO1 wild type and the pqsEind mutant. The AQs were extracted from overnight cultures grown in LB broth; where indicated (+), 1 mM IPTG was added to the growth medium. The average of the results from three independent experiments is shown and error bars represent two standard deviations from the mean.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2901523&req=5

fig01: A. Schematic representation of the pqs locus in P. aeruginosa PAO1 wild type and the IPTG-inducible pqsE strain, pqsEind. The Ω interposon (SmR/SpcR) is from plasmid pHP45Ω: the lacIQ repressor is derived, together with the Ptac promoter, from plasmid pME6032. B. Activity of the PpqsA::lux promoter fusion. The activity of the PpqsA promoter was monitored during growth in PAO1 wild type, pqsEind, rhlR and pqsEind rhlR double mutants. The maximal expression levels reached during the late exponential phase of growth are shown. Where indicated (+), 1 mM IPTG was added to the growth medium. Error bars are calculated from three independent experiments. C. Concentration of HHQ (grey bars) and PQS (white bars) determined by LC-mass spectrometry in PAO1 wild type and the pqsEind mutant. The AQs were extracted from overnight cultures grown in LB broth; where indicated (+), 1 mM IPTG was added to the growth medium. The average of the results from three independent experiments is shown and error bars represent two standard deviations from the mean.
Mentions: As pqsE influences the production of secondary metabolites such as pyocyanin and the rhamnolipids (Gallagher et al., 2002; Diggle et al., 2007; Farrow et al., 2008), it was not possible to determine whether the differentially regulated genes in the pqsA stimulon were affected as a consequence of a lack of AQs or PqsE. To address this question, we constructed a P. aeruginosa strain (pqsEind) in which the chromosomal pqsE gene was placed under the control of an IPTG-inducible promoter, such that the expression of pqsE is independent from the pqsABCD genes (Fig. 1A). The levels of pqsE expression in pqsEind and wild-type strains were compared by qRT-PCR analysis at an OD600 of 0.5 (early exponential phase) and 1.5 (late exponential phase). In both cases, when grown in the absence of IPTG the pqsEind strain expressed pqsE only at a basal level, while the provision of IPTG resulted in the premature induction and overexpression of pqsE with respect to the parental strain. pqsE transcription increased 9.6-fold at an OD600 0.5, and 18.1-fold at an OD600 1.5 with respect to the wild-type strain at the same OD600 (Appendix S1 and Fig. S2). The growth curve of each of the strains evaluated was identical with or without IPTG (data not shown).

Bottom Line: To gain insights into the relationship between the AQ stimulon, the PqsE stimulon and the regulatory function of PqsE, we constructed a pqsE inducible mutant (pqsEind) and compared the transcriptomes of the induced and uninduced states with a pqsA mutant.Furthermore, pqsE was required for swarming motility and virulence in plant and animal infection models in the absence of AQs, while mature biofilm development required both pqsA and pqsE.Taken together these data reveal that PqsE is a key regulator within the QS circuitry facilitating the environmental adaptation of P. aeruginosa.

View Article: PubMed Central - PubMed

Affiliation: School of Molecular Medical Sciences, Centre for Biomolecular Sciences, University Park, University of Nottingham, Nottingham NG7 2RD, UK.

ABSTRACT
The quorum sensing (QS) system of Pseudomonas aeruginosa constitutes a sophisticated genome-wide gene regulatory network employing both N-acylhomoserine lactone and 2-alkyl-4-quinolone (AQ) signal molecules. AQ signalling utilizes 2-heptyl-3-hydroxy-4-quinolone (PQS) and its immediate precursor, 2-heptyl-4-quinolone (HHQ). AQ biosynthesis requires the first four genes of the pqsABCDE operon and while the biochemical function of pqsE is not known, it is required for the production of secondary metabolites such as pyocyanin. To gain insights into the relationship between the AQ stimulon, the PqsE stimulon and the regulatory function of PqsE, we constructed a pqsE inducible mutant (pqsEind) and compared the transcriptomes of the induced and uninduced states with a pqsA mutant. Of 158 genes exhibiting altered expression in the pqsA mutant, 51% were also affected in the pqsE mutant. Following induction of pqsE, 237 genes were differentially expressed compared with the wild-type strain. In the pqsEind strain, pqsA was highly expressed but following induction both pqsA expression and AQ biosynthesis were repressed, revealing a negative autoregulatory role for PqsE. Furthermore, pqsE was required for swarming motility and virulence in plant and animal infection models in the absence of AQs, while mature biofilm development required both pqsA and pqsE. Taken together these data reveal that PqsE is a key regulator within the QS circuitry facilitating the environmental adaptation of P. aeruginosa.

Show MeSH
Related in: MedlinePlus