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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. Pyocyanin produced by PAO1 and both pqsEind and pqsA pqsEind mutants. Bacterial cultures were grown in LB broth (grey bars) or LB broth supplemented with 1 mM IPTG (white bars), and pyocyanin was extracted after 16 h growth (early stationary phase). B. Western blot analysis of Lectin A in cell extracts of PAO1 and both pqsEind and pqsA pqsEind mutants. Proteins were extracted from cultures grown for 16 h in LB broth to an OD600 of 2.5 (early stationary phase of growth), with (+) or without (−) 1 mM IPTG. An extract from P. aeruginosa PAO1 lecA mutant (lecA::lux) was used as a negative control. C. Swarming assays performed with PAO1 and both pqsEind and pqsA pqsEind mutants in the presence or absence of 1 fmM IPTG. D. Biofilm formation on stainless steel coupons by PAO1 and both pqsE and pqsA pqsEind mutants. A representative picture of the biofilm formed by each strain is also shown. For A and D, the average of the results from three independent experiments is reported with standard deviations.
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fig02: A. Pyocyanin produced by PAO1 and both pqsEind and pqsA pqsEind mutants. Bacterial cultures were grown in LB broth (grey bars) or LB broth supplemented with 1 mM IPTG (white bars), and pyocyanin was extracted after 16 h growth (early stationary phase). B. Western blot analysis of Lectin A in cell extracts of PAO1 and both pqsEind and pqsA pqsEind mutants. Proteins were extracted from cultures grown for 16 h in LB broth to an OD600 of 2.5 (early stationary phase of growth), with (+) or without (−) 1 mM IPTG. An extract from P. aeruginosa PAO1 lecA mutant (lecA::lux) was used as a negative control. C. Swarming assays performed with PAO1 and both pqsEind and pqsA pqsEind mutants in the presence or absence of 1 fmM IPTG. D. Biofilm formation on stainless steel coupons by PAO1 and both pqsE and pqsA pqsEind mutants. A representative picture of the biofilm formed by each strain is also shown. For A and D, the average of the results from three independent experiments is reported with standard deviations.

Mentions: Figure 2A and B shows that neither PAO1 pqsEind nor PAO1 pqsA pqsEind produce much pyocyanin or lectin when pqsE is not induced, while IPTG-dependent pqsE overexpression results in the production of almost 2.5 times the wild-type level of pyocyanin and substantially higher levels of Lectin A in both mutants, demonstrating that PqsE controls both virulence determinants in an AQ-independent manner. Despite the differential production of pyocyanin and Lectin A, the microarray data (Table 1) did not reveal major changes in the phz or lecA transcript levels in the pqsE-inducible strain compared with the wild type. This could either reflect a possible post-transcriptional regulatory role for PqsE or highlight an experimental limitation of the microarray technique employed.


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. Pyocyanin produced by PAO1 and both pqsEind and pqsA pqsEind mutants. Bacterial cultures were grown in LB broth (grey bars) or LB broth supplemented with 1 mM IPTG (white bars), and pyocyanin was extracted after 16 h growth (early stationary phase). B. Western blot analysis of Lectin A in cell extracts of PAO1 and both pqsEind and pqsA pqsEind mutants. Proteins were extracted from cultures grown for 16 h in LB broth to an OD600 of 2.5 (early stationary phase of growth), with (+) or without (−) 1 mM IPTG. An extract from P. aeruginosa PAO1 lecA mutant (lecA::lux) was used as a negative control. C. Swarming assays performed with PAO1 and both pqsEind and pqsA pqsEind mutants in the presence or absence of 1 fmM IPTG. D. Biofilm formation on stainless steel coupons by PAO1 and both pqsE and pqsA pqsEind mutants. A representative picture of the biofilm formed by each strain is also shown. For A and D, the average of the results from three independent experiments is reported with standard deviations.
© Copyright Policy - open-access
Related In: Results  -  Collection

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

fig02: A. Pyocyanin produced by PAO1 and both pqsEind and pqsA pqsEind mutants. Bacterial cultures were grown in LB broth (grey bars) or LB broth supplemented with 1 mM IPTG (white bars), and pyocyanin was extracted after 16 h growth (early stationary phase). B. Western blot analysis of Lectin A in cell extracts of PAO1 and both pqsEind and pqsA pqsEind mutants. Proteins were extracted from cultures grown for 16 h in LB broth to an OD600 of 2.5 (early stationary phase of growth), with (+) or without (−) 1 mM IPTG. An extract from P. aeruginosa PAO1 lecA mutant (lecA::lux) was used as a negative control. C. Swarming assays performed with PAO1 and both pqsEind and pqsA pqsEind mutants in the presence or absence of 1 fmM IPTG. D. Biofilm formation on stainless steel coupons by PAO1 and both pqsE and pqsA pqsEind mutants. A representative picture of the biofilm formed by each strain is also shown. For A and D, the average of the results from three independent experiments is reported with standard deviations.
Mentions: Figure 2A and B shows that neither PAO1 pqsEind nor PAO1 pqsA pqsEind produce much pyocyanin or lectin when pqsE is not induced, while IPTG-dependent pqsE overexpression results in the production of almost 2.5 times the wild-type level of pyocyanin and substantially higher levels of Lectin A in both mutants, demonstrating that PqsE controls both virulence determinants in an AQ-independent manner. Despite the differential production of pyocyanin and Lectin A, the microarray data (Table 1) did not reveal major changes in the phz or lecA transcript levels in the pqsE-inducible strain compared with the wild type. This could either reflect a possible post-transcriptional regulatory role for PqsE or highlight an experimental limitation of the microarray technique employed.

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