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Identification of new regulators controlling the production of 2-alkyl-4-quinolone (AQ) signal molecules in Pseudomonas aeruginosa
The quorum sensing (QS) cell-to-cell communication system of Pseudomonas aeruginosa represents a sophisticated gene regulatory network which controls virulence factor production as well as biofilm formation in response to cell density. This network employs both N-acylhomoserine lactones (AHLs) and 2-alkyl-4-quinolones (AQs) as signal molecules. HHQ and PQS are the main AQ signals in P. aeruginosa. These molecules have been found in the lungs of cystic fibrosis patients, and their contribution to virulence has been demonstrated in different animal and plant infection models. The synthesis of both HHQ and PQS requires the enzymes encoded by the pqsABCDE operon. To find new regulators which control the transcription of this operon, and consequently AQs production, random transposon mutagenesis was performed on a P. aeruginosa PAO1 PpqsA::lux reporter strain. Out of 5344 mutants screened, 7 mutants showing altered pqsA expression have been identified and characterized. Moreover, a molecular approach aimed at identifying new regulators directly binding to the pqsA promoter region has been undertaken.
Members of the Pseudomonas genus are oxidase positive, aerobic, non-spore forming, Gram-negative bacilli. The first pseudomonad was named Bacillus pyocyaneus  because of its ability to produce the blue pigment pyocyanin but the name was later changed to Pseudomonas aeruginosa. Nowadays, over 100 species comprise the genus Pseudomonas but the most well studied is Pseudomonas aeruginosa due to its high emergence in clinical environments.
P. aeruginosa is a Gram-negative, aerobic rod belonging to the class of Gamma proteobacteria. It is usually seen singularly, in pairs or in short chains and it has unipolar motility . A culture of P. aeruginosa is characterised by a sweet grape-like odour and as it can secret a variety of pigments, a blue-green colour is a common characteristic when pyocyanin is present. P. aeruginosa is able to grow at 37°C as well as at 42oC. Nutritionally, the organism is versatile and it can utilise a wide range of compounds for growth. The genome size of P. aeruginosa is quite large when compared to other prokaryotic organisms being 6.3 million base pairs . This genetic complexicity is probably the reason why the organism can adapt in a wide range of ecological niches.
Given the fact that P. aeruginosa can adapt to many environments and produces a variety of virulence factors it is perhaps surprising it rarely causes disease in healthy individuals . However, when the immune system is compromised, the bacterium can infect almost any site of the body including the lungs, eyes, blood and skin. In susceptible hosts, such as cystic fibrosis patients , P. aeruginosa chronic infection is responsible for high rates of morbidity and mortality . The impressive number of virulence factors produced by P. aeruginosa is the main reason for the cause of severe bloodstream infections and extensive tissue damage. Virulence determinants such as pili, rhamnolipids and flagella transport bacteria in desirable environments whereas exotoxin A, exoenzyme S, elastase and pyocyanin are secreted in order to provide nutrients for the pathogen, enable colonisation, interrupt the immune system of the host and stimulate host damage. Finally, siderophores such as pyoverdine and pyochelin are important in the uptake of iron from the host [6-10]. Despite the production of virulence factors, P.aeruginosa establishes biofilms especially in chronic infected patients to protect itself by the response of the host's immune system or antibiotics . Moreover, three types of motility displayed by P. aeruginosa and named swimming, twitching and swarming allow the bacterium to colonize host cell surfaces. These phenotypes expressed by P. aeruginosa are controlled by a sophisticated signalling system known as quorum sensing (QS).
QS is a gene regulatory mechanism which enables bacteria to communicate and co-ordinate gene expression depending on cell density via extracellular signals. Small signals known as "autoinducers" are produced by bacteria and when the signals reach a crucial threshold concentration, QS target genes can be activated or repressed . Approximately, 10% of the P. aeruginosa genome is controlled by QS. This regulatory system involves two main classes of signal molecules, N-acyl homoserine lactones (AHLs) and 2-alkyl-4-quinolones (AQs).
P. aeruginosa has two AHLs-based QS systems: the las and the rhl systems. The transcriptional regulator LasR and the autoinducer N-3-oxo-dodecanoyl homoserine lactone (3-oxo-C12-HSL) comprise the las system, whereas the transcriptional activator RhlR and the autoinducer N-butanoyl homoserine lactone (C4-HSL) constitute the rhl system. The major autoinducers 3-oxo-C12-HSL and C4-HSL are synthesised by the enzymes LasI and RhlI, respectively. The las and rhl systems are organised in a hierarchic structure in which the LasR/3-oxo-C12-HSL complex drives the expression of lasI, generating a positive feedback loop, and also induces the transcription of rhlI and rhlR, placing itself over the rhl system in the AHL-dependent QS hierarchy .
The major AQs which function as QS signals in P. aeruginosa are 2-heptyl-3-hydroxy-4-quinolone, known as Pseudomonas Quinolone Signal (PQS), 2-heptyl-4-quinolone (HHQ) and 2-heptyl-4-hydroxyquinoline N-oxide (HHQ-NO). Multiple genes are required for AQs synthesis and signal transduction. Most of these are arranged in the pqsABCDE operon which is controlled by the transcriptional regulator PqsR (MvfR) . The first four genes of this operon direct HHQ synthesis which is then oxidized to PQS via the action of PqsH, encoded by the pqsH gene. The biosynthesis of AQs involves the "head-to-head" condensation of anthranilate and Î²-ketodecanoic acid . Anthranilate is supplied via phnAB operon or kynABU genes and is primed by PqsA for entry into the AQ biosynthetic pathway. PqsB and PqsC are likely to be involved in the synthesis of long chain fatty acid molecules whereas PqsD acts as a condensing enzyme which may catalyze the condensation of anthranoyl-CoA with 3-oxo-acid or be implicated in the formation of a 3-oxo-acid precursor .
A positive feedback loop occurs within the AQ signalling pathway as PQS and HHQ can bind to PqsR transcriptional regulator and drive the expression of the pqsA promoter so up-regulating pqsABCDE expression. The pqsE gene in the pqsABCDE operon is not essential for the biosynthesis of PQS and HHQ but it is required for the AQ response . The actual function of PqsE is not yet understood but pqsE mutants were shown to have low production of pyocyanin, rhamnolipid and elastase. On the other hand overexpression of PqsE increased the production of pyocyanin, rhamnolipid and elastase when PQS and PqsR were not present. A recent study examined the regulatory function of pqsE and the results showed that PqsE is a key regulator within the QS system as it can regulate pqsA expression and AQ production. More specifically, the activity of the pqsA promoter (PpqsA) was shown to be increased in the absence of pqsE and reduced when pqsE was overexpressed .
PQS has also been demonstrated to act as an iron chelator , a pro-oxidant and as an inducer of the anti-oxidative stress response. It is also required for vesicle formation and biofilm maturation and it has been involved in an autolytic process at high cell population densities, which enables bacteria to use the released DNA as a component of the biofilm matrix . Mutants impaired in PQS production or more general in QS signalling exhibit less virulence in all animal models tested up to now and this correlates with the fact that QS is active during P. aeruginosa infections . Because of this reason, QS has been proposed as a promising drug target as by blocking QS system virulence of bacterial pathogens could be restricted.
The purpose of this study was to identify new regulators which control the production of AQs. This aim has been achieved through genetic and molecular approaches, like a random transposon mutagenesis and a promoter pull-down experiment. These analyses contributed to clarify the molecular mechanisms underlying the complex regulation of the QS circuit in P. aeruginosa, and provided new potential targets for the rational design of anti-Pseudomonas therapeutic agents.
MATERIALS AND METHODS
Bacterial strains, plasmids and growth conditions
Bacterial strains and plasmids used in this study are listed in Table 1. Luria-Bertani (LB) broth was routinely used for the growth of E. coli and P. aeruginosa. The strains were grown at 37oC with shaking at 200 r.p.m unless otherwise stated. The final concentration of antibiotics was: tetracycline (Tc) 200 Î¼g/ml (P. aeruginosa) or 10 Î¼g/ml (E. coli); gentamicin (Gm) 25 Î¼g/ml (P. aeruginosa) or 20 Î¼g/ml (E. coli). In order to select P. aeruginosa from E. coli after conjugation experiments, nalidixic acid (Nal) 15 Î¼g/ml was also added to LB agar plates. For long-term storage of bacterial strains, 400 Î¼l of a 50 % (v/v) glycerol sterile solution were added to 1 ml of the overnight bacterial culture. The resulting suspension of bacteria and glycerol was stored at -80o.
Table 1 - Bacterial strains and plasmids used in this study
Donor strain for the conjugative transfer of plasmids.
Simon et al. (1983)
Grant et al. (1990)
pqsA chromosomal deletion mutant derived from PAO1.
Reporter strain carrying the chromosomal insertion PpqsA::lux derived from PAO1 âˆ†pqsA.
PAO1 derivative in which pqsE expression is under the control of a Ptac promoter.
Aendekerk et al. (2005)
Fletcher et al., (2007)
Rampioni et al. (2010)
Vector which contains a Tn5 transposon for random mutagenesis in P.aeruginosa, GmR .
Preparation of E. coli S17.1ï¬pir electrocompetent cells
To prepare electrocompetent E. coli S17.1ï¬pir cells, 2 ml of an overnight culture grown in Nutrient Yeast Broth (NYB) at 37oC were diluted in 200 ml of fresh NYB in a 1 L flask and incubated at 37oC with shaking at 200 r.p.m. for 6 hrs. Cells were then centrifuged at 4oC, 10,000 r.p.m. for 5 min and washed twice with cold Transformation Buffer [10 % (vol/vol) glycerol, 1 mM 3-(N-morpholino)propanesulfonic acid, pH 7.2] before being resuspended in 1 ml of Transformation Buffer. E. coli S17.1ï¬pir cells were then flash-frozen in liquid nitrogen and stored at -80oC.
Random transposon mutagenesis
Random transposon mutagenesis was performed on the reporter strain P. aeruginosa PpqsA::lux. The suicide vector pLM1 was conjugated from the E. coli S17.1ï¬pir donor strain to the recipient strain P. aeruginosa PpqsA::lux. P. aeruginosa PpqsA::lux was grown overnight in LB medium at 42oC in order to repress the expression of restriction-modification systems that could decrease the conjugation efficiency. E. coli S17.1ï¬pir pLM1 was grown overnight in LB medium supplemented with Gm 20 Î¼g/ml at 37oC. 1 ml of each overnight culture were centrifuged at 4,000 r.p.m. for 5 min and washed twice in 1 ml of Phosphate Buffered Saline (PBS; 137 mM NaCl, 2.7 mM KCl, 8.1 mM Na2HPO4, 1.9 mM KH2PO4, pH 7.4). Pellets from each strain were then resuspended in 500 Î¼l of PBS and conjugation was achieved by mixing 500 Î¼l of donor cells with 500 Î¼l of recipient cells in a sterile tube. The mixed bacteria were again centrifuged and the remaining pellet was spotted onto an LB agar plate and incubated for 6 hrs at 37oC to allow mating to occur. Cells from the plate were then resuspended in PBS and plated onto LB agar plates supplemented with Gm 25 Î¼g/ml and Nal 15 Î¼g/ml in order to select only the P. aeruginosa PpqsA::lux mutants carrying the chromosomal inserted transposon.
Determination of cell density and bioluminescence
Colonies of mutants generated by random transposon mutagenesis were streaked on fresh LB agar plates supplemented with Gm 25 Î¼g/ml and Nal 15 Î¼g/ml and screened for altered bioluminescence emission compared to the wild type reporter strain with a photon-imaging camera. Mutants displaying increased or decreased bioluminescence were further analyzed by measuring their bioluminescence during the growth curve with an automated luminometer/spectrometer TECAN. Overnight LB cultures of P. aeruginosa mutants were diluted 1:500 in fresh LB broth and 300 Î¼l of the diluted cultures were grown in triplicate at 37oC in microtitre plates. Bacterial cell density and bioluminescence were measured every 30 min for 24 hrs as a function of optical density at 600 nm wavelength (OD600) and Relative Light Units (RLU), respectively.
Identification of the transposon insertion sites
In order to sequence the mutated genes, chromosomal DNA was extracted from the mutant strains using the Wizard Genomic DNA Purification Kit (Promega, UK) according to manufacturer's instruction. 5 Î¼l of the chromosomal DNA were digested with the restriction enzymes BamHI, NheI, PvuII, and XmaI at 37oC overnight. The restriction reactions were pulled in one tube and cleaned by phenol-chloroform-isoamyl alcohol extraction. The digested DNA in the aqueous phase was precipitated by adding 0.1 vol of NaOAc 3M and 3 vol of 100 % cold ethanol. DNA was then stored at -20oC for 2 hrs and centrifuged. The DNA pellet was washed with 70 % cold ethanol, air-dried, and resuspended in 17 Î¼l of H2O. Lastly, ligation reactions were carried out at 4oC for 24 hrs by adding to the DNA solution 1 Î¼l of T4 DNA Ligase (Promega, UK) and 2 Î¼l of T4 Ligation Buffer (Promega, UK).
5 Î¼l of the ligation mixture were mixed with 50 Î¼l of the E. coli S17.1ï¬pir electrocompetent cells and transferred in an electrode gap Gene Pulser cuvettes (Promega, UK). The electroporation pulse was set at 2.5 kV and the electroporation was delivered using the BioRad Gene Pulsar. 900 Î¼l of LB broth were then added to the electroporated cells. After 1 h of incubation at 37oC, cells were plated onto LB agar plates containing Gm 20 Î¼l/ml and incubated overnight at 37oC.
Plasmid DNA was extracted from the resulting transformed cells by using the QIAprep Spin Miniprep kit (Qiagen, UK), according to manufacturer's instruction. To identify the site of insertion of the transposons, the purified plasmids were sequenced with primers pLM1FW and pLM1RV (see Table 2) at the Genomic Sequencing Unit, Queen's Medical Centre, Nottingham, UK.
Table 2 - Oligonucleotide primers used in this study.
PpqsA FW Biotin
5' - TATGAATTCTGCAAATGGCAGGCGAGG - 3'
5' - [ B i o t i n ] TGCAAATGGCAGGCGAGG - 3'
5' - TATAAGCTTTGGACATGACAGAACGTTC - 3'
5' - AACAAGCCAGGGATGTAACG - 3'
5' - CAGCAACACCTTCTTCACGA - 3'
1 FW = Forward Primer
RV = Reverse Primer.
Pyocyanin levels were measured using an assay based on the method published by Essar et al. (1990). Optical density of the overnight bacterial cultures was initially measured at 600 nm wavelength. Pyocyanin was extracted from 5 ml of the same culture with 3 ml of chloroform. The chloroform fraction was then mixed with 2 ml of 0.2 N HCl, and the absorbance at 520 nm wavelength of the HCl solution was measured.
Swarming motility assay
Swarming motility was tested on Swarming Plates [0.5 % (wt/vol) Bacto agar (Difco), 0.8 % (wt/vol) Nutrient broth No.2 (Oxoid), 0.5 % (wt/vol) glucose]. 3 Î¼l of the tested bacterial cultures grown to stationary phase were spotted on the surface of the Swarming Plates and incubated overnight at 37oC.
HHQ and PQS extraction and quantification
The strains under investigation were grown overnight in 10 ml of LB broth at 37oC. AQs were extracted from 1 ml of the culture supernatant with 3 ml of acidified ethyl acetate, vortexed and centrifuged at 9000 rpm for 5 min. The organic phase was then transferred in a clean glass tube, dried and resuspended in 40 Î¼l of acidified ethyl acetate. PQS and HHQ levels in the extracts were determined by measuring with an automated luminometer/spectrometer TECAN light emission of the P. aeruginosa PAO1 âˆ†pqsA PpqsA::lux reporter strain incubated with 2 Î¼l of the extracts. As controls, 2 Î¼l of acidified ethyl acetate, 2 Î¼l of synthetic HHQ (60 ïM), or 2 Î¼l of synthetic PQS (60 ïM) were used.
Promoter pull-down experiment
Promoter templates were generated by PCR with the PpqsA FW primer labeled with biotin and the PpqsA RV listed in Table 2. Proteins were extracted from 400 ml of the PAO1 strain, of the PAO1 pqsE inducible strain and PAO1 pqsE inducible grown in presence of IPTG to an OD600 of 1.5. Lysis Buffer [100 mM NaCl, 10 mM Tris-HCl pH 8.0, 1 mM EDTA, 0.05 % (vol/vol) Triton X100] was prepared and used to wash bacterial pellets. After the addition of Lysozyme and the incubation of samples in 37oC for 30 min, cells were sonicated and centrifuged multiple times. Streptavidin coated paramagnetic beads (Dynabeads, Invitrogen) were washed with Wash Buffer (2 M NaCl, 10 mM Tris pH 7.5, 1 mM EDTA) and incubated on the magnetic rack for 2 min. This step was repeated for 3 times and before the incubation of the crude extracts with the Dynabeads, the protein extracts were supplemented with 1 mM PMSF and 50 Î¼g/ml salmon sperm aspecific DNA. PCR products were mixed with Streptavidin beads and incubated with the crude extract. Non-DNA binding proteins were removed with the magnetic separation. Dynabeads were again collected and washed with Lysis Buffer six times and then eluted with Elution Buffer [1.2 M NaCl, 10 mM Tris-HCl, pH 8.0, 1 mM EDTA, 0.05 % (vol/vol) Triton X100]. The eluted proteins were separated by SDS-PAGE, and selected bands were identified by MALDI-TOF analysis.
Generation of the promoter::mCherry reporter plasmids
In order to assess gene regulation at single cell level, the pqsA promoter region was cloned in the mini-CTXmCherry plasmid fused with the reporter gene mCherry. A fragment encompassing the pqsA promoter region was amplified with primers PpqsA FW and PpqsA RV (listed in Table 2). The PCR fragment was digested with EcoRI and HindIII and cloned into similarly digested mini-CTXmCherry, resulting in mini-CTX PpqsA::mCherry. This construct has been verified by restriction analysis and sequencing.
Identification of new genes involved in the regulation of PpqsA
To identify genes which regulate PpqsA activity, transposon mutagenesis was performed. Mutants carrying the PpqsA::lux fusion were screened for their light emission. The mutants were generated by conjugating the reporter strain PAO1 PpqsA::lux with E. coli S17.1Î»pir (pLM1). The pLM1 plasmid cannot be replicated in P. aeruginosa and it contains the Tn5 transposon. Tn5 is usually chosen because it can insert with little target sequence specificity and transposes with high affinity in Gram-negative bacteria. In this case it inserts into P. aeruginosa and produces mutants which were selected on LB agar plates supplemented with 25 Î¼g/ml gentamicin (Gm) and 15 Î¼l/ml nalidixic acid (Nal) because of the transposon resistance to gentamicin.
Multiple conjugations resulted in the generation of 5344 mutants which were streaked on fresh LB plates and screened for any alterations in their luminescence. 133 mutants displayed an increased or decreased luminescence when they were compared to the wild type reporter strain and these were assayed with the use of a TECAN luminometer measuring both luminescence (relative light units - RLU) and cell density (OD600). Any difference in the luminescence displayed by each mutant in the previous screening could be the result of a different growth on solid media, so luminescence was determined as a function of cell density in liquid media. Out of the 133 mutants further investigated, only 8 mutants showed a significant alteration of PpqsA expression in liquid media. Maximal promoter activity for these strains was initially calculated and plotted. As shown in Figure 1, some of the mutants had significant difference in maximal promoter activity when compared to the wild type strain. Specifically, mutant 59 displayed almost two times higher maximal promoter activity than the wild type, whereas PpqsA activity was completely abrogated in mutant 74. Results were further analysed by plotting RLU/OD600 against time. As shown in Figure 2, some mutants displayed different timing of PpqsA expression, so they produced a different pattern of promoter activation. The growth of each mutant can affect their bioluminescence profile, so growth curves were plotted and are reported in Figure 3.
Figure 1 - Maximal PpqsA promoter activity in P. aeruginosa wild type (wt) and selected mutant strains. Error bars represent mean SD from three independent experiments.
Figure 2 - PpqsA promoter activity during the growth curve of P. aeruginosa wild type (wt) and selected mutant strains. Each one of these graphs shows the expression of the parent strain PAO1 PpqsA::lux wild type and a selection of mutants. Error bars are calculated from three independent measurements.
Figure 3 -Growth curve of P. aeruginosa wild type (wt) and the tested mutant strains.
The transposon insertion site was mapped in the strains showing an altered PpqsA activity. Chromosomal DNA was extracted from the 8 mutants and then restricted using the enzymes BamHI, NheI, PvuII, and XmaI. The restriction reactions were pulled in one tube and the digested DNA was ligated and transformed in E. coli S17.1 Î»pir. The resulted plasmids were sequenced with primers pLM1 FW and pLM1 RV (Table 2). This allowed the identification of the gene disrupted by the transposon insertion for only 7 mutants, as the sequencing process for mutant 13 failed. The results are summarized in Table 3.
Probable TonB-dependent receptor
Amino acid biosynthesis and metabolism
Nucleotide biosynthesis and metabolism
Nucleotide biosynthesis and metabolism
DNA replication, recombination, modification and repair.
Table 3 - Identification of genes disrupted by the Tn5 transposon insertion in the different mutants. PA number, gene name, insertion site and proposed function are derived from the Pseudomonas Genome Project (www.pseudomonas.com).
Phenotypic characterization of selected mutants
Since pqsA is the first gene of the pqsABCDE operon, involved in the biosynthesis of HHQ and PQS, these two QS signal molecules were extracted from the mutant strains and quantified. As shown in Figure 4, HHQ and PQS levels are reduced in mutants 22, 47 and 74 with respect to the wild type strain, while the production of these molecules is increased of ~ 30 % in mutant 28. Interestingly, HHQ and PQS production in the selected mutants do not clearly correlated with the activity of PpqsA previously measured. For example, mutant 59 showed the higher PpqsA activity in the TECAN analysis, but it produces the same amount of HHQ and PQS measured in the wild type strain.
Since HHQ and PQS control the expression of important virulence-related phenotypes such as pyocyanin production and swarming motility, these two phenotypes were investigated in the selected mutant strains.
Pyocyanin levels measured in each mutant are reported in Figure 5. Some mutants, like 9 and 13, overproduced pyocyanin, while pyocyanin production was reduced in mutants 22, 47, and 59, and was completely abrogated in mutant 28 and 74. The decreased production of pyocyanin in mutant 74 was in line with the repression of PpqsA activity and HHQ and PQS production observed in this strain.
For what concerns swarming ability, as shown in Figure 6 mutants 13, 28, 47 and 59 were strongly impaired in this kind of surface motility, while mutants 22 and 74 displayed a swarming ability slightly decreased with respect to the wild type strain. Conversely, swarming was not affected in mutants 9 and 56. It was interesting to notice that mutant 74 overproduced pyocyanin with respect to the parental strain when grown on swarming plate, while this mutant was impaired in pyocyanin production in liquid media (see Figures 5 and 6).
Figure 4 - Activation level of the HHQ and PQS reporter strain when grown in presence of AQs extracted from the supernatants of P. aeruginosa wild type (wt) and selected mutant strains.
Figure 5 - Pyocyanin produced by P. aeruginosa wild type (wt) and selected mutants.
Figure 6 - Swarming motility of P. aeruginosa wild type and selected mutants.
Identification of regulatory factors directly controlling PpqsA transcription
To identify regulators which can bind directly to the pqsA promoter, a promoter pull-down experiment was performed. Promoter pull-down experiments involve interactions between a target promoter region labeled with biotin and crude cell extracts of the strain of interest. With the use of Streptavidin-coated beads, which display high affinity for biotin, the promoter DNA region can be easily recovered together with the regulators directly attached to it.
In this case the promoter region of pqsA was amplified by PCR and then incubated with crude extracts derived from the P. aeruginosa wild type strain or the P. aeruginosa pqsEind strain grown in presence or absence of IPTG. The latter strain behaves as a pqsE mutant when grown in LB, and as a PqsE over-expressing strain when IPTG is added to the media. These conditions were chosen since the activity of PpqsA has been previously demonstrated to increase in the absence of PqsE, and to be completely abrogated when PqsE is over-expressed. Streptavidin beads were used to recover the DNA fragment encompassing the pqsA promoter, and the transcriptional regulators directly bound to it were separated by SDS-PAGE. As shown in Figure 7, at least 5 proteins are bound to the pqsA promoter in the wild type condition, while the gel pattern is drastically altered when the same promoter is incubated with extracts from the PAO1 pqsEind strain. As expected, in the latter case the addition of IPTG to the media results in different proteins bound to the pqsA promoter region. The identification of selected bands by MALDI-TOF analysis is now in course.
1 2 3 4
Figure 7 - SDS-PAGE analysis of the proteins purified in the promoter pull-down experiment. The promoter region encompassing the pqsA promoter was incubated with crude extracts from P. aeruginosa PAO1 wild type (lane 2), P. aeruginosa PAO1 pqsEind (lane 3), or P. aeruginosa PAO1 pqsEind grown in presence of IPTG (lane 4). Lane 1, Prestained Broad Range Protein Ladder (NewEngland Biolabs).
Generation of the miniCTX-PpqsA::mCherry plasmid
In order to better characterize PpqsA activity in P. aeruginosa, the miniCTX-PpqsA::mCherry plasmid was generated. This plasmid will allow the insertion of the PpqsA::mCherry fusion in the chromosome of P. aeruginosa in single copy. The use of mCherry as a reporter gene instead of lux will result in a more accurate analysis of PpqsA expression, since fluorescence does not relies on the energetic status of the cells and it allows to monitor promoter activity at the single cell level.
P. aeruginosa employs a sophisticated co-operative behaviour known as quorum sensing (QS) which operates to assist environmental adaptation at cell population level. To further understand the regulatory mechanism which controls the production of the AQs signal molecules HHQ and PQS in P. aeruginosa, a transposon mutagenesis approach was performed to identify new regulators of the pqsABCDE operon.
The complexicity of the regulation of AQs synthesis is given by the impressive number of genes found to alter the transcription of the pqsA promoter (PpqsA) in this study. Out of only 5344 mutants screened in this study, corresponding to a genome coverage of ~ 60 %, 8 mutants were shown to affect the expression of PpqsA. This result, also considering that it is likely that the same insertion mutant has been considered more then one time in the first screening step, suggests that more then ten genes can be involved in PpqsA regulation.
Among the 8 mutants identified in this study to affect PpqsA expression, the transposon insertion site has been determined in 7 cases.
Mutant 9 has a mutation in the pfuA gene, a probable TonB-dependent receptor. The TonB complex can sense signals outside of the cell and it can transmit them in a way which leads to transcriptional activation of genes. The involvement of TonB in iron transport is interesting considering that PQS is not only a QS signal molecule, but it is also an iron chelator . This finding highlights how AQs production and iron uptake are interwoven in P. aeruginosa.
Mutant 22 has a mutation in the algC gene, which is involved in alginate production as well as in LPS synthesis. One of the factors which enable P. aeruginosa to persist in the lungs of CF patients is the alginate production . Alginate is also associated with biofilm development however it has been suggested that alginate is most likely an exopolysaccharide which is produced by the bacterium under stress conditions.
In mutant 28 the transposon was inserted in the carA gene, which encodes the alpha subunit of carbamoylphosphate synthetase involved in nucleotide biosynthesis. Carbamoylphosphate synthetase has been previously demonstrated to affect QS and biofilm formation in P. aeruginosa .
Mutant 47 has a mutation in purL, a gene involved in purine biosynthesis and metabolism , while in mutant 56 the transposon was inserted in the ORF PA3981. PA3981 encodes for a hypothetical with unknown function.
The most interesting mutants are 59 and 74. Surprisingly, these mutants have exactly the same site of transposon insertion in the dksA gene, a well characterized transcriptional regulator . dksA is one of the main regulators known to modulate AHL and AQ-dependent QS in P. aeruginosa and it is shown to negatively control rhl and to affect the production of elastase and rhamnolipid. Despite the same insertion site, the two mutants have completely different phenotypic characteristics. Mutant 59 has almost twice the maximal PpqsA activity with respect to the parental strain, while in mutant 74 PpqsA activity is abrogated. These two strains behave in a different way also for what concerns HHQ and pyocyanin production, and swarming ability. This controversial result suggest that the phenotypic differences observed in one of the two strains not only relies on dksA mutation, but is due to a secondary mutation occurred in the chromosome. The generation of a dksA clear deletion mutant will be performed to address this issue.
In some of the identified mutants the activity of PpqsA does not correlate with the expression of the phenotypes known to be controlled by HHQ and PQS. In mutant 59, for example, the increased PpqsA activity does not result in the increased production of AQs and pyocyanin, and a reduced swarming ability has been demonstrated for this strain. Mutants 47 and 74 are the only ones in which the reduced PpqsA transcription is reflected in a drastically lower level of AQs and pyocyanin production, and in a decreased swarming motility. This consideration highlights the complexity of the QS response in P. aeruginosa, and strengthens the concept that a fine tuning in the levels and timing of QS signal molecules production during the growth of P. aeruginosa is responsible for the control of pleiotropic phenotypes like pyocyanin production and swarming motility.
Moreover, the finding that genes involved in central metabolic processes like purine (purL) and nucleotide (carA) biosynthesis can affect AQs production, reinforce the idea that QS regulation does not only relies on cell density, but it is a complex regulatory circuit that integrates cell density sensing with metabolic and environmental stimuli.
This study lays the basis for a better understanding of the regulation of AQs production in P. aeruginosa. Different mutants with significant difference in PpqsA activity have been identified and they will be validated in the future by generating clear deletion mutants in the selected genes. The generation of the miniCTX-PpqsA::mCherry plasmid will allow to monitor PpqsA expression at the single cell level and during the biofilm mode of growth. Moreover, the identification of the regulators bound to the pqsA promoter region by MALDI-TOF analysis will provide more information on the protein directly affecting AQs biosynthesis. On the whole, this study represents a starting point for the identification of new promising targets for the production of anti-Pseudomonas drugs.
I would like to express my gratitude to my academic supervisors. I would like to thank my supervisor Prof. Miguel Cámara for providing me with the opportunity to work in this field. I am deeply indebted to my bench supervisor Dr. Giordano Rampioni for his endless guidance, stimulating support, interest and assistance in all aspects of the laboratory work which was carried out. I have furthermore to thank all the people working in the lab, for their advice and valuable hints which were of great help in challenging times.