A pyocyanin overproducer with insertional inactivation of ptsP gene was isolated from a mini-Mu insertion library in Pseudomonas aeruginosa PA68. The mutation was complemented by a functional ptsP gene in trans. The pyocyanin-overproducing phenotype was also found in a ptsP mutant constructed by gene replacement in the P. aeruginosa PAO1 strain. Reporter plasmids with PqscR-lacZ, PlasI-lacZ and PrhlI-lacZ were constructed and the β-galactosidase activity in the ptsP mutant/wild-type background was measured. The results showed that lack of Enzyme INtr (EINtr, encoded by ptsP) decreased transcription from the PqscR promoter and increased the activity of the PlasI and PrhlI promoters. Normally, QscR represses the quorum-sensing LasR-LasI and RhlR-RhlI systems involved in pyocyanin regulation. Our results showed that the ptsP gene has an important role in the regulation of pyocyanin production and that two quorum-sensing systems and their repressor QscR are involved in this regulation.
Pseudomonas aeruginosa is an important broad host range opportunistic pathogen . In humans, it is usually found associated with severe burns, cystic fibrosis, AIDS and cancer . The complex pathophysiology of infections associated with P. aeruginosa is due to its ability to produce a large number of virulence factors, including proteases, lipases, rhamnolipids and pyocyanin.
Most P. aeruginosa isolates produce pyocyanin. Pyocyanin is a blue-green redox-active secondary metabolite, belonging to a member of a large family of tricyclic compounds known as phenazines . Recent studies demonstrated that pyocyanin contributes to pathophysiological effects, such as cell respiration, ciliary beating , calcium homeostasis , inactivity of alpha1 protease inhibitor , inhibition of nitric oxide synthase , induction of neutrophil apoptosis , inflammatory response  and neutrophil-mediated tissue damage . In addition, pyocyanin enhanced the killing of Caenorhabditis elegans by a clinical P. aeruginosa isolate .
The genes for pyocyanin biosynthesis including operon phzA1B1C1D1E1F1G1, operon phzA2B2C2D2E2F2G2, gene phzM and gene phzS have been identified . Quorum-sensing systems, sigma factors and other regulators with high pleiotropism are involved in the regulation of the biosynthesis of pyocyanin and other secondary metabolites, exerting their influences directly or indirectly in different growth phases [13–24]. In the quorum-sensing network LasR-LasI, RhlR-RhlI and QscR (q uorum s ensing c ontrolled r epressor) are often involved in the adaptation of the organism to various environments. In this study, we found a new function of the ptsP gene (PA0337). It influences the transcription of qscR and the subsequent quorum-sensing systems in P. aeruginosa.
2Materials and methods
2.1Bacterial strains, plasmids and media
P. aeruginosa PA68 is a clinical isolate from the sputum of a patient with bronchiectasis . All bacterial strains and plasmids are listed in Table 1. The bacteria were cultured in Luria broth or on LB agar plates. For pyocyanin production, Pseudomonas broth  and Pseudomonas Isolation Agar were used. The antibiotics used were as follows: for Escherichia coli, 100 mg ml−1 ampicillin, 30 mg ml−1 kanamycin, 50 mg ml−1 tetracycline and 10 mg ml−1 gentamicin; for P. aeruginosa, 50 mg ml−1 kanamycin, 200 mg ml−1 tetracycline and 30 mg ml−1 gentamicin.
pDN19lacΩ containing the qscR promoter region as a 1.25 kb Eco RI–Bam HI insert
pDN19lacΩ containing the lasI promoter region as a 487 bp Eco RI–Bam HI insert
pDN19lacΩ containing the rhlI promoter region as a 559 bp Eco RI–Bam HI insert
2.2Enzymes and chemicals
Taq DNA polymerase, T4 DNA ligase, restriction enzymes and DNA molecular mass markers were purchased from TaKaRa (Dalian, Liaoning, China); Bacto-Peptone, tryptone, yeast extract and Pseudomonas Isolation Agar were purchased from Difco (Detroit, Michigan, USA). O-Nitrophenyl-β-d-galactopyranoside was purchased from Amresco (Solon, Ohio, USA).
2.3Screening for pyocyanin-overproducing mutants from a mini-Mu insertion library in P. aeruginosa strain PA68
Pyocyanin-overproducing mutants were screened from an artificial mini-Mu transposon (Km-Mu) insertion library of P. aeruginosa PA68 . Screening was done by streaking Mu transposon mutants on Pseudomonas Isolation Agar plates followed by incubation at 37 °C for 24 h. Streaks of pyocyanin positive strains are green, pyocyanin negative mutants are colorless and pyocyanin-overproducers are strongly green on this medium.
2.4Pyocyanin quantitation assay
P. aeruginosa strains were grown with aeration at 37 °C in Pseudomonas broth to maximize pyocyanin production. Each culture was inoculated from a fresh colony and adjusted to an optical density at 600 nm of 0.02 before incubation. Quantitation of pyocyanin production was done by extracting a 5 ml of culture with 3 ml of chloroform followed by mixing with 1 ml of 0.2 N HCl to give a red solution. The absorbance at 520 nm of this solution was a measure of the amount of extracted pyocyanin as described by Essar et al. .
2.5Recombinant DNA techniques and sequencing
Standard methods  were used for plasmid DNA isolation, genomic DNA preparation, restriction enzyme digestion, agarose gel electrophoresis and ligation. Briefly, genomic DNA of the pyocyanin mutant was digested with Bam HI (there is no Bam HI site in the artificial mini-Mu transposon), generating a fragment with a transposon attached to its genomic DNA flanks. This fragment was then cloned into the Bam HI site of plasmid pUC18 . DNA sequences of transposon borders were determined from this recombinant plasmid by using transposon-specific primers reading sequences outwards from within the transposon. Southern blotting was used to confirm that the insertion had occurred as single event (as described in ).
2.6trans-complementation of P. aeruginosa ptsP mutation
Primer PT1 (5′-TAGGATCCAGCGGGACTGAACACGGAG-3′) and primer PT2 (5′-TAGGATCCCTTGCGGGGAAAGCGTAA-3′) were used as the primers to amplify the complete ptsP gene from P. aeruginosa PA68. Bam HI restriction enzyme recognition sites were added to the ends of primers (shown in bold) and additional nucleotides were added to the 5′ ends to ensure efficient cleavage (shown in italic). A 2.38-kb amplification product was cloned into pUC18, yielding pUC18PT. The 2.38-kb DNA fragment from the Bam HI digestion of plasmid pUC18PT was purified with an agarose gel DNA purification kit (TaKaRa). Subsequently, this fragment was cloned into the Bam HI site of the broad-host-range vector pDN18 , leading to the construction of pDN18PT. The orientation of insertion was identified by analyzing the Kpn I restriction endonuclease map (there is one Kpn I site in the polylinker of pDN18 and one unique Kpn I site in the ptsP gene dividing this gene into 1.0 kb and 1.4 kb if it is cut with Kpn I). The plasmid containing the complete ptsP gene inserted in positive orientation relative to the T7 promoter was used for complementation of the ptsP gene mutation in B84.
2.7Insertional inactivation of ptsP gene by gene replacement in P. aeruginosa PAO1
Forward primer FPT1 (5′-CGGAATTCGCCGCTCTGGGTCAACAC-3′) and reverse primer FPT2 (5′-CGGAATTCGAACCGACCGAAAGGA-3′) (Eco RI site in bold) were used to amplify a 565-bp ptsP gene fragment from P. aeruginosa PAO1 . This fragment contains only one Bgl II site and was cloned into the Eco RI site of pUC18, yielding pUC18FPT. A gentamicin resistance gene cassette with Bam HI sticky ends (compatible ends for Bgl II) excised from plasmid pUC7G  was inserted into the unique Bgl II site within the ptsP gene fragment, generating pUC18FPTGm. This construct, which cannot replicate in P. aeruginosa, was utilized to generate a chromosomal mutation in the P. aeruginosa ptsP gene by marker exchange in strain PAO1.
2.8Construction of promoter-lacZ reporter gene fusions and β-galactosidase assays
The 1.25-kb region upstream of the qscR gene, presumed to contain the qscR promoter, was amplified by PCR using primers Q1 (5′-CGGAATTCGCAGCGAGTTGAAGACCA-3′) (Eco RI site in bold) and Q2 (5′-CGGGATCCAGTCGAGAACCAGGGAGAAGA-3′) (Bam HI site in bold). This PCR product was digested with Eco RI–Bam HI and was cloned in front of the promoterless lacZ gene of plasmid pDN19lacΩ, generating the PqscR-lacZ reporter plasmid, pDN19PQ. Subsequently, the PqscR-lacZ reporter plasmid was electroporated into both the wild-type strain PA68 and the ptsP mutant strain B84. Transformants were cultured in Pseudomonas broth for 18 h and subjected to β-galactosidase assays.
The PlasI-lacZ and PrhlI-lacZ reporter plasmids were constructed and transformed with the same strategy. To create the PlasI-lacZ reporter plasmid, pDN19PLI, primers LI1 (5′-CGGAATTCGGGCAGGTTCTCGCCATTC-3′) (Eco RI site in bold) and LI2 (5′-CGGGATCCAACCGAAAACCTGGGCTT-3′) (Bam HI site in bold) were used. The promoter region of the rhlI gene was amplified using primers RI1 (5′-CGGAATTCACAATTTGCTCAGCGTGCTTT-3′) (Eco RI site in bold) and RI2 (5′-CGGGATCCAGCCCTTCCAGCGATTCAG-3′) (Bam HI site in bold) to construct the PrhlI-lacZ reporter plasmid, pDN19PRI.
All constructs were verified by DNA sequencing. The β-galactosidase activity was measured as reported by Miller .
3Results and discussion
3.1Screening for pyocyanin-overproducing mutants from a mini-Mu transposon library of the P. aeruginosa PA68
Mutants from a pool of Mini-Mu insertion mutants of PA68 were inoculated individually on Pseudomonas Isolation Agar plates. Pyocyanin-overproducing mutants were screened by the strength of the green color of the streak cultures. One potential pyocyanin-overproducer with stronger green color compared to PA68, was isolated and named B84. Southern blotting analysis demonstrated that this mutant had only one copy of the Mu transposon integrated into the genome (data not shown).
3.2Sequence analysis of the DNA flanking the insertion of strain B84
The fragment including the mini-Mu transposon (Km-Mu) and the flanking regions was cloned from the pyocyanin-overproducer mutant B84. DNA sequencing of these flanking regions revealed that the inactive gene was ptsP. The complete nucleotide sequence of PA68 ptsP gene obtained with plasmid pUC18PT as sequencing template has been submitted to the GenBank Data Bank with accession number AY830124. Alignment of the sequence of ptsP gene in PA68 and PAO1 using BLAST analysis showed that the two sequences are more than 99% identical at the nucleotide level and 96% identical at the deduced amino acid level.
3.3Complementation of the inactive ptsP gene in strain B84
For trans-complementation, the complete ptsP gene was cloned into the plasmid pDN18, yielding pDN18PT and both plasmids were electroporated into the ptsP mutant strain B84, generating the strain B84/pDN18PT and the control strain B84/pDN18, respectively. Strains B84/pDN18PT, PA68, B84 and B84/pDN18 were cultured in Pseudomonas broth to measure their pyocyanin production in different growth phases. The results showed that B84 produced approximate six times more pyocyanin than PA68 in the late stationary phase (Fig. 1A). The mutation in B84 was complemented by plasmid pDN18PT, while the phenotype of the control strain B84/pDN18 did not change (Fig. 1A). This clearly showed that the ptsP mutation in B84 was responsible for the pyocyanin-overproduction phenotype.
3.4Construction of a ptsP mutant in the PAO1 strain
PA68 is a clinical P. aeruginosa strain. To confirm that knock-out of the ptsP gene results also in overproduction of pyocyanin in strain PAO1, a ptsP mutant of strain PAO1 was constructed by gene replacement. Plasmid pUC18FPTGm, which cannot replicate in Pseudomonas, carrying an insertionally inactivated ptsP gene fragment was constructed and introduced into PAO1 with gentamicin selection. In the obtained transformants, a double reciprocal recombination may have occurred, resulting in a replacement of the corresponding chromosomal copy of the ptsP gene with the mutant gene of the plasmid, giving rise to a ptsP mutant strain, PAO1-NPT. The replacement was confirmed by PCR from one transformant. From this putative ptsP mutant, a 2.4-kb fragment (0.6 kb ptsP gene fragment with a 1.8-kb gentamicin-resistance gene insertion) was amplified with PCR using primers FPT1 and FPT2 combined with PAO1-NPT genomic DNA as template, while a 0.6-kb fragment was amplified using same primers, with the PAO1 genomic DNA as template. The PAO1-NPT strain also overproduced pyocyanin in late stationary growth phase (Fig. 1B). These results further showed that the ptsP gene influences pyocyanin production in P. aeruginosa.
3.5Effect of the ptsP mutation on the expression of the qscR, lasI and rhlI genes using PqscR-lacZ, PlasI-lacZ and PrhlI-lacZ reporter plasmids
In P. aeruginosa, the expression of several virulence factors such as elastase, rhamnolipids and pyocyanin are regulated by quorum-sensing systems . Quorum-sensing is a regulation mechanism depending on the information of bacterial population delivered by small diffusible molecules which are produced by each individual bacterium . There are two quorum-sensing systems in P. aeruginosa, which have been extensively studied. These two systems are the LasR-LasI and RhlR-RhlI systems . Furthermore, it was recently reported that QscR encoded by gene qscR indirectly governs the timing of quorum-sensing-controlled gene expression by repressing the LasR-LasI and RhlR-RhlI systems at the level of both transcription and protein interaction [21,35]. One potential explanation for the pyocyanin-overproducing phenotype of the ptsP mutants in this study could be that the negative control on pyocyanin production exerted by ptsP depends on downregulation of quorum-sensing systems repressed by QscR. Therefore, we constructed PqscR-lacZ, PlasI-lacZ and PrhlI-lacZ reporter plasmids and transformed them into the wild-type strain PA68 and the ptsP mutant strain B84. Results of the β-galactosidase assays of these strains showed that the disruption of ptsP lead to a significant decrease (approximate 92%) in the expression of qscR and twofold or threefold increase in the expression of lasI and rhlI (Table 2). This result suggests that EINtr negatively controls pyocyanin production via QscR in P. aeruginosa.
Table 2. β-Galactosidase activity in strains carrying promoter-lacZ reporter plasmidsa
aThe promoter-lacZ reporter plasmids are listed at the top of each data column. The host strains are indicated in the far left column. β-Galactosidase activity of each transformant is shown as a percentage of the average activity in the wild-type background. 100%β-galactosidase activity represents, for PqscR-lacZ, 1110 Miller units; for PlasI-lacZ, 411 units; for PrhlI-lacZ, 866 units.
bThe values are means ± standard deviations.
cn, number of independent assays performed.
100.0 ± 8.7
100.0 ± 9.0
100.0 ± 9.6
PtsP mutant B84
8.1 ± 0.7
191.9 ± 15.8
228.2 ± 17.7
Gene ptsP encoding a phosphoenolpyruvate-protein phosphotransferase EINtr was firstly reported in E. coli. EINtr together with NPr and IIANtr encoded by the ptsO and ptsN genes, respectively, is a component of a phosphotranferase system important in the regulation of nitrogen metabolism and carbon metabolism . Since EINtr participates in a phosphate relay with NPr and IIANtr proteins, the phosphorylation status may allow it to serve as a branch point in different signal transduction systems . Additional functions of the ptsP gene have been discovered in other bacteria. In Azotobacter vinelandii, mutation in ptsP leads to a failure in poly-β-hydroxybutyrate metabolism and in the respiratory protection of nitrogenase under carbon-limiting conditions ; in Bradyrhizobium japonicum, EINtr is involved in oligopeptide transport ; in Legionella pneumophila and P. aeruginosa, the ptsP gene is a virulence factor [40,41].
The structure of EINtr is intriguing, consisting of two domains, an N-terminal domain is a GAF domain and a C-terminal domain is the PEP-utilizers domain (for phosphotranfer). GAF domain is ubiquitous sensory module found in signaling proteins in all phyla  and has a three-dimensional structure known to bind small molecules [43,44]. Although the ligand or the effector molecule of this N-terminal domain has not been identified, EINtr was suggested to serve a sensory function linking carbon and nitrogen metabolism .
Our study showed that also in P. aeruginosa the ptsP gene has regulatory functions and does influence quorum-sensing systems. The mechanism of this interaction needs further studies. Determination of the complex physiological functions of EINtr is an exciting challenge for the future.
We thank Dr. Harri Savilahti (University of Helsinki) providing us with mini-Mu transposon DNA and MuA transposase, Professor Shouguang Jin (University of Florida) for technical assistance and providing strain PAO1, plasmid pDN18, pDN19lacΩ and pUC7G. This work was supported by the National Natural Science Foundation of China (No. 30270075).