The Pseudomonas putida Lon protease is involved in N-acyl homoserine lactone quorum sensing regulation
© Bertani et al; licensee BioMed Central Ltd. 2007
Received: 18 April 2007
Accepted: 26 July 2007
Published: 26 July 2007
In Pseudomonas putida and Pseduomonas aeruginosa, the similar PpuR/RsaL/PpuI and LasR/RsaL/LasI acyl homoserine lactones (AHLs) quorum sensing (QS) systems have been shown to be under considerable regulation by other global regulators. A major regulator is the RsaL protein which strongly directly represses the transcription of the P. putida ppuI and P. aeruginosa lasI AHL synthases. In this study we screened a transposon mutant bank of P. putida in order to identify if any other regulators were involved in negative regulation of AHL QS.
In our screen we identified three Tn5 mutants which displayed overproduction of AHLs in P. putida strain WCS358. Two of the mutants had a Tn5 located in the rsaL gene, whereas in one mutant the transposon was located in the lon protease gene. Lon proteases play important roles in protein quality control via degradation of misfolded proteins. It was determined that in the P. putida lon mutant, AHL levels, PpuR levels and ppuI promoter activity all increased significantly; we therefore postulated that PpuR is a target for Lon. The Lon protease had no effect on AHL production in P. aeruginosa.
The Lon protease is a negative regulator of AHL production in P. putida WCS358. The Lon protease has also been shown by others to influence AHL QS in Vibrio fischeri and Agrobacterium tumefaciens and can thus become an important regulator of AHL QS timing and regulation in bacteria.
Quorum sensing (QS) is a common form of gene regulation based on cell-density involving intercellular communication relying on the production and response to signaling molecules [1, 2]. In Gram-negative bacteria, acyl-homoserine lactones (AHLs) are the most common signal molecules which were first described in the marine bacterium Vibrio fischeri as being involved in the cell-density dependent regulation of bioluminescence [1, 3]. The general mechanism of AHL QS relies on two proteins belonging to the LuxI and LuxR protein families. LuxI-family proteins are the major class of AHL synthase enzymes whereas LuxR-family proteins form complexes with AHLs which are then able to bind at specific DNA promoter sequences (called lux-type boxes) of QS regulated genes affecting their expression.
AHL QS has become a paradigm for bacterial communication having the common scheme that AHLs are produced at a basal level at low cell densities. At high cell densities, the concentrations of AHLs surpasses a certain threshold (corresponding to the "quorum" cell density) allowing interaction with the LuxR-family protein and the system then usually undergoes positive feedback through increase of expression of the luxI-family gene resulting in strong sudden activation of AHL QS. This cell-density dependent response has evolved as a means to provide advantages to a community of bacteria by synchronizing group behavior. AHL QS has been studied in several Gram-negative bacteria and the physiological processes controlled by this system are diverse but are often related to virulence in pathogenic organisms [1, 2, 4, 5].
The AHL QS systems are not always rigorously responding to cell-density as they are often integrated with other global regulatory responses and are thus influenced by other environmental factors [6–8]. Several systematic studies in Pseudomonas aeruginosa have shown that AHL QS is a global regulatory network controlling the expression of over 300 genes [9–11]. Recently other regulon studies in P. aeruginosa have demonstrated that the RpoS, VqsR and PprB global regulators are intimately interconnected with AHL QS regulons . This is probably why some AHL QS systems are themselves regulated in response to various stimuli ensuring a timely control at the appropriate environmental conditions. In fact, the regulation of AHL QS has been particularly studied in P. aeruginosa highlighting that the luxI/LuxI and luxR/LuxR family genes/proteins are themselves extensively regulated. Positive regulation of the lasI/R and rhlI/R, the two AHL QS systems homologs of luxI/R present in P. aeruginosa, occurs via transcriptional regulators such as GacA, Vfr and PprB (reviewed by [6–8]). At present however it is not known whether any of these positive regulators are acting directly on the AHL QS genes and the precise stimuli affecting these positive regulatory responses are also not clear. Other regulators repress AHL QS in P. aeruginosa likely to ensure that it is not activated at low cell-densities. Reports of these negative regulators include the H-NS like protein MvaT, the luxR-like orphan QscR, the post-transcriptional regulator RsmA, the alternative sigma factors RpoS and RpoN, and the newly characterized RsaL repressor (reviewed by [6–8]). Of these repressors only RsaL has been shown to regulate directly the AHL QS genes; more precisely it regulates the expression of the lasI AHL synthase by binding to its promoter repressing transcription [12, 13]. Interestingly we also reported that RsaL is a major negative regulator of AHL QS in Pseudomonas putida . Unlike in P. aeruginosa however, in strain WCS358 there is only one AHL QS system, designated PpuI/R, which produces and responds to N-3-oxo-dodecanoyl homoserine lactone (C12-3-oxo-AHL); this system is highly identical to the LasI/R system of P. aeruginosa [13, 14]. Localized in between the ppuI/R genes is the small rsaL repressor which when inactivated results in dramatic increase of ppuI expression and hence AHL production . In P. putida RsaL repression of the AHL synthase gene appears to be much stronger that in P. aeruginosa [12, 14].
In this study we were interested to investigate whether in P. putida WCS358 other negative ppuI/R (or PpuI/R) AHL QS regulators, either acting in concert with RsaL or independently, are present. By screening a Tn5 genomic mutant bank of strain WCS358 a negative regulatory mutant of AHL QS was identified and characterized to be inactivating a gene encoding for a Lon-like protease. It was demonstrated that this protease targeted PpuR thus affecting PpuR protein levels indicating that it is involved in regulating the AHL QS system.
Results and Discussion
Identification and characterization of AHL-overproducing mutants of P. putida WCS358
The P. putida Lon protease affects PpuR protein levels
Pseudomonas strains, plasmids and oligonucleotides used.
P. putida WCS358
Pseudomonas putida, wt
P. putida IBE4
lon1754::Tn5 of WCS358, Kmr
P. aeruginosa 18577
PA1803lon437::ISlacz/hah of PAO1, Tcr
Cloning vector, Ampr
Cloning vector, Ampr
Kmr Tra+ Mob+, ColE1 replicon
Expression vector, kmr
Broad-host-range vector, Gmr
pBluescriptKS carrying a 4.5 Kb BamHI/StuI fragment from IBE4 harboring some WCS358 and a fragment of Tn5, Ampr Kmr
pBluescriptKS carrying a 4.5 Kb EcoRI fragment from WCS358, Ampr
pBBRmcs5 carrying a 4.5 Kb EcoRI fragment from WCS358, Genr
pET28b derivative carrying ppuR of P. putida WCS358
pBBRmcs5 carrying the ppuR gene amplified from pPuR3586H with primers P0FW and P0RV
pMP220 promoter probe vector carrying the ppuI promoter region, Tcr
pMP220 promoter probe vector carrying the rsaL promoter region, Tcr
The P. aeruginosa Lon protease does not affect C12-3-oxo-AHL production
In this study we determined that the Lon protease is a negative regulator of AHL production in P. putida WCS358. AHL production and response in WCS358 occurs via the PpuR/RsaL/PpuI AHL QS system; this system is highly homologous to the LasR/RsaL/LasI system of P. aeruginosa. It was observed that in a Lon mutant, C12-3-oxo-AHL levels, PpuR levels and ppuI promoter activity all increase significantly; we therefore postulated that PpuR is a target for Lon. Unlike what occurs in P. putida WCS358, in P. aeruginosa Lon has no effect on AHL production. It was however observed that P. aeruginosa possesses in its genome another ORF with very similar features to Lon thus it cannot be excluded that other proteases could be involved in AHL QS regulation. As two other LuxR family regulators have been shown to be targeted by Lon, it is concluded that proteases could play an important role in AHL QS timing and regulation.
Bacterial strains, plasmids and media
The bacterial strains and plasmids used in this study are listed in Table 1. Pseudomonas putida WCS358 is a plant growth-promoting strain isolated from the rhizosphere of potato roots.Chromobacterium violaceum CVO26 is a double mini-Tn5 mutant derived from ATCC31532, this mutant is non-pigmented and production of the purple pigment can be induced by providing exogenous AHL inducer molecules . Escherichia coli DH5α and C. violaceum CV026 were grown in LB medium  at 37°C and 30°C respectively. Pseudomonas was grown in LB medium or M9 minimal medium  at 30°C. The following antibiotic concentrations were used: ampicillin (Amp) 100 μg/ml; kanamycin (Km) 100 μg/ml; nalidixic acid (Nx) 25 μg/ml; tetracycline (Tc) 10 μg/ml (E. coli), 40 μg/ml (Pseudomonas); chloramphenicol (Cm) 25 μg/ml (E. coli), 250 μg/ml (Pseudomonas); gentamycin (Gm) 10 μg/ml (E. coli), 40 μg/ml (Pseudomonas).
Recombinant DNA techniques
DNA manipulation as digestion with restriction enzymes, agarose gel electrophoresis, purification of DNA fragments, ligations with T4 ligase, end-filling with Klenow enzyme, hybridization, radioactive labeling by random priming and transformation of E. coli, were performed as described previously . Southern hybridizations were performed using N+Hybond membrane (Amersham Biosciences); plasmids were purified using the Jet star colums (Genomed, GmbH, Germany) or with the alkaline lysis method ; total DNA from Pseudomonas was isolated by Sarkosyl/Pronase lysis as described previously . Triparental matings between E. coli and P. putida were carried out with the helper strain E. coli DH5α (pRK2013) .
Isolation of an AHL over-expressing genomic mutant and cloning of a lon-like protease of P. putida WCS358
In order to identify P. putida WCS358 mutants that overproduce AHL, a Tn5 genomic mutant library was screened against the AHL biosensor strain C. violaceum CVO26. P. putida WCS358 induced only slightly pigmentation when streaked in close proximity to strain C. violaceum CVO26. This screening for AHL over-producers used here was previously employed to isolate rsaL mutants on P. putida WCS358 . 25,000 Tn5 genomic mutants of P. putida WCS358 were screened against CVO26 for AHL overproducer mutants as previously described [14, 15]. This led to the identification of one Tn5 mutant (designated P. putida IBE4), of which the Tn5 was not located in the rsaL gene and could induce strongly pigmentation of CVO26. The position of the Tn5 was determined after cloning from the mutant IBE4 chromosome a 4.2 kb BamHI-StuI fragment in pBluescript KS yielding pSCHIA5, which contained part of Tn5 (including the Km resistance gene) and part of adjacent WCS358 DNA. The adjacent DNA to the Tn5 was sequenced using as primer the oligonucleotide sequence Tn5ext (see Table 1) which was designed against the border of the Tn5 DNA sequence. The 4.2 kb BamHI-StuI fragment was then used as a probe to clone a 4.5 kb EcoRI from the chromosome of parent strain WCS358 in pBluescript KS yielding pB1A. This latter EcoRI fragment was sequenced (sequencing service, CRIBI, University of Padua, Padua, I) revealing that the Tn5 was positioned within an ORF of 2397 nucleotides encoding for a putative Lon-like protease of 799 amino acids (Figure 1).
Reporter gene fusion assay
β-galactosidase activities were determined during growth in LB medium essentially as described by Miller et al.,  with the modifications of Stachel et al. . All experiments were performed in triplicate and the mean value is given. The growth curves of all mutants were comparable to the one obtained for the parent strain.
Purification and quantification of C12-3-oxo-AHL
The purification, detection and characterization of AHLs were performed as previously described [23, 31]. Pseudomonas strains were grown O/N in M9 minimal medium supplemented with citric acid and the OD600 was measured. The spent culture supernatants were extracted two times with the same culture volume of ethyl acetate-0.1% acetic acid. The extracts were then dried and resuspended in ethyl acetate with an amount which resulted in 1 μl of final extract corresponding to 2 × 107 cells of the original culture. The quantity of C12-3-oxo-AHL in the extracts was determined using the specific 3-oxo-C12-AHL sensor P. putida SM17 (prsal220) as previously described . Briefly, P. putida SM17 is a double mutant of ppuI and rsaL genes consequently it does not produce the RsaL repressor and 3-oxo-C12-AHL. Adding exogenous 3-oxo-C12-AHL is quantified through β-galactosidase activity by using stain SM17 harboring (prsal220); this plasmid contains the PpuR-3-oxo-C12-AHL regulated rsaL promoter fused to promoterless lacZ gene. Overnight cultures of SM17(prsal220) were diluted in 10 ml of LB medium to an A660 of 0.1; the AHL extract to be quantified was then added and after 6 hours of growth β-galactosidase activity was determined. This 3-oxo-C12-AHL bacterial sensor has a linear dose response between 0.1 μM to 5 μM of 3-oxo-C12-AHL. Synthetic 3-oxo-C12-AHL was used as standard molecules (obtained from P. Williams, University of Nottingham, UK). The detection of the AHLs on the TLC plate was obtained overlaying the TLC plate with a thin layer of LB top-agar seeded with E. coli (pSB1075) as previously described [14, 31, 32].
PpuR antibodies and protein anaylsis
Antibodies against PpuR of P. putida were generated by injecting purified protein into rabbits. P. putida PpuR was purified as PpuR-His6 using expression plasmid pET28b (Novagen); ppuR was cloned as a 729 PCR fragment originated from primers FW121 and FW122 using P. putida WC358 genomic DNA as template and was cloned as a NcoI-HindIII fragment in pET28b yielding pPPUR3586H. This latter plasmid was introduced in E. coli BL21(DE3)pLys (Novagen) which then resulted in the isopropyl-β-D-thiogalactopyranoside-induced over-expression of PpuR tagged with six histidines at the C- terminus. The purification of PpuR-His6 was then carried out by Ni 2+ affinity under denaturing conditions according to the standard procedure suggested by the column manufacturer (Sigma-Aldrich, St. Louis, Mo.).
Proteins were transferred onto PVDF membrane (Immobilon-P; Millipore) using a tank system according to the manufacturer's instruction. The membrane was subjected to Western analysis using anti-PpuR polyclonal antibodies raised in rabbits and polyclonal goat anti rabbit immunoglobulins HRP (DakoCytomation, Glostrup, DK) and developed using Immun-Star HRP Substrate kit (BioRad laboratories, Hercules CA, USA). No significant cross-reaction of the polyclonal antibody against other P. putida WCS358 proteins was observed in this study.
DNA sequencing and nucleotide sequence accession numbers
All DNA sequences were performed at the CRIBI center (University of Padua, Italy) and the nucleotide sequence of the 4.414 EcoRI fragment has been deposited in GenBank/EMBL/DDBJ under the following accession number AM690373.
IB was supported by a fellowship from the Italian Cystic Fibrosis Research Foundation (Project FFC9/2005 financed by the Delegazione FFC di Vicenza). This work was in part supported by a grant from ISPESL, Rome, Italy (contract B/98-2DIPIA/03). This work was also supported by a grant from the Italian Ministry of University and Research PRIN-2006 entitled "Basi genetiche e molecolari della patogenicita' batterica". We thank the ICGEB staff of the animal house in their assistance in raising polyclonal PpuR antibodies and to Sara Ferluga for assistance in artwork.
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