A fleQ mutation and high intracellular c-di-GMP have nonadditive effects in causing elevated EPS gene expression

We have previously shown that high intracellular c-di-GMP results in elevated expression of a number of genes in P. aeruginosa PAO1, including genes for EPS biosynthesis (Hickman et al., 2005). An earlier study by Dasgupta et al. (2003) showed that a fleQ mutant of P. aeruginosa strain PAK had increased expression of many of the same genes that we found to be elevated in high c-di-GMP cells. To confirm and extend these findings in P. aeruginosa strain PAO1, we used reverse transcriptase PCR to compare levels of the EPS biosynthesis genes pelA (PA3064) and pslA (PA2231), as well as levels of hypothetical genes PA2441 and PA4625 in a fleQ mutant to those of the wild type and we also quantitated transcript levels in a wspF mutant. In our previous work we found that expression of pelA, pslA, PA2441, and PA4625 were elevated in the wspF mutant (Hickman et al., 2005). We found that the fleQ mutant had a 20-fold increased level of pelA transcript and a 2.8-fold increased level pslA transcript compared to wild type (Figure 2A and 2B). The levels of PA2441 and PA4625 transcription were also higher in a fleQ mutant compared to wild type (Figure 2C and 2D). A wspF strain with high intracellular c-di-GMP had levels of these transcripts that were comparable to those observed with the fleQ mutant. We also generated elevated levels of intracellular c-di-GMP in wild type and fleQ mutant cells by overexpressing the diguanylate cyclase encoded by PA1120 (Kulasakara et al., 2006). This also resulted in elevated transcript levels of pelA and pslA (Figure 2 A and B). Thus FleQ appears to negatively regulate transcription of pel, psl, PA2441, and PA4625 whereas high c-di-GMP appears to somehow stimulate transcription of these genes.

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Figure 2

FleQ is a negative regulator of genes that are also expressed at high c-di-GMP levels

Relative transcript levels for pelA (A), pslA (B), PA2441(C), PA4625 (D) and fleR (E) as assayed by quantitative RT-PCR are shown for wild type (PAO1), wspF mutant (high c-di-GMP), a fleQ mutant, a fleQ wspF mutant (high c-di-GMP), wild type expressing PA1120 from a plasmid (pJN1120), a fleQ mutant expressing PA1120, fleN mutant, and a fleN wspF mutant (high c-di-GMP). Strains with an asterisk indicate that these strains have high c-di-GMP levels and display phenotypes consistent with elevated c-di-GMP. Data points are the average of three independent biological samples. Error bars represent the standard deviations between replicates.

If FleQ and c-di-GMP influence gene transcription at the same point, then the effects of a fleQ mutation and high c-di-GMP on transcription should not be additive. Consistent with this, when we quantified transcript levels of pelA, pslA, PA2441, and PA4625 in a wspF fleQ double mutant we saw no increase in expression compared to either single wpsF or fleQ mutants (Figure 2).

FleN, a known antagonist of FleQ activation of flagella gene expression, also modulates pel and psl transcription

The activity of FleQ is known to be modulated by FleN (PA1454), which has been reported to bind to FleQ and dampen its ability to activate gene transcription (Dasgupta et al., 2000; Dasgupta and Ramphal, 2001). Strains lacking FleN have increased expression of flagella biosynthesis genes and fleN mutants are multiflagellated (Dasgupta et al., 2000). If FleN also dampens the ability of FleQ to repress transcription of EPS biosynthesis and other genes, then fleN mutants would be predicted to show decreased expression of genes that are negatively regulated by FleQ. In keeping with this, the transcript levels of pelA, pslA, PA2441, and PA4625 were 1.5, 2.8, 3.0, and 1.6 fold lower in a fleN mutant compared to wild type (Figure 2). The transcript levels of our target genes were lower in a wspF fleN double mutant than in a wspF mutant (a strain with elevated c-di-GMP) (Figure 2). However, the levels of the target gene transcripts were higher in the wspF fleN double mutant than in the fleN single mutant, indicating that the double mutant strain responded to c-di-GMP to modulate transcription. These results are consistent with a model where FleQ acts as a repressor of pel and psl gene transcription and FleN interacts with FleQ to antagonize its repressor activity.

Elevated c-di-GMP levels have small effects on transcription of flagella biosynthesis genes

Consistent with previously published results showing that FleQ is an important positive activator of flagella gene expression (Arora et al., 1997), we observed 20-fold lower levels of fleR transcript in the fleQ mutant compared to wild type (Figure 2E). The fleR transcript levels were 1.6 fold lower in a wspF mutant compared to wild type, indicating that elevated c-di-GMP has a much smaller effect on expression of flagella genes than on EPS biosynthesis genes. A wspF fleQ double mutant had the same low levels of fleR transcript as did a fleQ mutant alone. Loss of fleN resulted in an eight-fold increase in fleR transcript level (Figure 2E), consistent with previous reports (Dasgupta et al., 2000). A wspF fleN double mutant showed a 1.6 fold decrease in fleR transcript levels compared with the fleN mutant alone, indicating that the small effect of c-di-GMP on fleR transcript levels is observed in the presence or absence of the fleN gene (Figure 2E). Similar effects of elevated c-di-GMP on transcript levels of the flhA gene were also observed (data not shown). Since the effect of elevated c-di-GMP on the expression of flagella genes was small we decided to focus on the effects of c-di-GMP and FleQ on the pel operon.

fleQ or fleN mutations have minimal effects on c-di-GMP concentrations

One explanation for the effects of FleQ and FleN on expression of the pel and psl genes is that loss of FleQ or FleN affects c-di-GMP levels, which then influence gene transcription. In order to rule out this possibility we quantified c-di-GMP levels in strains lacking FleQ or FleN and compared them to the levels in wild-type cells. We measured c-di-GMP by liquid chromatography-mass spectrometry using a modification of a previously reported method (Thormann et al., 2006). We estimate from our measurements that the wild type strain PAO1 has an average intracellular concentration of 0.7 μM c-di-GMP and the wspF mutant has an intracellular concentration of c-di-GMP of about 3.0 μM. The fleQ and fleN mutants had intracellular c-di-GMP levels that were slightly lower than those of the wild-type strain (Figure 3). The intracellular concentration of c-di-GMP in the fleQ wspF double mutant was essentially the same as those measured in a strain with only a wspF mutation. Levels of c-di-GMP in the fleN wspF double mutant were slightly lower than in the wspF mutant alone. These small changes in c-di-GMP levels likely do not cause the large changes in gene expression that we observed in the fleQ and fleN mutants. This indicates that the effects of the fleQ mutation in causing elevated pel, psl, PA2441, and PA4625 transcription are not due to a secondary effect of this mutation causing increases in intracellular c-di-GMP.

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Figure 3

Loss of FleQ or FleN does not cause elevated c-di-GMP levels

C-di-GMP was extracted from whole cells and levels were measured as described in the Experimental Procedures. Strains are wild type (PAO1), a fleQ mutant, a wspF mutant, a fleQ wspF mutant, a fleN mutant, and a fleN wspF mutant. Data represent averages of three independent cultures. Error bars are the standard deviations between independent cultures.

FleQ binds to pel promoter DNA

Although there are relatively few examples of bacterial enhancer binding proteins like FleQ directly repressing transcription, it is not unprecedented for members this class of protein to act as transcriptional repressors (Chang et al., 2007; North et al., 1996; Weiss et al., 1991). To test the possibility that FleQ directly represses transcription, we purified FleQ and assayed its ability to bind to pelA promoter DNA in an electrophoretic mobility shift assay (EMSA). We incubated purified FleQ with a fragment of DNA spanning −284 to +30 base pairs relative to translational start of pelA. The addition of FleQ to reaction mixtures caused a shift in the mobility of the pel promoter DNA fragment (Figure 4A). FleQ did not affect the mobility of a 135 base pair fragment from the plasmid pUC19 that we included as a negative control (Figure 4A). Although FleQ was able to bind specifically to the pel promoter region, relatively large amounts of protein (500 nM) were required to see even a slight shift in DNA mobility, suggesting that FleQ is a relatively poor DNA binding protein. This is in agreement with previous reports showing that purified FleQ bound poorly to the promoters of several genes for flagella biosynthesis (Dasgupta and Ramphal, 2001; Jyot et al., 2002).

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Figure 4

FleQ binds to the pel promoter and binding is inhibited by c-di-GMP

A) Binding of FleQ or FleN to the pelA promoter in the absence and presence of ATP (10 μM). A fragment from the pUC19 vector is included as a negative control. The migration of unbound pelA promoter or pUC19 DNA though the gel is indicated by arrows. The concentrations of FleQ and FleN are indicated. B) The effect of FleN and ATP on FleQ-DNA complex formation. The FleQ and FleN proteins were provided in equimolar amounts at the concentrations indicated. ATP was added at a concentration of 10 μM where indicated. C) The effect of different nucleotides on FleQ-FleN-DNA complex formation. The nucleotide added and its concentration is indicated above each lane. D) Binding of FleQ and FleN to the pel promoter in the absence and presence of c-di-GMP. FleQ and FleN were provided in equimolar amounts at the concentrations indicated. c-di-GMP was added where indicated. All reactions contained 10 μM ATP.

FleN binds with FleQ to pel promoter DNA in an ATP-dependant manner

Previous work showed that FleN and FleQ physically interact with each other and that addition of FleN, together with FleQ, to EMSA reactions retarded the migration of fhlA promoter DNA through acrylamide gels more than did FleQ alone (Dasgupta and Ramphal, 2001). This was interpreted to mean that FleN binds to FleQ at the fhlA promoter to form a higher molecular weight complex. To test possible effects of FleN on pel promoter DNA migration though gels, we purified FleN and added it to our EMSA reactions either alone, or in equimolar amounts with full length FleQ. FleN alone had a barely detectable effect on pelA promoter migration in the EMSA assays (Figure 4A). FleN has a predicted nucleotide-binding site and is a predicted ATPase. When we added 10 μM ATP to FleN alone or to FleQ alone in gel shift reactions, we saw no effect of ATP on the ability of either of these proteins to bind the pelA promoter (Figure 4A). In contrast, the inclusion of FleN in reaction mixtures together with FleQ resulted in the formation of a complex with a slower mobility than that observed with FleQ alone when 10 μM ATP was present (Figure 4B). ADP also enhanced the ability of FleQ-FleN to shift pelA promoter DNA (Figure 4C). However the inclusion of AMP, GTP, or GMP in reactions had no effect on the ability of FleQ-FleN to shift the pelA promoter (Figure 4C).

C-di-GMP abrogates binding of the FleQ-FleN complex to pel promoter DNA

If c-di-GMP and FleQ regulate the transcription of the pel operon through a common system, then one would predict that the addition of c-di-GMP to EMSA reaction mixtures might influence the binding of FleQ and FleN to pel promoter DNA. We found that the addition of c-di-GMP at concentrations as low as 10 μM resulted in a decrease in the ability of FleQ-FleN to bind pel promoter DNA as assayed by retarded migration of the protein-DNA complex though gels (Figure 4D). These results indicate that FleQ-FleN constitute a c-di-GMP-responsive transcription regulatory complex. C-di-GMP addition did not influence the small shift in pelA mobility observed with 500 nM FleQ alone (data not shown).

We also tested if c-di-GMP addition affected the ability of FleQ-FleN to shift the promoter of the flagella genes fleSR. While FleQ-FleN shifted fleSR promoter DNA in the presence of ATP, we saw little to no effect of c-di-GMP on the ability of FleQ-FleN to shift fleSR promoter DNA (Supplementary Figure 1A, B). This result is consistent with our observation that elevated c-di-GMP resulted in a very small decrease of fleR transcript levels in vivo (Figure 2E).

Plasmid and strain construction

All primer sequences are available upon request. Plasmid pJNFleQ was constructed by ligating an EcoRI-SmaI fragment from plasmid pAT6 containing the FleQ gene into EcoRI-SmaI digested pJN105 (Tart et al., 2005). Plasmid pJN1120 was constructed by amplification of PA1120 with primers homologous to the 5′ and 3′ ends of PA1120 containing restriction sites for NheI and XbaI respectively. The PCR product was cloned into NheI-XbaI digested pJN105 and the resulting plasmid, pJN1120, was verified by sequencing. Plasmids were electroporated into wild-type PAO1 and the fleQ mutant using established protocols (Choi et al., 2006).

Deletions of pelA and pslBCD in a fleQ mutant were constructed using standard procedures for allelic exchange in P. aeruginosa. Two plasmids, pMPSL-KO1 and pMPELA, containing the deletion constructs (M. Starkey and M. Parsek, unpublished) were mated into a fleQ mutant and strains were selected on Pseudomonas isolation agar containing 50 gg/ml gentamycin. Double recombinant mutants were selected on LB plates containing 5% sucrose. Mutants were confirmed by PCR.

RNA isolation and real-time quantitative PCR

P. aeruginosa strains were grown in 5 ml cultures in test tubes overnight then subcultured twice into 125 ml baffled flasks to an OD₆₀₀ of 0.02. After the second subculture and subsequent growth, cells were harvested at an OD₆₀₀ of 0.4–0.6 for RNA isolation. RNA was isolated and cDNA synthesized using previously published procedures (Schuster et al., 2003). Primers for RT-PCR were designed using the program PrimerExpress (Applied Biosystems). Reactions were performed using SybrGreen master mix (Applied Biosystems) with 1 ng cDNA as template and 200 nM of each primer in a final reaction volume of 25 μl. Cycling parameters were 95°C for 10 min, followed by 40 cycles of 95°C for 15 sec and 60°C for 1 min. A final dissociation curve of all amplified products was performed as a quality control check. Standard curves for quantitation of transcripts were performed using dilutions of P. aeruginosa chromosomal DNA from 1 ×10⁻⁴ ng to 10 ng (Lequette et al., 2006). Transcript levels of all genes tested were normalized to transcript levels of the ampR gene (Lequette et al., 2006). Data presented are averages of three independent cultures grown on different days. Error bars represent the standard deviation between samples.

Quantitation of c-di-GMP levels by LC-MS

Cells were grown overnight in VBMM (Vogel-Bonner minimal medium) and subcultured the next day to an OD₆₀₀ of 0.02 in 125 ml baffled shake flasks containing 15 ml of VBMM. VBMM, a defined medium, was used for c-di-GMP quantitation because an unknown component of LB medium co-eluted with c-di-GMP and masked the c-di-GMP signal during LC-MS analysis. When cells reached an OD₆₀₀ of 0.4–0.6, 1 ml was removed, centrifuged for 1 min at 15,000 × g, and the supernatant was removed. The cell pellet was resuspended in 100 μl 0.6 M perchloric acid. Samples were incubated on ice for 30 min, and cell debris was removed by microcentrifugation at 4°C for 5 min at 15000 × g. Supernatants (100 μl) were removed and samples were neutralized by addition of 20 μl 2.5 M KHCO₃. The resulting precipitate was removed by centrifugation at 4°C for 5 min at 15,000 × g. These neutralized supernatants were stored at −80°C until analyzed by LC-MS. For protein determinations, three 1-ml samples of each culture were removed and centrifuged to pellet cells. Cell pellets were resuspended in 100 μl 1M NaOH and boiled in a water bath for 10 minutes. Samples were then stored on ice until protein was assayed. Standard Bradford protein assays were carried on all samples in duplicate using the Bio-Rad Protein assay reagent (Bio-Rad, Hercules, CA). Bovine serum albumin (BSA) containing 1 M NaOH was used as a standard.

Samples were analyzed at the University of Washington Mass Spectrometry Center using an HPLC/MS/MS system. Protocols for c-di-GMP separations and analysis by mass spectrometry were based on a previously published method (Thormann et al., 2006). Separation was achieved with an Acuity UPLC (Waters; Milford, MA) using a 2.1 × 50 mm Synergi Hydro RP column (Phenomenex; Torrance, CA). A gradient system was used starting from 98% aqueous (10 mM ammonium formate, pH 4.0) and 2% organic (acetonitrile). The aqueous went to 10% at 6.0 minutes, 95% at 6.5 minutes, 10% at 7.0 minutes and 98% at 7.5 minutes. Temperature was uncontrolled, flow was 0.3 ml/min and cycle time was 10 minutes. The extra step from high to low aqueous and back again between 6.5 and 7.5 minutes served to eliminate carry-over between injections. The c-di-GMP was detected by MS/MS multiple reaction monitoring (MRM) using a Premiere XL triple quadrapole mass spectrometer (MicroMass; Milford, MA) in positive electrospray ionization (API-ES+). The m/z 691>152 transition was used for quantitation; 691>248 and 691>540 were monitored as confirmatory signals. The collision energies were 30, 24, and 24 eV respectively; the cone voltages were 40 V in all cases. For a standard curve, 50, 100, 250, 500, and 1000 fmol pure c-di-GMP (Axxora, San Diego, CA) were analyzed by the above method. C-di-GMP levels are normalized to total protein per ml of culture. Data represent averages of three independent cultures. Error bars are the standard deviation between replicate cultures. Intracellular concentrations of c-di-GMP were estimated from this data assuming that 1 mg of protein corresponds to 6.7 × 10⁹ cells and that each P. aeruginosa cell has a volume of 1 × 10⁻¹⁵ liters.

Protein purification

FleQ, FleQ lacking its N-terminal domain (FleQ-trunc), and FleN were purified using the IMPACT system from NEB (Beverly, MA). The fleQ, fleQ-trunc, and fleN genes were each cloned into pTYB12 as NdeI-EcoRI fragments generating plasmids pFleQ-Int1, pFleQ-trunc-Int1, and pFleN-Int1. This generated N-terminal fusions of the Intein-Chitin binding domain (Intein-CBD) to FleQ, FleQ-trunc, and FleN, respectively. This resulted in the addition of Ala, Gly, and His residues to the N-terminus after cleavage of the Intein-CBD tag. Plasmids were transformed into E. coli ER2566 for expression of fusion proteins. For overexpression of the fusion proteins, E. coli carrying either pFleQ-Int1, pFleQ-trunc-Int1, or pFleN-Int1 were subcultured from overnights into 1 L of fresh LB in 3 L Fernbach flasks. Cultures were grown to an OD₆₀₀ of 0.4 and then shifted to 15°C. After 30 minutes at 15°C, 0.3 mM IPTG was added to induce expression of fusion proteins, and cultures were grown for 16 h. Cells were harvested by centrifugation at 5000 × g, pellets resuspended in column buffer with Triton X-100 (20 mM Tris-Cl pH 8.0, 250 mM KCl, 0.1 mM EDTA, 0.1% (v/v) Triton X-100), and cells were lysed by sonication. Lysates were clarified by centrifugation at 15,000 × g for 30 minutes. The solubility of the expressed proteins was checked by analyzing soluble and insoluble fractions by SDS-PAGE. Expression at 15°C prevented the insolubility of overexpressed FleN that had been previously described (Dasgupta and Ramphal, 2001).

Intein-CBD fusions were purified as previously described with the following modifications (Hickman et al., 2005). Proteins were loaded in column buffer plus Triton X-100 onto a 5 ml chitin bead column. Columns were washed with 50 ml column buffer plus Triton, then washed with 50 ml column buffer lacking Triton X-100. Cleavage of the Intein-CBD was induced by addition of column buffer containing 50 mM DTT or β-mercaptoethanol and incubating overnight at room temperature. When necessary, protein was concentrated using Amicon ultrafiltration devices (Millipore, Billerica, MA.) Proteins were dialyzed into column buffer containing 50% glycerol and stored at −20°C. Both FleQ and FleN were stable under these storage conditions for up to 2 months.

In vitro DNA binding analysis

The pel promoter region spanning −284 to +30 base pairs relative to translational start of pelA was PCR amplified. As a negative control a 135 base pair fragment of pUC19 vector was also PCR amplified. These DNA fragments were end labeled with T4 polynucleotide kinase and γ[³²P]-ATP. Equal amounts of each of these labeled DNA fragments (4 fmol, final total probe concentration 100 pM) were added to binding reactions with varying amounts of FleQ or FleQ lacking its N-terminal domain (FleQ-trunc) in binding buffer (10 mM Tris, pH 7.8, 8 mM magnesium acetate, 50 mM KCl, 5% glycerol, 250 ng/μl BSA, 20 μl total reaction volume). FleQ was incubated with DNA for 30 minutes on ice. Reactions carried out with FleN were performed as above with the addition of equimolar amounts of FleN and FleQ. Where indicated, 10 μM ATP or other nucleotides were also added to the reactions and incubated with the proteins for 30 minutes on ice prior to addition of DNA. Reactions containing c-di-GMP were performed as described above except c-di-GMP was incubated with proteins for 30 minutes before addition of DNA. All reactions mixtures were loaded onto a 5 % acrylamide gel containing 10 mM Tris-Cl pH 8.0, 400 mM glycine, 5 mM EDTA, and electrophoresed at 70 V at room temperature for 1–1.5 h. Gels were dried and exposed to a phosphorimaging screen overnight, visualized on a Storm phosphorimager (GE Healthcare, Pistcataway, NJ), and images analyzed using ImageQuant software (GE Healthcare, Pistcataway, NJ).

Public Health Service Grant GM56665 from the National Institute of General Medical Sciences supported this work. JWH was supported by a Cystic Fibrosis Foundation Postdoctoral Fellowship (R565-CR02). We would like to thank Thomas F. Kalhorn and the University of Washington Mass Spectrometry Center for their invaluable assistance with c-di-GMP quantitation. Plasmids pMPSL-KO1 and pMPELA were generous gifts from Melissa Starkey and M. Parsek. Purified QscR protein was a generous gift from Ken-Ichi Oinuma and E. P. Greenberg. We would also like to thank Amy L. Schaefer and Breck A. Duerkop for reading the manuscript and for insightful discussions.