Bacterial strains and growth conditions

E. coli strains used in this study are listed in Table 1. Strains were grown at 37°C in Luria-Bertani (LB) broth (10 g tryptone, 5 g yeast extract, 10 g NaCl per liter). Cultures of JM6733 containing plasmids were supplemented with 20 µg/mL chloramphenicol (BioExpress). JM6733 (ΔproQ::KAN), JM6753 (ΔproP::KAN), JM6754 (ΔproV::KAN), JM6755 (ΔproW::KAN), and JM6877 (ΔproX::KAN) were constructed by P1 transduction [18] of KEIO-collection mutants, JW5300, JW4072, JW2652, JW2653, and JW2654 respectively, into a clean BW25113 background [19]. JM6881 (ΔproVWX::KAN) was made using the λ_red recombination system [20]. JM6906 (ΔproP::FRT) was constructed by transformation of JM6753 (ΔproP::KAN) with pCP20, and subsequently, screening for sensitivity to kanamycin [21]. JM6926 (ΔproP::FRT, ΔproVWX::KAN) was constructed by P1 transduction of JM6906 by phage grown on JM6881. Genotypes were verified using primers flanking the genomic regions of interest (Table 2). Culture growth was monitored by measuring the absorbance at 600 nm.

Preparation of cell lysates for ribosomal fractionation

Isolation of ribosomal species was performed as previously described [22] with the following exceptions. Cultures of LB broth were inoculated with 1/200^th volume of a stationary overnight culture. For strain JM6733 (ΔproQ::KAN) containing the indicated plasmids, Isopropyl β-D-1-thiogalactopyranoside (IPTG) was added to a final concentration of 15 µM after the first 30 min of incubation to induce the expression from the lac promoter. Cell pellets were resuspended in 700 µL of lysis buffer containing 10 mM Tris pH 7.5, 10 mM MgCl₂, and 60 mM NH₄Cl per 100 mL of starting culture, flash frozen in liquid nitrogen, and stored at -80°C. 300 µm glass beads were added to frozen cells as they thawed. Cells were lysed by repeated vortexing for 1 min followed by 1 min on ice for a total time of 10 min. The lysate was cleared by centrifugation at 21,000 x g for 10 min at 4°C. For subunit dissociation experiments, cells were lysed without addition of chloramphenicol, in buffer containing 10 mM Tris pH 7.5, 1 mM MgCl₂, and 60 mM NH₄Cl and clarified as described. The absorbances of clarified lysates were measured at 260 nm.

MNase digestion of ribosomes

Lysates were prepared as previously described. 13 OD₂₆₀ units of lysate were transferred to a clean, 1.5 mL microcentrifuge tube. CaCl₂ was added to a final concentration of 5 mM. BSA (NEB) was added to a final concentration of 0.1 mg/mL. 4 µL of MNase (NEB) were added and the final reaction volume was brought up to 400 µL with lysis buffer. Digests were carried out at room temperature for 30 min. Mock digests were set up as controls, containing all reagents except MNase.

In vitro binding of ProQ and 70S ribosomes

70S ribosomes (NEB) were incubated with an equimolar ratio of purified ProQ in polysome lysis buffer, with or without equimolar P2 mRNA. Reactions were loaded onto sucrose gradients and separated as described.

Ribosomal fractionation

Ribosomal species were separated on sucrose gradients ranging from 10% to 47% sucrose in pollyalomar ultracentrifuge tubes (Beckman). Sucrose was dissolved in the following buffer: 100 mM NH4Cl, 10 mM Tris pH 7.5, 10 mM MgCl₂. 550 µL of 47% sucrose was added into the bottom of the tube and placed at -80°C until frozen. Next, 42% sucrose was added and the tube was placed back at -80°C until frozen. This method was repeated until the gradient was complete, using the following concentrations of sucrose: 47%, 42%, 37%, 32%, 27%, 22%, 17%, 12%, 10%. Gradients were stored at -80°C. Gradients were allowed to thaw completely at room temperature. 13 OD₂₆₀ units of lysate were applied to the top of each gradient. Gradients were balanced and submitted to ultracentrifugation in an SW50.1 (Beckman) rotor at 41K RPM for 1.5 h at 4°C. After ultracentrifugation, fractionation was performed as previously described [22]. Separation of dissociated subunits was done by ultracentrifugation on 20% sucrose cushions in an SW40TI (Beckman) rotor at 23 K RPM for 15 h at 4°C. Fractionation was performed as previously described.

Mutant His-ProQ constructs

Mutant constructs were made using pCA24N-ProQ from the ASKA collection [23] as a template for inside-out PCR. A SacI restriction endonuclease site was added to both primers (Table 2), allowing linear products from PCR amplification to be digested and self-ligated in frame to create pDS1-pDS5 (Table 1).

ProQ purification and Antibody production

A culture of JM6733 (ΔproQ::KAN), transformed with pCA24N-ProQ, was grown to late-logarithmic growth phase (OD₆₀₀ ^≈ 0.8) at 37°C in LB media. Production of a 6 histidine epitope-tagged ProQ was induced by addition of IPTG to a final concentration of 1mM, and the culture was shifted to ambient temperature with shaking. Cells were harvested 18 h post induction. Cells were resuspended in 8 mL of lysis buffer (10% glycerol, 20 mM Tris pH 8.0, 500 mM NaCl, 10 mM Imidazole, and 1 mM β-Mercaptoethanol) and complete EDTA-free protease inhibitors (Roche) per 1 g of wet pellet weight and disrupted by 5 passes through a french press. Insoluble debris was cleared via centrifugation at 15,000 RPM for 30 min at 4°C in an SA-600 rotor (Sorvall). Nickel affinity chromatography was performed using Ni-NTA Superflow resin (Qiagen) as per manufacturer’s directions with the following exceptions. After batch binding of 6His-ProQ the column was washed with 5 column volumes (CVs) of lysis buffer. Additional 5 CV washes were done with lysis buffer containing 1 M NaCl, as well as lysis buffer containing 20 mM and 30 mM Imidazole. 6His-ProQ was eluted from the column using 5 CVs of lysis buffer containing 500 mM Imidazole. Protein purity was monitored via SDS-PAGE, and the eluent was dialyzed overnight at 4°C in a low-salt buffer (10% glycerol, 20 mM Tris pH 8.0, 100 mM NaCl, 1 mM β-Mercaptoethanol). Cation exchange chromatography was performed using SP Sepharose Fast Flow resin (GE Healthcare). Cation exchange resin was equilibrated with 5 CVs of low salt buffer. Dialyzed 6His-ProQ was incubated with equilibrated resin for 30 min at room temperature with occasional agitation. After batch binding, the column was washed with 5 CVs of low salt buffer. 6His-ProQ was eluted from the column using a gradient of NaCl (100 mM to 1 M). 6His-ProQ purity was monitored via SDS-PAGE, and purified 6His-ProQ was again dialyzed against a low-salt buffer. After dialysis, 6His-ProQ was concentrated using a centrifugal filter with a 3 kDa molecular weight cutoff (Amicon), as per manufacturer’s directions. Purified 6His-ProQ was quantified using an ND-1000 spectrophotometer (NanoDrop Technologies, Inc.) and 600 µg was sent for antibody production in a rabbit host (Cocalico Biologicals, Inc).

Western-blot analysis of ribosomal fractionations

Denaturing SDS-PAGE loading buffer (50 mM Tris pH 6.8, 2% Sodium dodecyl sulfate (SDS), 10% glycerol, 1% β-Mercaptoethanol, 13 mM EDTA, 0.02% Bromophenol blue) was added to trichloroacetic acid (TCA) precipitated ribosomal fractions. Briefly, TCA was added to polysome gradient fractions to a final concentration of 15% and incubated at 4°C for 30 min. The precipitant was pelleted in a microcentrifuge at maximum speed for 10 min. The supernatant was removed and the pellet was washed twice with ice cold 100% acetone. Proteins were separated via SDS-PAGE and transferred to nitrocellulose (GE Water & Process Technologies). Protein detection was carried out using antibodies against ProQ (1:5000 in 5% dry milk in PBS + 0.1% Tween20) and ribosomal proteins L3 (1:5000 in same) and S2 (1:5000 in same). Antibodies to L3 and S2 were a generous gift from Catherine Squires.

Copy number calculation

proQ was cloned into vector pMCSG7 as described [24] using DS046 and DS047 (Table 2) to make pDS6 (Table 1), which adds an N-terminal 6-Histidine epitope tag, followed by a TEV cleavage site. ProQ was purified as previously described with the following exceptions. After the first nickel affinity column, elution fractions were pooled and dialyzed overnight in the presence of 6His-TEV protease. A second nickel affinity column was used to remove TEV protease and the flow through was collected. Fractions were analyzed via SDS-PAGE and pooled for cation-exchange chromatography. Purified ProQ was quantified using a Bradford assay (Biorad). Known amounts of ProQ, as well as whole cell lysate from a known number of wild type BW25113 E. coli cells were loaded onto SDS-PAGE and transferred to nitrocellulose for western-blot analysis using anti-ProQ antibodies. Band intensities were quantified using ImageJ and cellular concentration was calculated.

In vitro transcription

The proP genomic region was cloned into pBluescriptKS+ in two forms, starting at either -182 (proP P1, DS037, DS039) or -95 (proP P2, DS038, DS039). The rpoS genomic region was cloned into pBluescriptKS+ (rpoS-T7-forw_SacII, rpoS-T7-rev-PstI). PCR was performed to amplify the plasmid DNA for use as an in vitro transcription template. The forward primer maintained the integrity of the T7 polymerase dependent promoter (pBSIVTforward), and the reverse primer added a stop codon after glycine 245 for proP P1and P2 (DS076) and after tyrosine 147 for rpoS (rposIVTreverse), yielding mRNAs with sizes of 927 bp, 840 bp, and 1041 bp respectively. In vitro transcription was carried out as described previously [25], and the mRNAs were checked for quality using urea-acrylamide gel electrophoresis and TAE-agarose electrophoresis. The mRNA concentrations were determined by electrophoresis on a TAE-agarose gel and quantitative comparison to the RiboRuler High Range RNA Ladder (Thermo Scientific).

Tryptophan fluorescence

Purified ProQ was dialyzed into 10% glycerol, 20 mM HEPES pH 7.5, and 100 mM NaCl. The concentration was determined using Bradford assay. For binding experiments, ProQ was diluted to a final concentration of 20 nM in the same buffer plus 0.005% BRIJ35. Temperature was held constant at 20°C and tryptophan was excited at 295 nm and fluorescence was monitored at 355 nm using a QuantaMaster4 (Photon Technology International). The excitation wavelength was chosen to avoid any possible inner-filter effect stemming from the use of nucleic acids as ligands [26]. Increasing amounts of in vitro transcribed mRNAs were added to the reaction and the change in fluorescence was observed.

Biofilm assays

Plasmid pMR20-ProQ was constructed by cloning the amplicon from primers DS083 and DS084 into pMR20 [27] cut with KpnI and SacI (Table 2). Biofilm assays were performed with modifications of previous protocols [28,29] as follows. Strains were grown overnight in LB + 12.5 µg/mL tetracycline. 30 µL of the saturated overnight was inoculated into 1 mL of LB without salt (10 g tryptone, 5 g yeast extract) plus 12.5 µg/mL tetracycline, in a previously unused borosilicate test tube. Tubes were incubated at 25°C without shaking for 6 d. After 6 d, the media and loose cells were removed and the OD₆₀₀ was measured. Tubes were gently rinsed by submerging in deionized water 5 times and allowed to air dry. Biofilms were stained using 1% crystal violet for 20 min. Tubes were again rinsed by submerging in deionized water to reduce background staining. Biofilms were resuspended completely in 0.5% SDS and the OD₆₀₀ was measured. Relative biofilm formation was calculated as described previously [28].

ProQ associates with translating ribosomes

In order to verify the localization of ProQ on ribosomes, the soluble lysates from wild type E. coli (strain BW25113) were separated on sucrose density gradients (Figure 1A). Western-blot analysis of the resulting fractions revealed that ProQ predominantly associates with 70S and translating ribosomes, and to a lesser extent, at the top of the gradient and in fractions corresponding to the 30S ribosomal subunit. Antibodies against small and large ribosomal subunit proteins validated the assignment of the peaks in the UV-absorbance trace. Deletion of some ribosome-associated factors can lead to ribosomal defects and perturbation of the polysome profile [22]. Profiles from a ΔproQ strain were indistinguishable from wild type (data not shown), and therefore, ProQ is not likely a ribosome assembly factor, nor is it required for the translation of the majority of mRNAs in the cell.

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Figure 1. Ribosome association of ProQ under native and dissociating conditions.

Polysome profiles (254 nm) from wild type cell extracts in (A) associating conditions (10 mM Mg²⁺) and (B) dissociating conditions (1 mM Mg²⁺) are shown. Western-blot analysis of TCA-precipitated fractions using antibodies to ProQ, small ribosomal subunit protein 2 (S2), and large ribosomal subunit protein 3 (L3) are shown and aligned to the UV-absorbance trace. Whole cell extracts from wild type (BW25113) and ΔproQ (JM6733) are included, as is the soluble lysate (Load). UV-absorbance peaks correspond to (L to R): Free RNA/protein, 30S, 50S, 70S, and polysomes.

https://doi.org/10.1371/journal.pone.0079656.g001

Since the 30S and 50S ribosomal subunits work in tandem, but with separate roles during translation, identifying the subunit with which ProQ associates provides a clue as to the function of ProQ during translation. When isolated at lower concentrations of Mg²⁺, 70S ribosomes dissociate into 30S and 50S subunits [30]. At 1 mM Mg²⁺, the 70S and polysome species were predominantly dissociated, as revealed by the UV-absorbance trace and localization of S2 and L3 (Figure 1B). A large proportion of ProQ was found at the top of the gradient, but ProQ that did migrate into the gradient peaked with the 30S subunit but not with the 50S subunit. These data are consistent with the localization under non-dissociating conditions (Figure 1A). Thus, ProQ appears to preferentially associate with the 30S subunit.

The N-terminus and linker regions are necessary for ribosome association

ProQ is predicted to contain two structural domains, tethered by a positively charged linker region. The N- and C-termini have been modeled on the RNA binding proteins FinO and Hfq respectively [12,15]. Using available bioinformatic tools to predict secondary structure, disordered regions, and nucleic acid binding propensity [31–33], we assigned the boundaries of the linker region as amino acids E116-V180. Assignment of the lower bound was the most difficult. The nucleic acid binding predictor, BindN, indicated the presence of an unstructured positively charged domain starting at E116. The secondary structure predictor, PSIPRED, indicated the presence of an alpha helix until A124. Domain constructs were made using the 6HIS-ProQ plasmid from the ASKA collection [23] as a template. A schematic of each construct is provided (Figure 2A). To determine the regions of ProQ that are necessary for its association with the ribosome, each ProQ construct was expressed in a ΔproQ background, such that it was the only copy of ProQ in the cell. We could not detect a ProQ variant expressing only the C-terminal domain (proQ181-232). The distribution of ectopically expressed 6HIS-ProQ (Figure 2B) was similar to that of ProQ from wild type cells (Figure 1A); although, in addition to an association with 70S, polysome, and 30S fractions, there was a general increase in ProQ throughout the gradient. This broader distribution may reflect a higher than normal cellular concentration caused by exogenous expression or a decrease in affinity for translating ribosomes due to the N-terminal 6HIS-epitope tag. The high rate of speed and high G-forces produced during polysome fractionation can physically disrupt binding interactions, so addition of the epitope tag may cause dissociation of 6HIS-ProQ from ribosomes as it progresses through the gradient. Deletion of the C-terminal Hfq domain (proQΔ181C) did not affect association with the ribosomes (Figure 2B), and therefore, this part of the protein is not required for ribosome localization. In contrast, both the N-terminus and linker regions are required for ribosome association, as the majority of proQΔ115N and proQΔ124-180 were found at the top of the gradient. Consistently, removal of the C-terminus and linker domains together (proQ2-123) caused ProQ to be found at the top of the gradient. Moreover, the linker domain (proQ116-180) was not sufficient for ribosome association. These data suggest that both the N-terminal FinO domain and the linker regions are required for ribosome association of ProQ.

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Figure 2. Ribosome association of plasmid encoded proQ mutant constructs in polysome profiles.

(A) Schematic representation of proQ mutant constructs expressed from the IPTG inducible plasmid pCA24N. The predicted RNA binding region is colored in black, and amino-acid boundaries of each construct are labeled. (B) Mutant constructs were expressed as the lone copy of proQ. Western-blot analysis of TCA-precipitated fractions from the separation of ribosomal moieties on sucrose density gradients was performed. Whole cell extracts of cells after induction were included (Induced) along with soluble lysate (Load). Position of the 30S, 50S, 70S, and Polysome species are indicated based on western-blot localization of S2 (data not shown).

https://doi.org/10.1371/journal.pone.0079656.g002

ProQ interacts with the mRNA being translated

The comigration of ProQ into sucrose gradients with translating ribosomes could occur because of a physical interaction with the ribosome directly or because of an interaction with the mRNA bound by a 30S particle and/or 70S particle. To determine the nature of the ProQ association with ribosomes, we partially hydrolyzed the mRNA in cell lysates with micrococcal nuclease (MNase) under conditions that did not perturb the integrity of the highly structured, and relatively RNase insensitive, ribosome. As expected, the small ribosomal protein S2 was found associated with the remaining 70S and 2-mers, and no appreciable amount of S2 was found at the top of the gradient. This supports the claim that the integrity of these ribosomes was maintained. In mock treatments, mRNAs with up to 4 ribosomes are seen in sucrose gradients, and ProQ associates with all translating ribosomes (both the 70S and polysome forms) (Figure 3A). After treatment with MNase, the level of total polysomes was decreased concomitant with a large increase in free 70S ribosomes (Figure 3A) and consistent with cleavage of intra-ribosome mRNA. Strikingly, in contrast to a robust association of ProQ with 2-mer and 3-mer polysomes in untreated samples, ProQ was almost absent from these particles in the MNase treated samples. Additionally, a significant amount of ProQ was found at the top of the gradient, consistent with dissociation of ProQ from the ribosomes after disruption of mRNA. Thus, ribosome association of ProQ appears to be partially dependent on the mRNA being translated. In order to support this finding, we also performed in vitro binding experiments with mRNA-free 70S ribosomes. 70S ribosomes were incubated with equimolar purified ProQ, with or without proP P2 mRNA. After incubation, the reactions were applied to sucrose gradients and separated as before (Figure 3B). Though some ProQ is found in the gradient under the 70S peak, indicating a weak interaction with 70S ribosomes, the bulk of the ProQ is located in the fractions not corresponding to the 70S ribosomes. It is also worth noting that when proP mRNA was added, we consistently observed the concentration of ProQ in the gradient peaking around fraction 5. The most likely explanation for this result is that ProQ is binding to the proP mRNA and migrating to this region of the gradient.

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Figure 3. Ribosome association of ProQ after mRNA disruption.

(A) Cell lysates were left untreated (solid line) or treated with limited MNase (dashed line) and ribosomal species separated on sucrose gradients. The positions of ProQ and ribosomal protein S2 in the resulting TCA-precipitated fractions is determined by western-blot analysis. (B) mRNA free 70S ribosomes were mixed with equimolar amounts of purified ProQ without (foreground) or with (background) the addition of equimolar P2 mRNA before application to sucrose gradients. The position of ProQ and ribosomal protein S2 in the resulting TCA-precipitated fraction is determined by western-blot analysis.

https://doi.org/10.1371/journal.pone.0079656.g003

ProQ does not bind selectively to proP mRNA

It has been demonstrated that ProQ can bind to a model RNA substrate in vitro [15]. However, the model double-stranded substrate used previously was relatively small (39 bp in duplex form) compared to the size of an mRNA. To further understand the RNA binding properties of ProQ, binding experiments were performed using purified ProQ and in vitro transcribed mRNA substrates (Figure 4A). One possibility is that ProQ acts as a proP mRNA-specific translation factor. If true, we would predict that ProQ should interact preferentially with proP mRNA in vitro. We tested the ability of ProQ to bind to mRNA made from the proP P1 and P2 promoters, as well as an unrelated mRNA, rpoS (see methods). Under the conditions tested, ProQ showed no preference for proP mRNA transcribed from promoter P1 or P2, and the binding affinity for proP P1 and P2 were similar (17.0 ± 6.1 nM and 11.6 ± 2.7 nM, respectively; Figure 4). Interestingly, ProQ also bound tightly to rpoS mRNA (19.5 ± 7.8 nM; Figure 4). These slight differences among the K_D values are not sufficient to confer selectivity in vivo. Comparisons of our values to those previously reported for the model substrate are in agreement [15], though we report consistently lower K_D values. ProQ binds non-selectively to all mRNAs tested, but it does not bind to DNA, and we include the results as a negative control. To compliment the determination of the K_D values for specific mRNAs we sought to determine the cellular abundance of ProQ. Using quantitative western-blot analysis, we estimate the cellular copy number of ProQ to be 1.90 x 10³ ± 324 (95% confidence interval, data not shown). Assuming a cellular volume of 1.3 µm³ [34], the cellular concentration of ProQ is 2.43 ± 0.414 µM. This number is comparable to another estimate for the ProQ copy number obtained by high-throughput proteomic analysis of E. coli cytosolic proteins [35], and at approximately 2,000 copies/cell, ProQ is in the top 25% in terms of protein abundance.

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Figure 4. mRNA binding kinetics monitored by native tryptophan fluorescence.

(A) 20 nM ProQ was titrated with increasing amounts of in vitro transcribed mRNAs (“●” proP P1, “▲” proP P2, “■” rpoS). Tryptophan was excited at 295 nm and the fluorescence was monitored at 355 nm. The y-axis is provided in terms of normalized fluorescence (FL_F/FL_I), determined by dividing the fluorescence at each mRNA concentration (FL_F) by the fluorescence for 20nM protein only (FL_I). DNA yielded no measurable fluorescence shift and is included as a negative control (“□” DNA) (B) Summary of binding affinities of ProQ for in vitro transcribed mRNAs.

https://doi.org/10.1371/journal.pone.0079656.g004

Ribosome association of ProQ is not dependent on proP or proU

We have shown that ProQ is found on translating ribosomes and this association appears to be mediated through mRNA. Because the only well characterized phenotype for a proQ mutant is a decrease in proline uptake, we posit that ProQ is involved in the translation of a subset of mRNAs in the cell, including one or more of the proline transporter mRNAs. We therefore asked whether the ribosome association of ProQ was dependent on these mRNA transcripts. We first examined the localization of ProQ in a ΔproP mutant, as proP mRNA is the predicted target of ProQ. We found, however, that the ribosome association of ProQ was similar in a ΔproP strain to that seen in the wild type strain (Figures 1 and 5). A genetic interaction has been reported between proQ and the proW and proX loci within the proU operon [36] and therefore, these genes may be regulated by ProQ. For this reason, we examined ProQ localization in strain backgrounds deleted for each member of the proU operon, as well as a deletion of the entire proU operon (proVWX), and deletion of both proVWX and proP together. In all backgrounds tested, ProQ is repeatedly found associated with 70S particles and polysomes (Figure 5). Thus, ProQ ribosome association is not absolutely dependent on any of these genes, and the ribosome association of ProQ may be due to one or more additional cellular mRNAs.

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Figure 5. Ribosome association of ProQ in various strain backgrounds.

Cell lysates from various mutant backgrounds, as indicated, were separated by sucrose density ultracentrifugation and the localization of the ribosomal species (30S, 50S, 70s, polysomes) indicated. The localization of ProQ in the TCA-precipitated fractions is shown by immunoblot. Whole cell extracts from wild type, ΔproQ (JM6733), and the soluble lysate (Load) are included as indicated.

https://doi.org/10.1371/journal.pone.0079656.g005

ProQ is necessary for biofilm formation, independent of ProP

Having shown that ProQ localization to the ribosome is independent of all known potential interactions involved in proline transport, we sought to expand the ProQ target list by looking for new ΔproQ phenotypes. A high throughput study implicated ProQ in biofilm formation; though, it only reported it as a hit and did not verify the result through plasmid-based complementation [28]. Under the conditions tested, a proQ mutant strain was found to be about 50% deficient in biofilm formation compared to wild type cells (Figure 6). This phenotype was complemented by transforming ΔproQ cells with a low-copy plasmid containing the proQ gene, plus 500 bp of sequence upstream of the translation start site. To test if this phenotype was independent of the role of ProQ in ProP regulation, we examined biofilm formation in a proP mutant strain. This strain was able to form biofilms as well as wild type. As a result, we conclude that the decrease in biofilm formation is unique to a proQ mutant and is independent of the ProQ-ProP interaction, though the reason for this deficiency remains unclear.

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Figure 6. Biofilm formation defect in a ΔproQ strain.

Wild type (BW25113), ΔproQ (JM6733), and ΔproP (JM6753) strains were examined for formation of biofilms after 6 days. Strains were transformed with either empty vector (pMR20; shaded) or vector containing the proQ open reading frame, plus 500 bp of genomic sequence upstream of the translation start site (pMR20-ProQ; unshaded). Error bars represent 95% confidence intervals. A statistical difference is indicated (*) between wild type and ΔproQ (JM6733) with a p-value < 0.001.

https://doi.org/10.1371/journal.pone.0079656.g006