PutA Is Required for Virulence and Regulated by PruR in Pseudomonas aeruginosa

Pseudomonas aeruginosa, a Gram-negative opportunistic pathogenic bacterium, causes acute and chronic infections. Upon entering the host, P. aeruginosa alters global gene expression to adapt to host environment and avoid clearance by the host immune system. Proline utilization A (PutA) is a bifunctional enzyme, which converts proline to glutamate. Here we report that PutA was required for the virulence of P. aeruginosa in a murine acute pneumonia model. A putA mutant was more susceptible to oxidative stress compared to the wild type strain. An AraC/XylS family protein, PruR, directly bound to the upstream of −35 box in the putA promoter and activated putA expression. High concentration of proline in bacteria up-regulated pruR expression, which led to the activation of putA expression. As a feedback regulation, glutamate produced by PutA released PruR from the putA promoter and turned off the putA expression. PruR affected bacterial virulence through the regulation of the putA expression. Altogether, these data are the first to reveal that PutA plays an important role in the pathogenesis of P. aeruginosa, as well as to describe the genetic regulation of PutA in P. aeruginosa.

In a mouse acute pneumonia model, neutrophils are rapidly recruited to the infection site in response to the invading bacteria (Shaver and Hauser, 2004). Neutrophils are the major phagocytes that generate reactive oxygen species (ROS) to kill bacteria (Ziltener et al., 2016). In P. aeruginosa, the H 2 O 2 -responsive regulator OxyR senses oxidative stress and activates the expression of defensive genes, such as katA (catalase A), katB (catalase B), ahpB (alkyl hydroperoxide reductases B), and ahpCF (alkyl hydroperoxide reductases CF), to breakdown the ROS (Ochsner et al., 2000;Heo et al., 2010).
Proline is an important carbon and nitrogen source for bacterial growth (Wood, 1981;Kohl et al., 1988;Nagata et al., 2003). It also provides protection against osmotic, heat and oxidative stresses in prokaryotes as well as in eukaryotes (Csonka, system NtrY/NtrX up-regulates putA expression upon bacterial entry into host cells (Cheng et al., 2014). However, these genes are not present in the P. aeruginosa genome. In P. aeruginosa, pruR encoding an AraC/XylS family protein is located close to the putAP operon (Figure 1). The expression of putA gene is down-regulated in a pruR mutant strain, suggesting putA expression is regulated by the PruR (Nakada et al., 2002). Until now, no definitive analysis of the regulatory mechanisms by which P. aeruginosa can modulate putA expression has been reported.
In this article, to gain insights into the role of PutA in P. aeruginosa virulence, we constructed a putA mutant and determined its mortality in an acute pneumonia model. We also determined the regulatory mechanisms of putA expression by lacZ reporter assays, electrophoretic mobility shift assays and DNase I footprint assays. Overall, these data provide important information about the function and regulation of PutA in P. aeruginosa.

Ethics Statement
All animal studies complied with National and Nankai University guidelines regarding the use of animals in research. All animal experiment protocols have been approved by the institutional animal care and use committee of the College of Life Sciences of Nankai University (permit number NK-04-2012).
Plasmids used in this study are listed in Table S2. For DNA manipulation, standard protocols or manufacture instructions of commercial products were followed. Chromosomal gene mutation strains, putA and pruR, were generated as described previously (Hoang et al., 1998). Complemented strains, putA/ putA and pruR/pruR were obtained by integrating each gene including its promoter region into chromosome using pUC18Tmini-Tn7T-Gm (Choi and Schweizer, 2006). Insertion of an empty transposon (with the inverted repeats only) was not included, since previous researches have demenstrated that insertion in chromosome using this method did not change the characters of bacteria (Choi and Schweizer, 2006;Heacock-Kang et al., 2017;Munguia et al., 2017;Pletzer et al., 2017). Primers used for knock-out and complementation are listed in Table S3.

Murine Acute Pneumonia Model
Bacteria were grown in LB broth at 37 • C for overnight and then sub-cultured into fresh LB broth at 37 • C with aeration to OD 600 of 1.0. Bacterial cells were harvested by centrifugation and adjusted to 2 × 10 9 CFU/ml in sterile 1 × PBS (274 mM NaCl, 5.4 mM KCl [BBI life sciences], 20 mM Na 2 HPO 4 , 4 mM KH 2 PO 4 , pH 7.4). Female BALB/c mice (6-to 8-week old) (Academy of Military Medical Sciences, Beijing, China) were anesthetized with an intraperitoneal injection of 7.5% chloral hydrate (70 µl per mouse). Anesthetized mice were intranasally inoculated with 10 µl of bacterial suspension in each nostril, giving a total infection volume of 20 µl. Thirteen mice were used for each strain. The mice were monitored at least twice a day for 5 days (Weng et al., 2016).

H 2 O 2 Susceptibility Assay
H 2 O 2 susceptibility assay was performed as described with minor modification (Weng et al., 2016). Over-night cultures of P. aeruginosa strains were diluted in the indicated medium to OD 600 of 0.05 and cultured at 37 • C. When OD 600 reached 1.0, bacterial cells from 3 ml culture were collected and washed twice with sterile 1 × PBS. Then the bacterial cells were resuspended in 1 × PBS with 0.3% H 2 O 2 and incubated at 37 • C for 15 min. The live bacterial numbers were determined by serial dilution and plating.

Expression and Purification of Recombinant Protein
The DNA fragment encoding full-length PruR was amplified by PCR using P. aeruginosa PAK chromosomal DNA as a template and specific primers ( Table S3). The amplified fragment was digested with restriction enzymes and ligated into the same restriction enzyme-digested pET-28a(+). E. coli DH-5α cells were transformed with the ligation product. Plasmids were extracted using an Axyprep Plasmid Miniprep Kit (Axygen Biosciences, CA, USA) and the cloned fragment was confirmed by DNA sequencing. E. coli BL21(DE3) cells were then transformed with the resulting plasmid and induced to express the recombinant protein with isopropyl-thio-ß-Dgalactoside (IPTG). The recombinant PruR (rPruR) were Niaffinity purified from E. coli soluble fraction (Cheng et al., 2006

Construction of lacZ Fusions
For reporter assay in E. coli, lacZ fusions were constructed as described (Cheng et al., 2008). Briefly, DNA fragments were amplified by PCR using specific primers (Table S3) and inserted upstream of the promoter-less lacZ gene in pACYC184 (New England Biolabs, MA, USA). BL21(DE3) strain containing pET-28a(+) encoding rRruR, or pET-28a(+) alone (negative control) was transformed with the lacZ fusion constructs. After inducing the recombinant protein with 0.1 mM IPTG at 37 • C for 2 h, β-galactosidase activity was measured as described previously (Wang et al., 2007). Recombinant protein expression was confirmed by Western blot analysis using the anti-His-tag antibody (Sigma, MO, USA).
For reporter assay in P. aeruginosa, DNA fragment was amplified by PCR with specific primers (Table S3) and inserted upstream of the promoter-less lacZ gene in pDN19lacZΩ. P. aeruginosa strains were transformed with the lacZ fusion construct or pDN19lacZΩ vector (negative control). βgalactosidase activity was measured as described (Weng et al., 2016).

DNase I Footprint Analysis
DNase I footprint analysis was performed as described with minor modification (Zianni et al., 2006). A 184-bp DNA fragment upstream of the putA gene was amplified by PCR with primers shown in Table S3, except that the forward primer was labeled with 6-carboxyfluorescein (FAM). The FAM-labeled probe (300 ng) was incubated with 1 or 2 µg rPruR or 2 µg BSA (negative control) under the conditions described previously. Based on the results of DNase I (Takara, Dalian, China) optimization experiments, 0.09 U of DNase I was added to each reaction mixture and incubated at 25 • C for 5 min.
The reaction was terminated by heating the mixture at 80 • C for 10 min. The digested DNA fragments were extracted with phenol:chloroform:isoamyl alcohol (25:24:1) and precipitated with ethanol. The pellets were dissolved in 20 µl water. The sequences were then analyzed with Peak Scanner software v1.0 (Applied Biosystems, CA, USA) to convert the DNase I digestion maps into sequencing data to identify the exact sequences that were protected.

Proline Concentration Assay
Proline concentration in bacterial cells was determined as described (Shabnam et al., 2016). Harvested bacterial cells were resuspended in 150 µl water and boiled for 10 min. After centrifugation at 14,000 rpm for 5 min at 4 • C, 100 µl of the supernatant was incubated with 100 µl of acid-ninhydrin (0.25 g ninhydrin dissolved in 6 ml glacial acetic acid and 4 ml 6 M phosphoric acid) and 100 µl of glacial acetic acid at 100 • C for 1 h. The reaction was stopped by incubation on ice, and the mixture was extracted with 400 µl toluene. The toluene phase was separated, and the OD 520 was measured to determine the concentration of proline in the extract.

Quantitative RT-PCR
Total RNA was isolated from bacteria using an RNeasy Minikit (Tiangen Biotech, Beijing, China). The cDNA was synthesized from total RNA using random primers and PrimeScript Reverse Transcriptase (TaKaRa). For quantitative PCR, cDNA was mixed with 4 pmol of forward and reverse primers (Table S3) and SYBR Premix Ex TaqTM II (TaKaRa) in a total reaction volume of 20 µl. The results were determined using a CFX Connect Real-Time system (Bio-Rad).

Statistical Analysis
Statistical analyses were performed with the GraphPad Prism software. Survival data were analyzed with the log-rank (Mantel-Cox) test. Other results were analyzed by the Student's t-test (two-tailed).

PutA Is Essential for P. aeruginosa Infection in a Mouse Acute Pneumonia Model
Given that little information is currently available about the role of the proline utilization system in P. aeruginosa infection, we sought to define the function of PutA by assessing the requirement of PutA for P. aeruginosa virulence. We infected mice with a wild type PAK strain (Weng et al., 2016), a putA mutant strain or a putA/putA complemented strain in an acute pneumonia model. Compared to the wild type PAK strain, the putA mutant strain caused significantly lower mortality (P < 0.01; Figure 2). Complementation with a putA gene driven by its native promoter restored the bacterial virulence (Figure 2). The growth rates of these three strains showed no differences when cultured on LB broth or MMP medium supplemented with glucose and (NH 4 ) 2 SO 4 ( Figure S1). These results indicated that FIGURE 2 | Role of PutA in P. aeruginosa infection in a mouse acute pneumonia model. Mice were inoculated intranasally with 4 × 10 7 CFU bacteria of indicated strains. The mice were monitored for 5 days after the infection. The data were from 13 mice per strain. *p < 0.01, compared to the wild type PAK strain or the putA/putA complemented strain by log-rank (Mantel-Cox) test.
PutA is required for the virulence of P. aeruginosa in the murine acute pneumonia model.

P. aeruginosa putA Mutant Strain Is More Sensitive to Oxidative Stress Compared to the Wild Type PAK Strain in Vitro
It has been shown previously that bacterial PutA are linked to the resistance against oxidative stress (Zhang et al., 2015). ROS produced by neutrophils is one of the major mechanisms of host defense against bacteria (Shaver and Hauser, 2004;Ziltener et al., 2016). To test the hypothesis that PutA in P. aeruginosa is required for the ability of bacteria to cope with oxidative stress, we treated the wild type PAK strain, the putA mutant strain or the putA/putA complemented strain with 0.3% H 2 O 2 at 37 • C for 15 min. Survival rate of the putA mutant strain was significantly lower than that of wild type PAK strain (Figure 3). Complementation of putA gene rescued the mutant strain from the increased sensitivity to H 2 O 2 -mediated killing (Figure 3). These experiments indicated that proline utilization is involved in bacterial defense against oxidative stress, which aids bacteria to establish infection in hosts.

PruR Activates putA Expression
Since PutA is essential for P. aeruginosa virulence, we then investigated the regulation of putA expression. The only available information is that in a pruR mutant P. aeruginosa strain, putA expression was down-regulated, suggesting putA expression is under the control of PruR (Nakada et al., 2002). We examined whether PruR transcriptionally activates the putA expression. Fragments (F1, F2, F3) derived from putA promoter region with various lengths were inserted upstream of a promoterless lacZ gene in a pACYC184 plasmid ( Figure 4A). The fusion plasmids were transformed into E. coli BL21(DE3) strain harboring a plasmid (pPruR) expressing recombinant FIGURE 3 | Roles of PutA in bacterial resistance to oxidative stress. Indicated strains were treated with 0.3% H 2 O 2 at 37 • C for 15 min and the numbers of live bacteria were determined by serial dilution and plating. The values reflect bacterial numbers of each strain relative to that of wild type PAK strain. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *P < 0.01 compared to the wild type PAK strain or the putA/putA complemented strain by student's t-test.
P. aeruginosa PruR (rPruR) or an empty pET-28a(+) vector. Induction of rPruR by IPTG resulted in a significant increase in β-galactosidase activity in the strain harboring F1-lacZ fusion or F2-lacZ fusion, but not in the strain harboring F3-lacZ fusion ( Figure 4B), indicating that PruR binds to F1 and F2 fragments of putA promoter and trans-activates putA expression. The expression of rPruR was confirmed by Western blot analysis ( Figure 4B). Also we examined whether PruR activates the expression of PA0781 gene, which is located upstream of the putA in diverging direction. Our β-galactosidase assay results showed PruR could not activate the expression of PA0781 gene ( Figure S2).

PruR Binds Directly to the putA Promoter
To examine whether PruR directly binds to the putA promoter and determine the binding site, we preformed EMSA using fragments derived from different positions in the putA promoter region ( Figure 4A). Upon incubation with rPruR, shifted bands were detected for fragment F1, F2, F4, and F6, but not for F3 and F5, indicating that PruR bound to the putA promoter region and the binding site was located in F6, whose location is from −79 to −27 counting from the transcriptional start site (Figures 4C, 5B).
Next we performed DNase I protection assays to reveal the DNA sequence to which PruR binds. The protected region of the sense strand was determined by comparing the sequence of a DNA sample protected by 1 or 2 µg rPruR to the sequence of an unprotected DNA sample incubated with 2 µg BSA ( Figure 5A). The region protected by rPruR was located from −71 to −41 counting from the putA transcriptional start site, which was in F6 fragment and right upstream of −35 box ( Figure 5B). To confirm the PruR binding site, fragments F7 (containing the binding site) and F8 (without the binding site) were inserted upstream of a promoter-less lacZ gene in a pACYC184 plasmid (Figure 5B). Also we mutated CC (−71, −70 from the transcriptional start site) into AT in F7-lacZ fusion (F7MU, Figure 5B). Induction of rPruR by IPTG resulted in a significant increase in β-galactosidase activity in the strain harboring F7-lacZ fusion, but not in the strain harboring F7MU-lacZ fusion or F8-lacZ fusion (Figure 5C).

Proline and Glutamate Are Signals Controlling putA Expression
To determine the signals regulating the putA expression, we constructed reporter plasmid by inserting putA promoter upstream of a promoter-less lacZ gene in a pDN19lacZ plasmid and transformed the resulting fusion construct into the wild type PAK strain, the putA mutant strain or the putA/putA complemented strain. These strains were cultured in LB broth at 37 • C for overnight. Due to the lack of proline utilization, proline concentration was significantly higher in the putA mutant strain than that in the wild type PAK strain or the putA/putA complemented strain (Figure 6A). At the same time, the putA expression was activated as β-galactosidase activity was significantly increased in the putA mutant strain (Figure 6B), suggesting the high proline concentration is an activation signal for putA expression.
To confirm the effect of proline on the putA expression, the wild type PAK strain was cultured in MMP medium supplemented with different concentration of proline. The addition of proline resulted in increased levels of β-galactosidase activity in a dose-dependent manner ( Figure 6C and Figure S3). To further investigate the regulation of the putA expression, the effect of glutamate, which is the product of PutA enzyme activity, was examined. The high level of β-galactosidase activity induced by proline was repressed by the addition of glutamate ( Figure 6C). However, adding proline into the binding reaction did not increase the affinity of rPruR to the putA promoter ( Figure 6D). Meanwhile, glutamate released rPruR from the putA promoter in a dose-dependent manner ( Figure 6D). The expression of pruR and putA was highly up-regulated in MMP medium supplemented with 25 mM proline, but did not show significant changes in MMP medium supplemented with 25 mM glutamate ( Figure 6E). The expression of pruR showed no significant difference in MMP supplemented with 25 mM proline and in MMP supplemented with 25 mM proline and 25 mM glutamate. However, the expression of putA in MMP supplemented with both proline and glutamate was significantly lower than that in MMP supplemented with proline ( Figure 6E). Furthermore, the expression of pruR was highly up-regulated in the putA mutant strain compared to that of the wild type PAK strain or the putA/putA complemented strain ( Figure 6F). All together, these results indicated that are shown. Three fragments, F1-3, corresponding to various regions upstream of putA gene were inserted upstream of the promoter-less lacZ gene in pACYC184 for reporter gene assay. The direction of each fragment is indicated by an arrow. Six fragments, F1-6, corresponding to various regions upstream of putA gene were used for EMSA. Designation and length of each fragment is shown on the left. (B) PruR activates the putA expression. β-galactosidase assays were used to measure the transcriptional activities of lacZ reporter fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *P < 0.01 compared to no IPTG induction or the empty pET-28a(+) vector by student's t-test. Western blot analyses of samples from the β-galactosidase assays were performed using an anti-His-tag antibody to verify the expression of rPruR. The blots below the graphs are representative blots for three independent experiments. The position of rPruR is indicated by an arrowhead. (C) PruR binds to the putA promoter. Lane 1, DNA probe (50 ng); lane 2, DNA probe incubated with rPruR (2 µg). Shifted bands are indicated by arrowheads. The length (bp) of each fragment is shown above each panel. The gel was stained with ethidium bromide.
high concentration of proline up-regulates pruR expression, which in turn activates putA expression, and high glutamate concentration reduces the affinity of PruR to putA promoter, then turns off the expression of putA. We also examined whether PruR is auto-regulated. Our EMSA and β-galactosidase assay results shown in Figure S2 demonstrated that PruR neither bound to its own promoter region, nor activated its expression.

PruR Affects P. aeruginosa Virulence Through the Regulation of the putA Expression
To evaluate the roles of PruR in the virulence of P. aeruginosa, we infected mice with the wild type PAK strain, a pruR mutant strain or a pruR/pruR complemented strain in the acute pneumonia model. Compared to the wild type PAK strain, , and F8 were inserted upstream of the promoter-less lacZ gene in pACYC184 for reporter gene assay. Designation and length of each fragment is shown on the left. (C) PruR binds to the protected region and activates the putA expression. β-galactosidase assays were used to measure the transcriptional activities of lacZ reporter fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *P < 0.01 compared to no IPTG induction or the empty pET-28a(+) vector by student's t-test. Western blot analyses of samples from the β-galactosidase assays were performed using an anti-His-tag antibody to verify the expression of rPruR. The blots below the graphs are representative blots for three independent experiments. The position of rPruR is indicated by an arrowhead.
the pruR mutant strain caused significantly lower mortality (P < 0.01; Figure 7A). Complementation with a pruR gene driven by its native promoter restored the bacterial virulence ( Figure 7A). The growth rates of these three strains showed no differences when cultured on LB broth or MMP medium supplemented with glucose and (NH 4 ) 2 SO 4 ( Figure S4). These results indicated that PruR is required for the virulence of P. aeruginosa in the murine acute pneumonia model.
We further examined the resistance of the pruR mutant strain to oxidative stress. The wild type PAK strain, the pruR mutant strain or the pruR/pruR complemented strain was treated with 0.3% H 2 O 2 at 37 • C for 15 min. Survival rate of the pruR mutant strain was significantly lower than those of the wild type PAK strain and the pruR/pruR complemented strain ( Figure 7B). Overexpression of a putA gene rescued the pruR mutant strain from the increased sensitivity to H 2 O 2mediated killing (Figure 7B). These experiments indicated that the P. aeruginosa PruR affects bacterial survival under oxidative stress through the regulation of the putA expression.

DISCUSSION
In this study, we demonstrated that PutA is required for the virulence of P. aeruginosa in a murine acute pneumonia model. Further experiments demonstrated that PruR directly binds to FIGURE 6 | Proline and glutamate are the signals regulating the putA expression. (A) Proline concentration in bacterial cells. Indicated strains were cultured in LB broth for overnight. Proline concentration in bacterial cells was measured. The values reflect proline amount relative to total bacterial protein amount. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *P < 0.01 compared to wild type PAK strain, or the putA/putA complemented strain by student's t-test. (B) The putA expression is up-regulated in the putA mutant strain. β-galactosidase assays were used to measure the transcriptional activities of lacZ reporter fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *P < 0.01 compared to wild type PAK strain, or the putA/putA complemented strain, or the putA mutant strain harboring pDN19lacZΩ vector by student's t-test. (C) The effects of proline and glutamate on the putA expression. The wild type PAK strain was cultured in MMP medium supplemented with different concentrations of proline and glutamate at 37 • C for overnight. β-galactosidase assays were used to measure the transcriptional activities of the putA promoter-lacZ fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. (D) EMSA for dose-dependent effects of proline and glutamate on the binding of PruR to the putA promoter. DNA fragment F4 (50 ng) was incubated alone (-) or with 2 µg rPruR (PR) or with 2 µg rPruR and proline or glutamate at different concentrations (lanes 1-3: 25, 50, and 100 mM, respectively). Shifted bands are indicated by an arrowhead. Black triangles show proportions of concentration of proline or glutamate. (E) The expression of pruR and putA in different media. RNA samples for quantitative RT-PCR were prepared from the wild type (Continued) FIGURE 6 | PAK strain cultured in different media at 37 • C for overnight. -, MMP medium only; Pro, MMP medium supplemented with 25 mM proline; Glu, MMP medium supplemented with 25 mM glutamate; Pro+Glu, MMP medium supplemented with 25 mM proline and 25 mM glutamate. The expression of pruR or putA is normalized against that of 16S rRNA. Values relative to the amount cultured in MMP medium are shown. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *P < 0.01 by student's t-test. (F) The expression of pruR in different strains. RNA samples for quantitative RT-PCR were prepared from indicated strains cultured in LB broth at 37 • C for overnight. The pruR expression is normalized against that of 16S rRNA. Values relative to the amount of the wild type PAK strain are shown. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *P < 0.01 compared to the wild type PAK strain or the putA/putA complemented strain by student's t-test.
the putA promoter. High concentration of proline in bacteria upregulates pruR expression. Then PruR activates putA expression. As a feedback regulation, glutamate produced by PutA turns off the putA expression. PruR affected bacterial virulence through the regulation of putA expression. It is for the first time to reveal that the proline utilization plays an important role in the pathogenesis of P. aeruginosa, as well as to describe the genetic regulation of the Put system in P. aeruginosa.
Proline is a multifunctional amino acid playing important roles in carbon and nitrogen metabolism, protein synthesis, and protection against various environmental stresses such as drought, osmotic stress, and oxidative stress (Barnett and Naylor, 1966;Csonka, 1981a;Wood, 1988;Chen and Dickman, 2005;Krishnan et al., 2008;Szabados and Savoure, 2010;Natarajan et al., 2012;Liang et al., 2013). Wild type P. aeruginosa exhibits higher resistance to H 2 O 2 stress than the putA mutant strain. And complementation of putA gene could restore oxidative stress protection. These results indicate that proline also provides resistance against oxidative stress in P. aeruginosa which is a PutA dependent phenomenon.
OxyR plays important roles in the regulation of oxidative stress responsive genes in E. coli and P. aeruginosa (Zheng et al., 2001;Jo et al., 2015). Oxidation of two conserved cysteine residues in OxyR results in formation of an intra-molecular disulfide bond, which leads to the binding of OxyR to promoters of target genes, then activates their expression (Jo et al., 2015). In E. coli, OxyR regulates the expression of responsive genes, such as katG (hydroperoxidase I), ahpCF (peroxiredoxin AhpCF), grxA (glutaredoxin I), and trxC (thioredoxin 2), to protect bacteria against ROS (Zheng et al., 2001). Under oxidative stress, proline utilization by PutA changes bacterial intracellular redox condition, which activates OxyR, leading to increased expression of katG, grxA, and trxC (Zhang et al., 2015). In P. aeruginosa, OxyR regulates the expression of katA, katB, ahpB, and ahpCF to defend against host produced ROS (Ochsner et al., 2000;Lee et al., 2005;Heo et al., 2010). Proline protection against oxidative stress of P. aeruginosa might also depend on OxyR regulation. This possibility is currently being investigated by our group.
In the absence of proline, PutA protein of E. coli, S. typhimurium, or P. putida represses the expression of putA gene and the divergent putP gene (Muro-Pastor and Maloy, 1995;Vílchez et al., 2000;Becker and Thomas, 2001). In contrast, P. aeruginosa PutA lacks the DNA binding domain and is unlikely auto-regulated. In A. tumefaciens and R. capsulatus PutR acts as a transcriptional activator of putA. Adding proline into culture medium leads to the transcriptional activation of putA through PutR. Subsequently PutR suppresses putR expression (Keuntje et al., 1995;Cho and Winans, 1996). However, P. aeruginosa does not encode PutR homolog. Our results showed that PruR binds to the putA promoter region and activates the putA expression. Proline did not change the affinity of PruR to the binding site, instead, high intracellular proline concentration up-regulated the pruR expression. Also we found that PruR is not auto-regulated. These results suggest that there might be other unknown mechanisms involved in proline signal detection and pruR regulation, which then controls putA expression.
In B. abortus, the two-component system NtrY/NtrX acts as a redox sensor sensing oxidative tension, and regulates the expression of nitrogen respiration enzymes (Carrica Mdel et al., 2012). In E. chaffeensis, NtrY/NtrX regulates the expression of PutA and GlnA, which converts glutamate to glutamine by nitrogen assimilation (Cheng et al., 2014). P. aeruginosa however does not encode NtrY/NtrX, but NtrB/NtrC, which is the homologous pair to NtrY/NtrX. In E. coli, the sensor kinase NtrB detects the concentration changes of α-ketoglutarate and glutamine through a signal transduction protein P(II) and phosphorylates the cognate response regulator NtrC, which then regulates the expression of nitrogen metabolism genes, including glnA (Ninfa et al., 2000;Dixon and Kahn, 2004;Lilja et al., 2004;Forchhammer, 2008;Hervás et al., 2009). In P. aeruginosa genome, P(II) protein is encoded by glnK gene. Proline is an important nitrogen source for bacterial growth (Wood, 1981;Kohl et al., 1988;Nagata et al., 2003). Proline accumulation might affect the concentration of α-ketoglutarate and glutamine, which can be converted from proline. P(II) might detect these changes and activate pruR expression through the NtrB/NtrC system. Our group is investigating this hypothesis now.
The AraC/XylS family proteins consist of two domains, a nonconserved domain, which seems to be involved in effector/signal reorganization and dimerization, and a conserved DNA binding domain that contains two helix-turn-helix DNA binding motifs (Gallegos et al., 1997). E. coli AraC binds to araFGH promoter, facilitates recruitment of RNA polymerase and isomerization to open complex, and prevents improper binding of RNA polymerase (Johnson and Schleif, 2000). Our results showed that P. aeruginosa PruR binding site was located from −71 to −41 counting from transcriptional start site in the putA promoter region, which is right upstream of the −35 box. Interestingly PruR binding was affected by glutamate concentration. It is possible that PruR initiates putA expression by RNA polymerase recruitment, and the non-conserved domain of PruR senses the intracellular glutamate concentration. Our results showed that high concentration of glutamate did not release PruR from the putA promoter completely, suggesting there might be additional regulatory mechanisms involved. To fully elucidate FIGURE 7 | Role of PruR in P. aeruginosa infection in a mouse acute pneumonia model and bacterial resistance to oxidative stress. (A) Mice were inoculated intranasally with 4 × 10 7 CFU bacteria of indicated strains. The mice were monitored for 5 days after the infection. The data were from 13 mice per strain. *p < 0.01, compared to the wild type PAK strain or the pruR/pruR complemented strain by log-rank (Mantel-Cox) test. (B) Indicated strains were treated with 0.3% H 2 O 2 at 37 • C for 15 min and the numbers of live bacteria were determined by serial dilution and plating. The values reflect bacterial numbers of each strain relative to that of the wild type PAK strain. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. *P < 0.01 compared to the wild type PAK strain, the pruR/pruR complemented strain or the pruR/pUCP20-putA PutA over-expression strain by student's t-test.
the mechanism of PruR regulation on PutA expression, further studies on the interaction among PruR, RNA polymerase, and glutamate are warranted.

AUTHOR CONTRIBUTIONS
ZC, RZ, and XF: conceived and designed the experiments and wrote the paper; RZ, XF, XW, XP, CL, RS, YJ, and FB: performed the experiments; ZC, RZ, XF, SJ, and WW: analyzed the data.

FUNDING
This work was supported by National Science Foundation of China (31741009 and 81670766), Science and Technology Committee of Tianjin (15JCYBJC53900 and 15JCZDJC33000), the State Key Laboratory of Medicinal Chemical Biology (Nankai University) (2017011), and the Fundamental Research Funds for the Central Universities. The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

SUPPLEMENTARY MATERIAL
The Supplementary Material for this article can be found online at: https://www.frontiersin.org/articles/10.3389/fmicb. 2018.00548/full#supplementary-material Table S1 | Bacterial strains used in this study. Table S2 | Plasmids used in this study. Table S3 | Oligonucleotides used in this study. Figure S1 | The growth rate of the putA mutant strain shows no differences to that of the wild type PAK strain or the putA/putA complemented strain. Indicated strains were cultured in (A) LB broth or (B) MMP medium containing 1.0% (w/v) glucose and 0.1% (w/v) (NH 4 ) 2 SO 4 at 37 • C. Figure S2 | PruR does not activate the expression of PA0781 and pruR. (A) PruR does not activate the expression of PA0781 and pruR. β-galactosidase assays were used to measure the transcriptional activities of lacZ reporter fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. Western blot analyses of samples from the β-galactosidase assays were performed using an anti-His-tag antibody to verify the expression of rPruR. The blots below the graphs are representative blots for three independent experiments. The position of rPruR are indicated by arrowheads. (B) PruR does not bind to the pruR promoter. Lane 1, DNA probe (50 ng); lane 2, DNA probe incubated with rPruR (2 µg). The length (bp) of the fragment is shown above the panel. The gel was stained with ethidium bromide. Figure S3 | The effects of proline on the putA expression. The wild type PAK strain was cultured in MMP medium supplemented with different concentrations of proline at 37 • C for overnight. β-galactosidase assays were used to measure the transcriptional activities of the putA promoter-lacZ fusions. Data indicate the means ± standard deviations from three independent experiments performed in triplicate. Figure S4 | The growth rate of the pruR mutant strain shows no difference to that of the wild type PAK strain or the pruR/pruR complemented strain. Indicated strains were cultured in (A) LB broth or (B) MMP medium containing 1.0% (w/v) glucose and 0.1% (w/v) (NH 4 ) 2 SO 4 at 37 • C.