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The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5
Biochemical and Biophysical Research Communications xxx (2017) 1e8
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The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf5 Menggang Li, Jinyong Yan, Yunjun Yan* Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2017 Accepted 7 April 2017 Available online xxx
Pseudomonas lipases are well studied enzymes. However, few studies have been conducted to explore the mechanism underlying the regulation of lipases expression. AlgR, a global regulator, controls the expression of multiple genes, regulates bacterial peristalsis, and participates in the regulation of quorumsensing (QS) system, and so on. In this study, the effect of AlgR on lipase expression was investigated by knocking out the algR and rsmZ genes or overexpressing them. It is found out that AlgR can regulate the expression of lipA at both transcriptional and translational levels, but the transcriptional level was dominant. AlgR is also able to regulate the expression of rsmX/rsmY/rsmZ. Additionally, using algR/rsmZ double gene knock-out, it showed that AlgR could directly bind to the promoter sequence of rsmZ to regulate lipA activity. In conclusion, this study for the first time indicates that AlgR directly binds to rsmZ to regulates the expression of lipA via regulating transcription of rsmZ, and mainly regulates the expression of lipA at transcriptional level in P. protegens Pf-5. © 2017 Elsevier Inc. All rights reserved.
Keywords: P. protegens Pf-5 AlgR rsmZ Regulation
1. Introduction Lipases are one important group of industrial enzymes, widely found in a variety of animals, plants, and microorganisms. However, the main source of commercial lipases is microbe [1,2]. Currently, lipase is widely used in food [3,4], detergent [5,6], pollution control [7], and bio-energy [8,9], as well as in other industrial fields [10,11]. But conventional breeding and optimization of medium and fermentation conditions may not address the problem of low production of bacterial lipases. Therefore, it is necessary to elucidate the molecular mechanism underlying the regulation of gene expression so as to find more satisfactory solutions to this bottleneck. Previous studies have shown that bacterial lipases expression is regulated by the two-component regulatory system, for example, in Pseudomonas alcaligenes, lipase expression is directly regulated by LipQ-LipR (lipQ/R) [12]. Whereas, in bacteria P. aeruginosa, CbrACbrB (cbrA/B) is a two-component regulator of lipase expression [13]. In addition, las, rhl and other two-component regulatory
* Corresponding author. E-mail address: [email protected] (Y. Yan).
systems also regulate the expression of quorum-sensing (QS) system and indirectly regulate lipase expression [14e16]. A study also showed that in Pseudomonas protegens Pf-5, the GacS-GacA twocomponent regulatory system mediated lipA expression via rsmE rather than rsmA [17]. However, in P. protegens Pf-5, whether there are other lipase regulators remains to be further studied. On the other hand, algR is a widely studied regulatory gene. Earlier studies found that the two-component regulatory system of AlgZ/AlgR in P. aeruginosa regulates the production of alginate [18,19]. As the studies progressed, researchers found that AlgR also regulates the expression of many other proteins [20]. In P. aeruginosa, AlgR activates the transcription of the fimUpilVWXY1Y2E operon, regulates the action of the type IV pilus, and affects the twitching motility [21]. Moreover, AlgR was also found to be involved in the expression of some genes in the QS system. For example, AlgR inhibits the transcription of rhl [22]. In addition, AlgR also regulates the expression of some proteins in the T3ss system. It was found that AlgR indirectly regulates the expression of T3ss through two pathways. One pathway is to control Vfr (virulence factor regulator), and the other pathway is through the indirect regulation of rsmA/Y/Z expression to mediate T3ss system [23]. AlgR also modulates rhamnolipid production and motility [24].
http://dx.doi.org/10.1016/j.bbrc.2017.04.034 0006-291X/© 2017 Elsevier Inc. All rights reserved.
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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M. Li et al. / Biochemical and Biophysical Research Communications xxx (2017) 1e8
Nevertheless, the function of AlgR in P. protegens Pf-5 has not been reported, thus it still remains unclear whether algR can regulate the gene expression of lipase. Therefore, in this study the function of algR in P. protegens Pf-5 was systematically investigated and its effect on the regulation of the expression of lipase was further elucidated.
Tek, Doraville, GA, USA) based on the protocols of manufacturer. Synthetic Oligonucleotide primers were ordered through Anygene Biological Technology Co., Ltd., Wuhan, China. Shanghai Sunny Biotechnology Co., Ltd. (Shanghai, China) conducted the DNA sequencing service. Standard methods were used for all molecular biological methods that are not described in detail.
2. Materials and methods
2.2. Gene knockout and complementation of algR and rsmZ in P. protegens
2.1. Bacteria, plasmids, and culture conditions The bacteria and plasmids used in this study are listed in Table 1. P. protegens was cultivated at 28 C and E. coli was incubated at 37 C in LB (solid medium plus 1.5% agar). Antibiotic concentrations of P. protegens and E. coli were as follows: ampicillin 100 mg/ml, gentamicin 50 mg/ml, and kanamycin 40 mg/ml. Sucrose was used at a concentration of 10% when gene knockout was performed using the suicide plasmid pJQ200SK. ONPG (orthonitrophenyl-ß-D-galactopyranoside) was used at 4 mg/ml and IPTG (isopropyl-b-Dthiogalactopyranoside) was used at 0.5 mM. Taqaq (TaKaRa), KOD Plus DNA polymerase (TaKaRa), RNA reverse transcriptase, restriction endonucleases, DNA ligase, DNA gel extraction, and plasmid preparation were performed using commercial kits (Omega Bio-
The upstream sequences (1000bp and 560bp) of the initiation sites of algR and the rsmZ gene and the downstream sequences of the termination sites (900bp and 400bp, respectively) were ligated by fusion PCR, digested with XbaI/BamHI, and then ligated with the suicide plasmid pJQ200SK [25] to construct the pJQDalgR and pJQDrsmZ. The knockout vectors pJQDalgR and pJQDrsmZ were then transferred into P. protegens, respectively, and mutants were selected on 10% sucrose LB plates. P. protegens integrated with plasmid pJQ200SK was unable to grow on 10% sucrose plates. Thus, the double-recombination of the strains lost the plasmid pJQ200SK. PCR and sequencing were then used to determine the success of the mutants with algR and rsmZ knockout, which were named as Pf6003 and Pf6285, respectively. The algR and rsmZ double gene
Table 1 List of the bacteria and plasmids used in this study. Strain/plasmid
plasmid Description
Reference or source
E. coli BL21(DE3) BL/pET-28a BL/pET-algR P. protegens Pf-5 Pf6003 Pf6285 Pf6100 Pf-5F3 Pf-5F4 Pf6003F3 Pf6003F4 Pf6285F3 Pf6285F4 Pf6100F3 Pf6100F4 Plasmids Triparental mating, pRK2073 pJQ200SK pJQDalgR Overexpression pBBR1MCS-5 pBBR1Km pBBRKm pBBR-algR pBBR-rsmX pBBR-rsmY pBBR-rsmZ pBBRK-algR pBBRK-rsmX pBBRK-rsmY pBBRK-rsmZ pET-28a
Top10 mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 araD139(araleu)7697galU galK rpsL(Strr) endA1 nupG F ompT hsd SB(rB mB) dcm gal(DE3) BL21(DE3) with pET-28a; Kmr BL21(DE3) with pET- algR; Kmr
Invitrogen Novagen This study This study
Rhizosphere isolate; Apr DalgR derivative of Pf-5; Apr DrsmZ derivative of Pf-5; Apr DalgR/D rsmZ derivative of Pf-5; Apr pJQ003 conjugated into Pf-5; Gmr pJQ004 conjugated into Pf-5; Gmr pJQ003 conjugated into Pf6003; Gmr pJQ004 conjugated into Pf6003; Gmr pJQ003 conjugated into Pf6285; Gmr pJQ004 conjugated into Pf6285; Gmr pJQ003 conjugated into Pf6100; Gmr pJQ004 conjugated into Pf6100; Gmr
[37] This This This This This This This This This This This
Helper plasmid for triparental mating; Spr
[26]
Suicide vector with sacB counterselectable marker used for homologous recombination; Gmr pJQ200SK carrying a 2.747-kb XbaI/BamHI insert with a deletion in the coding region of algR; Gmr
[25] This study
Broad-host-range vector; Gmr NcoI-BgIII-digested kanamycin resistance cassettesubcloned in pBBR1MCS-5 digested with the same endonucleases pBBR1Km with a 1280-bp BamHI-XbaI fragment harboring lacIq-Plac; Kmr pBBR1MCS-5 with a 762-bp BamHI-HindIII fragment harboring the codingregion of algR; Kmr pBBR1MCS-5 carrying a 183-bp BamHI-HindIII fragment harboring the coding region of rsmX; Kmr pBBR1MCS-5 carrying a 135-bp BamHI-HindIII fragment harboring the coding region of rsmY; Kmr pBBR1MCS-5 carrying a 145-bp BamHI-HindIII fragment harboring the coding region of rsmZ; Kmr pBBRKm with a 752-bp BamHI-HindIII fragment harboring the coding region of algR; Kmr pBBRKm carrying a 183-bp BamHI-HindIII fragment harboring the coding region of rsmX; Kmr pBBRKm carrying a 135-bp BamHI-HindIII fragment harboring the coding region of rsmY; Kmr pBBRKm carrying a 145-bp BamHI-HindIII fragment harboring the coding region of rsmZ; Kmr Expression vector carrying an N-terminal His tag thrombin-T7 tag configuration plus an optional C-terminal His tag sequence; Kmr pET-28a carrying a 762-bp NdeI-HindIII fragment harboring the coding region of algR; Kmr
[27] This study This study This study This study This study This study This study This study This study This study Novagen
pET-algR Plasmid-borne lacZ fusion pBBR003 pBBR1MCS-5 derivative with a translational lipA-lacZ fusion; Gmr pBBR004 pBBR1MCS-5 derivative with a transcriptional lipA-lacZ fusion; Gmr Chromosome-borne lacZ fusion pJQ003 pJQ200SK derivative with a translational lipA-lacZ fusion; Gmr pJQ004 pJQ200SK derivative with a transcriptional lipA-lacZ fusion; Gmr
study study study study study study study study study study study
This study This study This study This study This study
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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knockout was achieved using the same method. pJQDrsmZ was transferred into Pf6003, and then the rsmZ gene knockout mutant was selected. algR and rsmZ double gene knockout mutant was named as Pf6100. In the present study, pRK2073 [26] was used as a helper plasmid, and the plasmid was transferred into P. protegens using tri-parental hybridization. To construct a complementation strain of algR gene after knock-out, recombinant expression plasmid pBBR-algR was created. The plasmid pBBR-algR was constructed by ligating the 762bp base sequence containing the algR gene and the promoter sequence into the Pseudomonas-E. coli shuttle plasmid pBBR1MCS-5 [27] after BamHI/HindIII digestion. pBBR-algR was then transferred into the Pf6003 mutant to generate the complementation strain Pf6003/pBBR-algR. The plasmid pBBRK-algR was constructed by ligating the 762bp base sequence containing the algR gene and the promoter sequence into plasmid pBBRKm after BamHI/HindIII digestion. The same methods were utilized to construct pBBR-rsmX, pBBR-rsmY, pBBR-rsmZ, pBBRKrsmX, pBBRK-rsmY, and pBBRK-rsmZ. Pf6285/pBBR-rsmZ and Pf6100/pBBR-rsmZ were also constructed using the same method.
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2.5. Expression and purification of AlgR protein The 765bp DNA fragment containing the entire algR ORF sequence (747bp) was amplified using PCR with Pf-5 as a template. After digestion with NdeI-HindIII, the fragment was ligated with the expression vector pET28a to construct the algR expression vector pET28a-algR (Table 1). PET28a-algR was transferred to E. coli BL21 (DE3) and cultured in LB liquid medium at 37 C until OD z 0.8. 0.5 mM IPTG was added and transferred to 16 C for 20 h, and then centrifuged, the cells were harvested and resuspended in nickel A buffer (20 mM imidazole, 300 mM NaCl, 25 mM Tris-HCl, pH 8.0), which was supplemented with 1 mg/ml1 leupeptin, 1 mg/ml1 aprotinin and 50 mM phenyl methyl sulfonyl. Cell suspension solution was then shaken at 4 C for 30 min slowly, and ultrasonic cell disruptor was used to lyse cells. The cell lysate was loaded onto a nickel-nitrilotriacetic acid agarose column (GE Healthcare). The column was then washed twice using 5 ml nickel buffer, and then AlgR protein was eluted with the elution buffer containing 500 mM imidazole. And then, the purified AlgR protein was stored in a buffer containing 20 mM Tris-HCl), 200 mM NaCl, 1 mM dithiothreitol (DTT), and 1 mM EDTA.
2.3. Construction of promoter-lacZ reporter gene In order to study the regulation of lipase by algR, the reporter gene of promoter-lacZ was constructed by fusing lipase lipA promoter sequence with lacZ sequence. We used PCR to amplify from lacZ from E. coli BL21 (DE3) genomic DNA. The amplicons of 'lacZ (Bp 22 to 3110 relative to translation start site) does not have SD sequence and the first seven codons. The wild-type lacZ (bp 18 to 3110 relative to translation start site) has SD sequence. After being digested by BamHI and HindIII, 'lacZ and lacZ were then cloned into plasmid pBBR1MCS-5 to generate translational fusion plasmid (pBBR1MCS-5), PBBR001, and transcriptional fusion plasmid pBBR002, respectively. We used PCR to amplify from lipA. After digested with KpnI and HindIII, the amplicons of lipA (bp 613 to 18 relative to translation start site) were cloned into plasmid pBBR001 to generate plasmid pBBR003. Similarly, amplicons lipA' (bp 613 to 12 relative to translation start site) were cloned into plasmid pBBR002 to generate plasmid pBBR004 (Table 1). Finally, after pBBR003 and pBBR004 being digested with SphI and BamHI, lipA-lacZ、lipA'-'lacZ was cloned into plasmid pJQ200SK to generate plasmids pJQ003, pJQ004, respectively (Table 1).
2.4. RT-PCR analysis P. protegens was cultured to an OD600 of approximately 5.5 and RNA was then extracted by an RNA kit (CWBIO, Beijing, China). Total RNA (2ug) was reverse transcribed with random hexamer primers using a Thermo Scientific Revert Aid first-strand cDNA synthesis kit. An ABI 7500 Real Time PCR machine (Applied Biosystems, Foster City, CA, USA) and its default program (2 min at 50 C and 10 min at 95 C followed by 40 cycles at 94 C for 15 s, and at 60 C for 60 s) were employed for qRT-PCR with a reaction mixture volume of 20 mL in an optical 96-well plate. 10 mL of SYBR Green Master Mix (Roche), 10 pM of each primer, 10 ng of final cDNA and 6.4 mL of RNase-free water were added to the reaction mixture. A control was also included in each plate with 2.0 mL of RNase-free water as a template. Three technical replicates were contained in each plate. Specificity verification of the PCR amplification dissociation and the PCR efficiency curves were determined for each candidate reference gene prior to the qRT-PCR evaluation of these genes in Pf-5. Using rpoD as an internal reference, the differences of mRNA expression were then determined.
2.6. Electrophoretic mobility shift assay Electrophoretic mobility shift assay (EMSA) analysis was conducted with the Thermo Scientific Light Shift Chemiluminescent DNA EMSA kit. Oligonucleotide probes were first synthesized, labeled using a Biotin 30 End DNA Labeling Kit (Thermo Scientific) and annealed. The binding reactions for probes and protein were carried out based on the manufacturer's protocol. Then, proteineDNA complexes were separated on native polyacrylamide gels (6%). Finally, EMSA Kit was used to detect the biotin-labeled probes. 2.7. b-Galactosidase activity assay The ß-galactosidase activity assay was based on a method of Miller. In brief, the ß-galactosidase activity was normalized to the optical density at 600 nm (OD 600) of the bacterial culture in Miller units. 0.1 mM isopropyl-ß-D -thiogalactopyranoside (IPTG) was added to cultures to induce expression of strains containing pBBR1MCS-5 or pET-28a derivatives.
2.8. Lipase activity assay Since LipA is an intracellular lipase [28], the activity of LipA is measured as the activity of whole cell lipase. The bacterial samples were prepared according to the method provided in the literature and the activity of Lipase was determined by pNPC, which contains 2.9 ml of Tris-HCl Buffer (50 mM, pH ¼ 9.0) and 30 ml of pNPC (10 mM pNPC in acetonitrile). The mixture was preheated at 55 C for 5 min and then 70 ml of the cell sample was added. The reaction was centrifuged at 12,000 rpm for 2 min at 4 C and the supernatant was taken for OD410 absorbance. The amount of enzyme needed to release 1 mmol of p-nitrophenol/minute by 1.0 ml sample with OD600 of 1.0 was defined as one unit of lipase activity. The activity of the lipase was expressed as U/ml OD600. 2.9. Statistical analysis All experiments were performed in triplicate, and the data were analyzed by unpaired Student's t tests, and differences with a P value of 0.05 were considered statistically significant.
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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3. Results 3.1. AlgR regulates the expression of lipA at the transcriptional level In P. protegens Pf-5, there is no classical QS regulatory system [29]. So far, the regulation of lipase expression is still not well studied. In order to further understand the relationship between algR and lipA, we conducted algR gene knockout, and constructed algR gene knockout mutant, Pf6003. The effect of algR on lipase expression was analyzed by measuring the activities of b-galactosidase and the whole cell lipase. The results were presented in Fig. 1A. It showed that the growth of Pf-5 was not altered by the knockout of algR. The growth curves of the wild-type, mutant, and complementation strains were consistent, indicating that the growth of the bacteria was not changed after knockout of algR gene. However, by measuring the lipase activity and b-galactosidase activity of the wild and mutant strains, it was found out that the activity of the whole cell lipase was significantly decreased after knockout of algR (Fig. 1B). To investigate whether AlgR regulates
lipA expression through mediating lipA promoter activity, we constructed lipA-lacZ, as well as fusion reporter strains of lipA'-'lacZ, Pf5F3, Pf-5F4 and Pf6003F3, Pf6003F4, respectively. The results were given in Fig. 1C. As it showed that the activity of b-galactosidase of the mutant Pf6003 was lower than that of the wild-type Pf-5, both at the transcriptional level and at the translation level. While the alteration in transcriptional level was more obvious, indicating that AlgR mainly control lipA promoter activity in transcriptional level, then regulating the expression of lipA gene. On the contrary, by measuring lipase and b-galactose activities after overexpression of AlgR in complementation mutant strains, it was found out that the activities of lipase and b-galactosidase were restored to the level of the wild-type bacteria (Fig. 2A and B). Furthermore, RT-PCR results showed that the expression of lipA in the mutant Pf6003 was also lower than that in the wild strain (Fig. 1D). These results indicate that AlgR regulates the expression of by affecting the activity of lipA promoter. That is to say, AlgR regulates the expression of lipA at both transcriptional level and translation level, but mainly at the transcriptional level.
Fig. 1. Effects of algR mutation on lipA expression. A. Growth curve of Pseudomonas sp. Overnight culture of bacteria were inoculated in 50 ml LB medium. The initial OD value was adjusted to about 0.1. Bacteria culture was placed in 200 rpm shaker at 28 C, and OD value was determined once every 2 h. B. Relative activity of whole cell lipase in wild-type (Pf-5) and algR mutant (Pf6003). The whole cell lipase activity was measured after the bacteria were cultured in a 50 ml LB medium to a stationary phase. C. b-galactosidase activity in wild-type (Pf-5) and algR mutant (Pf6003) at transcriptional level (Pf-5F4, Pf6003F4) and translational level (Pf-5F3, Pf6003F3). The bacteria were incubated in a 50 ml LB medium to a stationary phase and the enzyme activity of b-galactosidase was determined. D. RT-PCR of relative quantification of lipA expression in Pf-5 and Pf6003. The mRNA expression of lipA gene was measured after the bacteria were incubated to the stationary phase. All experiments were performed in triplicate, and the mean values ± standard deviations are indicated.*, P < 0.05 compared with the control.
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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whether AlgR regulated the expression of lipA by rsmX/rsmY/rsmZ. We first expressed the AlgR protein through the purified expression system of peT-28a, and then explored whether AlgR protein bound to the rsmX/rsmY/rsmZ promoter sequence using EMSA (Fig. 4C). The result showed that AlgR protein could bind directly to the promoter region of rsmZ, but not to the promoter sequences of rsmX and rsmY. However, the expression of rsmX/rsmY was also decreased in combination with the expression of rsmX/rsmY/rsmZ, indicating that the expression of rsmX/rsmY/rsmZ was altered by AlgR protein. However, it had greater effect on rsmZ. In addition, the results of overexpression of rsmX, rsmY, rsmZ (Fig. 3A and B) was consistent with the results of EMSA, further demonstrating that AlgR may regulate the expression of lipA by regulating rsmZ. 3.4. AlgR regulates lipA expression through rsmZ To further determine whether AlgR affects the expression of lipA by rsmZ, we knocked out rsmZ in the wild-type Pf-5 and the mutant Pf6003 to construct rsmZ mutant strains Pf6285 (Pf-5 (DrsmZ)) and Pf6100 (DalgR/DrsmZ). We hypothesize if AlgR regulates the expression of lipA via rsmZ, LipA activity will be robustly decreased after knockout of algR and rsmZ. If we overexpressed rsmZ in algR mutant pf6003, the LipA activity should be partially restored. As shown in Fig. 4A and B. The results are consistent with our hypothesis. The activity of b-galactosidase and the activity of whole cell lipase are significantly lowered after double knock-out of algR and rsmZ than single knockout of algR. Additionally, overexpression of rsmZ in the algR mutant really restored LipA enzyme activity to the wild-type level. Moreover, the activity of LipA was significantly increased after overexpression of rsmZ. These results all suggests that AlgR binds to rsmZ to directly regulate the expression of lipA. 4. Discussion Fig. 2. Effects of algR overexpression on expression of lipA. A. Relative activity of whole cell lipase in wild-type (Pf-5) and algR mutant (Pf6003). The whole cell lipase activity was measured after the bacteria were cultured in a 50 ml LB medium to a stationary phase. Pf-5/pBBR, wild-type with pBBRM1CS-5; Pf-5/pBBR-algR, wild-type overexpressing algR; Pf6003/pBBR, algR mutant with pBBRM1CS-5; Pf-5/pBBR-algR complementary algR mutant. B. Influence of algR overexpression on expression of a chromosome-borne lipA-lacZ Transcriptional fusion in different strains. Bacteria were cultured in 50 ml LB medium to the stationary phase. The enzyme activity of bgalactosidase was then determined. Pf-5F4/pBBR,wild-type with pBBRM1CS-5; Pf-5F4/ pBBR-algR, wild-type overexpressing algR; Pf6003F4/pBBR, algR mutant with pBBRM1CS-5; Pf-5F4/pBBR-algR, complementary algR mutant. All experiments were performed in triplicate, and the mean values ± standard deviations are indicated. *, P < 0.05 compared with the control.
3.2. AlgR affects rsmX/rsmY/rsmZ expression Previous studies have shown that AlgR modulates the T3ss system through indirect regulation of rsmA/rsmY/rsmZ transcription in P. aeruginosa [23]. However, no report have been conducted in Pf5. The results showed that the transcription level of rsmX/rsmY/ rsmZ was decreased to some extent after algR knockout, and the expression of rsmZ was reduced the most (see Fig. 3C).
3.3. AlgR binds directly to the rsmZ promoter sequence To study the interaction between algR and lipA, AlgR protein was expressed and the binding site of AlgR protein was explored using EMSA. We found that rsmX/rsmY/rsmZ was involved in the regulation of lipase in Pf-5, whereas in P. aeruginosa, AlgR indirectly regulates rsmX/rsmY/rsmZ, which regulates the expression of the related genes. It was found that rsmA/rsmY/rsmZ sequence homology was not high in Pf-5 and P. aeruginosa. Thus, we examined
Recent studies have shown that AlgR is a global transcriptional regulator that controls not only the production of alginate but also the twitching motility [19]. Global transcriptome analysis indicated that it also controlled many other different genes, including those associated with QS, type IV pili, type III secretion system, anaerobic metabolism, cyanide [30], and rhamnolipid production [19,31]. However, the details of how these genes are regulated by AlgR have not been well studied, and especially there are fewer reports on the regulation of lipase expression by AlgR. In order to investigate whether AlgR regulates the expression of lipase in genus Pseudomonas, we knocked out the algR gene in P. protegens Pf-5 and constructed mutant Pf6003. We found that the activity of lipase decreased significantly after knockout of algR (Fig. 1B). Meanwhile, we also overexpressed algR in the wild-type Pf-5 and the mutant Pf6003. It was found that complementation of algR in the mutant Pf6003 restored the activity of lipase and overexpression of algR enhanced the activity of lipase in the wild-type Pf-5 (Fig. 2A and B). In addition, after examining the growth curve, the algR knockout did not alter the growth of the bacteria strain (Fig. 1A). These results indicate that the activity of lipA in Pf-5 is regulated by AlgR. Moreover, our results further showed that AlgR regulates promoter activity both at transcriptional level and translational level (Fig. 1C), but regulates lipase expression mainly at transcriptional level. After knockout of algR, RT-PCR analysis showed that the expression of lipA was also reduced (Fig. 1D). Small RNAs (sRNAs) play an important role in the regulation of gene expression in gram-negative bacteria [32e34]. In P. protegens CHA0, which is similar to Pf-5, GacS/GacA regulates the expression of genes related to secondary metabolism via rsmX, rsmY and rsmZ [35]. In P. aeruginosa PAO1, GacA directly regulates transcription of rsmY and rsmZ to regulate up to hundreds of gene expression [36]. It
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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Fig. 3. Effects of rsmX, rsmY, rsmZ on lipase expression. A. Relative activity of whole cell lipase LipA. Pf-5/pBBR, wild type; Pf-5/pBBR-rsmX, wild-type overexpressing rsmX; Pf-5/ pBBR-rsmY, wild-type overexpressing rsmY; Pf-5/pBBR-rsmZ, wild-type overexpressing rsmZ. The whole cell lipase activity was measured after the bacteria were cultured to the stationary phase. The activity of rsmX, rsmY, rsmZ, and lipA were increased, but the activity of lipase increased more after overexpression of rsmY and rsmZ. B. Influence of rsmX, rsmY, rsmZ overexpression on expression of a chromosome-borne lipA-lacZ transcriptional fusion in WT strains Pf-5. The b-galactosidase activity of each strain was measured in the stationary phase after inoculation into 50 ml LB medium.C. RT-PCR of relative quantification of rsmX, rsmY, rsmZ in Pf-5 and Pf6003. The mRNA expression levels of rsmX, rsmY, and rsmZ were measured after the bacteria were cultured in the stationary phase. All experiments were performed in triplicate, and the mean values ± standard deviations are indicated. *, P < 0.05 compared with the control.
is also reported that AlgR regulates T3ss by indirectly regulating the expression of rsmA/Y/Z [23]. However, the mechanisms of AlgRmediated regulation of rsmA, rsmY, rsmZ are still unclear. In Pf-5, in order to investigate whether rsmX, rsmY and rsmZ are involved in the regulation of lipA expression via algR, we first examined the expression of rsmX, rsmY, rsmZ after knockout of algR (Fig. 3C). It was found that the transcription levels of rsmX, rsmY, rsmZ were all decreased after knockout of algR, and the decrease of rsmZ expression was the highest. On the contrary, the activities of lipase and b-galactosidase were significantly increased after overexpression of rsmX, rsmY and rsmZ in Pf-5 cells, suggesting that rsmX, rsmY and rsmZ all participate in the regulation of the expression of lipA (Fig. 4A and B). Moreover, EMSA results (Fig. 4C) showed that AlgR bound to rsmZ directly, rather than to rsmX, rsmY, to regulate the expression of lipA. To further validate the results, a double-knock out strain of algR/rsmZ and a single knock-out strain of rsmZ were constructed to analyze the regulation of rsmZ via measuring the activities of the whole-cell lipase (LipA) and bgalactosidase (Fig. 4A and B), it was found that the activity of LipA and the promoter activity of lipA were much lower in the doubleknock out strain than that of the single gene knockout.
Additionally, in the algR mutant strain pf6003, supplement expression of rsmZ could restore the activity of the enzyme to a level close to that of the wild strain (Fig. 4A and B), suggesting that algR mainly regulates the expression of lipA by controlling rsmZ transcription. Our results for the first time clearly described the interaction between algR and rsmX, rsmY, and rsmZ. In addition, our previous studies have shown that rsmE, rather than rsmA, directly binds to the lipA promoter to activate lipA translation [17]. Therefore, future research is needed to investigate the interaction between rsmX, rsmY, rsmZ and rsmA, rsmE, and dissect the mechanism underlying the regulation of lipA expression via algR. In conclusion, the present study has demonstrated that, in P. protegens Pf-5, AlgR directly binds to rsmZ to regulates the expression of lipA via regulating transcription of rsmZ, and mainly regulates the expression of lipA at transcriptional level.
Conflict of interest disclosure The authors disclose no potential conflicts of interest.
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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Fig. 4. AlgR regulates the lipase expression of Pf-5 via rsmZ. A. Enzyme activity of b-galactosidase after algR and rsmZ single or double knock-out. Pf-5F4, wild-type strain; Pf6003F4, ⊿algR; Pf6285F4, ⊿rsmZ; Pf6100F4, ⊿algR/DrsmZ; Pf6003F4/pBBRK-rsmZ, rsmZ overexpression in ⊿algR mutant; Pf-5/pBBRK-rsmZ, rsmZ overexpression in wild type. b-galactosidase activity was determined after the bacteria were cultured to the stationary phase. As shown in the results, b-galactosidase activity was much lower after double knockout. In addition, overexpression of rsmZ in the algR mutant enhanced b-galactosidase activity even slightly higher than the wild bacteria, indicating that effects of AlgR on lipase expression can be compensated by the expression of rsmZ. B. Relative activity of whole cell lipase LipA after algR and rsmZ single or double knockout. Pf-5, wild-type strain; Pf6003, ⊿algR; Pf6285, ⊿rsmZ; Pf6100, ⊿algR/DrsmZ; Pf6003/pBBRK-rsmZ, rsmZ overexpression in ⊿algR mutant; Pf-5/pBBRK-rsmZ, rsmZ overexpression in wild type. The whole cell lipase activity was measured after the bacteria were cultured to the stationary phase. As shown in the results, the relative activity of lipase was lower after double knockout than that of one gene knockout, indicating that algR and rsmZ both regulate the expression of lipase. In addition, rsmZ can partially compensate the effects of algR gene knockout. Meanwhile, after overexpression of rsmZ, lipase activity was higher than the wild strains. C. The EMSA results of binding of AlgR to the rsmX/Y/Z promoter sequence. NC (Negetive Control) and PC (Positive Control) were positive and negative control systems to validate EMSA system; rsmX, rsmX promoter sequence; rsmX þ AlgR, rsmX promoter sequence and AlgR protein. rsmY, rsmY promoter sequence; rsmY þ AlgR, rsmY promoter sequence and AlgR protein. rsmZ, rsmZ promoter sequence; rsmZ þ AlgR: rsmZ promoter sequence and AlgR protein. The 1 nM biotin-labeled DNA probe was incubated with purified AlgR protein in 20 ml binding buffer, and the AlgR-DNA complexes and free DNAs were cross-linked to the membrane by a 320-nm UV-light cross-linking instrument. The biotin-labeled bands were detected by the Thermo Scientific chemiluminescent nucleic acid detection module. All experiments were performed in triplicate, and the mean values ± standard deviations are indicated. *, P < 0.05 compared with the control.
Acknowledgements
Transparency document
This work is financially supported by the National Natural Science Foundation of China (No J1103514), the National High Technology Research and Development Program of China (2011AA02A204), the Innovation Foundation of Shenzhen Government (JCYJ20120831111657864), the National Natural Science Foundation of Hubei Province (No. 2015CFA085). Many thanks are indebted to Analytical and Testing Center of Huazhong University of Science and Technology for their valuable assistances in lipase activity measurement.
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.04.034.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2017.04.034.
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The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf5 Menggang Li, Jinyong Yan, Yunjun Yan* Key Laboratory of Molecular Biophysics, Ministry of Education, College of Life Science and Technology, Huazhong University of Science and Technology, Wuhan 430074, China
a r t i c l e i n f o
a b s t r a c t
Article history: Received 15 March 2017 Accepted 7 April 2017 Available online xxx
Pseudomonas lipases are well studied enzymes. However, few studies have been conducted to explore the mechanism underlying the regulation of lipases expression. AlgR, a global regulator, controls the expression of multiple genes, regulates bacterial peristalsis, and participates in the regulation of quorumsensing (QS) system, and so on. In this study, the effect of AlgR on lipase expression was investigated by knocking out the algR and rsmZ genes or overexpressing them. It is found out that AlgR can regulate the expression of lipA at both transcriptional and translational levels, but the transcriptional level was dominant. AlgR is also able to regulate the expression of rsmX/rsmY/rsmZ. Additionally, using algR/rsmZ double gene knock-out, it showed that AlgR could directly bind to the promoter sequence of rsmZ to regulate lipA activity. In conclusion, this study for the first time indicates that AlgR directly binds to rsmZ to regulates the expression of lipA via regulating transcription of rsmZ, and mainly regulates the expression of lipA at transcriptional level in P. protegens Pf-5. © 2017 Elsevier Inc. All rights reserved.
Keywords: P. protegens Pf-5 AlgR rsmZ Regulation
1. Introduction Lipases are one important group of industrial enzymes, widely found in a variety of animals, plants, and microorganisms. However, the main source of commercial lipases is microbe [1,2]. Currently, lipase is widely used in food [3,4], detergent [5,6], pollution control [7], and bio-energy [8,9], as well as in other industrial fields [10,11]. But conventional breeding and optimization of medium and fermentation conditions may not address the problem of low production of bacterial lipases. Therefore, it is necessary to elucidate the molecular mechanism underlying the regulation of gene expression so as to find more satisfactory solutions to this bottleneck. Previous studies have shown that bacterial lipases expression is regulated by the two-component regulatory system, for example, in Pseudomonas alcaligenes, lipase expression is directly regulated by LipQ-LipR (lipQ/R) [12]. Whereas, in bacteria P. aeruginosa, CbrACbrB (cbrA/B) is a two-component regulator of lipase expression [13]. In addition, las, rhl and other two-component regulatory
* Corresponding author. E-mail address: [email protected] (Y. Yan).
systems also regulate the expression of quorum-sensing (QS) system and indirectly regulate lipase expression [14e16]. A study also showed that in Pseudomonas protegens Pf-5, the GacS-GacA twocomponent regulatory system mediated lipA expression via rsmE rather than rsmA [17]. However, in P. protegens Pf-5, whether there are other lipase regulators remains to be further studied. On the other hand, algR is a widely studied regulatory gene. Earlier studies found that the two-component regulatory system of AlgZ/AlgR in P. aeruginosa regulates the production of alginate [18,19]. As the studies progressed, researchers found that AlgR also regulates the expression of many other proteins [20]. In P. aeruginosa, AlgR activates the transcription of the fimUpilVWXY1Y2E operon, regulates the action of the type IV pilus, and affects the twitching motility [21]. Moreover, AlgR was also found to be involved in the expression of some genes in the QS system. For example, AlgR inhibits the transcription of rhl [22]. In addition, AlgR also regulates the expression of some proteins in the T3ss system. It was found that AlgR indirectly regulates the expression of T3ss through two pathways. One pathway is to control Vfr (virulence factor regulator), and the other pathway is through the indirect regulation of rsmA/Y/Z expression to mediate T3ss system [23]. AlgR also modulates rhamnolipid production and motility [24].
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Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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Nevertheless, the function of AlgR in P. protegens Pf-5 has not been reported, thus it still remains unclear whether algR can regulate the gene expression of lipase. Therefore, in this study the function of algR in P. protegens Pf-5 was systematically investigated and its effect on the regulation of the expression of lipase was further elucidated.
Tek, Doraville, GA, USA) based on the protocols of manufacturer. Synthetic Oligonucleotide primers were ordered through Anygene Biological Technology Co., Ltd., Wuhan, China. Shanghai Sunny Biotechnology Co., Ltd. (Shanghai, China) conducted the DNA sequencing service. Standard methods were used for all molecular biological methods that are not described in detail.
2. Materials and methods
2.2. Gene knockout and complementation of algR and rsmZ in P. protegens
2.1. Bacteria, plasmids, and culture conditions The bacteria and plasmids used in this study are listed in Table 1. P. protegens was cultivated at 28 C and E. coli was incubated at 37 C in LB (solid medium plus 1.5% agar). Antibiotic concentrations of P. protegens and E. coli were as follows: ampicillin 100 mg/ml, gentamicin 50 mg/ml, and kanamycin 40 mg/ml. Sucrose was used at a concentration of 10% when gene knockout was performed using the suicide plasmid pJQ200SK. ONPG (orthonitrophenyl-ß-D-galactopyranoside) was used at 4 mg/ml and IPTG (isopropyl-b-Dthiogalactopyranoside) was used at 0.5 mM. Taqaq (TaKaRa), KOD Plus DNA polymerase (TaKaRa), RNA reverse transcriptase, restriction endonucleases, DNA ligase, DNA gel extraction, and plasmid preparation were performed using commercial kits (Omega Bio-
The upstream sequences (1000bp and 560bp) of the initiation sites of algR and the rsmZ gene and the downstream sequences of the termination sites (900bp and 400bp, respectively) were ligated by fusion PCR, digested with XbaI/BamHI, and then ligated with the suicide plasmid pJQ200SK [25] to construct the pJQDalgR and pJQDrsmZ. The knockout vectors pJQDalgR and pJQDrsmZ were then transferred into P. protegens, respectively, and mutants were selected on 10% sucrose LB plates. P. protegens integrated with plasmid pJQ200SK was unable to grow on 10% sucrose plates. Thus, the double-recombination of the strains lost the plasmid pJQ200SK. PCR and sequencing were then used to determine the success of the mutants with algR and rsmZ knockout, which were named as Pf6003 and Pf6285, respectively. The algR and rsmZ double gene
Table 1 List of the bacteria and plasmids used in this study. Strain/plasmid
plasmid Description
Reference or source
E. coli BL21(DE3) BL/pET-28a BL/pET-algR P. protegens Pf-5 Pf6003 Pf6285 Pf6100 Pf-5F3 Pf-5F4 Pf6003F3 Pf6003F4 Pf6285F3 Pf6285F4 Pf6100F3 Pf6100F4 Plasmids Triparental mating, pRK2073 pJQ200SK pJQDalgR Overexpression pBBR1MCS-5 pBBR1Km pBBRKm pBBR-algR pBBR-rsmX pBBR-rsmY pBBR-rsmZ pBBRK-algR pBBRK-rsmX pBBRK-rsmY pBBRK-rsmZ pET-28a
Top10 mcrA (mrr-hsdRMS-mcrBC) 80lacZM15 lacX74 recA1 araD139(araleu)7697galU galK rpsL(Strr) endA1 nupG F ompT hsd SB(rB mB) dcm gal(DE3) BL21(DE3) with pET-28a; Kmr BL21(DE3) with pET- algR; Kmr
Invitrogen Novagen This study This study
Rhizosphere isolate; Apr DalgR derivative of Pf-5; Apr DrsmZ derivative of Pf-5; Apr DalgR/D rsmZ derivative of Pf-5; Apr pJQ003 conjugated into Pf-5; Gmr pJQ004 conjugated into Pf-5; Gmr pJQ003 conjugated into Pf6003; Gmr pJQ004 conjugated into Pf6003; Gmr pJQ003 conjugated into Pf6285; Gmr pJQ004 conjugated into Pf6285; Gmr pJQ003 conjugated into Pf6100; Gmr pJQ004 conjugated into Pf6100; Gmr
[37] This This This This This This This This This This This
Helper plasmid for triparental mating; Spr
[26]
Suicide vector with sacB counterselectable marker used for homologous recombination; Gmr pJQ200SK carrying a 2.747-kb XbaI/BamHI insert with a deletion in the coding region of algR; Gmr
[25] This study
Broad-host-range vector; Gmr NcoI-BgIII-digested kanamycin resistance cassettesubcloned in pBBR1MCS-5 digested with the same endonucleases pBBR1Km with a 1280-bp BamHI-XbaI fragment harboring lacIq-Plac; Kmr pBBR1MCS-5 with a 762-bp BamHI-HindIII fragment harboring the codingregion of algR; Kmr pBBR1MCS-5 carrying a 183-bp BamHI-HindIII fragment harboring the coding region of rsmX; Kmr pBBR1MCS-5 carrying a 135-bp BamHI-HindIII fragment harboring the coding region of rsmY; Kmr pBBR1MCS-5 carrying a 145-bp BamHI-HindIII fragment harboring the coding region of rsmZ; Kmr pBBRKm with a 752-bp BamHI-HindIII fragment harboring the coding region of algR; Kmr pBBRKm carrying a 183-bp BamHI-HindIII fragment harboring the coding region of rsmX; Kmr pBBRKm carrying a 135-bp BamHI-HindIII fragment harboring the coding region of rsmY; Kmr pBBRKm carrying a 145-bp BamHI-HindIII fragment harboring the coding region of rsmZ; Kmr Expression vector carrying an N-terminal His tag thrombin-T7 tag configuration plus an optional C-terminal His tag sequence; Kmr pET-28a carrying a 762-bp NdeI-HindIII fragment harboring the coding region of algR; Kmr
[27] This study This study This study This study This study This study This study This study This study This study Novagen
pET-algR Plasmid-borne lacZ fusion pBBR003 pBBR1MCS-5 derivative with a translational lipA-lacZ fusion; Gmr pBBR004 pBBR1MCS-5 derivative with a transcriptional lipA-lacZ fusion; Gmr Chromosome-borne lacZ fusion pJQ003 pJQ200SK derivative with a translational lipA-lacZ fusion; Gmr pJQ004 pJQ200SK derivative with a transcriptional lipA-lacZ fusion; Gmr
study study study study study study study study study study study
This study This study This study This study This study
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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knockout was achieved using the same method. pJQDrsmZ was transferred into Pf6003, and then the rsmZ gene knockout mutant was selected. algR and rsmZ double gene knockout mutant was named as Pf6100. In the present study, pRK2073 [26] was used as a helper plasmid, and the plasmid was transferred into P. protegens using tri-parental hybridization. To construct a complementation strain of algR gene after knock-out, recombinant expression plasmid pBBR-algR was created. The plasmid pBBR-algR was constructed by ligating the 762bp base sequence containing the algR gene and the promoter sequence into the Pseudomonas-E. coli shuttle plasmid pBBR1MCS-5 [27] after BamHI/HindIII digestion. pBBR-algR was then transferred into the Pf6003 mutant to generate the complementation strain Pf6003/pBBR-algR. The plasmid pBBRK-algR was constructed by ligating the 762bp base sequence containing the algR gene and the promoter sequence into plasmid pBBRKm after BamHI/HindIII digestion. The same methods were utilized to construct pBBR-rsmX, pBBR-rsmY, pBBR-rsmZ, pBBRKrsmX, pBBRK-rsmY, and pBBRK-rsmZ. Pf6285/pBBR-rsmZ and Pf6100/pBBR-rsmZ were also constructed using the same method.
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2.5. Expression and purification of AlgR protein The 765bp DNA fragment containing the entire algR ORF sequence (747bp) was amplified using PCR with Pf-5 as a template. After digestion with NdeI-HindIII, the fragment was ligated with the expression vector pET28a to construct the algR expression vector pET28a-algR (Table 1). PET28a-algR was transferred to E. coli BL21 (DE3) and cultured in LB liquid medium at 37 C until OD z 0.8. 0.5 mM IPTG was added and transferred to 16 C for 20 h, and then centrifuged, the cells were harvested and resuspended in nickel A buffer (20 mM imidazole, 300 mM NaCl, 25 mM Tris-HCl, pH 8.0), which was supplemented with 1 mg/ml1 leupeptin, 1 mg/ml1 aprotinin and 50 mM phenyl methyl sulfonyl. Cell suspension solution was then shaken at 4 C for 30 min slowly, and ultrasonic cell disruptor was used to lyse cells. The cell lysate was loaded onto a nickel-nitrilotriacetic acid agarose column (GE Healthcare). The column was then washed twice using 5 ml nickel buffer, and then AlgR protein was eluted with the elution buffer containing 500 mM imidazole. And then, the purified AlgR protein was stored in a buffer containing 20 mM Tris-HCl), 200 mM NaCl, 1 mM dithiothreitol (DTT), and 1 mM EDTA.
2.3. Construction of promoter-lacZ reporter gene In order to study the regulation of lipase by algR, the reporter gene of promoter-lacZ was constructed by fusing lipase lipA promoter sequence with lacZ sequence. We used PCR to amplify from lacZ from E. coli BL21 (DE3) genomic DNA. The amplicons of 'lacZ (Bp 22 to 3110 relative to translation start site) does not have SD sequence and the first seven codons. The wild-type lacZ (bp 18 to 3110 relative to translation start site) has SD sequence. After being digested by BamHI and HindIII, 'lacZ and lacZ were then cloned into plasmid pBBR1MCS-5 to generate translational fusion plasmid (pBBR1MCS-5), PBBR001, and transcriptional fusion plasmid pBBR002, respectively. We used PCR to amplify from lipA. After digested with KpnI and HindIII, the amplicons of lipA (bp 613 to 18 relative to translation start site) were cloned into plasmid pBBR001 to generate plasmid pBBR003. Similarly, amplicons lipA' (bp 613 to 12 relative to translation start site) were cloned into plasmid pBBR002 to generate plasmid pBBR004 (Table 1). Finally, after pBBR003 and pBBR004 being digested with SphI and BamHI, lipA-lacZ、lipA'-'lacZ was cloned into plasmid pJQ200SK to generate plasmids pJQ003, pJQ004, respectively (Table 1).
2.4. RT-PCR analysis P. protegens was cultured to an OD600 of approximately 5.5 and RNA was then extracted by an RNA kit (CWBIO, Beijing, China). Total RNA (2ug) was reverse transcribed with random hexamer primers using a Thermo Scientific Revert Aid first-strand cDNA synthesis kit. An ABI 7500 Real Time PCR machine (Applied Biosystems, Foster City, CA, USA) and its default program (2 min at 50 C and 10 min at 95 C followed by 40 cycles at 94 C for 15 s, and at 60 C for 60 s) were employed for qRT-PCR with a reaction mixture volume of 20 mL in an optical 96-well plate. 10 mL of SYBR Green Master Mix (Roche), 10 pM of each primer, 10 ng of final cDNA and 6.4 mL of RNase-free water were added to the reaction mixture. A control was also included in each plate with 2.0 mL of RNase-free water as a template. Three technical replicates were contained in each plate. Specificity verification of the PCR amplification dissociation and the PCR efficiency curves were determined for each candidate reference gene prior to the qRT-PCR evaluation of these genes in Pf-5. Using rpoD as an internal reference, the differences of mRNA expression were then determined.
2.6. Electrophoretic mobility shift assay Electrophoretic mobility shift assay (EMSA) analysis was conducted with the Thermo Scientific Light Shift Chemiluminescent DNA EMSA kit. Oligonucleotide probes were first synthesized, labeled using a Biotin 30 End DNA Labeling Kit (Thermo Scientific) and annealed. The binding reactions for probes and protein were carried out based on the manufacturer's protocol. Then, proteineDNA complexes were separated on native polyacrylamide gels (6%). Finally, EMSA Kit was used to detect the biotin-labeled probes. 2.7. b-Galactosidase activity assay The ß-galactosidase activity assay was based on a method of Miller. In brief, the ß-galactosidase activity was normalized to the optical density at 600 nm (OD 600) of the bacterial culture in Miller units. 0.1 mM isopropyl-ß-D -thiogalactopyranoside (IPTG) was added to cultures to induce expression of strains containing pBBR1MCS-5 or pET-28a derivatives.
2.8. Lipase activity assay Since LipA is an intracellular lipase [28], the activity of LipA is measured as the activity of whole cell lipase. The bacterial samples were prepared according to the method provided in the literature and the activity of Lipase was determined by pNPC, which contains 2.9 ml of Tris-HCl Buffer (50 mM, pH ¼ 9.0) and 30 ml of pNPC (10 mM pNPC in acetonitrile). The mixture was preheated at 55 C for 5 min and then 70 ml of the cell sample was added. The reaction was centrifuged at 12,000 rpm for 2 min at 4 C and the supernatant was taken for OD410 absorbance. The amount of enzyme needed to release 1 mmol of p-nitrophenol/minute by 1.0 ml sample with OD600 of 1.0 was defined as one unit of lipase activity. The activity of the lipase was expressed as U/ml OD600. 2.9. Statistical analysis All experiments were performed in triplicate, and the data were analyzed by unpaired Student's t tests, and differences with a P value of 0.05 were considered statistically significant.
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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3. Results 3.1. AlgR regulates the expression of lipA at the transcriptional level In P. protegens Pf-5, there is no classical QS regulatory system [29]. So far, the regulation of lipase expression is still not well studied. In order to further understand the relationship between algR and lipA, we conducted algR gene knockout, and constructed algR gene knockout mutant, Pf6003. The effect of algR on lipase expression was analyzed by measuring the activities of b-galactosidase and the whole cell lipase. The results were presented in Fig. 1A. It showed that the growth of Pf-5 was not altered by the knockout of algR. The growth curves of the wild-type, mutant, and complementation strains were consistent, indicating that the growth of the bacteria was not changed after knockout of algR gene. However, by measuring the lipase activity and b-galactosidase activity of the wild and mutant strains, it was found out that the activity of the whole cell lipase was significantly decreased after knockout of algR (Fig. 1B). To investigate whether AlgR regulates
lipA expression through mediating lipA promoter activity, we constructed lipA-lacZ, as well as fusion reporter strains of lipA'-'lacZ, Pf5F3, Pf-5F4 and Pf6003F3, Pf6003F4, respectively. The results were given in Fig. 1C. As it showed that the activity of b-galactosidase of the mutant Pf6003 was lower than that of the wild-type Pf-5, both at the transcriptional level and at the translation level. While the alteration in transcriptional level was more obvious, indicating that AlgR mainly control lipA promoter activity in transcriptional level, then regulating the expression of lipA gene. On the contrary, by measuring lipase and b-galactose activities after overexpression of AlgR in complementation mutant strains, it was found out that the activities of lipase and b-galactosidase were restored to the level of the wild-type bacteria (Fig. 2A and B). Furthermore, RT-PCR results showed that the expression of lipA in the mutant Pf6003 was also lower than that in the wild strain (Fig. 1D). These results indicate that AlgR regulates the expression of by affecting the activity of lipA promoter. That is to say, AlgR regulates the expression of lipA at both transcriptional level and translation level, but mainly at the transcriptional level.
Fig. 1. Effects of algR mutation on lipA expression. A. Growth curve of Pseudomonas sp. Overnight culture of bacteria were inoculated in 50 ml LB medium. The initial OD value was adjusted to about 0.1. Bacteria culture was placed in 200 rpm shaker at 28 C, and OD value was determined once every 2 h. B. Relative activity of whole cell lipase in wild-type (Pf-5) and algR mutant (Pf6003). The whole cell lipase activity was measured after the bacteria were cultured in a 50 ml LB medium to a stationary phase. C. b-galactosidase activity in wild-type (Pf-5) and algR mutant (Pf6003) at transcriptional level (Pf-5F4, Pf6003F4) and translational level (Pf-5F3, Pf6003F3). The bacteria were incubated in a 50 ml LB medium to a stationary phase and the enzyme activity of b-galactosidase was determined. D. RT-PCR of relative quantification of lipA expression in Pf-5 and Pf6003. The mRNA expression of lipA gene was measured after the bacteria were incubated to the stationary phase. All experiments were performed in triplicate, and the mean values ± standard deviations are indicated.*, P < 0.05 compared with the control.
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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whether AlgR regulated the expression of lipA by rsmX/rsmY/rsmZ. We first expressed the AlgR protein through the purified expression system of peT-28a, and then explored whether AlgR protein bound to the rsmX/rsmY/rsmZ promoter sequence using EMSA (Fig. 4C). The result showed that AlgR protein could bind directly to the promoter region of rsmZ, but not to the promoter sequences of rsmX and rsmY. However, the expression of rsmX/rsmY was also decreased in combination with the expression of rsmX/rsmY/rsmZ, indicating that the expression of rsmX/rsmY/rsmZ was altered by AlgR protein. However, it had greater effect on rsmZ. In addition, the results of overexpression of rsmX, rsmY, rsmZ (Fig. 3A and B) was consistent with the results of EMSA, further demonstrating that AlgR may regulate the expression of lipA by regulating rsmZ. 3.4. AlgR regulates lipA expression through rsmZ To further determine whether AlgR affects the expression of lipA by rsmZ, we knocked out rsmZ in the wild-type Pf-5 and the mutant Pf6003 to construct rsmZ mutant strains Pf6285 (Pf-5 (DrsmZ)) and Pf6100 (DalgR/DrsmZ). We hypothesize if AlgR regulates the expression of lipA via rsmZ, LipA activity will be robustly decreased after knockout of algR and rsmZ. If we overexpressed rsmZ in algR mutant pf6003, the LipA activity should be partially restored. As shown in Fig. 4A and B. The results are consistent with our hypothesis. The activity of b-galactosidase and the activity of whole cell lipase are significantly lowered after double knock-out of algR and rsmZ than single knockout of algR. Additionally, overexpression of rsmZ in the algR mutant really restored LipA enzyme activity to the wild-type level. Moreover, the activity of LipA was significantly increased after overexpression of rsmZ. These results all suggests that AlgR binds to rsmZ to directly regulate the expression of lipA. 4. Discussion Fig. 2. Effects of algR overexpression on expression of lipA. A. Relative activity of whole cell lipase in wild-type (Pf-5) and algR mutant (Pf6003). The whole cell lipase activity was measured after the bacteria were cultured in a 50 ml LB medium to a stationary phase. Pf-5/pBBR, wild-type with pBBRM1CS-5; Pf-5/pBBR-algR, wild-type overexpressing algR; Pf6003/pBBR, algR mutant with pBBRM1CS-5; Pf-5/pBBR-algR complementary algR mutant. B. Influence of algR overexpression on expression of a chromosome-borne lipA-lacZ Transcriptional fusion in different strains. Bacteria were cultured in 50 ml LB medium to the stationary phase. The enzyme activity of bgalactosidase was then determined. Pf-5F4/pBBR,wild-type with pBBRM1CS-5; Pf-5F4/ pBBR-algR, wild-type overexpressing algR; Pf6003F4/pBBR, algR mutant with pBBRM1CS-5; Pf-5F4/pBBR-algR, complementary algR mutant. All experiments were performed in triplicate, and the mean values ± standard deviations are indicated. *, P < 0.05 compared with the control.
3.2. AlgR affects rsmX/rsmY/rsmZ expression Previous studies have shown that AlgR modulates the T3ss system through indirect regulation of rsmA/rsmY/rsmZ transcription in P. aeruginosa [23]. However, no report have been conducted in Pf5. The results showed that the transcription level of rsmX/rsmY/ rsmZ was decreased to some extent after algR knockout, and the expression of rsmZ was reduced the most (see Fig. 3C).
3.3. AlgR binds directly to the rsmZ promoter sequence To study the interaction between algR and lipA, AlgR protein was expressed and the binding site of AlgR protein was explored using EMSA. We found that rsmX/rsmY/rsmZ was involved in the regulation of lipase in Pf-5, whereas in P. aeruginosa, AlgR indirectly regulates rsmX/rsmY/rsmZ, which regulates the expression of the related genes. It was found that rsmA/rsmY/rsmZ sequence homology was not high in Pf-5 and P. aeruginosa. Thus, we examined
Recent studies have shown that AlgR is a global transcriptional regulator that controls not only the production of alginate but also the twitching motility [19]. Global transcriptome analysis indicated that it also controlled many other different genes, including those associated with QS, type IV pili, type III secretion system, anaerobic metabolism, cyanide [30], and rhamnolipid production [19,31]. However, the details of how these genes are regulated by AlgR have not been well studied, and especially there are fewer reports on the regulation of lipase expression by AlgR. In order to investigate whether AlgR regulates the expression of lipase in genus Pseudomonas, we knocked out the algR gene in P. protegens Pf-5 and constructed mutant Pf6003. We found that the activity of lipase decreased significantly after knockout of algR (Fig. 1B). Meanwhile, we also overexpressed algR in the wild-type Pf-5 and the mutant Pf6003. It was found that complementation of algR in the mutant Pf6003 restored the activity of lipase and overexpression of algR enhanced the activity of lipase in the wild-type Pf-5 (Fig. 2A and B). In addition, after examining the growth curve, the algR knockout did not alter the growth of the bacteria strain (Fig. 1A). These results indicate that the activity of lipA in Pf-5 is regulated by AlgR. Moreover, our results further showed that AlgR regulates promoter activity both at transcriptional level and translational level (Fig. 1C), but regulates lipase expression mainly at transcriptional level. After knockout of algR, RT-PCR analysis showed that the expression of lipA was also reduced (Fig. 1D). Small RNAs (sRNAs) play an important role in the regulation of gene expression in gram-negative bacteria [32e34]. In P. protegens CHA0, which is similar to Pf-5, GacS/GacA regulates the expression of genes related to secondary metabolism via rsmX, rsmY and rsmZ [35]. In P. aeruginosa PAO1, GacA directly regulates transcription of rsmY and rsmZ to regulate up to hundreds of gene expression [36]. It
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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Fig. 3. Effects of rsmX, rsmY, rsmZ on lipase expression. A. Relative activity of whole cell lipase LipA. Pf-5/pBBR, wild type; Pf-5/pBBR-rsmX, wild-type overexpressing rsmX; Pf-5/ pBBR-rsmY, wild-type overexpressing rsmY; Pf-5/pBBR-rsmZ, wild-type overexpressing rsmZ. The whole cell lipase activity was measured after the bacteria were cultured to the stationary phase. The activity of rsmX, rsmY, rsmZ, and lipA were increased, but the activity of lipase increased more after overexpression of rsmY and rsmZ. B. Influence of rsmX, rsmY, rsmZ overexpression on expression of a chromosome-borne lipA-lacZ transcriptional fusion in WT strains Pf-5. The b-galactosidase activity of each strain was measured in the stationary phase after inoculation into 50 ml LB medium.C. RT-PCR of relative quantification of rsmX, rsmY, rsmZ in Pf-5 and Pf6003. The mRNA expression levels of rsmX, rsmY, and rsmZ were measured after the bacteria were cultured in the stationary phase. All experiments were performed in triplicate, and the mean values ± standard deviations are indicated. *, P < 0.05 compared with the control.
is also reported that AlgR regulates T3ss by indirectly regulating the expression of rsmA/Y/Z [23]. However, the mechanisms of AlgRmediated regulation of rsmA, rsmY, rsmZ are still unclear. In Pf-5, in order to investigate whether rsmX, rsmY and rsmZ are involved in the regulation of lipA expression via algR, we first examined the expression of rsmX, rsmY, rsmZ after knockout of algR (Fig. 3C). It was found that the transcription levels of rsmX, rsmY, rsmZ were all decreased after knockout of algR, and the decrease of rsmZ expression was the highest. On the contrary, the activities of lipase and b-galactosidase were significantly increased after overexpression of rsmX, rsmY and rsmZ in Pf-5 cells, suggesting that rsmX, rsmY and rsmZ all participate in the regulation of the expression of lipA (Fig. 4A and B). Moreover, EMSA results (Fig. 4C) showed that AlgR bound to rsmZ directly, rather than to rsmX, rsmY, to regulate the expression of lipA. To further validate the results, a double-knock out strain of algR/rsmZ and a single knock-out strain of rsmZ were constructed to analyze the regulation of rsmZ via measuring the activities of the whole-cell lipase (LipA) and bgalactosidase (Fig. 4A and B), it was found that the activity of LipA and the promoter activity of lipA were much lower in the doubleknock out strain than that of the single gene knockout.
Additionally, in the algR mutant strain pf6003, supplement expression of rsmZ could restore the activity of the enzyme to a level close to that of the wild strain (Fig. 4A and B), suggesting that algR mainly regulates the expression of lipA by controlling rsmZ transcription. Our results for the first time clearly described the interaction between algR and rsmX, rsmY, and rsmZ. In addition, our previous studies have shown that rsmE, rather than rsmA, directly binds to the lipA promoter to activate lipA translation [17]. Therefore, future research is needed to investigate the interaction between rsmX, rsmY, rsmZ and rsmA, rsmE, and dissect the mechanism underlying the regulation of lipA expression via algR. In conclusion, the present study has demonstrated that, in P. protegens Pf-5, AlgR directly binds to rsmZ to regulates the expression of lipA via regulating transcription of rsmZ, and mainly regulates the expression of lipA at transcriptional level.
Conflict of interest disclosure The authors disclose no potential conflicts of interest.
Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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Fig. 4. AlgR regulates the lipase expression of Pf-5 via rsmZ. A. Enzyme activity of b-galactosidase after algR and rsmZ single or double knock-out. Pf-5F4, wild-type strain; Pf6003F4, ⊿algR; Pf6285F4, ⊿rsmZ; Pf6100F4, ⊿algR/DrsmZ; Pf6003F4/pBBRK-rsmZ, rsmZ overexpression in ⊿algR mutant; Pf-5/pBBRK-rsmZ, rsmZ overexpression in wild type. b-galactosidase activity was determined after the bacteria were cultured to the stationary phase. As shown in the results, b-galactosidase activity was much lower after double knockout. In addition, overexpression of rsmZ in the algR mutant enhanced b-galactosidase activity even slightly higher than the wild bacteria, indicating that effects of AlgR on lipase expression can be compensated by the expression of rsmZ. B. Relative activity of whole cell lipase LipA after algR and rsmZ single or double knockout. Pf-5, wild-type strain; Pf6003, ⊿algR; Pf6285, ⊿rsmZ; Pf6100, ⊿algR/DrsmZ; Pf6003/pBBRK-rsmZ, rsmZ overexpression in ⊿algR mutant; Pf-5/pBBRK-rsmZ, rsmZ overexpression in wild type. The whole cell lipase activity was measured after the bacteria were cultured to the stationary phase. As shown in the results, the relative activity of lipase was lower after double knockout than that of one gene knockout, indicating that algR and rsmZ both regulate the expression of lipase. In addition, rsmZ can partially compensate the effects of algR gene knockout. Meanwhile, after overexpression of rsmZ, lipase activity was higher than the wild strains. C. The EMSA results of binding of AlgR to the rsmX/Y/Z promoter sequence. NC (Negetive Control) and PC (Positive Control) were positive and negative control systems to validate EMSA system; rsmX, rsmX promoter sequence; rsmX þ AlgR, rsmX promoter sequence and AlgR protein. rsmY, rsmY promoter sequence; rsmY þ AlgR, rsmY promoter sequence and AlgR protein. rsmZ, rsmZ promoter sequence; rsmZ þ AlgR: rsmZ promoter sequence and AlgR protein. The 1 nM biotin-labeled DNA probe was incubated with purified AlgR protein in 20 ml binding buffer, and the AlgR-DNA complexes and free DNAs were cross-linked to the membrane by a 320-nm UV-light cross-linking instrument. The biotin-labeled bands were detected by the Thermo Scientific chemiluminescent nucleic acid detection module. All experiments were performed in triplicate, and the mean values ± standard deviations are indicated. *, P < 0.05 compared with the control.
Acknowledgements
Transparency document
This work is financially supported by the National Natural Science Foundation of China (No J1103514), the National High Technology Research and Development Program of China (2011AA02A204), the Innovation Foundation of Shenzhen Government (JCYJ20120831111657864), the National Natural Science Foundation of Hubei Province (No. 2015CFA085). Many thanks are indebted to Analytical and Testing Center of Huazhong University of Science and Technology for their valuable assistances in lipase activity measurement.
Transparency document related to this article can be found online at http://dx.doi.org/10.1016/j.bbrc.2017.04.034.
Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.bbrc.2017.04.034.
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Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034
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Please cite this article in press as: M. Li, et al., The Pseudomonas transcriptional regulator AlgR controls LipA expression via the noncoding RNA RsmZ in Pseudomonas protegens Pf-5, Biochemical and Biophysical Research Communications (2017), http://dx.doi.org/10.1016/ j.bbrc.2017.04.034