Chaperone Assisted Expression And Purification Of Siderophore Monoxygense From Shewanella Putrefaciens-95
Putrescine monoxygenase (PMO) (EC 1.14.13.59) is the initial enzyme in the biosynthesis of putrebactin and it catalyzes the hydroxylation of putrescine to N-hydroxyl putrescine, the precursor for synthesis of a siderophore putrebactin. Although many Flavin monoxygenases genes (FMOs) and N-hydroxylating monoxygenases (NMOs) genes have been successfully over expressed in order to study the physiological roles in siderophore biosynthesis but still scanty efforts have been made to unravel the molecular, structural and kinetic and aspects. In this investigation we have cloned the complete 1518bp coding sequence of pubA for Putrescine monoxygenase from Shewanella putrefaciens 95 (SpPMO) that encodes the corresponding protein comprising of 505 amino acid residues. The deduced (SpPMO) protein showed (53 and 36%) sequence identity with other characterized bacterial NMOs from Erwinia amylovora and Gordonia rubripertincta respectively. Chaperone assisted heterologous expression of SpPMO in pET151/D-Topo expression vector under the control of bacteriophage T7 promoter permitting a high level IPTG dependant expression system produced a 54 kDa recombinant SpPMO protein with His6 tag at its N-terminus. The expression of recombinant SpPMO was confirmed by western blotting using anti-his6 antibody. The purified protein showed FAD and NADPH depending N-Hydroxylating activity. This study has paved a way to understand the steps involved in the biochemical pathway of putrebactin synthesis which can be further investigated by studying its kinetic mechanism and physiological role in siderophore biosynthesis.
Introduction
Iron is an essential nutrient and is typically available at concentrations below minimal quantity to support growth of microbial pathogens in mammalian hosts. As iron is present in insoluble ferric hydroxide form and further availability is reduced because of mammalian iron –binding proteins such as ferritin and lacto ferritin sequestering them. Secretion and production of siderophores by pathogens such as Mycobacterium tuberculosis, Aspergillus fumigates, Shewanella putrefaciens leads to combating the iron deficiency during infection. These compounds are low molecular weight organic compounds that act as metal chelators involved in iron uptake. The role of siderophores is to scavenge ferric iron from the host in order to facilitate the bacteria to grow. Biosynthesis of siderophores involve the action of novel flavin–containing N-hydroxylating monoxygenases (NMOs) and the coding gene identified as pubA, residing within a conserved five gene cluster in the genomes of Shewanella species leading to the formation of siderophore named putrebactin.
Homologous NMOs are present in a number of other siderophore-producing organisms and disruption of these monooxygenases in microorganisms such as Burkholderia cepacia, and Aspergillus fumigates resulted in depletion of effective persistence and colonization and have been reported to be vital for virulence. Substantial cloning and purification work of monoxygenases has been obtained in recent years. Expression and purification of the genes encoding NMOs in Mycobacterium tuberculosis and Mycobacterium smegmatis have been identified as MbtG and MbsG, involved in the production of siderophore mycobactin whereas, the modulation of the activity of MbsG is reported in the biosynthesis of hydroxamate containing siderophores. IucD gene coding for Lysine hydroxylase was the first enzyme expressed and purified for the production of aerobactin siderophore.
Cloning and expression of a fungal ornithine hydroxylase from Aspergillus fumigates using pVP56K vector along with kinetic mechanism was reported. Heterologous expression, purification and characterization of an l-Ornithine N5 hydroxylase involved in pyovirdine siderophore biosynthesis in Pseudmonas aeruginosa were reported. Further, substantial structural data of NMOs has been obtained in recent years especially with reference to SidA, PvdA, NbtG giving a rational understanding of the biochemical properties, specifically to uncoupling and stabilization of the co-factor FAD with the monoxygenase.
Purification and characterization of non recombinant or native 1-napthol-2-hydroxylase from carbaryl- degrading Pseudomonas strain c4 and characterization of the ornithine hydroxylation step in albachelin biosynthesis (AMO) has been reported in recent years. The N-terminus fusion of AMO with an 8xHIS tag was necessary to obtain a soluble protein. Additionally the tag allowed the use of Immobilized Affinity Chromatography (IMAC).
Herein we report the cloning, chaperone assisted heterologous expression and kinetic characterization of SpPMO. In this study, the coding sequence of SpPMO expressed in E. coli is characterized by bioinformatic analysis. Though, till date there have been reports of heterologous expression of N-hydroxylating Monooxygenases (NMOs), Baeyer-Villiger-Monooxygenases (BVMOs) and Flavin containing Monooxygenases (FMOs) belonging to class B monooxygenases from various organisms, to the best of our knowledge, this is the first report relating to chaperone assisted heterologous expression of Shewanella putrefaciens PMO gene in E. coli. Materials and MethodsBacterial strains, vectors and chemicals: Shewanella putrefaciens 95 JCM (20190) (equivalent to ATCC 8071) was purchased from the Japan Collection of Microorganisms JCM Japan. Phusion high fidelity PCR master mix was obtained from New England Biolabs (England). Restriction enzymes were obtained from Merck. The pET151/D-Topo vector, E. coli TOP10 and E. coli BL21 Star (DE3) chemically competent cells and Purelink genomic DNA mini Kit were purchased from Thermo Fischer Scientific (USA). The plasmid pGroES/EL was purchased from Takara Bio Inc. DNA sequencing was performed in Eurofins Genomics India Private Limited, Bangalore. Protein purification was performed on AKTA prime Plus FPLC (GE Healthcare) using His Trap 5 mL FF and Sephadex G25 columns. Quick gel extraction and Mini prep kits were purchased from Qiagen. Luria -Bertani (LB) Agar, SDS, Agarose, Acrylamide, Bisacrylamide, Putrescine, Lysine , NADPH, FAD and Western blotting chemicals were purchased from Sigma Aldrich.
Primary antibody: Anti His Monoclonal (1:4000) Secondary antibody: Anti Mouse IgG- HRP Conjugated (1: 30000) Substrate: ECL. Dual colour Precision markers for SDS-PAGE were purchased from Bio-Rad.
Cloning of full length SpPMO gene: Genomic DNA of Shewanella putrefaciens 95 JCM (20190) was isolated and purified using PurelinkTM genomic DNA extraction kit using an overnight grown culture of S. putrefaciens 95 in LB media. The purity and concentration was checked by Nanodrop and agarose gel electrophoresis. SpPMO gene was amplified by PCR using the genomic DNA from Shewanella putrefaciens 95 as template. The forward 5’-C A C C G T G A C T A C A C T A C A A A G G G A A A T T G A 3’ and reverse primer 5’- T T A T T G C G C C T C C T T A T T C G 3’ were designed using Oligo perfectTM Designer software (Invitrogen) tool based on the SpPMO gene coding sequence available in NCBI database and synthesized from Sigma. The PCR mixture (25 µl) contained 125 ng of genomic DNA, Phusion high fidelity PCR master mix (12µL) and 2µM of each primer. The reaction conditions consisted of an initial denaturation step of 98oC for 2 min followed by 30 cycles of denaturation at 98oC for 30 sec, annealing at 58o C for 60 sec and extension at 72oC for 30 sec and a final extension at 72 oC for 10 min. The 1522-bp PCR product containing 1518 SpPMO gene was separated on a 1% agarose gel and visualized by staining with SYBR safe stain and extracted from the gel using a QIA quick gel extraction kit. Topo cloning reaction was carried out by mixing purified PCR product with the expression vector (pET151/D-Topo vector) in the ratio 1:1, 2:1 and used to transform One Shot Top10 chemically competent E.coli cells. The transformation mix was plated on LB plates supplemented with ampicillin (100 µg/ml). About 5-8 colonies were picked and cultured overnight in LB medium containing ampicillin antibiotic. Plasmid DNA was isolated using the PureLink™ HQ Mini Plasmid purification kit. The isolated plasmids were analyzed by EcoRI restriction digestion and PCR analysis performed to confirm the presence of the insert. The sequences of the four positive clones (C1, C2, C3 and C4) were determined by Sanger DNA sequencing method (Eurofins Genomics India Pvt Ltd, Bengaluru). C2 clone was chosen to express full length expression of SpPMO in the chemically competent host E. coli BL21 Star (DE3).
Bioinformatic analysis of SpPMO protein: SpPMO coding sequence was translated to amino acid sequence by ExPASy tool from EMBL-EBI. ClustalW was used to do multiple sequence alignment of SpPMO with other PMOs, FMOs, and BVMO. Evolutionary tree with selected proteins was built by neighbour joining method using MEGA7. 1000 replicates were taken to achieve confidence level for the branches by using bootstrap analysis. The motifs were identified using ScanProsite tool. Phyre2 and I-TASSER server was used to predict tertiary structure. Ligand binding sites were obtained using COACH and COFACTOR servers. The SAVES validated the obtained 3D model. Heterologous Expression of SpPMO in E. coli: The expression host E. coli BL21 Star (DE3) cells containing the pET151/D-Topo-SpPMO plasmid clone C2 were plated onto LB plates supplemented with 100μg/ml of ampicillin. A single colony from the plates was used to inoculate four 25 ml LB medium containing 100μg/mL of ampicillin. The culture was incubated overnight at 370C with continuous shaking at 220 rpm. Sixteen 500ml flasks, each containing 250 ml of LB media containing 100μg/ml of ampicillin and 1% glucose were inoculated with overnight culture and kept at 370C with agitation at 220 rpm until the OD at 600 nm reached 0.5-0.8 (mid log phase ). IPTG concentration of 0.5-1 mM was added to all the culture flasks for induction. The temperature was then reduced to 200C and grown for 14 h at 180 rpm. The cells were harvested by centrifugation at 8000 rpm for 20 min. The resulting cell paste (~ 50 g) was stored at -800C till further use. Co expression of recombinant SpPMO in E. coli: Chemically competent E. coli BL21 Star (DE3) cells were cotransformed with pGroES/EL (10ng) and pET151/D-Topo-SpPMO (5ng) plasmids by heat-shock method for 45 sec at 42oC. The transformation mix was plated on LB plates containing ampicillin (100 µg/ml) and chloramphenicol (40µg/ml) each and grown at 37oC. The positive transformants were used for growing pre culture and used to inoculate 4L LB media supplemented with appropriate antibiotics. Induction of chaperones was done with L-arabinose (0.5 mg/mL) after 2 h of inoculation while that of SpPMO was done with 0.5mM IPTG when the OD 600 nm reached to 0.6-0.7. The cells were grown for overnight at 20oC in a shaking incubator at 180 rpm. All samples taken were processed for determination of cell biomass (O.D. 600nm) and quantification of protein expression (per O.D.600 of the cells) by SDS– PAGE (17). A control culture without induction was also run in parallel.
Purification of recombinant Sp PMO: The induced co transformed E.Coli BL21 star (DE3) cells had 25g wet weight were harvested by centrifugation at 8000 rpm , 15 min at 4˚C and re suspended in 25mL of buffer A (25mM HEPES, 300mM NaCl, 5% glycerol, pH 7.5 , PMSF 1mM, Dnase 25ug/mL, lysozyme 25ug/mL) and incubated on ice for 30 minutes. The cells were lysed by Qsonica Ultra sonication (70% Amplitude, 10min, 10 sec pulses with 20 sec delays). The pellet was removed by centrifugation at 13,000g for 30 min and the supernatant was stored at -20˚C until further use. The resulting supernatant was loaded onto His60 column (10 ml, Clontech) previously equilibrated with buffer A and the same buffer was used to wash the column until no protein was detected in the flow through. Elution was performed using a 0-300 mM imidazole gradient at a flow rate of 5 ml/min. The active fractions were pooled, concentrated and applied to Q-sepharose columns (5 ml GE Healthcare) equilibrated with buffer A. Elution was performed using a 40 ml gradient of 0-0.8M NaCl. Fractions containing PMO were pooled, concentrated and loaded onto to a Superdex-200 (16/60 GE Healthcare). Elution was performed using buffer A and a single peak with active fractions were collected and concentrated. The concentrate sample containing SpPMO-6His protein was dialyzed overnight against 20 mM HEPES pH 7.5, 10 μM FAD buffer. Fractions containing SpPMO activity were pooled and concentrated (Millipore). All purification steps were performed at 40C. The dialysed sample was analysed by SDS–PAGE to determine purity and Western blot analysis was performed to identify PMO-6His protein using anti-HisHRP Ab at 1:2000 dilution.
Western blot analysis: The purified Sp PMO along with the controls run on 4-12% polyacralamide gel (Invitrogen) as described above was transferred to nitrocellulose membrane and developed according to Cold Spring Harbor protocols. The transferred SpPMO was developed using penta His Tag antibody (Invitrogen) and developed using Tetramethylbenzidine (TMB) liquid substrate system (Sigma). Absorbance Spectra: The spectrum of purified SpPMO (30µM) in 100mM phosphate buffer (pH 7.5) was recorded using Perkin Elmer spectrophotometer. The flavin component was extracted by following the published procedure. An extinction coefficient at 450 nm of 12,600 M-1cm-1 was calculated for the FAD bound to SpPMO from the extinction coefficient at 450 nm for free FAD, of 11300 M-1cm-1. SpPMO Activity Assay. The formation of the product N-hydroxy putrescine by SpPMO was assayed by a variation of published Csaky Iodine oxidation procedure.
