Iron plays an essential role in the growth and re-production of most microorganisms, since a substan-tial fraction of enzymes requires the metal ion as their catalysis centers. To be more specifically, a systematic analysis of 310 redox-dependent enzymes data shows that 30% of enzymes contain metal iron as redox cen-ters. And iron acts as a cofactor involved in most cellu-lar processes such as electron transfer, RNA synthesis and resistance to reactive oxygen intermediates [1, 2]. Although iron is the fourth most abundant element on the earth's surface, its bioavailability is limited in aque-ous environments . The majority of Fe3+ forms fer-ric oxide hydrate complexes (Fe2O3·nH2O) in the exis-tance of oxygen and water at the physiological pH. The concentration of free ferric ions is from 10−9 mol/L to 10−18 mol/L since these complexes are quite stable . In order to acquire the ferric ions in environments, bac-teria, fungi and plants secrete the low-molecular-weight secondary metabolites termed siderphores (200−2000 Da) as iron chelating agents to facilitate absorption of the iron in vitro [5-7].
Pseudomonas aeruginosa is responsible for chronic infections in the pathogenesis of cystic fibrosis (CF) . Under iron-deficient environments, Pseudomonas aeruginosa can produce the yellow-green, uorescent, water-soluble pigments termed pyoverdine (Fig. 1) to ac-quire ferric ions. Pyoverdines have extremely high affin-ity for Fe3+ ion with stability constant in general around 1032 mol/L . Although pyoverdines were discovered more than 120 years ago , the function of iron acqui-sition was identified by Meyer and Abdallah until 1978 . And in the late 1980s and early 1990s, the struc-ture of pyoverdines were analyzed using the NMR and mass spectrometry techniques [12-15]. Pyoverdine not only plays the role as iron-scavenger in Pseudomonas aeruginosa, but also regulates the production of at least three virulence factors including exotoxin A, an endo-protease, and pyoverdine itself, all of them are major contributors for the bacterial infections . Further-more, to some extent, pyoverdine can also protect the bacteria from metal toxicity and the ROS which lead to deadly harm to microbe [4, 7, 17]. As for application, pyoverdine can serve as biological recognition elements for the uorescent detection such as ferric ion, furazoli-done and copper ion in environment [18-20].
In previous research, the classical or stopped-ow spectrophotometry analysis suggested that the reac-tion of the free pyoverdine chelates with the mono-hydroxylated Fe3+ species Fe(OH)2+ to form the ferric-pyoverdine complexes, and these data was supported by the dissociative Eigen-Wikin mechanism [9, 21]. How-ever the in situ infrared spectroscopy confirmed that pyoverdine catechol ligand can directly interact with the TiO2 and Fe2O3 surfaces by the covalent bonding aqueous environment [22, 23]. So it remains unknown for the mechanism of the pyoverdine acquiring the in-soluble iron.
In this work, we used the laser light scattering (LLS) to detect the interactions between the pyoverdine and insoluble ferric hydroxide colloids. Our experiments also reveal that iron plays a highly significant role in Pseudomonas aeruginosa growth and pyoverdine production.II. EXPERIMENTAL PROCEDURES A. Microorganisms and pyoverdine production
The strains used in this experiment were the Pseu-domonas aeruginosa ATCC 15692 and the Pseu-domonas aeruginosa PAO1. The iron-limited culture medium termed synthetic succinate medium (SSM) had following composition: 7.86 g/L K2HPO4·3H2O, 3 g/L KH2PO4, 1 g/L (NH4)2SO4, 0.2 g/L MgSO4·7H2O, 4 g/L succinic acid. The medium was adjusted to pH=7.0 by 1 mol/L NaOH before sterilization . The 1 L culture medium was dispensed into 1 L conical asks, each of them contained 200 mL of medium. The asks were inoculated 10 mL of Pseudomonas aerugi-nosa ATCC 15692 which grew to exponential-phase in aerobic condition at 37 ℃ and 220 r/min in a shaker-incubator. After 17 h, the culture medium was cen-trifuged at 10000×g for 10 min at 4 ℃. In order to get the bacteria-free crude pyoverdine solution, the su-pernatant was filtered by 0.22 μm membranes (Merck Millipore) .B. Pyoverdine isolation and purification
Pyoverdine isolation and purification were carried out as a published procedure with some modifications . In brief, the cell-free supernatant was buffered with 1 mol/L HEPES (N-2-hrydroxyethylpiperazine-N′-ethanesulfonic acid) buffer to pH=7.0 and then applied to a chelating Sepharose fast ow column (1.6×2.5 cm, 5 mL; GE Healthcare). This col-umn was pre-saturated with CuSO4 and equilibrated with 20 mmol/L HEPES buffer (pH=7.0) contain-ing 100 mmol/L NaCl. The eluent ow rate was set at 300 mL/h. The column was then washed with 50 mL of 20 mmol/L HEPES buffer and eluted with 20 mmol/L acetate buffer (pH=5.0) containing 100 mmol/L NaCl. Per 5 mL fraction was collected and the A400 absorbance spectroscopy was measured by NanoDrop 2000 Spectrophotometer (Thermo SCI-ENTIFIC) to determine pyoverdine-Cu containing. The pyoverdine-containing fractions were pooled separately and lyophilized.
Each of the fractions dried pyoverdine was dissolved in 1 mL 10 mmol/L EDTA and then applied to a Sephadex G-15 column (1 cm×80 cm, GE Healthcare) that had been pre-equilibrated with deionized water. The column was eluted with ultrapure water at a ow rate of 20 mL/h and 15 fractions (4 mL) were collected and the absorbance of UV-Vis spectrum (190−1000 nm) was monitored. The fractions with the highest ab-sorbance at 385 nm were chosen as purified-pyoverdine and the samples were pooled, lyophilized, and stored at −20 ℃.C. Preparation of the samples for DLS characterization
The purified-pyoverdine was dissolved into 0.1 mol/L phosphate buffered saline (PBS) to final concentration to 25, 50, 100 μmol/L at physiological pH 7.4 and then the dust in solution was filtered by 0.45 μm mem-brane (Millipore). The absorption spectrum of the free pyoverdine was measured by UV-Vis spectrum at 25 ℃. The concentration of the free-pigment was cal-culated using the extinction coefficients λmax=385 nm and ε=16500 (mol/L)−1cm−1 . And the Fe3+ source was obtained from the 1 mmol/L FeCl3 solution pH 2.0 which was filtered by 0.45 μm membrane. In a typical experiment, a proper volume of the dust-free FeCl3 was directly added into the dust-free free pyoverdine PBS buffer to start the biodegradation of ferric hydroxide.D. Laser light scattering
A modified commercial LLS spectrometer (ALV/DLS/SLS-5022F) equipped with an ALV5000 multi-τ digital time correlator and a cylindrical solid-state He-Ne laser (UNIPHASE, out power=22 mW at λ0=632.8 nm) as the light source was used. In dynamic LLS, the self-beating mode of the intensity-intensity time auto-correlation function G(2)(t, q) was measured where t is related to the delay time and q represents the scattering vector [25-27]. The line-width distribution G(Γ) is accompanied with the analysis of G(2)(t, q). For a pure diffusive relaxation, Γ is connected with the translational diffusion coefficient distribution G(D) or a hydro-dynamic radius distribution f(Rh) via the Stock-Einstein equation: Rh=(kBT/6πη0)/D, where kB, T and η0 are the Boltzmann constant, the absolute temperature and the solvent viscosity, respectively . According to the definition of Rayleigh scatter ratio Rvv(q) which is concerned with the scatter particles property, size and concentration. In the dilute solution, Rvv(q) can be related to the weight-average molar mass Mw, the second virial coefficient A2, and the root-mean square z-average radius
Where dn=dC, n, NA, λ0 represent the specific refrac-tive index increment, Avogadros number, the solvent re-fractive index, and the vacuum light wavelength. Since when C→0 and q→0, Rvv≈KCMw . In this study, we used a fixed angle (30°) to obtain all data and the concentrations of Fe3+ and free-pyoverdine were both at micro molar level.E. Bacteria growth rate and pyoverdine production measurement
The various contents of iron culture mediums were prepared by adding different volume of 1 mmol/L FeCl3 (pH=2.0) into the solution containing 50 μmol/L free pyoverdine or not SSM. In order to assure that the initial quantity of bacteria was consistent, the strain of Pseudomonas aeruginosa PAO1 was cultured to exponential-phase in aerobic condition at 37 ℃ and 220 r/min. The 10 μL of the bacteria was inoculated into the 1 mL fresh SSM and the growth rate of bacte-ria was monitored every 2 h by measuring the optical density at 600 nm (OD600) by eppendorf biophotometer plus (Thermo Fisher SCIENTIFIC).III. RESULTS AND DISCUSSION A. Characterization of the free-pyoverdine
The purified pyoverdine can be characterized with the mtrix-assisted laser desorption ionization time of ight mass spectrometry (MALDI-TOF MS) (Fig. 2) to obtain the intact siderphore molar mass and the UV-Vis spectrum (Fig. 3) to identify free-pyoverdine. The MALDI-TOF MS gave compound molecular ion M+ at m/z=1334.5003 which was coincided with the sim-ulation of the free pyoverdine via Chemoffice 2015 and the previous reports . And the absorption spectrum of free pyoverdine in water solution could be observed at 385 nm while the Fe-pyoverdine complexes had the largest peak at 403 nm according to the reported data in Ref., so we acquire the metal free pyoverdine.B. The stability of iron hydroxide colloids
Considerable evidence suggests that iron participates in many bacterial processes while the amount of free iron in aerobic aqueous environments (pH=7.0) is less than 10−17 mol/L . To understand the ferric hy-droxide colloids stability, dynamic laser lighting scat-ter (DLS) was applied to observe the different con-tents of iron in the 0.1 mol/L PBS buffer (pH=7.40) at room temperature. Subsequently, the sizes of insol-uble Fe(OH)3 colloids were measured for more than 12 h (Fig. 4 (a) and (b)). The average hydrodynamic ra-dius
Since the ferric hydroxide colliods are very stable in environment, the free Fe3+ is extremely restricted for bacterial uptake. To understand the relationship be-tween the iron and the Pseudomonas aeruginosa growth better, we measured the bacterial population OD600 every 2 h in different Fe3+ contents SSM which were supplemented or not with 50 μmol/L free pyoverdine. Figure 5(a) shows that iron-rich environments help the bacteria to multiply vice versa. The amount of bacteria in iron-rich mediums (final contents of 5 μmol/L FeCl3) were more than 22 times compared to iron-limited medi-ums (final contents of 0 μmol/L FeCl3) after 10 h in-cubation. Interestingly, exogenous free pyoverdine in different iron contents SSM could promote bacteria re-production and narrowed the gap in growth rate of the various iron contents (Fig. 5(b)). The results suggest that the Pseudomonas aeruginosa proliferation rate in-creased with the iron contents increasing even in the ferric ions scarce conditions and free pyoverdine can fa-cilitate bacterial reproduction. This observation indi-cates that bacteria can make full use of insoluble iron and bacteria may secrete some substances to dissolve ferric hydroxides for iron acquisition.D. Pyoverdine dissolves insoluble iron
To test this hypothesis, we first addressed the ques-tion, do bacteria always secreted the pyoverdine no mat-ter the amount of iron contained in the culture medi-ums. The pyoverdine UV-Vis absorbtion was measured in bacterial centrifugal supernatant uid after 14 h in-cubation. Figure 6 shows that the pyoverdine concen-tration without addition FeCl3 medium was five times higher than addition FeCl3 to final iron content of 5 μmol/L in SSM. These results indicate that bacterial pyoverdine secretion was reduced with the increasing iron contents in SSM. The data also suggest that even in iron-rich medium, bacteria still needs to synthesis pyoverdine in order to dissolve the ferric hydroxide col-loids.
The chelation of pyoverdine and the ferric ion was identified by various groups . Whether the interac-tion between the pyoverdine with insoluble ferric hy-droxide remain mysterious. In order to observe the interaction between the pyoverdine and ferric hydrox-ide colloids, we used the dynamic laser light scatter-ing to obtain the real-time data of the complexes
In this work, we reveal that pyoverdine can directly interact with the Fe(OH)3 colloids to form new com-plexes and gradually degraded themselves. Iron con-tents in the environments play an important role in the Pseudomonas aeruginosa growth and pyoverdine production. Even in the iron-rich environment, Pseu-domonas aeruginosa still need pyoverdine to dissolve the insoluble iron. To understand the mechanism of the pyoverdine combining with various types of iron can help us to prevent and treat the Pseudomonas aerugi-nosa infection.V. ACKNOWLEDGMENTS
We thank professor Fan Jin of University of Sci-ence and Technology of China provided idea and de-signed the experiments. This work was supported by the National Program on Key Basic Research Project (No.2012CB933802) and the National Natural Science Foundation of China (No.21274141, No.21104071).
|||Andreini C, Bertini I, Cavallaro G, L. Holliday G, and M. Thornton J, J. Biol. Inorg. Chem. 13 , 1205 (2008). DOI:10.1007/s00775-008-0404-5|
|||Braun V, Biol. Chem. 378 , 779 (1997).|
|||A. Mies K, I. Wirgau J, and L. Crumbliss A, BioMetals 19 , 115 (2006). DOI:10.1007/s10534-005-4342-1|
|||Miethke and M. A. Marahiel M, Microbiol. Mol. Biol. Rev. 71 , 413 (2007). DOI:10.1128/MMBR.00012-07|
|||Cornelis P, Appl. Microbiol. Biotechnol. 86 , 1637 (2010). DOI:10.1007/s00253-010-2550-2|
|||C. Hider and X. Kong R, Nat. Prod. Rep. 27 , 637 (2010). DOI:10.1039/b906679a|
|||J. Schalk I, Hannauer M, and Braud A, Environ. Microbiol. 13 , 2844 (2011). DOI:10.1111/j.1462-2920.2011.02556.x|
|||R. Govan and V. Deretic J, Microbiol. Rev. 60 , 539 (1996).|
|||G. Anne-Marie A, Blanc S, Rachel N, Z. Ocaktan A, and A. Abdallaht M, Inorg. Chem. 33 , 6931 (1994).|
|||J. Schalk and L. Guillon I, Environ. Microbiol. 15 , 1661 (2013). DOI:10.1111/emi.2013.15.issue-6|
|||M. M. a. M. Abdallah J, J. Gen. Microbiol. 107 , 319 (1978). DOI:10.1099/00221287-107-2-319|
|||Briskot G, Taraz K, and Budzikiewicz H, Liebigs Annalen Der Chemie 1989 , 375 (1989). DOI:10.1002/(ISSN)1099-0690|
|||Demange P, Wendenbaum S, Linget C, Mertz C, T. Cung M, Dell A, and A. Abdallah M, Biol. Met. 3 , 155 (1990). DOI:10.1007/BF01140574|
|||Mohn G, Taraz K, and Budzikiewicz H, Z. Naturforsch B45 , 1437 (1990).|
|||Gipp S, Hahn J, Taraz K, and Budzikiewicz H, Z. Naturforsch C46 , 534 (1991).|
|||L. Lamont I, A. Beare P, Ochsner U, I. Vasil A, L. Vasil M, and R. Soc. London Proc, Ser A99 , 7072 (2002).|
|||Braud A, Hoegy F, Jezequel K, Lebeau T, and J. Schalk I, Environ. Microbiol. 11 , 1079 (2009). DOI:10.1111/emi.2009.11.issue-5|
|||Barrero J, Camara C, Perez-Conde M, San Jose C, and Fernandez L, Analyst 120 , 431 (1995). DOI:10.1039/AN9952000431|
|||Yin K, Zhang W, and Chen L, Biosens. Bioelectron. 51 , 90 (2014). DOI:10.1016/j.bios.2013.07.038|
|||Yin K, Wu Y, Wang S, and Chen L, Sens. Actuators B 232 , 257 (2016). DOI:10.1016/j.snb.2016.03.128|
|||Eigen and R. Wilkins M, Adv. Chem. Ser. 1 , 55 (1965).|
|||Y. Hamish J, Upritchard G, J. Bremer P, L. Lamont I, and James McQuillan A, Langmuir 23 , 7189 (2007). DOI:10.1021/la7004024|
|||J. B. Michael J. McWhirter P, L. Lamont Iain, and J. McQuillan A, Langmuir 19 , 3575 (2003). DOI:10.1021/la020918z|
|||X. a. W. S. Kisaalita R, Appl. Environ. Microbiol. 64 , 1472 (1998).|
|||Chu B, Laser Scattering, 2nd Edn.. New York: Academic Press (1991).|
|||Brown W, Light Scattering:Principles and Development. Oxford: Oxford University Press (1996).|
|||J. Berne and R. Pecora B, Dynamic Light Scattering:with Applications to Chemistry, Biology, and Physics. New York: Courier Corporation (1976).|
|||Gan Z, T. Fung J, Li M, Zhao Y, G. Wang S, and Wu C, Macromolecules 32 , 590 (1999). DOI:10.1021/ma981121a|
|||D. Weinberg E, Microbiol. Rev. 42 , 45 (1978).|
|||Meyer and J. Hornsperger J, Microbiology 107 , 329 (1978).|