Calcium Involved Directional Organization of Polymer Chains in Polyester Nanogranules in Bacterial Cells

Soil bacteria accumulate polyesters (typically poly([R]-3-hydroxybutyrate (PHB), in which one end of the chain terminates with a carboxyl group) in the form of hydrated, amorphous nanogranules in cells. However, it is not clear what drives the structure of these biomaterials inside bacterial cells. Here, we determined that calcium guides intracellular formation of PHB nanogranules. Our systematic study using the surface zeta potential measurement and the carboxyl-specific SYTO-62 dye binding assay showed that the terminal carboxyl is not exposed to the granule surface but is buried inside native “unit-granules” comprising the mature granule. Extracellular Ca2+ was found to mediate the formation of these PHB unit-granules, with uptaken Ca2+ stored inside the granules. Comparative [Ca2+]-dependent fluorescence spectroscopy revealed that the native granules in Cupriavidus necator H16 act as a Ca2+ storage system, presumably for the regulation of its cytosolic Ca2+ level, but those from recombinant Escherichia coli do not. This study reveals intimate links between Ca2+ and native granule formation, and establishes a novel mechanism that intracellular PHB granules function as Ca2+ storage in order to relieve soil bacteria from Ca2+ stress.

Culture conditions. C. necator H16 was cultivated for PHB homopolymer accumulation in PHA synthesis medium containing 20 g/L of fructose as the sole carbon source for 72 h at 30°C.
H. pseudoflava was cultivated to accumulate P(HB-co-HV) copolymers in PHA synthesis medium containing an appropriate amount of γ-valerolactone plus 10 g /L of glucose as carbon sources for 72 h at 30°C. PHV homopolymer was accumulated in P. denitrificans grown on PHA synthesis medium containing 10 ml/L of n-valeric acid as the sole carbon source for 60 h at 30°C. For the PHB accumulation in E. coli, two-step cultivation was carried out. E. coli DH10B:pBAD-Ae-pha (C. necator PHA synthesis operon) and E. coli DH10B:pMMB-pha (Acinetobacter PHA synthesis operon) were cultivated in 500 mL of LB medium, respectively, and after 12 h, the cells were transferred to 500 mL of 10 g/L glucose containing LB medium and cultivated for 48 h at 37°C. One hundred μg/mL of ampicillin and 34 μg/mL of chloramphenicol were added to the medium for the maintenance of plasmids pBAD-Ae-pha and pMMB-pha, respectively. To investigate the roles of Ca 2+ in C. necator H16, C. necator H16 cells were cultivated in NR medium or PHA synthesis medium containing 20 g/L of fructose as the sole carbon source or 5 g/L of citric acid and 5 g/L of crotonic acid as the cosubstrate in the presence of 0 ~ 25 mM CaCl2 at 30°C .

PHA isolation and characterization.
For the extraction of PHA, the cells were harvested by centrifugation (4,000 x g for 7 min), washed with methanol, and dried overnight under a vacuum at ambient temperature. PHA was extracted from dried cells with hot chloroform in a Pyrex Soxhlet apparatus for 6 h. The solvent extract concentrated by vacuum rotary evaporation was precipitated in rapidly stirred 10 volumes of cold methanol. The isolated PHA was purified by reprecipitation and was dried overnight under a vacuum at ambient temperature. Quantitative determination of the monomer units in PHA was determined by analyzing the methyl esters, which were recovered from a sulfuric acid/methanol treatment of the PHA, using a Hewlett-Packard HP5890 Series II gas chromatograph (GC) equipped with a HP-1 capillary column and a flame ionization detector (7,8). A typical GC run condition was as follows: initial temperature 80°C , 2 min; heating rate, 8°C /min; final temperature 180°C , 0.5 min; carrier (He) flow rate, 3 mL/min; injector temperature, 230°C ; detector temperature, 280°C . The standardization of each GC peak was made against the PHA of known structure characterized by quantitative nuclear magnetic resonance (NMR) analyses [3,4]. The 1 H-NMR analyses of PHA samples were carried out on a Bruker-DRX 500 MHz spectrometer (Germany). The spectra of the samples were recorded at room temperature in CDCl3. The integration of the split spectral signals was performed with standard software.
Enzyme isolation and purification: P. stutzeri BM190 was precultured in NB medium for 12 h, enzymes were obtained by growing the precultured cells in PHB degradation medium (pH 7.5) consisted of 1.7 g/L KH2PO4, 6.2 g/LNa2HPO4·12H20, 1.0 g/L (NH4)2SO4, 0.5 g/L MgSO4·7H2O, 0.1 g/L FeCl3·6H2O, 0.05 g/L CaCl2·2H2O. The strains were cultivated under aerobic conditions at 30°C, 170 rpm in the medium containing 0.15% PHB granules as the sole carbon source. The culture supernatant was applied to a Phenyl-Toyopearl (Tosoh Co., Tokyo, Japan) hydrophobic interaction chromatography column equilibrated with 50 mM Tris-HCl buffer (pH 7.5). Fractions of enzyme with high activity were collected and dialyzed against distilled water. All the enzyme purification steps were carried out at 4°C.

Enzymatic degradation of PHA granules:
The activity of PHB depolymerase was assayed spectrophotometrically by measuring the initial decrease in the optical density (O.D.) of the PHA granules at 660 nm. All of the PHA granules were suspended in 50 mM Tris-HCl buffer (pH 8.0) containing 1 mM MgCl2, and their initial absorbance was 3.0 ± 0.03 at 660 nm. The reaction was initiated by the addition of 0.24 µg/mL (final concentration) of purified enzyme suspended in 50 mM Tris-HCl containing 1 mM MgCl2 (pH 8.0). The decrease in turbidity of the PHB polymer was monitored at 660 nm in a shaker bath at 37°C using a spectrophotometer (Hewlett Packard UV 8452A). As a first order of approximation, the decrease in the turbidity of the PHB granules was analyzed in terms of first-order kinetics, and the degradation rate constant k1 was calculated [5].
Determination of the enzymatic hydrolysis products. The water-soluble products after complete enzymatic degradation of artificial and native P(HB-co-HV) granules were analyzed using a Shimadzu LC-10A high performance liquid chromatography (HPLC) system equipped with a gradient controller and an SPD-10A UV spectrophotometric detector [6]. The reaction mixtures after enzymatic degradation were harvested by centrifugation (10,000 x g, 10 min) and the clear supernatants were collected and filtered throughout 0.2 μm syringe filter (Minisart, Sartorius Stedim Biotech, Germany) to eliminate residual granule debris. Twenty microliters of the supernatant was injected into the Aminex HPX-87H Ion Exclusion column (300 mm ⅹ7.8 mm, Bio-Rad) [7] and then was eluted with 0.5 mM H2SO4 solution at a flow rate of 0. The spectra of the samples were recorded at room temperature in CDCl3. The integration of the split spectral signals was performed with standard software.
Differential scanning calorimeter (DSC). Thermal transitions of the end-capped PHB polyesters were measured under nitrogen flow of 50 mL/min by using a differential scanning calorimeter (DSC) (TA Instruments, New Castle, DE), DSC Q200 equipped with a cooling accessory. Samples of 10-15 mg were encapsulated in aluminum pans and heated from -50 to 150°C at a rate of 10°C/min (first scan). The samples were maintained at 150°C for 1 min and then cooled to -50°C. They were then reheated from -50 to 150°C at a rate of 10°C/min (second scan). The melting temperature (Tm) was taken at the peak of the melting endotherm.
Gel permeation chromatography (GPC). GPC measurements were performed with polymer solution in chloroform (1%, w/v), filtered via 0.45 mm Costar Syrfil filters. The molecular weights were determined using an Agilent 1100 series Gel Permeation Chromatography system (Agilent, Santa Clara, CA), consisting of a series of three PLgel columns (105, 103 , and 102), an Agilent G1310A Isocratic pump, an Agilent 1047A RI detector, an Agilent G1316A column compartment, and an Agilent G1311A vacuum degasser, at a flow rate of 1.0 mL/min. The injection volume of the samples was 60 ml. Chloroform was used as the mobile phase. The run temperature was 30°C . The molecular weight of the polymers were determined relative to polystyrene standards (Polymer Laboratories, Amherst, MA), having a narrow molecular weight distribution.
Determination of particle size. Particle sizes and size distributions were determined by the light scattering method (DLS-8000; Otsuka Electronics Co., Osaka, Japan). The mean particle size of the PHA granules was determined in triplicate and the average values were calculated.
Determination of remaining fructose and NH4 + . Remaining fructose was determined using 3,5-dinitrosalicylic acid (DNS) method [9]. One milliliter of sample was taken from the supernatant and diluted 50-100 fold. The diluted sample was transferred into a test tube, and was added with 3 ml of DNS reagent. The test tube containing 4 ml mixture was placed in the boiling water bath for 5 min and cooled to room temperature. The absorbance of this reaction mixture was measured at 550 nm (A550) on an X-ma 1000 UV/VIS spectrophotometer (Human Science, Korea). Then, five 1 ml standards containing 0.2, 0.4, 0.6, 0.8 and 1.0 mg of fructose, respectively were prepared according to the same procedure as in the above sample solution. The standard curve obtained showed a linear relation between the absorbance and fructose concentration over the concentration range of 0.1-1.0 mg/mL fructose. As the absorbance of the samples were compared to that of the standard curve, the amount of remaining fructose in the sample was determined.
Remaining NH4 + was measured with Nessler's reagent [10]. Nessler reaction was performed by adding 1 mL of Nessler's reagent to 1 mL of the culture supernatant samples. The mixed reaction solution was stirred well, and kept standing for at least 10 min at room temperature. The absorbance of the reaction mixture was measured at 450 nm (A450). From the standard curve showing a linear relation between the absorbance and NH4 + concentration over the concentration range of 0.01-0.03 mg/mL NH4 + solution, the absorbance of samples was compared to that of the standard curve and then the amount of remaining NH4 + in the sample was determined.
Determination of PHB molecular weight. PHB molecular weight was calculated from the Mark-Howink equation which gives a relation between molecular weight and intrinsic viscosity. is related to the methine proton (CH3-CH(OH)-) in the terminal hydroxyl containing group. NMR peak ratio analysis showed that in the purified PHB-1-octadecanol sample, ~20% of the hydroxyl groups was replaced by alkenic proton and ~80% of free hydroxyl group was retained. The fact that the sum of the peak areas for the olefinic proton and methine proton in the opposite end of PHB are almost equal to the half of the peak area at 3.99 ppm peak ascribing to the hydroxyl adjoining methylene in 1-octadecanol revealed that more than 98% of the terminal carboxyl groups were esterified with 1-octadecanol. Mn was calculated to be ~3000 from 1 H-NMR data  HPLC analysis of the enzymatic degradation products of native PHB nanogranules accumulated in various phaZ mutants and phaZC). The cyclized 3HB dimer was initially assumed as a Ca 2+ chelating ligand. But only 3HV monomer and dimer was detected and the peak at 53 min was not observed in the degradation of P(3HV) artificial granules with BM190 WT enzyme even though native P(3HV) granules isolated from Paracoccus denitrificans contained 3.138 mg Ca 2+ /g PHA. Therefore, we concluded that the tiny amount of cyclic 3HB dimer appeared at 53 min was ascribable to a byproduct of the enzymatic degradation reaction of PHB and it was excluded from a candidate molecule for Ca 2+ chelating ligand in our assumption.
h P(3HH-co-3HO-co-3HD) copolymer composed of 3HO as major monomer. The above data showed that the genes related with intracellular PHB degradation have no effect on Ca 2+ controlled PHB accumulation. b) The undetermined deficit in the elemental analysis was assumed to be due to oxygen atom.

Supplementary
c) ICP-AES analysis method was used.