Bacterial strains and media
A. denitrificans A41(deposited in WDCM567 as JCM9787) was cultured in Luria-Bertani (LB) medium (Sambrook et al. 2001) or C medium, which contained (per liter) 5 g of (NH4)2SO4, 2.93 g of KH2PO4, 5.87 g of K2HPO4, 0.3 g of MgSO4·7H2O, 2 g of NaCl, 0.03 g of CaCl2, 0.01 g of FeSO4· H2O, 0.6 mg of NiSO4·7H2O, and 2 mL of a trace element solution; the pH was adjusted to 7.0 using NaOH. The trace element solution contained (per liter) 4 mg of MoO3, 28 mg of ZnSO4·5H2O, 2 mg of CuSO4·5H2O, 4 mg of H3BO3, 4 mg of MnSO4·5H2O, and 4 mg of CoCl2·6H2O. Biphenyl was added to the C medium at a final concentration of 0.5% (not dissolved) or 0.3% as the sole carbon source, and cultures were incubated on a reciprocal shaker at 120 rpm and 30°C. Antibiotics were adequately used as described previously (Sambrook et al. 2001)
For the one-step cultivation, the concentrations of nitrogen sources were altered to achieve a nutrient-deficiency condition that induced PHA production. For this, we used C medium containing 0.5% biphenyl as the sole carbon source and (NH4)2SO4 as the nitrogen source at concentrations changed by 1/10 (N-100), 1/20 (N-50), 1/40 (N-25) and 1/100 (N-10) of the original value (N-1000, C/N ratio = 2). These altered media had corresponding C/N ratios of 21, 41, 83, and 166, respectively.
In the two-step cultivation protocol, the first growth step, which was aimed at gaining cell mass, was performed on LB complex medium until the stationary growth phase was reached, and the second culture used C medium (without any nitrogen source) containing 0.5% biphenyl for PHA production. In between the two phases, cells were harvested using centrifugation and resuspended in the second medium.
In an ideal case, cells were grown using C medium containing 0.3% biphenyl as the first culture, then harvested after 36 h, and their initial dry cell weight was adjusted to 1.38 g/L for resuspension in C medium without nitrogen sources containing 0.3% biphenyl. This medium was then used for growth aiming PHA production.
Cells were harvested at several time points (from 6 to 108 h) by centrifugation at 4°C and were then washed with distilled water and lyophilized.
Polymer analysis using gas chromatography (GC) and gel permeation chromatography (GPC)
The cellular PHA content and polymer composition were determined by analyzing freeze-dried cells using GC. Approximately 15 mg of dry cells were subjected to methanolysis with a solution consisting of 1.7 mL methanol, 0.3-mL 98% sulfuric acid, and 2.0 mL chloroform at 100°C for 140 min to convert the constituents to their methyl esters (Braunegg et al. 1978). The addition of 1 mL water to the reaction mixture induced phase separation. The lower chloroform layer was used for GC analysis on a B353B system (GL Sciences, Eindhoven, The Netherlands) equipped with an InertCap1 capillary column (30 m x 0.25 mm) and a flame-ionization detector.
The polymers accumulated in cells were extracted using chloroform for 48 h at room temperature and purified by reprecipitation with methanol. Their molecular mass was measured using a GPC 20A GPC system and 10A refractive index detector (Shimadzu, Kyoto, Japan) with Shodex K-806M and K-802 columns. Chloroform was used as the eluent at a flow rate of 0.8 mL/min, and samples were applied at 1.0 mg/mL. Polystyrene standards with low polydispersity were used in the construction of a calibration curve.
Biphenyl consumption analysis
Solid-state biphenyl was sterilized using ultraviolet radiation for 5 min on a clean benchtop. Strain A41 was cultured in a test tube with a screw cap containing 10 mL of 0.5% biphenyl (w/v). To evaluate the residual amount of biphenyl in triplicate at nine time points: 12 h, 24 h, 36 h, 48 h, 60 h, 72 h, 84 h, 96 h, and 108 h, the fraction of biphenyl was carried out as follows. To each culture sample, 10 mL of ethyl acetate (Wako Japan Co. Ltd., Osaka, Japan) was added and mixed well, and samples were then subjected to centrifugation (3,300 × g for 10 min.) using an MX-300 centrifuge (TOMY Co. Ltd., Tokyo, Japan). The supernatants were filtered with a polyvinylidene fluoride (PVDF) filter (13 mm diameter, pore size 0.22 µm, Merck Millipore, Burlington, USA). Samples were subjected to high-performance liquid chromatography (HPLC) for measuring the consumption of biphenyl as a solid compound during bacterial cultivation. The following set-up was used in our experiments: Gilson 305 and 306 pumps (Gilson, Lewis Center, USA); an Ascentis C18 15 cm × 4.6 mm, 5 µm column (SUPELCO, Apex Scientific, Kildare, Ireland), detection at 246 nm; and solvent composition consisting of H2O and acetonitrile (ratios of 3 and 7), and a flow rate of 1.0 mL/min. For biphenyl quantification, a benzene calibration curve was used as an internal reference.
Transmission electron microscopy for the observation of polymer accumulation
Cells were harvested, washed twice in 0.1 M phosphate buffer (pH 7.0), and fixed in 2% (w/v) glutaraldehyde in the same solution. Subsequently, cells were suspended in 2% (w/v) OsO4 for 1 h, gradually dehydrated in ethanol [50%, 70%, 80%, 90%, and 99.5% (v/v); 15 min each], and embedded in Quetol651. Ultrathin sections (thickness 70 nm) were cut with a microtome using a Diatome diamond knife. The sections were collected using 400-mesh cupper grids coated with a carbon layer and observed in a Jeol‐2010 electron microscope (Jeol Ltd., Akishima, Japan)
Genetic analysis
The functionality of the phaC gene from A. denitrificans A41 was evaluated using a gene disruption technique. The primers 5'-ATTCTAGACTGGACCCGGAATGCAAC-3' and 5'-ATGGTACCCATCCCGCCTGTAACGTAAG-3' were used to amplify the phaC1 (ade) gene from the A. denitrificans A41 genome. The resulting amplicon was used as a probe for a subsequent colony hybridization. According to the available genome information for A41, the phaC1B1R locus can be efficiently cloned as a 5,997-bp KpnI fragment. The bacterial chromosome was, therefore, digested with the KpnI endonuclease (Takara, Shiga, Japan), a gel fragment containing approximately 6-kb DNA was cut out, and the extracted DNA fragments were cloned into the pUC19 vector (Takara, Shiga, Japan). The DNA library obtained here was subjected to colony hybridization (Grunstein and Wallis 1979). Positive colonies selected from the library were used as candidates. Recombinant plasmids were extracted from these candidates and confirmed using Southern blot analysis (Southern 2006).
The tetracycline resistance gene, derived from the pBR322 plasmid (Takara, Shiga, Japan), was cloned into the phaC1 gene within the KpnI fragment on vector pUC19. The resulting plasmid was digested with KpnI, transformed into A. denitrificans A41 by electroporation (Micro Pulser, BIO-RAD, Hercules, USA), and candidates for the phaC1 disruptant strain were selected on LB plates containing tetracycline. Colonies were selected from these plates, grown in LB medium containing tetracycline, and chromosomal DNA was extracted as described previously (Sambrook et al. 2001). The phaC1-disrupted strains were confirmed and selected using Southern blot analyses.
Bioinformatic analysis of the A41 genes
The pha genes and their neighboring sequences in the A41 genome were analyzed using the IMC GE software (available at https://www.insilico-biology.com). Nucleotide and amino acid (aa) sequences were retrieved from the National Center for Biotechnology Information (NCBI) database, and a phylogenetic tree was constructed based on the partial amino acid sequence (~ 596 aa) of PhaC using the maximum-likelihood method. Bootstrap values (1000 replicates) are shown for each branch.
Genome sequence accession number
Draft genome sequences of Achromobacter denitrificans A41 were deposited into the DNA Data Bank of Japan with accession numbers BLWG01000001-BLWG01001041