Identification and characterization of a novel extracellular polyhydroxyalkanoate depolymerase in the complete genome sequence of Undibacterium sp. KW1 and YM2 strains

Tomohiro Morohoshi; Taishiro Oi; Tomohiro Suzuki; Shunsuke Sato

doi:10.1371/journal.pone.0232698

Abstract

Polyhydroxyalkanoate (PHA) is a biodegradable polymer that is synthesized by a wide range of microorganisms. One of the derivatives of PHA, poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) has flexible material properties and low melting temperature. We have previously demonstrated that PHBH is degradable in a freshwater environment via the formation of biofilm on the surface of the PHBH film. Undibacterium sp. KW1 and YM2, which were isolated from the biofilm present on the PHBH film in the freshwater sample, were shown to have PHBH-degrading activity. In this study, the complete genome sequence of KW1 and YM2 revealed that the extracellular PHA depolymerase gene, designated as phaZ_UD, was present in their chromosomes. Sequence analysis revealed that PhaZ_UD contained four domains: a signal peptide, catalytic domain, linker domain, and substrate-binding domain. Escherichia coli harboring a PhaZ_UD-expressing plasmid showed PHBH-degrading activity in LB medium containing 1 wt% PHBH powder. The recombinant His-tagged PhaZ_UD from KW1 and YM2 was purified from the culture supernatant and shown to have PHBH-degrading activity at the optimum temperature of 35 and 40°C, respectively. When the degradation product in the PHBH solution was treated with PhaZ_UD and assayed by LC-TOF-MS, we detected various oligomer structures, but no more than pentamers, which consist of 3-hydroxybutyrate and 3-hydroxyhexanoate. These results demonstrated that PhaZ_UD may have an endo-type extracellular PHA depolymerase activity.

Citation: Morohoshi T, Oi T, Suzuki T, Sato S (2020) Identification and characterization of a novel extracellular polyhydroxyalkanoate depolymerase in the complete genome sequence of Undibacterium sp. KW1 and YM2 strains. PLoS ONE 15(5): e0232698. https://doi.org/10.1371/journal.pone.0232698

Editor: Pradeep Kumar, North Eastern Regional Institute of Science and Technology, INDIA

Received: January 19, 2020; Accepted: April 19, 2020; Published: May 5, 2020

Copyright: © 2020 Morohoshi et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Data Availability: All relevant data are within the paper and its Supporting Information files.

Funding: T.M. reports a grant from the Kaneka Corporation (Hyogo, Japan), during the conduct of the study; and S.S. is an employee of the Kaneka Corporation. The funder provided support in the form of salaries for author SS, but did not have any additional role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. The specific roles of these authors are articulated in the ‘author contributions’ section.

Competing interests: TM reports receiving grant supports from Kaneka Corporation and SS is an employee of the Kaneka Corporation. The other authors have declared that no competing interest exist. These do not alter our adherence to PLOS ONE policies on sharing data and materials.

Introduction

In modern society, plastic pollution is a serious problem with serious effects on the environment and wildlife [1]. Especially, pollution by smaller pieces of plastic, called microplastic, has become a serious problem [2]. Microplastics are defined as particles less than 5 mm in size, which are generated from the plastic debris fragmented by UV radiation in the aquatic environment [2]. The use of biodegradable plastics is one of the solutions to prevent the generation of microplastics in the aquatic environment. Biodegradable plastics can be broken down by various bacteria, taken as a carbon source, and finally converted into CO₂ [3]. Many kinds of oil- and bio-based biodegradable plastics have been developed and used to solve this serious environmental problem [3]. One of them, polyhydroxyalkanoate (PHA), is synthesized by a wide range of bacteria from renewable resources such as sugars, plant oils, glycerol, and others [4, 5]. It has been demonstrated that PHA is degradable in natural environments by PHA-degrading bacteria, which produce the extracellular PHA-degrading enzyme under both anaerobic and aerobic conditions [6]. For instance, poly(3-hydroxybutyrate) (PHB), one of the representative PHA derivatives, can be degraded by PHA depolymerase, which catalyzes the ester hydrolysis of PHB and converts it to 3-hydroxybutyrate (3HB) monomers or oligomers [7].

Poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) (PHBH) is a random copolymer of 3HB and 3-hydroxyhexanoate (3HH) and has flexible material properties and a low melting temperature, which is advantageous due to lower temperatures needed for pyrolysis [8]. We have previously demonstrated that the PHBH film is degradable in both seawater and freshwater environments [8, 9]. In freshwater environments, PHBH-degrading bacteria isolated from the biofilm formed on the surface of the PHBH film were assigned to the genus Acidovorax, Undibacterium, and Chitinimonas [9]. Notably, the analysis of the bacterial community in the biofilms formed on the PHBH films showed that the genera Acidovorax and Undibacterium were predominant. Although the genus Acidovorax has been previously shown to be PHA-degrading bacteria [10, 11], there is no report of the genus Undibacterium as having this activity. Some bacterial strains, which are proposed as novel species within the genus Undibacterium, have been isolated from the freshwater samples or freshwater species [12, 13]. However, the physiological properties, genomic information, and genetic application of the genus Undibacterium have yet to be reported. In our previous study, we isolated two PHBH-degrading strains, Undibacterium sp., KW1 and YM2, from the Japanese lakes Kawaguchi-ko and Yamanaka-ko, respectively [9]. In this study, to elucidate the PHBH-degrading activity of the KW1 and YM2 strains, their complete genome was sequenced and a novel PHBH-degrading gene was identified. Moreover, we investigated the enzymatic activity of the purified PHBH-degrading enzyme.

Materials and methods

Bacterial strains, plasmids, compounds, and growth conditions

Escherichia coli was grown at 37°C in Luria-Bertani (LB) medium. Undibacterium sp. KW1 and YM2 were grown at 30°C in R2A medium (Difco, Tokyo, Japan). Solid bacterial media were prepared by adding agar to a final concentration of 1.5%. Antibiotics were added as required, at final concentrations of 100 and 20 μg/mL for ampicillin and chloramphenicol, respectively. The PHBH powder (KANEKA Biodegradable Polymer^™ X131A: 3HHx = 6 mol%) was obtained from the Kaneka Corporation, Hyogo, Japan.

Genome sequencing

The genomic DNA of the KW1 and YM2 strains was extracted using the DNeasy Blood & Tissue Kit (Qiagen, Tokyo, Japan). Genome sequencing was performed on the PacBio RSII platform (Pacific Biosciences, Menlo Park, CA) using libraries prepared with the SMRTbell Template Prep Kit 1.0 (Pacific Biosciences) by Macrogen Japan Corp. (Kyoto, Japan). The sequencing reads were assembled using the Canu version 1.6 software [14]. Prediction of putative coding sequences and gene annotations was performed using the Microbial Genome Annotation Pipeline (MiGAP) from the DNA Data Bank of Japan (DDBJ). Protein-coding sequences were predicted using a combination of MetaGeneAnnotator [15], RNAmmer [16], tRNAScan [17], and BLAST [18].

Cloning of the phaZ gene from the Undibacterium strains

The phaZ genes from the KW1 and YM2 strains were amplified by PCR with KOD FX Neo DNA polymerase (Toyobo, Osaka, Japan) and the following primer sets: 5ʹ-TCTGGATCCCCAGGAATACGGACGTATGTCCACCC-3ʹ and 5ʹ-TCTAAGCTTCAGTAAAGGCTCAGGAGCAGACACGC-3ʹ for KW1, and 5ʹ-TCTGGATCCGGAATACGGAAGTATGTCCACCCTGG-3ʹ and 5ʹ-TCTAAGCTTCTGACTGCGCCTTATCAGATTCAGGG-3ʹ for YM2. The PCR was performed using the following cycling parameters: 98°C for 10 s and 68°C for 1 min for 30 cycles. The PCR fragments were digested with the BamHI and HindIII (underlined) enzymes and inserted into the same restriction sites of the low copy vector pSTV28 (Takara Bio, Shiga, Japan). The constructed plasmids were transformed into E. coli DH5α cells and used for the PHBH degradation assay. The PHBH powder was mixed with LB medium at a concentration of 1 mg/mL and sonicated to obtain better dispersion. After being autoclaved, 10 mL of agar medium were poured into 60 mm plastic Petri dishes. E. coli cells harboring the phaZ-expressing plasmid were streaked onto the center of PHBH-containing LB plates. After incubation for one week at 30°C, the development of clear zones around the colonies was considered as degradation of PHBH.

Expression and purification of His-tagged PhaZ

For the construction of the expression plasmid for His-tagged PhaZ from the KW1 and YM2 strains, the phaZ genes were amplified using the KOD FX Neo DNA polymerase and following primer sets: 5ʹ-TCTCATATGAATCATATCAAGACCATGTTGCGG-3ʹ and 5ʹ-TCTGGATCCTCAATGATGATGATGATGATGAGGGCAATTACCAATGATG-3ʹ for KW1, and 5ʹ-TCTCATATGAACAAGATCAAGATTATCTTGCAG-3ʹ and 5ʹ-TCTGGATCCTCAATGATGATGATGATGATGGGGGCAATTACCAATGATG-3ʹ for YM2. The PCR was performed using the following cycling parameters: 98°C for 10 s and 68°C for 1 min for 30 cycles. The pGEX28 plasmid, which is based on the low-copy vector pSTV28, was constructed for the expression of His-tagged PhaZ under the tac promoter in this study (S1 Fig). The PCR products were digested by the NdeI and BamHI (underlined) enzymes and inserted into the same restriction sites on the pGEX28 plasmid to construct the pGEX28-KW1 (for PhaZ from KW1) and pGEX28-YM2 (for PhaZ from YM2) plasmid, respectively.

For the expression and purification of His-tagged PhaZ, a colony of E. coli BL21(DE3) harboring the pGEX28-KW1 or pGEX28-YM2 plasmid was directly inoculated into 100 mL of LB medium containing chloramphenicol. Cells were cultivated for approximately 8 h at 30°C until they reached the late exponential growth phase. Then, isopropyl-β-d-thiogalactoside (IPTG) was added at a final concentration of 500 μM. After incubation for 12 h at 30°C, the cell-free supernatant was prepared by centrifugation. Protein purification was performed using the ÄKTA start system (GE Healthcare, Tokyo, Japan). The filtrated cell-free supernatant was loaded onto a HisTrap affinity chromatography column (GE Healthcare) equilibrated with a loading buffer (20 mM sodium phosphate buffer, 500 mM NaCl, pH 7.4) containing 20 mM imidazole and eluted with the loading buffer containing 500 mM imidazole after washing off unbound bacterial proteins. The expression and purity of the His-tagged PhaZ were confirmed by SDS-PAGE.

Assay of PHBH-degrading activity

To evaluate the PHBH-degrading activity of the purified PhaZ protein, a PHBH suspension was prepared, with a final concentration of 1 mg/mL in 10 mM Tris-HCl buffer (pH 8.0) and 1 mM CaCO₃, and sonicated to obtain a better dispersion. To estimate the optimal temperature for the PHBH-degrading activity of PhaZ, 30 μL of the purified PhaZ solution was mixed with 270 μL of the PHBH solution. After incubation at 20 to 55°C for 1 h, 700 μL of distilled water was added. The PHBH-degrading activity was evaluated by measuring the decrease in turbidity at 600 nm and the maximum value was set to 100%. The experiment was repeated at least three times.

LC-TOF-MS analysis of PHBH degradation products produced by PhaZ

A total of 500 μL of the purified PhaZ solution was mixed with 4.5 mL of the 5 mg/mL PHBH solution and statically incubated at 30°C until the turbidity disappeared. The chemical composition of the PHBH degradation products was analyzed by LC-TOF-MS. The PHBH degradation products were analyzed in negative ionization mode using a NexeraX2 (Shimadzu, Kyoto, Japan) coupled with a maXis4G mass spectrometer (Bruker Daltonics, Bremen, Germany) with an ACQUITY UPLC HSS T3 column (1.8 μm, 2.1 × 150 mm; Nihon Waters K.K., Tokyo, Japan) under the following conditions: ionization method; APCI method, column oven; 50°C, mass range; 50–1550 m/z. The mobile phase was 0.1% formic acid and an acetonitrile gradient and the flow rate was 0.5 mL/min.

Nucleotide sequence accession number

The complete genome of Undibacterium sp. KW1 was deposited in the DDBJ/ENA/GenBank databases under the accession numbers AP018439 (chromosome) and AP018440 (plasmid pUKW01). The complete genome of Undibacterium sp. YM2 was deposited in the DDBJ/ENA/GenBank databases under the accession numbers AP018441 (chromosome) and AP018442 (plasmid pUYM01).

Results and discussions

The complete genome sequence revealed the presence of the PHA-depolymerase gene in Undibacterium strains

To identify the genes involved in the degradation of PHBH, we obtained the complete genome sequences of the KW1 and YM2 strains using the PacBio RSII platform. For the KW1 strain, we obtained 105,851 reads with an average length of 10,942 bp, which sequenced approximately 1.16 Gbp. After assembly, the genome of the KW1 strain included a single chromosome and one plasmid. The complete circular chromosome and the endogenous plasmid pUKW01 were 6,557,365 and 135,601 bp in size, respectively. For the YM2 strain, 145,259 reads with an average length of 10,896 bp were produced, which sequenced approximately 1.58 Gbp. After assembly, the genome of the YM2 strain also included a single chromosome and one plasmid. The complete circular chromosome and the endogenous plasmid pUYM01 were 6,484,812 and 175,363 bp in size, respectively. The complete genome of one genus Undibacterium strain, Undibacterium parvum DSM 23061, has been previously sequenced (accession number CP034464). The complete genome of DSM 23061 was shorter than that of KW1 and YM2 and did not contain the endogenous plasmid. The general features of the complete genome of the KW1 and YM2 strains are shown in Table 1.

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Table 1. The general features of the genome of Undibacterium sp. KW1 and YM2, and U. parvum DSM 23061.

https://doi.org/10.1371/journal.pone.0232698.t001

Subsequently, the presence of a PHA depolymerase gene homolog was investigated in the complete genomes of the KW1 and YM2 strains. We found that the amino acid sequences of UNDKW_2839 and UNDKW_4104 from KW1, and UNDYM_2916 and UNDYM_4081 from YM2 showed high similarities to the known PHA depolymerase. Generally, crystalline PHA-based polymers, such as the PHBH film, can be degraded by extracellular PHA depolymerase but not the intracellular version of this enzyme [10]. The results of the SignalP analysis [19] showed that the UNDKW_4104 sequence from the KW1 strain and the UNDYM_4081 sequence from the YM2 strain were predicted to be extracellular proteins with an N-terminal signal peptide (SP) of 26 amino acid residues. Therefore, UNDKW_4104 and UNDYM_4081 were identified as the extracellular PHA depolymerase gene involved in PHBH degradation and designed as the phaZ_UD gene. On the other hand, since the UNDKW_2839 sequence from the KW1 strain and the UNDYM_2916 sequence from the YM2 strain did not contain N-terminal signal peptides, it is possible that these proteins may act as intracellular PHA depolymerases.

In silico characterization of the PhaZ_UD homolog from the family Oxalobacteraceae

The known PHA depolymerases are composed of a catalytic domain (CD) at the N terminus, substrate-binding domain (SBD) at the C terminus, and linker domain (LD) connecting the two previously mentioned domains [20]. Sequence analysis revealed that the putative PhaZ_UD contained four domains, SP, CD, LD, and SBD (Fig 1). PHA depolymerases have a catalytic triad as the active site and the catalytic serine is embedded in a GXSXG sequence motif known as a lipase box [7]. The catalytic triad Ser165, Asp240, and His299 was found in the PhaZ_UD protein and Ser165 was also one of the residues in the lipase box-like sequence (GLSSG) (Fig 1). In general PhaZ sequences, the LD domain showed a high degree of homology to the fibronectin type III domain [21]. PROSITE motif scan [22] revealed the presence of a fibronectin type III domain homolog in the PhaZ protein from the KW1 and YM2 strains. The conserved motif sequence (sxxxHxxAGRa) was observed in all SBDs in known PhaZ sequences [21]. The SBD-like sequences (SNYAHVQAGRA) were also found at the C-termini in PhaZ_UD (Fig 1). These results demonstrate that the features of previously reported PHA depolymerases were well-conserved in PhaZ_UD.

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Fig 1. Amino acid sequence and alignment of PhaZ_UD and PhaZ homologs from the family Oxalobacteraceae.

The putative SP, CD, LD, and SBD were marked in blue, orange, yellow, and green, respectively. The predicted cleavage site of the N-terminal signal peptide was labeled by an arrow. The putative catalytic triad was marked by asterisks. The conserved lipase box sequence was shown by an overline. The conserved motif sequence of SBD domain was shown by a dotted underline. PhaZ_UD were compared with extracellular PhaZ homologs from the family Oxalobacteraceae, Duganella sp. HH101 (UniProt accession no. A0A1E7WFX8), Janthinobacterium sp. HH01 (L9PFG0), and Massilia sp. PDC64 (A0A1G7ANH7).

https://doi.org/10.1371/journal.pone.0232698.g001

The results of the BLAST search revealed that PhaZ_UD showed high similarity (over 80%) to a PhaZ homolog from bacteria belonging to the family Oxalobacteraceae, namely Duganella sp. HH101, Janthinobacterium sp. HH01, and Massilia sp. PDC64 (Fig 1). Similarly to PhaZ_UD, the PhaZ homologs of the family Oxalobacteraceae also conserved the SP, catalytic triad (Ser, Asp, and His), lipase box-like sequence, and SBD motif sequence (Fig 1). To investigate the distribution of the PhaZ homolog in the family Oxalobacteraceae, we searched for the presence of the phaZ gene homolog in the complete genome of 24 strains belonging to this family in the NCBI GenBank database. The results of the BLAST search revealed that the phaZ gene homolog was present in the complete genome of some strains belonging to the genus Massilia, Janthinobacterium, and Herbaspirillum, but not in Collimonas and Oxalobacter (S2 Fig). Although the complete genome sequence of the Undibacterium parvum type strain DSM 23061 possesses the putative intracellular PHA depolymerase gene (EJN92_20730), the extracellular PHA depolymerase gene homolog, which contains an SP at its N-terminus, was not found in that genome. The length of the chromosome of the DSM 23061 strain (approximately 4.9 Mbp) was shorter than that of the KW1 and YM2 strains (approximately 6.5 Mbp) (Table 1). It is possible that the chromosome of the DSM 23061 strain was shortened due to the process of evolution and, therefore, lost the extracellular PHA depolymerase gene.

The PhaZ_UD proteins have PHBH-degrading activity

As the KW1 and YM2 strains were isolated from a freshwater environment as PHBH-degrading bacteria, the PhaZ_UD proteins from the two strains were evaluated for their PHBH-degrading activities. Because intracellular expression of PhaZ_UD from the KW1 and YM2 strains in E. coli cell caused a growth defect in our experimental conditions, the phaZ_UD gene from the KW1 and YM2 strains was subcloned into the low-copy pSTV28 vector based on the p15A origin. E. coli harboring the PhaZ_UD-expressing plasmid were checked for PHBH-degrading activity on LB medium containing 1 wt% PHBH powder. After incubation for one week at 30°C, the development of clear zones around the colonies was observed (S3 Fig). On the other hand, E. coli harboring the putative intracellular PHA depolymerase genes, UNDKW_2839 from the KW1 strain and UNDYM_2916 from the YM2 strain, did not show PHBH-degrading activity (S3 Fig). These results demonstrated that PhaZ_UD may function as an extracellular PHA depolymerase and be involved in the degradation of PHBH in the parent strains, Undibacterium sp. KW1 and YM2.

Expression, purification, and characterization of PhaZ_UD

To obtain the purified PhaZ_UD protein, the recombinant plasmid for expressing PhaZ_UD, which was fused with a histidine tag at the C-terminus, was constructed and transformed into E. coli BL21(DE3) cells. Since the His-tagged PhaZ_UD preserved its SP sequence at N-terminal, the His-tagged PhaZ_UD were secreted and therefore purified from the culture supernatant. The results from the SDS-PAGE revealed that the overexpressed products were approximately 56 kDa in size, as expected (Fig 2A). To confirm whether the two PhaZ_UD proteins have PHBH-degrading activity, the purified His-tagged PhaZ_UD was mixed with PHBH solution and incubated at 30°C. After 4 h incubation, the turbidity of the PHBH solution treated with His-tagged PhaZ_UD was significantly decreased (Fig 2B). These results demonstrated that the PhaZ_UD proteins have PHBH-degrading activity and that the protein is secreted.

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Fig 2. Purification and characterization of His-tagged PhaZ_UD.

(A) Purification of His-tagged PhaZ_UD from the culture supernatant of the KW1 and YM2 strains. Lane M, protein molecular weight marker (Nihon Genetics, Tokyo, Japan); lane 1, cell-free culture supernatant; and lane 2, purified His-tagged PhaZ_UD from KW1 or YM2. Samples were analyzed by SDS-PAGE in a 10% polyacrylamide gel. The approximately 56 kDa protein band of PhaZ_UD was indicated by an arrow. (B) The PHBH-degrading activity of His-tagged PhaZ_UD. A total of 200 μL of purified PhaZ_UD sample was mixed with 800 μL of 5 mg/mL PHBH solution in a 1.5 mL microtube. After incubation for 4 h at 30°C, PHBH-degrading activity was observed as a drastic decrease in turbidity in the samples mixed with PhaZ_UD.

https://doi.org/10.1371/journal.pone.0232698.g002

To estimate the optimal temperature for the PHBH-degrading activity of PhaZ_UD, the PHBH powder was incubated with PhaZ_UD at various temperatures. His-tagged PhaZ_UD from the KW1 and YM2 strains displayed over 80% of its maximum activity between 30 and 40°C and the optimum temperatures of PhaZ_UD from the KW1 and YM2 strains were 35 and 40°C, respectively (Fig 3). On the other hand, the relative activity was greatly reduced at over 45°C. In a previous study, the enzymatic properties of the PHA depolymerase from Alcaligenes faecalis AE122 and Acidovorax sp. DP5, which belonged to the order Burkholderiales, were characterized [10, 23]. Since the optimum temperature for PHA-degrading activity of the proteins from A. faecalis AE122 and Acidovorax sp. DP5 were 55 and 40°C, respectively, the optimum temperature of PhaZ_UD was similar to that of Acidovorax sp. We have demonstrated that the genus Acidovorax and Undibacterium are the dominant PHBH-degrading bacteria in the biofilms formed on the PHBH film in freshwater samples [9]. Therefore, it was assumed that PHBH-degrading enzymes with similar properties were secreted by the Undibacterium and its related bacteria in the biofilm, which were then used to degrade the PHBH films.

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Fig 3. Assay for the optimal temperature of PhaZ_UD.

The purified PhaZ_UD solution from the KW1 (A) and YM2 (B) strains was mixed with 1 mg/mL PHBH solution and incubated at temperatures ranging from 20 to 50°C. After incubation for 1 h, PHBH-degrading activity was evaluated by measuring the decrease in turbidity at 600 nm. The maximum activity of each PHBH degradation was defined as 100%. The experiments were performed at least three times and error bars indicate standard deviations.

https://doi.org/10.1371/journal.pone.0232698.g003

PhaZ_UD works as an endo-type PHA depolymerase

To elucidate the mechanism of PHBH degradation by PhaZ_UD, we investigated the degradation products in the PHBH solution treated with PhaZ_UD from the KW1 strain by LC-TOF-MS. The His-tagged PhaZ_UD-treated PHBH solution was used for LC-TOF-MS assay just before its turbidity completely disappeared. The detected PHBH monomers or oligomers, which was derived from PHBH degradation by PhaZ_UD, were listed in Table 2. In this assay, oligomers with a molecular weight of 1,000 or more were not detected. The detected oligomers with a higher molecular weight were tetramers and pentamers, which consisted of two 3HB and 3HH, and two 3HB and three HH, respectively. The detected trimers contained three types of composition, which were 3HB-3HH-3HB, 3HH-3HB-3HH, and 3HH-3HH-3HB. The detected dimers contained 3HB-3HH and 3HB-3HB, but not 3HH-3HH. The detected monomers contained both 3HB and 3HH. Due to the fact that oligomers of various lengths were detected as the degradation product in the PHBH solution treated with PhaZ_UD, it was suggested that PhaZ_UD may have at least an endo-type PHA depolymerase activity, and might have an exo-type PHA depolymerase and/or PHA oligomer hydrolase activity. We used a PHBH powder containing 3HH at a concentration of 6 mol% in this study. Although 3HB-3HB bonds are present at a higher ratio than 3HH-3HB and 3HH-3HH, all three structure of the detected trimers contained a 3HH unit and the 3HB-3HB-3HB trimer and 3HH-3HH dimer was not detected in the PhaZ_UD-treated PHBH solution. These features suggested that PhaZ had a lower hydrolysis activity against PHBH oligomers that contained multiple 3HH units.

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Table 2. Chemical formula, ionization mode, theoretical and measured m/z value, and retention time for detected metabolites.

https://doi.org/10.1371/journal.pone.0232698.t002

Conclusions

In this study, we demonstrated that the genome sequences of the Undibacterium sp. KW1 and YM2 contain the phaZ gene homolog and that PhaZ_UD degrades PHBH by acting as an endo-type PHA depolymerase. The genus Undibacterium belongs to the family Oxalobacteraceae, which is a phylogenetically diverse aquatic bacterium family [24]. Although the family Oxalobacteraceae includes the genera Oxalobacter, Collimonas, Massilia, Duganella, Janthinobacterium, Herbaspirillum, and Undibacterium [24], it has not been reported that these genera have extracellular PHA-degrading activity and possess the phaZ gene homolog in their genome. The bacterial strains belonging to the genus Undibacterium has been isolated from the various aquatic environments. However, the role of genus Undibacterium in such environments remains unclear. We have previously demonstrated that the genus Undibacterium was predominant in the biofilm formed on the PHBH films in freshwater samples obtained from various places in Japan [9]. In the biofilm, it is possible that the genus Undibacterium produce extracellular PHA depolymerase, degrade PHBH films, and uptake PHBH oligomers with reduced molecular weight as a carbon source. Therefore, in aquatic environments where the genus Undibacterium might be ubiquitous, PHBH-based polymers may be an attractive choice, as this type of biodegradable might not remain as microplastic due to degradation.

Supporting information

S1 Fig. Vector map of pGEX28.

https://doi.org/10.1371/journal.pone.0232698.s001

(TIF)

S2 Fig. Phylogenetic tree based on the complete sequences of chromosome of 24 strains belonged to the family Oxalobacteraceae in NCBI GenBank database.

The name of bacterial strains, which have the phaZ gene homolog in their complete genome, was shown in blue.

https://doi.org/10.1371/journal.pone.0232698.s002

(TIF)

S3 Fig. PHBH-degrading activity of E. coli harboring pUC118-based plasmids.

PHBH-degrading activity was detected on the LB agar plate containing 1 mg/mL PHBH powder after incubation for one week at 30°C. The development of clear zones around the colonies was evaluated as a degradation of PHBH.

https://doi.org/10.1371/journal.pone.0232698.s003

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S1 Table. Raw data for Fig 3.

https://doi.org/10.1371/journal.pone.0232698.s004

(XLSX)

S1 Raw Images.

https://doi.org/10.1371/journal.pone.0232698.s005

(PDF)

References

1. Jambeck JR, Geyer R, Wilcox C, Siegler TR, Perryman M, Andrady A, et al. Plastic waste inputs from land into the ocean. Science. 2015;347: 768–771. pmid:25678662
- View Article
- PubMed/NCBI
- Google Scholar
2. Bakir A, O’Connor IA, Rowland SJ, Hendriks AJ, Thompson RC. Relative importance of microplastics as a pathway for the transfer of hydrophobic organic chemicals to marine life. Environ Pollut. 2016;219: 56–65. pmid:27661728
- View Article
- PubMed/NCBI
- Google Scholar
3. Tokiwa Y, Calabia BP. Biodegradability and biodegradation of polyesters. J Polym Environ. 2007;15: 259–267.
- View Article
- Google Scholar
4. Anderson AJ, Haywood GW, Dawes EA. Biosynthesis and composition of bacterial poly(hydroxyalkanoates). Int J Biol Macromol. 1990;12: 102–105. pmid:2078525
- View Article
- PubMed/NCBI
- Google Scholar
5. Guo-qiang C, Qiong W, Kai Z, Peter HY. Functional polyhydroxyalkanoates synthesized by microorganisms. Chinese J Polym Sci. 2000;18: 389–396.
- View Article
- Google Scholar
6. Lu J, Takahashi A, Ueda S. 3-Hydroxybutyrate oligomer hydrolase and 3-hydroxybutyrate dehydrogenase participate in intracellular polyhydroxybutyrate and polyhydroxyvalerate degradation in Paracoccus denitrificans. Appl Environ Microbiol. 2014;80: 986–993. pmid:24271169
- View Article
- PubMed/NCBI
- Google Scholar
7. Knoll M, Hamm TM, Wagner F, Martinez V, Pleiss J. The PHA Depolymerase Engineering Database: A systematic analysis tool for the diverse family of polyhydroxyalkanoate (PHA) depolymerases. BMC Bioinformatics. 2009;10: 89 pmid:19296857
- View Article
- PubMed/NCBI
- Google Scholar
8. Morohoshi T, Ogata K, Okura T, Sato S. Molecular characterization of the bacterial community in biofilms for degradation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) films in seawater. Microbes Environ. 2018;33: 19–25. pmid:29386425
- View Article
- PubMed/NCBI
- Google Scholar
9. Morohoshi T, Oi T, Aiso H, Suzuki T, Okura T, Sato S. Biofilm formation and degradation of commercially available biodegradable plastic films by bacterial consortiums in freshwater environments. Microbes Environ. 2018;33: 332–335. pmid:30158390
- View Article
- PubMed/NCBI
- Google Scholar
10. Vigneswari S, Lee TS, Bhubalan K, Amirul AA. Extracellular polyhydroxyalkanoate depolymerase by Acidovorax sp. DP5. Enzyme Res. 2015;2015: 212159. pmid:26664741
- View Article
- PubMed/NCBI
- Google Scholar
11. Volova TG, Prudnikova SV, Vinogradova ON, Syrvacheva DA, Shishatskaya EI. Microbial degradation of polyhydroxyalkanoates with different chemical compositions and their biodegradability. Microb Ecol. 2017;73: 353–367. pmid:27623963
- View Article
- PubMed/NCBI
- Google Scholar
12. Kampfer P, Irgang R, Busse HJ, Poblete-Morales M, Kleinhagauer T, Glaeser SP, et al. Undibacterium danionis sp. nov. isolated from a zebrafish (Danio rerio). Int J Syst Evol Microbiol. 2016;66: 3625–3631. pmid:27307140
- View Article
- PubMed/NCBI
- Google Scholar
13. Chen WM, Hsieh TY, Young CC, Sheu SY. Undibacterium amnicola sp. nov., isolated from a freshwater stream. Int J Syst Evol Microbiol. 2017;67: 5094–5101. pmid:29058644
- View Article
- PubMed/NCBI
- Google Scholar
14. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27: 722–736. pmid:28298431
- View Article
- PubMed/NCBI
- Google Scholar
15. Noguchi H, Taniguchi T, Itoh T. MetaGeneAnnotator: detecting species-specific patterns of ribosomal binding site for precise gene prediction in anonymous prokaryotic and phage genomes. DNA Res. 2008;15: 387–396. pmid:18940874
- View Article
- PubMed/NCBI
- Google Scholar
16. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35: 3100–3108. pmid:17452365
- View Article
- PubMed/NCBI
- Google Scholar
17. Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25: 955–964. pmid:9023104
- View Article
- PubMed/NCBI
- Google Scholar
18. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215: 403–410. pmid:2231712
- View Article
- PubMed/NCBI
- Google Scholar
19. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8: 785–786. pmid:21959131
- View Article
- PubMed/NCBI
- Google Scholar
20. Ohura T, Kasuya K-I, Doi Y. Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains. Appl Environ Microbiol. 1999;65: 189–197. pmid:9872779
- View Article
- PubMed/NCBI
- Google Scholar
21. Kasuya K-i, Takano T, Tezuka Y, Hsieh WC, Mitomo H, Doi Y. Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1. Int J Biol Macromol. 2003;33: 221–226. pmid:14607367
- View Article
- PubMed/NCBI
- Google Scholar
22. Sigrist CJ, Cerutti L, Hulo N, Gattiker A, Falquet L, Pagni M, et al. PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform. 2002;3: 265–274. pmid:12230035
- View Article
- PubMed/NCBI
- Google Scholar
23. Kita K, Ishimaru K, Teraoka M, Yanase H, Kato N. Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122. Appl Environ Microbiol. 1995;61: 1727–1730. pmid:7646009
- View Article
- PubMed/NCBI
- Google Scholar
24. Eder W, Wanner G, Ludwig W, Busse H-J, Ziemke-Kägeler F, Lang E. Description of Undibacterium oligocarboniphilum sp. nov., isolated from purified water, and Undibacterium pigrum strain CCUG 49012 as the type strain of Undibacterium parvum sp. nov., and emended descriptions of the genus Undibacterium and the species Undibacterium pigrum. Int J Syst Evol Microbiol. 2011;61: 384–391. pmid:20305060
- View Article
- PubMed/NCBI
- Google Scholar

[ref1] 1. Jambeck JR, Geyer R, Wilcox C, Siegler TR, Perryman M, Andrady A, et al. Plastic waste inputs from land into the ocean. Science. 2015;347: 768–771. pmid:25678662
View Article
PubMed/NCBI
Google Scholar

[2] View Article

[3] PubMed/NCBI

[4] Google Scholar

[ref2] 2. Bakir A, O’Connor IA, Rowland SJ, Hendriks AJ, Thompson RC. Relative importance of microplastics as a pathway for the transfer of hydrophobic organic chemicals to marine life. Environ Pollut. 2016;219: 56–65. pmid:27661728
View Article
PubMed/NCBI
Google Scholar

[6] View Article

[7] PubMed/NCBI

[8] Google Scholar

[ref3] 3. Tokiwa Y, Calabia BP. Biodegradability and biodegradation of polyesters. J Polym Environ. 2007;15: 259–267.
View Article
Google Scholar

[10] View Article

[11] Google Scholar

[ref4] 4. Anderson AJ, Haywood GW, Dawes EA. Biosynthesis and composition of bacterial poly(hydroxyalkanoates). Int J Biol Macromol. 1990;12: 102–105. pmid:2078525
View Article
PubMed/NCBI
Google Scholar

[13] View Article

[14] PubMed/NCBI

[15] Google Scholar

[ref5] 5. Guo-qiang C, Qiong W, Kai Z, Peter HY. Functional polyhydroxyalkanoates synthesized by microorganisms. Chinese J Polym Sci. 2000;18: 389–396.
View Article
Google Scholar

[17] View Article

[18] Google Scholar

[ref6] 6. Lu J, Takahashi A, Ueda S. 3-Hydroxybutyrate oligomer hydrolase and 3-hydroxybutyrate dehydrogenase participate in intracellular polyhydroxybutyrate and polyhydroxyvalerate degradation in Paracoccus denitrificans. Appl Environ Microbiol. 2014;80: 986–993. pmid:24271169
View Article
PubMed/NCBI
Google Scholar

[20] View Article

[21] PubMed/NCBI

[22] Google Scholar

[ref7] 7. Knoll M, Hamm TM, Wagner F, Martinez V, Pleiss J. The PHA Depolymerase Engineering Database: A systematic analysis tool for the diverse family of polyhydroxyalkanoate (PHA) depolymerases. BMC Bioinformatics. 2009;10: 89 pmid:19296857
View Article
PubMed/NCBI
Google Scholar

[24] View Article

[25] PubMed/NCBI

[26] Google Scholar

[ref8] 8. Morohoshi T, Ogata K, Okura T, Sato S. Molecular characterization of the bacterial community in biofilms for degradation of poly(3-hydroxybutyrate-co-3-hydroxyhexanoate) films in seawater. Microbes Environ. 2018;33: 19–25. pmid:29386425
View Article
PubMed/NCBI
Google Scholar

[28] View Article

[29] PubMed/NCBI

[30] Google Scholar

[ref9] 9. Morohoshi T, Oi T, Aiso H, Suzuki T, Okura T, Sato S. Biofilm formation and degradation of commercially available biodegradable plastic films by bacterial consortiums in freshwater environments. Microbes Environ. 2018;33: 332–335. pmid:30158390
View Article
PubMed/NCBI
Google Scholar

[32] View Article

[33] PubMed/NCBI

[34] Google Scholar

[ref10] 10. Vigneswari S, Lee TS, Bhubalan K, Amirul AA. Extracellular polyhydroxyalkanoate depolymerase by Acidovorax sp. DP5. Enzyme Res. 2015;2015: 212159. pmid:26664741
View Article
PubMed/NCBI
Google Scholar

[36] View Article

[37] PubMed/NCBI

[38] Google Scholar

[ref11] 11. Volova TG, Prudnikova SV, Vinogradova ON, Syrvacheva DA, Shishatskaya EI. Microbial degradation of polyhydroxyalkanoates with different chemical compositions and their biodegradability. Microb Ecol. 2017;73: 353–367. pmid:27623963
View Article
PubMed/NCBI
Google Scholar

[40] View Article

[41] PubMed/NCBI

[42] Google Scholar

[ref12] 12. Kampfer P, Irgang R, Busse HJ, Poblete-Morales M, Kleinhagauer T, Glaeser SP, et al. Undibacterium danionis sp. nov. isolated from a zebrafish (Danio rerio). Int J Syst Evol Microbiol. 2016;66: 3625–3631. pmid:27307140
View Article
PubMed/NCBI
Google Scholar

[44] View Article

[45] PubMed/NCBI

[46] Google Scholar

[ref13] 13. Chen WM, Hsieh TY, Young CC, Sheu SY. Undibacterium amnicola sp. nov., isolated from a freshwater stream. Int J Syst Evol Microbiol. 2017;67: 5094–5101. pmid:29058644
View Article
PubMed/NCBI
Google Scholar

[48] View Article

[49] PubMed/NCBI

[50] Google Scholar

[ref14] 14. Koren S, Walenz BP, Berlin K, Miller JR, Bergman NH, Phillippy AM. Canu: scalable and accurate long-read assembly via adaptive k-mer weighting and repeat separation. Genome Res. 2017;27: 722–736. pmid:28298431
View Article
PubMed/NCBI
Google Scholar

[52] View Article

[53] PubMed/NCBI

[54] Google Scholar

[ref15] 15. Noguchi H, Taniguchi T, Itoh T. MetaGeneAnnotator: detecting species-specific patterns of ribosomal binding site for precise gene prediction in anonymous prokaryotic and phage genomes. DNA Res. 2008;15: 387–396. pmid:18940874
View Article
PubMed/NCBI
Google Scholar

[56] View Article

[57] PubMed/NCBI

[58] Google Scholar

[ref16] 16. Lagesen K, Hallin P, Rodland EA, Staerfeldt HH, Rognes T, Ussery DW. RNAmmer: consistent and rapid annotation of ribosomal RNA genes. Nucleic Acids Res. 2007;35: 3100–3108. pmid:17452365
View Article
PubMed/NCBI
Google Scholar

[60] View Article

[61] PubMed/NCBI

[62] Google Scholar

[ref17] 17. Lowe TM, Eddy SR. tRNAscan-SE: a program for improved detection of transfer RNA genes in genomic sequence. Nucleic Acids Res. 1997;25: 955–964. pmid:9023104
View Article
PubMed/NCBI
Google Scholar

[64] View Article

[65] PubMed/NCBI

[66] Google Scholar

[ref18] 18. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ. Basic local alignment search tool. J Mol Biol. 1990;215: 403–410. pmid:2231712
View Article
PubMed/NCBI
Google Scholar

[68] View Article

[69] PubMed/NCBI

[70] Google Scholar

[ref19] 19. Petersen TN, Brunak S, von Heijne G, Nielsen H. SignalP 4.0: discriminating signal peptides from transmembrane regions. Nat Methods. 2011;8: 785–786. pmid:21959131
View Article
PubMed/NCBI
Google Scholar

[72] View Article

[73] PubMed/NCBI

[74] Google Scholar

[ref20] 20. Ohura T, Kasuya K-I, Doi Y. Cloning and characterization of the polyhydroxybutyrate depolymerase gene of Pseudomonas stutzeri and analysis of the function of substrate-binding domains. Appl Environ Microbiol. 1999;65: 189–197. pmid:9872779
View Article
PubMed/NCBI
Google Scholar

[76] View Article

[77] PubMed/NCBI

[78] Google Scholar

[ref21] 21. Kasuya K-i, Takano T, Tezuka Y, Hsieh WC, Mitomo H, Doi Y. Cloning, expression and characterization of a poly(3-hydroxybutyrate) depolymerase from Marinobacter sp. NK-1. Int J Biol Macromol. 2003;33: 221–226. pmid:14607367
View Article
PubMed/NCBI
Google Scholar

[80] View Article

[81] PubMed/NCBI

[82] Google Scholar

[ref22] 22. Sigrist CJ, Cerutti L, Hulo N, Gattiker A, Falquet L, Pagni M, et al. PROSITE: a documented database using patterns and profiles as motif descriptors. Brief Bioinform. 2002;3: 265–274. pmid:12230035
View Article
PubMed/NCBI
Google Scholar

[84] View Article

[85] PubMed/NCBI

[86] Google Scholar

[ref23] 23. Kita K, Ishimaru K, Teraoka M, Yanase H, Kato N. Properties of poly(3-hydroxybutyrate) depolymerase from a marine bacterium, Alcaligenes faecalis AE122. Appl Environ Microbiol. 1995;61: 1727–1730. pmid:7646009
View Article
PubMed/NCBI
Google Scholar

[88] View Article

[89] PubMed/NCBI

[90] Google Scholar

[ref24] 24. Eder W, Wanner G, Ludwig W, Busse H-J, Ziemke-Kägeler F, Lang E. Description of Undibacterium oligocarboniphilum sp. nov., isolated from purified water, and Undibacterium pigrum strain CCUG 49012 as the type strain of Undibacterium parvum sp. nov., and emended descriptions of the genus Undibacterium and the species Undibacterium pigrum. Int J Syst Evol Microbiol. 2011;61: 384–391. pmid:20305060
View Article
PubMed/NCBI
Google Scholar

[92] View Article

[93] PubMed/NCBI

[94] Google Scholar

Figures

Abstract

Introduction

Materials and methods

Bacterial strains, plasmids, compounds, and growth conditions

Genome sequencing

Cloning of the phaZ gene from the Undibacterium strains

Expression and purification of His-tagged PhaZ

Assay of PHBH-degrading activity

LC-TOF-MS analysis of PHBH degradation products produced by PhaZ

Nucleotide sequence accession number

Results and discussions

The complete genome sequence revealed the presence of the PHA-depolymerase gene in Undibacterium strains

In silico characterization of the PhaZUD homolog from the family Oxalobacteraceae

The PhaZUD proteins have PHBH-degrading activity

Expression, purification, and characterization of PhaZUD

PhaZUD works as an endo-type PHA depolymerase

Conclusions

Supporting information

S1 Fig. Vector map of pGEX28.

S2 Fig. Phylogenetic tree based on the complete sequences of chromosome of 24 strains belonged to the family Oxalobacteraceae in NCBI GenBank database.

S3 Fig. PHBH-degrading activity of E. coli harboring pUC118-based plasmids.

S1 Table. Raw data for Fig 3.

S1 Raw Images.

References

In silico characterization of the PhaZ_UD homolog from the family Oxalobacteraceae

The PhaZ_UD proteins have PHBH-degrading activity

Expression, purification, and characterization of PhaZ_UD

PhaZ_UD works as an endo-type PHA depolymerase