Abstract
Polyhydroxyalkanoate (PHA) synthase is a key enzyme for PHA production in microorganisms. The class IV PHA synthase is composed of two subunits: PhaC and PhaR. The PhaR subunit, which encodes the phaR gene, is only present in class IV PHA synthases. Therefore, the phaR gene is used as a biomarker for bacteria that contain a class IV PHA synthase, such as some Bacillus spp. The phaR gene was developed to screen phaR-containing Bacillus spp. The phaR screening method involved two steps: phaR gene amplification by PCR and phaR amplicon detection using a DNA lateral flow assay. The screening method has a high specificity for phaR-containing Bacillus spp. The lowest amount of genomic DNA of B. thuringiensis ATCC 10792 that the phaR screening method could detect was 10 pg. This novel screening method improves the specificity and sensitivity of phaR gene screening and reduces the time and cost of the screening process, which could enhance the opportunity to discover good candidate PHA producers. Nevertheless, the screening method can certainly be used as a tool to screen phaR-containing Bacillus spp. from environmental samples.
Keywords: DNA lateral flow, PHA synthase, phaR, PHA, Bacillus
Introduction
Plastics play a critical role in daily life because of their attractive properties and wide range of applications [1]. The increased rate of plastic consumption has led to environmental problems because plastic waste is difficult to manage as its degradation rate is very low in the environment. The cost of plastic waste management has increased because of the requirement for landfill space and energy for recycling processes. Broken-down plastic waste produces microplastics that are known to harm wild animals, especially marine organisms, which mistake them for food. Microplastics can also be consumed by humans via drinking water and food. Although the risk to humans is still unclear, the smallest microplastics can enter the bloodstream [2]. Thus, biodegradable plastics have been developed to overcome environmental issues caused by traditional plastics. Biodegradable plastics can be mineralized by microorganisms in the environment [3]. Some bioplastics can be produced from bacteria and archaea, such as polyhydroxyalkanoate (PHA), polylactic acid (PLA) and polybutylene succinate (PBS) [4, 5]. Among bioplastics, PHAs are easily degraded to carbon dioxide and water, or to water and methane depending on the concentration of oxygen [4]. PHAs are a promising biomaterial for the development of environmentally friendly biodegradable plastics. PHAs can be synthesized by many bacteria and some archaea under imbalanced growth conditions such as excess carbon and limited availability of essential nutrients including nitrogen, phosphorus, and oxygen for growth [5-6]. The key enzymes in PHA polymerization are PHA synthases, which can be classified into four main classes depending on their structures, substrate specificities and subunit components [6]. The class IV PHA synthase is encoded by the phaC and phaR genes and catalyzes the polymerization of short-chain-length monomers (C3 - C5) [7]. Class IV PHA synthases can be found mostly in bacteria that belong to the genus Bacillus [8]. The genus Bacillus is one of the largest bacterial groups of the family Bacillaceae. The Bacillus genus contains 379 species and 7 subspecies [9-10]. Bacillus are found in various types of environments, such as air, soil and water. Most of the bacterial species in the Bacillus genus are nonpathogenic bacteria [11]. Bacillus spp. are reported to be the most versatile PHA producers [12, 13]. They can use a wide variety of carbon sources for PHA production, including corn steep liquor [14], crude glycerol [15], molasses [14] and wastewater from the sugar industry [8]. Some bacterial strains of the Bacillus genus, such as B. cereus [16], B. megaterium [14], B. mycoides [17], B. subtilis [8] and B. thuringiensis [15], have been reported as candidate PHA producers. PHA-producing Bacillus are of interest due to their ability to use a wide variety of substrates for growth and to produce PHAs. This will be a very economical method for bioplastic production [15].
There are a variety of methods available to identify PHA producers. Functional screening methods such as Sudan Black staining [18], Nile Blue A staining and direct staining of bacterial colonies by fluorescence dye [19] are useful methods for the detection of PHA granules that have been accumulated in cells. However, these functional screening methods require time, specific media and growth conditions and the screening of candidate microorganisms for PHA production. False negative results were observed during screening when the candidate microorganisms were grown in nonoptimized media or conditions [20].
Thus, polymerase chain reaction (PCR) can be used as a genetic screening method for the detection of important gene markers of PHA-production pathways [21, 22]. The PCR method shows high specificity to the target biomarker genes and allows amplification of the target genes at low concentrations [21, 22]. However, postanalysis including gel electrophoresis is required after PCR. This step is time consuming and requires specific equipment. Thus, a simple and fast method for amplicon detection, such as a DNA lateral flow assay, can be applied instead of gel electrophoresis.
As mentioned above, the combination of PCR and a DNA lateral flow assay was developed as a method for the detection of the phaR gene of PHA-producing Bacillus spp.
Materials and Methods
Bacterial Strains and Culture Method
Bacillus cereus ATCC 14579 and Bacillus thuringiensis ATCC 10792 were purchased from American Type Culture Collection (ATCC). Cupriavidus necator DSM 428, Pseudomonas aeruginosa DSM 19880, genomic DNA of Allochromatium vinosum DSM 180 and genomic DNA of Haloquadratum walsbyi DSM 16854 were obtained from the German Collection of Microorganisms and Cell Cultures (DSMZ). Escherichia coli ATCC 25922 and Staphylococcus aureus ATCC 25923 were obtained from the Department of Microbiology, Faculty of Pharmacy, Mahidol University. Bacteria were inoculated in LB broth and incubated at a temperature between 30-37°C with shaking at 250 rpm overnight.
Primer Design for the phaR Gene of the Bacillus spp.
The phaR gene sequences of bacteria were obtained from the GenBank genome database. A multiple sequence alignment was performed with complete phaR gene sequences based on a progressive alignment method using Clustal X version 1.81 [23]. The aligned sequences of the phaR gene were used as input data for phylogenetic tree analysis. The conserved regions of the phaR gene were selected for the primers designed. A neighbor-joining (NJ) tree of the phaR gene was constructed using MEGA version 6 [24]. The phaR phylogenetic tree was visualized by TreeView version 1.6 [25].
PCR Analysis
Genomic DNA (gDNA) was extracted using a DNeasy Blood and Tissue Kit (Qiagen, Germany). The concentration of gDNA was determined with a Nanodrop spectrophotometer (Thermo, USA). B. cereus ATCC 14579 and B. thuringiensis ATCC 10792 were used as positive control samples because each strain contained the phaR gene. The phaR primers were purchased from Bio Basic Canada Inc. The forward primer (BGPhaR-F) contained biotin at the 5' end and the reverse primer (BGPhaR-R) contained FITC at the 5' (Table 1). The size of the PCR product was approximately 122 bp. The PCR was performed in a total volume of 50 μl consisting of 40.2 μl of deionized water, 5 μl of 10×Vi Buffer S (Vivantis, Malaysia), 2 μl of 80 μM dNTPs (Vivantis), 1 μl of 10 μM BGPhaR-F (Bio Basic), 0.5 μl of 10 μM BGPhaR-R (Bio Basic), 1 μl of 10 ng/μl DNA template and 0.3 μl of 5 U/μl Taq DNA polymerase (Vivantis). The PCR conditions started with an initial step at 94°C for 2 min, followed by 35 cycles of 94°C for 30 s, 54°C for 30 s, and 72°C for 30 s, and a final step of 72°C for 1 min. PCR was performed on a T100 Thermal Cycler (Bio-Rad, USA). The reproducibility of the PCRs was examined by testing three replicates of genomic DNA by each assay and repeating the experiment five times. The amplicon was analyzed by gel electrophoresis in 1% w/v of agarose gel at 80 V for 35 min. GeneRuler 100 bp Plus DNA Ladder (Thermo) was used as the DNA marker for agarose gel electrophoresis. PCR products were visualized with a UV transilluminator (Syngene, England).
Table 1.
The primers used to screen the phaR gene of Bacillus spp.
| Primer | Sequence (5’→3’) | Length (bp) | 5’ Labeled |
|---|---|---|---|
| BGPhaR-F | GATCCAYTWCAAGCATGGAAA | 21 | Biotin |
| BGPhaR-R | TTCAAATCTAGAACRYTKCCCAT | 23 | FITC |
Specificity Test of the phaR Screening Method
The details of the microorganisms used in this study are shown in Table 2. B. cereus ATCC 14579 and B. thuringiensis ATCC 10792 were used as positive controls because they are able to produce PHAs and contain the phaR gene. A no-template control (NTC) reaction was performed using deionized water instead of the DNA template. E. coli ATCC 25922 and S. aureus ATCC 25923 were used as negative controls for group 1 (NC1) because they cannot produce PHAs and lack the phaR gene. Negative control group 2 (NC2) included A. vinosum DSM 180, C. necator DSM 428, H. walsbyi DSM 16854 and P. aeruginosa DSM 19880, which are all capable of producing PHAs but lack the phaR gene. The phaR screening method consisted of two steps, amplification and detection. First, the phaR gene of Bacillus spp. was amplified by PCR. Then, the phaR amplicon was observed on the HybriDetect DNA lateral flow assay (Milenia Biotec, Germany). The specificity of the phaR screening method was depended on the amplification step. PCR amplification was positive when the amplicon contained biotin and FITC at its flanking region. The DNA lateral flow assay was set up according to the instructions provided with the kit. One microliter of PCR product was added to the DNA lateral flow strip. The results appeared on the DNA lateral flow strip within one minute. One line on the strip (control line) indicated that no amplification of the phaR gene was detected. The phaR amplicon was detected when two lines appeared on the strip (control line and test line).
Table 2.
Specific details of the microorganisms used in this study.
| No | Species | PHA synthase | phaR | Designated |
|---|---|---|---|---|
| 1 | B. cereus ATCC 14579 | Class IV | Yes | Positive control |
| 2 | B. thuringiensis ATCC 10792 | Class IV | Yes | Positive control |
| 3 | E. coli ATCC 25922 | - | No | NC1 |
| 4 | S. aureus ATCC 25923 | - | No | NC1 |
| 5 | C. necator DSM 428 | Class I | No | NC2 |
| 6 | P. aeruginosa DSM 19880 | Class II | No | NC2 |
| 7 | A. vinosum DSM 180 | Class III | No | NC2 |
| 8 | H. walsbyi DSM 16854 | Class III | No | NC2 |
Sensitivity Test of the phaR Screening Method
Genomic DNA of the positive control samples was diluted to 1,000 pg/μl, 100 pg/μl and 10 pg/μl. Each dilution of the gDNA was used as a DNA template for the PCR as previously described. The PCR products were examined with 1% w/v agarose gel electrophoresis and the DNA lateral flow assay (Milenia Biotec).
Screening phaR Gene of Bacillus spp. from Environmental Samples
Soil samples were collected from a municipal landfill of Yangon, Myanmar (16°52'22.1"N 96°11'56.0"E). The soil samples were kept in 50 ml sterile tubes and stored at 4°C. One gram of soil was resuspended in 10 ml of LB broth. Then, 100 μl of the suspension was spread onto LB agar (Titan, India) and cultured at 30°C overnight. Different single colonies were selected based on their morphological characteristics. The selected colonies were grown in LB broth at 30°C with shaking at 250 rpm overnight. The gDNA of the isolated strains was obtained using a DNeasy Blood and Tissue Kit (Qiagen). Then, the phaR genes were amplified by the PCR method, and the amplicon was detected on the HybriDetect DNA lateral flow strip (Milenia Biotec).
Sequencing of the phaR amplicon was performed using the dideoxy method (1st Base, Singapore). The partial sequences of the phaR gene were subjected to multiple sequence alignment based on the progressive alignment method using Clustal X version 1.81 [23]. The NJ tree of the phaR gene was constructed based on the maximum composite likelihood model using MEGA version 6 [24].
Results and Discussion
Primer Design for the phaR Gene of the Bacillus spp.
The details of the phaR gene that were selected for this study are shown in Table S1. The length of the phaR genes of the Bacillus spp. were 483 to 600 bp. The NJ tree of the phaR gene (Fig. S1) was constructed based on the maximum composite likelihood model. The conserved region of phaR genes was used to design primers. Based on the consensus sequence of the alignment, the conserved region of position 70-91 was selected for the design of the forward primer, and the conserved region of position 169-192 was selected for the design of the reverse primer (Fig. S2). The details of the phaR primers are shown in Table 1.
PCR Analysis
PCR was performed for validation of the phaR primers (Table 1). The PCR was optimized to achieve the highest specificity of the primers and the shortest analysis. The PCR successfully amplified 122 bp of the phaR gene of B. cereus ATCC 14579 and B. thuringiensis ATCC 10792, and less than 100 min was required for the PCR analysis. Nonspecific amplification and primer dimers were not observed.
Specificity Test of the phaR Screening Method
The PCR products of the phaR amplification are shown in Fig. 1. There was no PCR product for NTC, NC1 and NC2. No nonspecific amplifications or primer dimers were observed in any PCR amplifications. The PCR products were tested with a DNA lateral flow assay, and these results are shown in Fig. 2. The control line and test line were observed with DNA lateral flow when tested with the amplicons of the positive controls (B. cereus ATCC 14579 and B. thuringiensis ATCC 10792). However, only the control line was observed with DNA lateral flow when tested with the amplicons of NC1 and NC2.
Fig. 1. Analysis of the specificity of the phaR PCR.
(M) DNA marker, (N) no-template control, (P1) positive control (B. cereus ATCC 14579), (P2) positive control (B. thuringiensis ATCC 10792), (1) E. coli ATCC 25922, (2) S. aureus ATCC 13565, (3) A. vinosum DSM 180, (4) C. necator DSM 48, (5) H. walsbyi DSM 16854, and (6) P. aeruginosa DSM 19880.
Fig. 2. Detection of the phaR amplicon with the DNA lateral flow assay.
(N) No-template control, (P1) positive control (B. cereus ATCC 14579), (P2) positive control (B. thuringiensis ATCC 10792), (1) E. coli ATCC 25922, (2) S. aureus ATCC 13565, (3) A. vinosum DSM 180, (4) C. necator DSM 48, (5) H. walsbyi DSM 16854, and (6) P. aeruginosa DSM 19880.
Sensitivity Test of the phaR Screening Method
The phaR amplicon of B. cereus ATCC 14579 is shown in Fig. 3 with the agarose gel electrophoresis and Fig. 4 with the DNA lateral flow assay. The lowest concentration of the DNA template that the PCR and DNA lateral flow assay could detect was 100 pg. The phaR amplicon of B. thuringiensis ATCC 10792 is shown in Fig. 5 with the agarose gel electrophoresis and Fig. 6 with the DNA lateral flow assay. The lowest concentration of the DNA template that the PCR and DNA lateral flow assay could detect was 10 pg.
Fig. 3.
Analysis of the sensitivity of the phaR PCR for B. cereus ATCC 14579; (M) DNA marker, (N) notemplate control, (1) 1,000 pg, (2) 100 pg and (3) 10 pg.
Fig. 4.
Detection of the phaR amplicon of B. cereus ATCC 14579 with the DNA lateral flow assay; (N) notemplate control, (1) 1,000 pg, (2) 100 pg and (3) 10 pg.
Fig. 5.
Analysis of the sensitivity of the phaR PCR for B. thuringiensis ATCC 10792, (M) DNA marker, (N) notemplate control, (1) 1,000 pg, (2) 100 pg, (3) 10 pg and (4) 1 pg.
Fig. 6.
Detection of the phaR amplicon of B. thuringiensis ATCC 10792 with the DNA lateral flow assay; (N) no-template control, (1) 1,000 pg, (2) 100 pg, (3) 10 pg and (4) 1 pg.
Screening phaR Gene of Bacillus spp. from Environmental Samples
Sixteen isolated strains were obtained from the soil of a municipal landfill of Yangon, Myanmar. However, seven bacterial strains contained the phaR gene (Table 3) after screening with the phaR PCR combined with the DNA lateral flow assay. The phaR amplicon of the positive clones was sequenced, and phylogenetic analysis was performed. The phaR genes of the isolated strains were affiliated with bacterial strains in the genus Bacillus (Fig. 7).
Table 3.
The phaR-containing strains isolated from the soil of a municipal landfill in Yangon, Myanmar.
| No | Strain | Closet relative species | % Similarity |
|---|---|---|---|
| 1 | YGMW1 | B. thuringensis | 96 |
| 2 | YGMW2 | B. thuringensis | 96 |
| 3 | YGMW3 | B. thuringensis | 96 |
| 4 | YGMW4 | B. thuringensis | 96 |
| 5 | YGMW7 | B. thuringensis | 94 |
| 6 | YGMW9 | B. thuringensis | 95 |
| 7 | YGMW10 | B. thuringensis | 95 |
Fig. 7. Neighbor-joining tree of the phaR gene of Bacillus spp. that were isolated from the soil of a municipal landfill of Yangon, Myanmar.
The values associated with nodes correspond to the bootstrap value in %.
Discussion
Bacillus spp. are very attractive candidates for PHA production. However, more than three hundred species of the Bacillus genus still have not been investigated for PHA production. Screening PHA-producing Bacillus spp. using classical microbiological methods is a time-consuming process that requires appropriate media and optimal growth conditions for inducing PHA accumulation in cells [26]. Thus, Shamala and colleagues [26] developed a semi-nested PCR method for detection of the phaC gene, which encodes the PhaC subunit of the class IV PHA synthase [6]. The primers for the semi-nested PCR were designed from a single nucleotide sequence of the phaC gene of B. megaterium. This method successfully amplified the phaC gene of the ten standard Bacillus strains [26]. The class IV PHA synthase is a heterodimer that contains PhaC and PhaR subunits [6]. The PhaR subunit is presented only in class IV PHA synthase. It is encoded by the phaR gene, which can be used as a biomarker for class IV PHA synthases. In the current study, sixty-four phaR gene sequences from Bacillus strains were used to design primers. This ensured that the primers covered the conserved regions of the phaR gene of Bacillus spp. This may have increased the possibility of successful rates of phaR screening from PHA-producing Bacillus spp. Moreover, the phaR PCR required less time than the phaC semi-nested PCR that was described by Shamala and colleagues [26]. Because there is only one step for amplification of the phaR gene, the amplicon can be immediately detected with the DNA lateral flow assay. Therefore, the phaR screening method comprises two major steps: phaR gene amplification by PCR and phaR amplicon detection with a DNA lateral flow assay. The phaR screening results demonstrated highly specific detection of the phaR gene in B. cereus ATCC 14579 and B. thuringiensis ATCC 10792 (Figs. 1-2). Based on the results of the sensitivity test of the phaR screening method, the lowest amount of gDNA (B. thuringiensis ATCC 10792) that the screening method could detect was 10 pg (Figs. 5-6). The phaR screening method can be applied to screen Bacillus spp. isolated from soil samples (Table 3). The similarity of the phaR genes of the isolated Bacillus strains was 94-96%, and the closest relative was B. thuringiensis (Table 3). Kumar and colleagues have reported that B. thuringiensis EGU45 can utilize high concentrations of crude glycerol for PHA co-polymer production and can produce PHAs in the medium, which contains high nitrogen concentrations [15].
The phaR screening method is a novel assay for screening phaR-containing Bacillus spp. The chance of discovering a novel phaR gene or PHA-producing Bacillus spp. may increase with the use of the phaR screening method. Nevertheless, the phaR screening method can certainly be used as a tool for screening for phaR-containing Bacillus spp. that have been isolated from environmental samples.
Supplemental Material
Supplementary data for this paper are available on-line only at http://jmb.or.kr.
Acknowledgments
This study is part of a Ph.D. thesis at Mahidol University. This work was supported in part by grants from the Chulabhorn Graduate Institute (to M.Y.), and the Thailand Research Fund (TRF) Grant [Grant RSA6080061] (to S.M.).
Footnotes
Conflict of Interest
The authors have no financial conflict of interest to declare.
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Supplementary Materials
Supplementary data for this paper are available on-line only at http://jmb.or.kr.