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. 2017 May 25;4(2):52.
doi: 10.3390/bioengineering4020052.

Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria

Eduarda Morgana da Silva Montenegro  1 , Gabriela Scholante Delabary  2 , Marcus Adonai Castro da Silva  3 , Fernando Dini Andreote  4 , André Oliveira de Souza Lima  5
Affiliations

Molecular Diagnostic for Prospecting Polyhydroxyalkanoate-Producing Bacteria

Eduarda Morgana da Silva Montenegro et al. Bioengineering (Basel). .

Abstract

The use of molecular diagnostic techniques for bioprospecting and microbial diversity study purposes has gained more attention thanks to their functionality, low cost and quick results. In this context, ten degenerate primers were designed for the amplification of polyhydroxyalkanoate synthase (phaC) gene, which is involved in the production of polyhydroxyalkanoate (PHA)-a biodegradable, renewable biopolymer. Primers were designed based on multiple alignments of phaC gene sequences from 218 species that have their genomes already analyzed and deposited at Biocyc databank. The combination of oligos phaCF3/phaCR1 allowed the amplification of the expected product (PHA synthases families types I and IV) from reference organisms used as positive control (PHA producer). The method was also tested in a multiplex system with two combinations of initiators, using 16 colonies of marine bacteria (pre-characterized for PHA production) as a DNA template. All amplicon positive organisms (n = 9) were also PHA producers, thus no false positives were observed. Amplified DNA was sequenced (n = 4), allowing for the confirmation of the phaC gene identity as well its diversity among marine bacteria. Primers were also tested for screening purposes using 37 colonies from six different environments. Almost 30% of the organisms presented the target amplicon. Thus, the proposed primers are an efficient tool for screening bacteria with potential for the production of PHA as well to study PHA genetic diversity.

Keywords: biopolymer; bioprospecting; environmental diversity.

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Conflict of interest statement

The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Analysis of the gene sequences used to design the primers, and their phylogenetic classification by the Megan 4 program.
Figure 2
Figure 2
Positioning of the forward and reverse primers accordingly to the consensus sequence.
Figure 3
Figure 3
phaC gene amplification. Line M: ladder ʎ-hind III. (A) phaC amplicons for combinations of primers with positive results (organism LAMA 677): 1. phaCF1/phaCR1; 2. phaCF2/phaCR1; 3. phaCF3/phaCR1. (B) phaC amplicons in: 1. Bacillus pumilus; 2. B. thurigiensis; 3. B. megaterium; 4. B. cereus ATCC; 5. C. necator. (C) phaC amplicons in: 1. Pseudomonas sp. (LAMA 572); 2. LAMA 677; 3. Halomonas hydrothermalis (LAMA 685); 4. LAMA 691; 5. LAMA 694; 6. Micrococcus luteus (LAMA 702); 7. LAMA 726; 8. LAMA 729; 9. LAMA 737; 10. LAMA 748; 11. Brevibacterium sp. (LAMA 758); 12. LAMA 760; 13. LAMA 790; 14. Halomonas sp. (LAMA 761); 15. LAMA 765; 16. LAMA 896.
Figure 3
Figure 3
phaC gene amplification. Line M: ladder ʎ-hind III. (A) phaC amplicons for combinations of primers with positive results (organism LAMA 677): 1. phaCF1/phaCR1; 2. phaCF2/phaCR1; 3. phaCF3/phaCR1. (B) phaC amplicons in: 1. Bacillus pumilus; 2. B. thurigiensis; 3. B. megaterium; 4. B. cereus ATCC; 5. C. necator. (C) phaC amplicons in: 1. Pseudomonas sp. (LAMA 572); 2. LAMA 677; 3. Halomonas hydrothermalis (LAMA 685); 4. LAMA 691; 5. LAMA 694; 6. Micrococcus luteus (LAMA 702); 7. LAMA 726; 8. LAMA 729; 9. LAMA 737; 10. LAMA 748; 11. Brevibacterium sp. (LAMA 758); 12. LAMA 760; 13. LAMA 790; 14. Halomonas sp. (LAMA 761); 15. LAMA 765; 16. LAMA 896.
Figure 4
Figure 4
Sub-tree alignment of sequenced amplicons (LAMA 677, LAMA 737, LAMA 748, and LAMA 760), showing organisms with more genetic similarity. The analysis were through the software Geneious V.5.5.3.
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