Rapid detection of polyhydroxyalkanoate-accumulating bacteria isolated from the environment by colony PCR

• Published in: Microbiology (Society for General Microbiology) 2000-08, Vol.146 (8), p.2019-2025
• Main Authors: Sheu, Der-Shyan, Wang, Yun-Ting, Lee, Chia-Yin
• Format: Article
• Language: English
• Subjects: Acyltransferases - genetics; Acyltransferases - metabolism; Bacteria - genetics; Bacteria - isolation & purification; Bacteria - metabolism; Base Composition; Base Sequence; Biological and medical sciences; Biology of microorganisms of confirmed or potential industrial interest; Biotechnology; DNA Primers - genetics; DNA, Bacterial - chemistry; DNA, Bacterial - genetics; Environmental Microbiology; Evaluation Studies as Topic; Fundamental and applied biological sciences. Psychology; Genes, Bacterial; Hydroxy Acids - metabolism; Mission oriented research; Physiology and metabolism; polyhydroxyalkanoic acid; Polymerase Chain Reaction - methods; Polymerase Chain Reaction - statistics & numerical data; Ralstonia eutropha; Sensitivity and Specificity
• ISSN: 1350-0872; 1465-2080
• DOI: 10.1099/00221287-146-8-2019

Graduate Institute of Agricultural Chemistry, National Taiwan University, 1, Sec. 4, Roosevelt Rd, Taipei 106, Taiwan 1 Author for correspondence: Chia-Yin Lee. Tel: [phone] ext. 2816. Fax: [phone]. e-mail: m477{at}ccms.ntu.edu.tw Colony PCR and semi-nested PCR techniques were employed for screening polyhydroxyalkanoate (PHA) producers isolated from the environment. Three degenerate primers were designed based on multiple sequence alignment results and were used as PCR primers to detect PHA synthase genes. Optimized colony PCR conditions were achieved by adding 3% DMSO combined with 1 M betaine to the reaction mixture. The sensitivity limit of the colony PCR was 1 x 10 5 viable cells for Ralstonia eutropha . Nineteen PHA-positive bacteria were used to evaluate this PCR protocol; fifteen of the nineteen could be detected by colony PCR, and the other four could be detected by applying semi-nested PCR detection following colony PCR. In a preliminary screening project, 38 PHA-positive strains were isolated from environmental samples by applying the PCR protocol, and their phenotype was further confirmed by Nile blue A staining assay. By combining the colony PCR and semi-nested PCR techniques, a rapid, reliable and highly accurate detection method has been developed for detecting PHA producers. This protocol is suitable for screening large numbers of environmental isolates. The PHA accumulation ability of well-separated colonies isolated from environmental samples can be directly validated by PCR with no further culturing or chromosomal DNA extraction procedures. In addition to its application to the screening of wild-type isolates, the individual PCR-amplified product is also suitable as a specific probe for PHA operon cloning. The results suggest that the application of this PCR protocol for rapid detection of PHA producers from the environment is plausible. Keywords: colony PCR, semi-nested PCR, polyhydroxyalkanoates, PHA synthase, degenerate primers Abbreviations: PHA, polyhydroxyalkanoate