Graduate Institute of Agricultural Chemistry, National Taiwan University, 1, Sec. 4, Roosevelt Rd, Taipei 106, Taiwan1
Author for correspondence: Chia-Yin Lee. Tel: +886 2 2363 0231 ext. 2816. Fax: +886 2 2366 0581. e-mail: m477{at}ccms.ntu.edu.tw
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ABSTRACT |
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Keywords: colony PCR, semi-nested PCR, polyhydroxyalkanoates, PHA synthase, degenerate primers
Abbreviations: PHA, polyhydroxyalkanoate
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INTRODUCTION |
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There are many phenotypic detection methods for detecting intracellular PHA granules which are applied to the screening of PHA producers, including Sudan Black staining (Schlegel et al., 1970 ) and Nile blue A staining (Ostle & Holt, 1982
), which result in dark blue or fluorescent granules. Although these methods are quite sensitive, it is rather time-consuming and labour-intensive work to screen a large number of environmental isolates. Alternative staining methods have recently been developed for directly staining colonies (Kranz et al., 1997
) or growing bacteria on plates containing Nile blue A or Nile red (Spiekermann et al., 1999
), resulting in fluorescent colonies that can be visualized by UV illumination. These colony-staining methods are suitable for screening large numbers of strains. However, appropriate carbon sources should be used, and a long culture time (3 d) is required for PHA granule accumulation. In addition, these methods cannot distinguish between bacteria that accumulate PHA granules and those that accumulate lipid compounds.
In this study, a genotypic detection method, which circumvents the major drawbacks inherent in phenotypic detection methods described above, was devised.
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METHODS |
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Nucleotide sequence analysis.
The nucleotide sequences of 13 PHA synthase (phaC) genes were collected for sequence analysis, derived from the following Gram-negative bacteria: Acinetobacter sp. (Schembri et al., 1994 ), Aeromonas caviae (Fukui & Doi, 1997
), Ralstonia eutropha (formerly known as Alcaligenes eutrophus) (Peoples & Sinskey, 1989
), Alcaligenes sp. (GenBank accession no. U78047), Methylobacterium extorquens (Valentin & Steinbüchel, 1993
), Paracoccus denitrificans (Ueda et al., 1996
), Pseudomonas aeruginosa (Timm & Steinbüchel, 1992
) (containing two phaC genes), P. oleovorans (Huisman et al., 1991
) (containing two phaC genes), Rhizobium etli (Cevallos et al., 1996
), Rhizobium meliloti (Tombolini et al., 1995
) and Zoogloea ramigera (GenBank accession no. U66242). Multiple sequence alignment was achieved by employing the SeqWeb software allowing a GapWeight of 10 and a GapLengthWeight of 5.
Synthetic oligonucleotide primers.
Three degenerate primers (all 26-mers) were designed and synthesized according to the multiple sequence alignment results. The sequences were 5'-A T C A A C A A ( G G G / A ) T ( T T / A ) C T A C ( A A /G)TC( C C / T)T(CC/G)GACCT-3' (designated phaCF1, corresponding to nt 741766 of R. eutropha phaC), 5'-GT(CCC/GG)TTC( G G G / A A ) T ( G G G / C C ) ( A A A / G G ) T ( C C / G ) ( T T / A ) ( C CC/GG)CTGGCGCAACCC-3' (designated phaCF2, corresponding to nt 846871 of R. eutropha phaC) and 5'-AGGTA G T T G T ( T T / C ) G A C ( C C C / G G ) ( A A A / C C ) ( A A A / C C)(GGG/A)TAG (TTT/G)TCCA-3' (designated phaCR4, corresponding to nt 12371212 of R. eutropha phaC).
Colony PCR.
The optimized colony PCR reaction mixture contained 1xPCR amplification buffer [20 mM (NH4)2SO4, 72·5 mM Tris/HCl, 0·1% Tween 20, pH 9·0], 2·5 mM MgCl2, 200 µM each deoxynucleotide triphosphate, 2·5 µM each primer, 1·25 U Supertherm DNA polymerase (LPI) in 50 µl PCR reaction mixture. A final concentration of 100 µg ml-1 of acetylated BSA (New England BioLabs), 3% dimethyl sulfoxide (DMSO) (Sigma) and 1 M betaine (Sigma) as PCR additives were also added to the reaction mixture. Colonies approximately 1 mm in diameter were picked up with a sterilized toothpick and directly transferred to the PCR tube as DNA templates. The thermal cycle programme, run on a GeneAmp PCR system 9700 (Perkin Elmer) consisted of one cycle of 94 °C for 10 min, 51 °C for 2 min, 72 °C for 2 min, and 35 cycles of 94 °C for 20 s, 57 °C for 45 s (decreased by 1 s per cycle), 72 °C for 1 min, and then incubation at 72 °C for 5 min, and a final incubation at 4 °C.
Sensitivity of colony PCR.
Tenfold serial dilutions of R. eutropha overnight cultures were prepared in nutrient broth. Aliquots of each dilution were subjected to plate counting to determine bacterial cell concentration. Other aliquots of each dilution were centrifuged, and the cell pellet was washed once with sterilized deionized water and then used as DNA template for PCR.
Detection of PCR products.
PCR-amplified DNA fragments were observed by agarose gel electrophoresis in 1·3% agarose gels (FMC). Ten microlitres of each amplification mixture and the molecular mass marker (HaeIII digest of X174 DNA) were subjected to agarose gel electrophoresis and ethidium bromide staining. The amplified DNA fragments were visualized by UV illumination.
Southern hybridization.
To confirm the amplified DNA fragment, 10 µl of colony PCR products was subjected to 1·3 % agarose gel electrophoresis and then transferred to a Hybond-N+ membrane (Amersham) by alkaline Southern transfer (Reed & Mann, 1985 ). An internal oligonucleotide probe, phaCF2, was labelled with [
-32P]ATP by 5'-end labelling with T4 polynucleotide kinase (New England BioLabs) as a probe (1·5 ng ml-1). The membranes were air-dried and hybridized at 38 °C overnight in 1xSSC buffer containing 1% SDS, 1% casein (Merck), 0·5 mg ml-1 of denatured herring sperm DNA (Boehringer Mannheim) and 50% (v/v) deionized formamide (Clontech). At low stringency, the buffer and hybridization temperature were the same as those above except for the lack of formamide. After hybridization, the membranes were rinsed with 2xSSC and 2xSSC/0·1% SDS at 38 °C, followed by 0·5xSSC/0·1% SDS at 25 °C. Then the membranes were autoradiographed with X-ray film (Fuji) and exposed at -70 °C for an appropriate length of time.
Semi-nested PCR.
Semi-nested PCR was done with the primers phaCF2 and phaCR4, with a predicted product of 406 bp, to further confirm the colony PCR results. For positive products, 1 µl of 100-fold-diluted colony PCR products was subjected to semi-nested PCR. For negative products, 1 µl of undiluted colony PCR products was directly supplied as DNA templates for semi-nested PCR. The 50 µl PCR mixture contained 1x PCR amplification buffer, 1·5 mM MgCl2, 200 µM each deoxynucleotide triphosphate, 2 µM each primer, 2% DMSO and 0·5 U Supertherm DNA polymerase. The thermal cycle programme, run on a GeneAmp PCR system 9700 (Perkin Elmer), was 94 °C for 5 min (initial denaturation), 25 cycles of 94 °C for 15 s, 57 °C for 15 s, 72 °C for 30 s, and then incubation at 72 °C for an additional 5 min, with final incubation at 4 °C.
Isolation and screening of environmental strains.
The screening procedure for PHA producers from the environment was modified from Gomez et al. (1996) . Serial dilutions of active sludge were prepared in MSM. Aliquots of the dilution series were spread onto MSM plates containing 1% (w/v) sucrose (Sigma), 1·5% (w/v) sodium gluconate (Wako), 1% (w/v) glycerol (Sigma) or 0·1% (w/v) sodium octanoate (Wako) as carbon sources and were cultured at 30 °C overnight. The grown colonies were individually streaked onto MSM plates containing appropriate carbon sources to obtain well-separated single colonies. The PHA producer candidates were directly screened by colony PCR and semi-nested PCR from these well-separated colonies.
Nile blue A staining.
PHA-positive strains identified by PCR were cultured in 50 ml MSM containing an appropriate carbon source at 30 °C with reciprocal shaking at 150 r.p.m. for 3 d. The bacterial cells were stained with Nile blue A and observed as described by Ostle & Holt (1982) . The fluorescence microscope used was an Olympus AX 70; the excitation filter, barrier filter and dichroic mirror sets were BP450480, BA515 and DM500, respectively. The staining results were photographed in colour on Kodak Kodacolor print film (400 ASA).
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RESULTS |
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Sensitivity of colony PCR
A dilution series of R. eutropha cells was prepared as DNA templates for testing the sensitivity of the colony PCR. The detection limit of the colony PCR was approximately 1x105 viable cells (Fig. 2). On the basis of the results, colonies approximately 1 mm in diameter, containing ~1x107 viable cells, were picked up as DNA templates for colony PCR in the following experiments.
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DISCUSSION |
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The sizes of products obtained by colony PCR and semi-nested PCR were consistent with the predicted results except those of A. latus and C. acidovorans, for which sizes were larger than predicted (Fig. 3, lanes 3 and 4). Recently, the phaC genes of A. latus (Choi et al., 1998
) and C. acidovorans (Sudesh et al., 1998
) have been cloned and sequenced. Aligning the primer positions, the corresponding products, 593 and 518 bp, would be amplified from A. latus by primer pairs phaCF1-phaR4 and phaCF2-phaCR4, respectively. In C. acidovorans, the corresponding products were 621 and 555 bp by the above primer pairs. The predictive products were larger than the former; however, they obviously matched our agarose gel analysis results.
Thirty-eight PHA-positive strains isolated from the environment by PCR were further confirmed by Nile blue A staining assay. Among these isolates, 87% (33/38) possessed significant PHA accumulation ability, which strongly supports the accuracy of the genotype screening results. However, there are still five isolates (13%) for which no PHA granules were detected by Nile blue A staining. This outcome may be due to an inappropriate carbon source used in the culture medium or to a low yield of PHA granule accumulation resulting in negative detection by Nile blue A staining. These results also reflected the possibility that there may be some bacterial isolates that harbour a non-functional PHA synthase gene. The PHA-positive strains identified included five Gram-positive and five Gram-variable strains. This suggested that the colony PCR protocol is also applicable for detecting Gram-positive PHA producers.
It is often difficult to carry out gene cloning of PHA operons due to the lack of a universal probe. The degenerate primers used in this study were capable of amplifying a partial DNA fragment of phaC from all 19 reference strains, including strains from which pha operons have not yet been cloned (e.g. A. vinelandii and A. chroococcum). In addition, these individual PCR products are very suitable for use as specific probes for genomic library screening. The PCR protocol can also be regarded as a universal method to prepare a PHA-synthase-specific probe from individual strains for gene cloning. Alternatively, genomic library screening can also be rapidly achieved by using colony PCR directly. The colony PCR technique proposed in this study will significantly accelerate the discovery of new PHA operons.
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ACKNOWLEDGEMENTS |
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Received 3 February 2000;
revised 24 April 2000;
accepted 12 May 2000.