179R and 179L Primer Paper

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O R I G I N A L P A P E R

D. K. Y. Solaiman R. D. Ashby T. A. Foglia

Rapid and specic identication of medium-chain-lengthpolyhydroxyalkanoate synthase gene by polymerase chain reaction

Received: 16 August 1999 / Received revision: 23 December 1999 / Accepted: 4 January 2000

Abstract A polymerase chain reaction (PCR) protocolwas developed for the specic detection of genes coding

for type II polyhydroxyalkanoate (PHA) synthases. Theprimer-pair, I-179L and I-179R, was based on the highlyconserved sequences found in the coding regions ofPseudomonas phaC1 and phaC2 genes. Puried genomicDNA or lysate of colony suspension can serve equallywell as the target sample for the PCR, thus aording asimple and rapid screening of phaC1/C2-containingmicroorganisms. Positive samples yield a specic 540-bpPCR product representing partial coding sequences ofthe phaC1/C2 genes. Using the PCR method, P. cor-rugata 388 was identied for the rst time as a medium-chain-length (mcl)-PHA producer. Electron microscopicstudy and PHA isolation conrmed the production of

mcl-PHA in P. corrugata 388. The mcl-PHA of thisorganism has a higher molecular weight than that ofsimilar polymers produced by other pseudomonads.

Introduction

Polyhydroxyalkanoates (PHAs) are biodegradablepolymers synthesized by many microorganisms(Steinbu chel 1991). With few exceptions, these polymersare usually accumulated as inclusion bodies when theorganism is grown under conditions in which the carbon

substrate is in excess but another nutrient component,such as nitrogen, phosphorus, sulfur or oxygen, is

limited (Anderson and Dawes 1990). PHAs are generallygrouped into two classes depending on the carbon

chain length of the b-hydroxy ester monomers. Short-chain-length (scl-)PHAs contain monomer repeat-unitsof 35 carbon atoms, whereas medium-chain-length(mcl-)PHAs are composed of monomer repeat-units of614 carbon atoms (Lee 1996).

The genetic organization of PHA biosynthesis genesvaries among PHA-producing organisms. Three majorclasses of PHA biosynthesis loci have been described(Poirier et al. 1995; Steinbu chel and Fu chtenbusch1998). In the rst class (type I system), as typied by thepha locus of Ralstonia eutropha (formerly Alcaligeneseutrophus), the gene encoding the PHA synthase (phbC)is adjacent to phbA and phbB. These respectively code

for b-ketothiolase and acetoacetyl-CoA reductase, twoenzymes closely linked to the biosynthesis of scl-PHA.The second class (type II) of PHA genetic system con-sists of two synthase genes (phaC1 and phaC2) separatedby the gene coding for the depolymerization of PHA(phaZ). The type II system is commonly found in mcl-PHA-producing pseudomonads. The type III PHAbiosynthesis gene cluster is found in Chromatium vino-sum, Synechocystis spp, and Thiocystis violacea. In theseorganisms, the synthase enzyme is composed of twopolypeptide subunits encoded by phbE and phbC genes.The phbA and phbB genes are also located in this locus,but are usually transcribed divergently from the phbE

and phbC genes. Recently, McCool and Cannon (1999)characterized the pha locus of Bacillus megaterium andsuggested that it represented type IV PHA biosynthesisgenetic organization.

Methods for identifying PHA-producing organismsabound (Spiekermann et al. 1999; Takagi and Yamane1997). The majority of these methods, however, employlipophilic dyes to stain the polymers or cause them touoresce. Although highly sensitive, these reagents alsoreact with other lipid inclusion bodies and thus are notspecic for PHA. Furthermore, the production of PHAis often dependent on specic growth conditions(Anderson and Dawes 1990). If such conditions are not

Appl Microbiol Biotechnol (2000) 53: 690694 Springer-Verlag 2000

Mention of brand or rm names does not constitute an endorse-ment by the U.S. Department of Agriculture over others of asimilar nature not mentioned.

D. K. Y. Solaiman (&) R. D. Ashby T. A. FogliaU.S. Department of Agriculture, Agricultural Research Service,Eastern Regional Research Center, 600 East Mermaid Lane,Wyndmoor, PA 19038, USAe-mail: [email protected].: +1-215-2336476Fax: +1-215-2336559


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met and the polymer is not produced, then the dye-basedscreening would fail to identify the microorganisms ashaving PHA-producing capability. Timm et al. (1994)described a Southern-blot hybridization method foridentifying PHA synthase genes. Their procedureallowed for broad-spectrum detection of both the phbC-and the phaC-type PHA synthase genes. In this com-munication, we report a rapid and sensitive polymerasechain reaction (PCR) procedure for the specic detec-tion of phaC-type genes. The procedure was tested ina screening experiment, resulting in the rst descriptionof Pseudomonas corrugata as a mcl-PHA producingorganism.

Materials and methods

Bacteria and growth conditions

Bacteria were obtained from the following sources: ATCC(Manassas, Va.), ARS Culture Collection (Peoria, Ill.), Dr. R.Gross (Polytechnic University, Brooklyn, N.Y.), and Dr. W. Fett(ERRC-ARS-USDA, Wyndmoor, Pa.). Cells were grown either inLuria medium (1% w/v tryptone; 0.5% w/v yeast extract; 0.5% w/vNaCl) or tryptic soy broth (Difco, Detroit, Mich.). The corre-sponding solid media were prepared in 1.21.5% (w/v) agar. Cellswere grown at 30 C, with 250 rpm rotary shaking for the liquidcultures.

Molecular biology procedures

Genomic DNA was isolated by using a Wizard Genomic DNAPurication Kit (Promega, Madison, Wis.). Template DNA sam-ples for use in colony PCR were prepared as follows: a singlecolony on solid growth medium was picked with a sterile toothpickinto 50 ll MilliQ water in a 500-ll Eppendorf tube. The cell sus-

pension was heated to 95 C for 10 min. Five microliters of thelysate was used in colony PCR (50 ll total volume).

PCR was performed in a GeneAmp PCR System 9700 (PEApplied Biosystems, Foster City, Calif.). Forward (I-179L; 5-ACAGATCAACAAGTTCTACATCTTCGAC-3) and reverse(I-179R; 5-GGTGTTGTCGTTGTTCCAGTAGAGGATGTC-3) primers, custom-ordered from Life Technologies (Gaithersburg,Md.), were based on two highly conserved sequences deduced froma multiple alignment analysis of the pseudomonad phaC genes.While I-179L is also homologous to a sequence region in the type I

phbC genes, the I-179R is highly specic only to the type II phaCgenes. Taq DNA polymerase and ELONGASE enzyme mix (bothfrom Life Technologies) were used following the manufacturer'sprotocol. PCR products were analyzed by agarose gel electropho-resis in TBE buer (0.089 M tris-base, 0.089 M boric acid, 0.002 MNa-EDTA).

PCR cloning and sequencing

PCR products were separated by electrophoresis on agarose gelcontaining 4 lg crystal violet/ml in TAE buer (50 mM tris-ace-tate, pH 8; 1 mM EDTA). The desired fragment was excised andeluted from the gel using the GENECLEAN II kit (BIO101, LaJolla, Calif.). The puried PCR fragment was subcloned into apT7Blue-3 vector using a Perfectly Blunt cloning kit (Novagen,Madison, Wis.). The recombinant DNA was used to transformEscherichia coli DH5a (Life Technologies). White transformantswere selected on solid Luria medium containing 100 lg ampicillin/ml and pre-spread with both 35 ll of 5-bromo-4-chloro-3-indolyl-b-D-galactopyranoside (50 mg/ml) and 20 ll of 100 mM isopropyl-b-D-thioglucopyranoside. Plasmid DNA was puried from the

transformants with the Wizard Minipreps system (Promega). Se-quencing reactions with double-stranded DNA template werecarried out using an ABI PRISM BigDye Terminator Cycle Se-quencing Ready Reaction kit (PE Applied Biosystems). Sequencedata were acquired and analyzed on an ABI PRISM 310 GeneticAnalyzer (PE Applied Biosystems). Bioinformatic analysis of DNAsequences was performed with BLAST (Altschul et al. 1997),BLAST2 (Tatiana and Madden 1999) and Omiga 1.1.3 (Oxford

Molecular Group, Beaverton, Ore.) programs.

PHA isolation and characterization

Pseudomonas cultures were grown in 1-l Erlenmeyer asks con-taining 500 ml of E* medium (Brandl et al. 1988) supplementedwith glucose (0.5% w/v) and/or oleic acid (0.5% v/v). Cells weregrown at 30 C with 200250 rpm orbital-shaking for 72 h. Whenneeded, an aliquot was removed and processed for transmissionelectron microscopic imaging as previously described (Solaimanet al. 1999). PHAs were extracted from the cells as previouslydescribed (Solaiman et al. 1999). The monomer repeat-unit com-position and molecular mass of the polymer were determined bygas chromatography/mass spectroscopic analysis and gel perme-ation chromatography, respectively (Cromwick et al. 1996).

Results

PCR detection of phaC sequences

We rst developed the PCR protocol using genomic-DNAs puried from P. resinovorans, P. oleovorans,P. putida, P. citronellolis, and P. saccharophila. Theseorganisms have been reported to produce mcl-PHA(Brandl et al. 1988; Cromwick et al. 1996; Eggink et al.1993; Solaiman et al. 1999; Timm and Steinbuchel1990). Our results showed that with the exception of

P. saccharophila, a distinct 540-bp PCR product wasobtained (Fig. 1). The size of the PCR product agreeswith the length of the phaC1 and phaC2 genes anked bythe I-179L/I-179R primer-pair. When ELONGASE, for-mulated by its manufacturer for long PCR amplicationwas used, an additional ca. 3.4-kb amplicon was

Fig. 1 Polymerase chain reaction (PCR) detection of phaC1/C2genes. Puried genomic DNA and regular Taq DNA polymerasewere used in the reactions. Lane 1 No DNA, lane 2 Pseudomonasresinovorans NRRL B-2649, lane 3 P. oleovorans NRRL B-14683,lane 4 P. putida KT2442, lane 5 P. citonellolis NRRL B-2504, lane 6P. saccharophila NRRL B-628, lane 7 DNA size markers (k-DNA/HindIII + /X174/HaeIII)

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observed (data not shown). This long amplicon repre-sents the DNA sequence anked by the I-179L andI-179R annealing sites in the phaC1 and phaC2 genes,respectively, and included the entire phaZ gene.

The PCR protocol developed using the puriedgenomic-DNAs was then tested for the detection ofphaC gene sequences in lysates prepared by heating thesuspension of bacterial colonies. The results (Fig. 2)showed that the characteristic 540-bp PCR product wasproduced even from the crude cell-lysates of microor-ganisms shown to contain phaC1 and phaC2 genes(Fig. 1). Results in Fig. 2 also show that P. viridiavaand P. uorescens indeed harbor the type-II pha locus.Production of PHA in these two organisms had previ-ously been documented (Steinbu chel 1991). Interesting-ly, unlike P. oleorovans NRRL B-14683, the presence ofpseudomonad-type phaC genes was not detected instrain NRRL B-14682 using the present PCR protocol.These PHA synthase genes were not detected in an oil-degrading Pseudomonas sp. ATCC 21909.

Using this rapid and specic PCR method, severalmicroorganisms representing pseudomonads from dif-ferent rRNA homology groups were screened for thepresence of the type-II pha gene locus. Data in Table 1show that among the organisms screened, only P. cor-rugata 388 [belonging to the non-uorescent rRNAhomology group I pseudomonad (Young et al. 1992)]contains the phaC1- and phaC2-type genes.

Sequence analysis of the amplicon from P. corrugata

The 540-bp PCR product from P. corrugata sample was

subcloned and sequenced. A BLASTX search using thecloned 540-bp P. corrugata sequence as query producedamino acid sequence matches with more than 35 PHA

and poly-b-hydroxybutyrate (PHB) synthases (data not

shown). The alignments having the most homology werethose with the PHA synthase 2 (PhaC2) of Pseudomonassp. 613 [GenBank (GB) AB014758], P. resinovorans(GB AF129396), P. putida (GB AF042276), P. oleovo-rans (Swiss-Prot P26496), and P. aeruginosa (ProteinInformation Resource S29307). Apparently, the partic-ular bacterial clone chosen for the sequence analysiscontained an amplied segment of the P. corrugataphaC2 gene.

Characterization of mcl-PHA of P. corrugata

When visualized by transmission electron microscopicimaging, P. corrugata 388 grown for 72 h under PHA-inducing conditions exhibited the characteristic poly-mer-containing inclusion bodies (Fig. 3). Isolation of thepolymer from the P. corrugata culture produced PHAat a crude yield of 49% cell dry weight. Compositionanalysis of the PHA showed that the major repeat-unitswere 3-hydroxyoctanoate (C8:0; 47.0 1.0 mol%),3-hydroxydecanoate (C10:0; 24.5 0.5 mol%), and3-hydroxytetradecenoate (C14:1; 16.5 0.5 mol%).Results of gel permeation chromatography indicatedthat the weight-average and number-average molecularmass of the P. corrugata PHA were 735 105 kDa and

181 45 kDa, respectively. The polydispersity of thepolymer was calculated as 4.19 0.46.

Discussion

The PCR screening protocol described in this paper is arapid, simple, and specic method for identifying mcl-PHA-producing microorganisms. The method is espe-cially important for the identication and verication oforganisms that harbor mcl-PHA biosynthesis genes.Lipophilic dye-based screening procedures (Spiekermannet al. 1999; Takagi and Yamane 1997) are useful and

Fig. 2 PCR detection of phaC1/C2 genes in bacterial lysates. Lane 1No bacterial lysate, lane 2 P. resinovorans NRRL B-2649, lane 3P. oleovorans NRRL B-14682, lane 4 P. oleovorans NRRL B-14683,lane 5 P. putida KT2442, lane 6 P. putida ATCC 17391, lane 7P. citronellolis NRRL B-2504, lane 8 P. viridiava ATCC 13223,lane 9 P. uorescens ATCC 17824, lane 10 Pseudomonas sp. ATCC21909, lane 11 P. saccharophila NRRL B-628, lane M DNA sizemarkers

Table 1 PCR detection of type II PHA synthase genes in selectedpseudomonads. PCR was performed using cell lysates preparedby boiling of the resuspension of bacterial colony as describedin Materials and methods. Bacterial strains were obtained fromDr. William Fett, ERRC

Bacterial strain rRNA-DNAHomology group

phaC1/C2a

Comamonas acidovoransATCC 15668

III )

C. testosteroni ATCC 11996 III )Pseudomonas corrugata 388 I +P. stutzeri ATCC 17588 I )P. andropogonis 27 II )P. andropogonis 11 II )Ralstonia solanacearum K60 II )

a Determined as indicated by the presence or absence of 540-bpPCR amplicon

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direct for visualizing the presence of PHA inclusion

bodies in microbes. However, these methods cannotdierentiate between scl-PHA, mcl-PHA, and other lipidmaterials such as triacylglycerols and waxes. Further-more, since PHA biosynthesis often depends on specicgrowth conditions (Anderson and Dawes 1990), thestaining methods could overlook potential PHAproducers that do not contain the polymer at the time ofscreening. Moreover, in contrast to elaborate and time-consuming hybridization methods (Schembri et al. 1994;Timm et al. 1994), this PCR procedure also provides ameans for quick, simple, and mcl-PHA-specic detectionof the type II PHA biosynthesis genes, using only celllysate as the source of DNA template.

Numerous microorganisms have been reported tosynthesize PHAs. In many cases, the type of PHAproduced was not clearly dened. For example, P. cor-rugata is characterized as a PHB producer based on Nileblue A staining (Sutra et al. 1997). However, the repeat-unit composition of the polymer had not previously beencharacterized. In this study, we conclusively showed thatP. corrugata contains the type II PHA biosynthesis genelocus and proceeded to conrm the production of mcl-PHA by this organism. Using a similar approach, otherorganisms previously reported as PHA producers cannow be veried with respect to the type of their PHAbiosynthesis genes and presumably the class of polymer

itself. The simultaneous existence of type I PHA genescannot be ruled out by the present PCR procedure.However, in conjunction with broad-specicity DNA-hybridization methods (Schembri et al. 1994; Timmet al. 1994), this PCR procedure can serve as a powerfultool for characterizing PHA-biosynthesis gene loci inmicroorganisms.

Randomly selected pseudomonads from the threerRNA-DNA homology groups were screened for thepresence of type II PHA synthase genes (Table 1).The results showed that only P. corrugata harbors thephaC1/C2 genes, further supporting the observation thatmcl-PHAs are mostly produced by Pseudomonasbelonging to the rRNA-DNA homology group I(Steinbu chel 1991). We also observed that strain varia-tion is important in determining the PHA-producingpotential of a bacterial species. For example, whilephaC1/C2 genes were detected (Table 1) in the mcl-PHA-producing P. oleovorans NRRL B-14683 (ATCC29347;Tf41l, Schwartz), these type II PHA synthase

genes were not found in P. oleovorans NRRL B-14682(ATCC 13474). A separate experiment conrmed thatmcl-PHA was not synthesized by strain NRRL B-14682(data not shown). Similarly, our results showed thatP. stutzeri ATCC 17588 did not harbor the phaC1/C2genes (Table 1). He et al. (1998), however, reported thatP. stutzeri 1317 was an mcl-PHA producer. Some of theorganisms listed in Table 1 (e.g., Comamonas acidovo-rans, C. testosteroni, and R. solanacearum) have beenreported as PHA producers (Steinbu chel 1991). Thefailure to detect the specic PCR amplicon in thesemicroorganisms may be indicative of strain variation asdescribed above. Alternatively, these organisms may

contain non-type II PHA synthase genes that would notbe detected by the present PCR procedure. A multiplexPCR method that combines the current phaC1/C2-spe-cic primer-pair with non-type II pha-specic PCRoligomers should allow for simultaneous detection orverication of all classes of PHA synthase genes.

An important discovery in this study is that P. cor-rugata contains phaC1/C2 genes and produces amcl-PHA. Even though this species is classied as apolyhydroxyalkanoate-accumulating microbe, it waslong assumed to synthesize PHB (Sutra et al. 1997).Results from this study, however, conclusively show thata mcl-PHA is produced by this bacterium. More im-

portantly, the study showed that the PHA polymerproduced by P. corrugata has the highest molecularweight value among the unsaturated mcl-PHAs reportedto date (Ashby and Foglia 1998; Brandl et al. 1988).Accordingly, this bacterial species may be an importantorganism for the production of high molecular weightmcl-PHAs.

Acknowledgements We thank Nicole Cross, Rob DiCiccio, andLaurie Fortis for technical assistance; Dr. Peter H. Cooke andDouglas Soroka (Microscopic Imaging Group, Core TechnologiesUnit, ERRC) for acquiring the electron microscopic images ofP. corrugata; and Dr. Alberto Nunez for performing GC/MSanalyses.

Fig. 3 Thin-section electron microscopic images of P. corrugata 388.Cells were cultured in chemically dened E* medium supplementedwith glucose and oleic acid. Incubation was at 30C for 72 h with250 rpm rotary shaking

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179R and 179L Primer Paper