Improvement of the Thermostability and Activity of a Pectate Lyase by Single Amino Acid Substitutions, Using a Strategy Based on Melting-Temperature-Guided Sequence Alignment

In the vast number of random mutagenesis experiments that havetargeted protein thermostability, single amino acid substitutionsthat increase the apparent melting temperature (T_m) of the enzymemore than 1 to 2°C are rare and often require the creationof a large library of mutated genes. Here we present a casewhere a single beneficial mutation (R236F) of a hemp fiber-processingpectate lyase of Xanthomonas campestris origin (PL_Xc) produceda 6°C increase in T_m and a 23-fold increase in the half-lifeat 45°C without compromising the enzyme's catalytic efficiency.This success was based on a variation of sequence alignmentstrategy where a mesophilic amino acid sequence is matched withthe sequences of its thermophilic counterparts that have establishedT_m values. Altogether, two-thirds of the nine targeted singleamino acid substitutions were found to have effects either onthe thermostability or on the catalytic activity of the enzyme,evidence of a high success rate of mutation without the creationof a large gene library and subsequent screening of clones.Combination of R236F with another beneficial mutation (A31G)resulted in at least a twofold increase in specific activitywhile preserving the improved T_m value. To understand the structuralbasis for the increased thermal stability or activity, the variantR236F and A31G R236F proteins and wild-type PL_Xc were purifiedand crystallized. By structure analysis and computational methods,hydrophobic desolvation was found to be the driving force forthe increased stability with R236F.

INTRODUCTION

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INTRODUCTION

Improving the thermostability of a protein or enzyme is desirablefor commercially viable bioprocesses that prolong product shelflife, increase energy efficiency, and save costs (30). Unfortunately,there are no simple rules for improving thermostability, themechanisms of which include the formation of disulfide bridges,hydrophobic or aromatic interactions, contact order, hydrogenbonding, ion pairing, dimer-dimer interaction, and a preponderanceof glutamine usage (32, 36, 41). A critical and challengingtask in thermostability engineering is, therefore, choosinga method whereby promising results can be obtained quickly andefficiently. Both rational (structure-based, including molecularmodeling) and randomized or irrational (directed evolution orrandom and combinatorial engineering) approaches to proteinthermostability improvement have been applied with varying degreesof success (11, 19, 30). Notable single amino acid substitutionsthat have led to dramatic increases in melting temperatures(T_m; the temperature at which 50% of the protein is unfolded),by 20°C and 24°C, respectively, were identified in theDrosophila protein drk SH3 domain (24), and in a bacterial malatedehydrogenase (4). However, these successes were made possibleonly through a good understanding of the thermodynamics andkinetics of folding, or through knowledge of the electrostaticinteractions in the dimer-dimer interface of the tetramericprotein. Otherwise, single amino acid substitutions that increasethe apparent T_m of a given enzyme more than 1 to 2°C arerare (19).

In this study, we selected a bacterial-genome-mined pectatelyase sequence as a template in order to evolve added thermostabilityand/or catalytic activity using a newly improvised strategythat does not require the construction of a large library ofclones, as in directed evolution experiments, or specializedcomputational skills, as in rational protein engineering approaches.We further characterized the beneficial variants at the biochemicaland structural levels. Pectate lyases (EC 4.2.2.2) are secretedenzymes produced by a variety of plant-pathogenic bacteria andtherefore are best studied as virulence factors (for a review,see reference 15). Biotechnologically, these pectinolytic enzymes,as well as various endopolygalacturonases, are useful rettingor bioscouring reagents for the processing of natural bast fibers(e.g., hemp and flax), and cotton fabric (1, 2, 28, 35).

MATERIALS AND METHODS

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MATERIALS AND METHODS

Molecular cloning and site-directed mutagenesis.
Standard methods were used for isolation of plasmid DNA, cloning,and transformation (33). The Xanthomonas campestris pectatelyase II (PL_Xc) gene (NP_638163) was amplified from its genomicDNA (ATCC 33913) and cloned into pSD80, an isopropyl-β-D-thiogalactopyranoside(IPTG)-inducible expression vector, at the EcoRI and HindIIIrestriction sites (34). This cloning is part of a larger genome-miningeffort for pectinase-encoding sequences (Z. Xiao, J. Boyd, S.Grosse, E. Coupe, and P. C. K. Lau, unpublished data). The recombinantplasmid (pSD80PLII) and resultant derivatives were transformedinto Escherichia coli strain Rosetta 2 (Novagen). Site-directedmutagenesis was performed using the QuikChange mutagenesis kit(Stratagene, La Jolla, CA) according to its instruction manual.The mutagenic oligonucleotide primers are listed in Table S1in the supplemental material. The expected mutations were confirmedby gene sequencing using a BigDye DNA sequencing kit (AppliedBiosystems) with an automated DNA sequencer (model 377; ABIPrism).

Multiple sequence alignment.
The ClustalW program (http://www.ebi.ac.uk/clustalw/) was usedto align the X. campestris pectate lyase II (NP_638163) sequence(9) with the four available thermostable pectate lyase sequences,selected by screening of more than a thousand candidates inthe NCBI database for their established thermostability in reportedliterature. The thermostable candidate sequences are accessionnumbers CAD56882 from Bacillus licheniformis (3), BAA96478 fromBacillus sp. strain P-4-N (14), BAB40336 from Bacillus sp. strainTS-47 (37), and AAD35518 from Thermotoga maritima MSB8 (18),with optimal temperatures of 65 to 69, 70, 70, and 90°C,respectively.

Calculation of the success rate of the mutation.
The success rate of the mutation, expressed as a percentage,is calculated as (number of improved variants x 100)/(totalnumber of screened clones) (see Table S2 in the supplementalmaterial).

Protein expression and fermentation.
Single colonies of E. coli Rosetta 2 harboring the X. campestrispectate lyase II gene and the desired mutations (V26A, A31G,L64I, Y66V, K69T, F70I, Q123R, V187I, R236F, and the A31G R236Fdouble mutation) were taken from plates (LB medium containing50 µg/ml ampicillin and 30 µg/ml chloramphenicol)and grown overnight at 30°C in the same medium (startingwith 5 ml; afterwards the colonies were transferred to 500 mlof medium). Fermentations to produce appropriate amounts ofpectate lyase (up to 1 g of protein for each variant) were carriedout in a New Brunswick Scientific BioFlo 110 Benchtop fermentorsystem using defined medium (31). Fermentors with a workingvolume of 6 liters were inoculated with 300 ml of each preculture(giving a starting optical density at 600 nm of about 0.3),which had been grown overnight at 30°C on LB medium. Glucose(0.7%) was used as the sole carbon source. The pH was maintainedautomatically using NH₄OH (29%) and H₃PO₄ (5 M). One milliliterof antifoam was added after sterilization. After a batch phaseof about 9 h, a feed with 0.22 ml/min of glucose (50% solution)and MgSO₄ (20 g/liter) was started. Culture was induced a fewminutes later by addition of 1 mM IPTG, and the temperaturewas reduced to 25°C. After 16 h (optical density at 600nm, about 25), the fermentors were brought to 10°C and cellswere harvested subsequently. Cells were centrifuged (at 6,000x g for 20 min at 4°C), resuspended in phosphate buffer(20 mM; pH 7.0), and broken via two passages in a microfluidizeroperating at 12,000 lb/in². Unbroken cells and debris were removedby filtration using a hollow fiber membrane (pore size, 0.22µm; on an Amicon DC 10 ultrafiltration system), and thefiltrate was kept at 4°C for further processing.

Protein purification.
The cleared cell extract (about 1 liter) was loaded onto anSP-Sepharose FF column (XK50/20) (GE Healthcare) previouslyequilibrated with 20 mM sodium phosphate buffer (pH 7.0). Thecolumn was washed with the same buffer, and bound protein waseluted using a linear NaCl gradient from 0 to 200 mM. Activefractions were pooled and concentrated using a YM 3 membranein an Amicon stirring cell. Protein levels during chromatographywere monitored at 280 nm, and concentrations were determinedusing the Bradford assay (5).

Pectate lyase assay and kinetics.
The pectate lyase activity was determined by measuring the increasein the absorbance at 232 nm of polygalacturonic acid (PGA) (8).Fifty microliters of enzyme and 50 µl of 0.5% PGA in PLbuffer (50 mM Tris-HCl buffer [pH 8.5] containing 0.5 mM CaCl₂)were incubated at 50°C for 30 min. The reaction was stoppedby addition of 20 µl of 0.35 M HCl. One hundred microlitersof reaction mixture was used to determine the absorbance at232 nm. One unit of pectate lyase was defined as the amountof enzyme producing 1 µmol of unsaturated product in 1min under the assay conditions. The amount of degraded productsof pectin was determined by the increase in absorbance at 232nm (8). The molar extinction coefficient for the unsaturatedproduct at 232 nm is 4,600 M^–1 cm^–1. All data wereaverages of triplicate measurements. The kinetic parametersK_m, V_max, and k_cat were determined at the optimal temperaturesof the purified enzymes.

Gel electrophoresis and zymogram staining.
Two hundred nanograms of protein samples was heated for 10 minat 45°C in sample loading buffer before being applied toa 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis(SDS-PAGE) gel containing 0.1% PGA. After electrophoresis, proteinbands were stained with Coomassie blue. For zymogram staining,the gel was soaked in 2.5% Triton X-100 for 30 min and washedwith PL buffer for 30 min. After 20 h of incubation at 40°Cin PL buffer containing 0.1% PGA, the gel was stained with 0.05%(wt/vol) ruthenium red for 20 min and washed with water for60 min.

Stability measurement.
To determine the half-life of inactivation, the pectate lyaseswere incubated in PL buffer at 45°C. Samples were takenat various time points, followed by the pectate lyase assay.Purified proteins were adjusted to about 0.5 mg/ml in 10 mMphosphate buffer, pH 7.0, for determining the circular dichroism(CD) spectrum and T_m. Thermal denaturation experiments wereperformed at a temperature increase of 40°C per h. The datawere collected with a JASCO J-710 CD spectrometer at a wavelengthof 222 nm, where maximal signal difference was observed.

Protein crystallization and structure determination.
Initial crystallization conditions were identified using thePEGs (Nextal, Montreal, Canada), Classic Suite I and II (Qiagen),screens, with the sitting drop vapor diffusion set up at 20°C.Numerous chemical conditions yielded triclinic crystals overnight.The conditions were reproduced and optimized using the hangingdrop vapor diffusion method in 24-well Linbro plates (HamptonResearch). The best conditions found were 20 to 30% (wt/vol)polyethylene glycol 3350 and various buffers with pHs rangingfrom 5.5 to 8.5. The crystals belong to the primitive triclinicspace group P1, with unit cell dimensions of 47.2 Å fora, 53.2 Å for b, 73.0 Å for c, 71.7° for ${alpha}$ , 80.0°for β, 69.0° for ${gamma}$ , and two protein molecules in theasymmetric unit. The Matthews coefficient (23) V_m is 2.20 Å³Da^–1, corresponding to a solvent content of 44%. The cryoprotectantsolution used consisted of mother liquor supplemented with 10to 12% (wt/vol) glycerol, and data were collected at 100 K.Diffraction data extended to 2.0, 2.1, and 1.9 Å resolutionfor wild-type, R236F, and A31G R236G proteins, respectively.The images were processed with the HKL2000 program package (27).The structure of wild-type PL_Xc was solved by molecular replacementusing the MolRep program (38) and the structure of Erwinia chrysanthemipectate lyase (22) (PDB entry 1AIR) as a search model. Freeatom density modification was implemented using ARP/wARP (29)to improve the electron density maps, and the protein modelwas built manually using COOT (12). The model was refined usingREFMAC5 (26) with the TLS option, with each of the monomerstreated separately. The structures of the R236F and A31G R236Fvariants were solved and refined in the same manner as for wild-typePL_Xc, whose structure was used as a search model for molecularreplacement. Refinement statistics are summarized in Table S4in the supplemental material for the three structures solved.The models were validated with PROCHECK (20).

Computational method.
The stabilities (i.e., the free energies of folding) of pectatelyase variants relative to that of the wild-type protein werecalculated with the FOLD-X program (13), using the crystal structureof the wild-type protein determined in this study (chain A).The FOLD-X program employs a first-principle-based energy functionthat was shown to be able to predict folding free energies witha squared-correlation coefficient of 0.69 and a standard deviationof 0.81 kcal/mol, within a data set of more than 1,000 mutationsfrom various proteins. The effect of the R236F mutation on stabilitywas further verified by performing a FOLD-X calculation usingthe crystal structure of the R236F variant (chain A) and simulatingits mutation to the wild-type protein. All crystallographicwater molecules and observed phosphate ions were removed. Possiblewater bridges between protein atoms were implicitly taken intoaccount during FOLD-X calculations. Point mutations were introducedby the FOLD-X program with the mutate function of the WHAT IFprogram (42), which optimizes the mutated side chains with respectto rotamer distributions (7) and hydrogen bond networking (16).The analyses of relative stabilities in terms of contributionsfrom van der Waals, hydrogen bonding, electrostatic, and waterbridge interaction energies, hydrophobic and polar desolvation,and main-chain and side-chain entropies, were based on the FOLD-Xcalculations. Clash energies were not included in the calculationof folding free energies.

Protein structure accession numbers.
The coordinates of the structures of wild-type, R236F, and A31GR236F PL_Xc have been deposited in the Protein Data Bank withthe codes 2OX3, 2OXZ, and 2OY1, respectively.

RESULTS AND DISCUSSION

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RESULTS AND DISCUSSION

Sequence characteristics of a new pectate lyase in comparison to its thermophilic counterparts.
The predicted pectate lyase NP_638163 entry of Xanthomonas campestrispv. campestris ATCC 33913 in the CAZy database (PL_Xc) is a biochemicallyuncharacterized entity except for its annotated sequence classificationas a member of the polysaccharide lyase family 1 of proteins(http://www.cazy.org/fam/PL1.html). We established the T_m ofPL_Xc to be 48°C, certifying it as a mesophilic protein (seebelow). Figure 1 shows a multiple alignment of the predictedamino acid sequence of PL_Xc with four available sequences ofthermostable PLs, whose optimal temperatures have been establishedas ranging from 65 to 90°C. Interestingly, only nine aminoacid positions were invariant in all four thermostable PLs butnot in PL_Xc. Pairwise comparison of the subject and databasePL_Xc sequences ranging from 212 to 301 amino acids gave only25 to 36% identity, indicating low sequence homology among thisgroup of proteins.

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FIG. 1. Multiple alignment of protein sequences of thermostable pectate lyases and thermolabile PL_Xc in family PL1. The signal peptide sequences of the respective proteins are not shown. The numbering of each sequence starts from the initiation codon. Conserved catalytic sites (K199, R230, R235), conserved calcium binding sites (D137, D175, D179), the core structure of the parallel β-helix (vWiDH region), and sites conserved in all thermostable PL1 pectate lyases (CAD56882 from Bacillus licheniformis, BAA96478 from Bacillus sp. strain P-4-N, BAB40336 from Bacillus sp. strain TS-47, AAD35518 from Thermotoga maritima MSB8) but variant in PL_Xc (NP_638163) are shaded.

Characterization of PL_Xc and its variants.
We set out to test whether any of the nine variable positionscould contribute to added thermostability and/or catalytic activityin PL_Xc when they were replaced by the corresponding residueof a thermophilic counterpart (Table 1). A PGA assay was usedto determine the activity and thermal inactivation of PL_Xc andits variants in crude cell extracts. One mutation, R236F, increasedthe T_m by 6°C and extended the half-life at 45°C 23-fold.Three other mutations, F70I, Q123R, and V187I, reduced thermostabilitiesby 18 to 53% (Fig. 2). Thus, four out of the nine positionsappeared to be crucial for the thermostability of PL_Xc. Of theother mutations, the Y66V substitution resulted in a loss ofactivity, while the A31G variant was fivefold more active thanits parent enzyme, although its thermostability was not substantiallychanged. Combining the best attributes of A31G and R236F ina double variant, the A31G R236F variant, resulted in a 5-foldimprovement in activity and a 12-fold increase in thermostabilityover those of the wild-type enzyme (Table 1). All in all, two-thirdsof the amino acid substitutions (A31G, Y66V, F70I, Q123R, V187I,and R236F) substantially affected either the thermostabilityor the catalytic activity of the PL_Xc enzyme, indicating a highsuccess rate of identifying susceptible residues without havingto generate a large library.

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TABLE 1. Thermostabilities and activities of PL_Xc and its variants

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FIG. 2. Thermostability of PL_Xc and its variants.

Enzyme purification and kinetics.
The wild-type enzyme and the A31G, R236F, and A31G R236F variantswere characterized further by purification to apparent homogeneitywith a single cation-exchange SP-Sepharose column yielding 40%recovery of the respective proteins. These proteins appearedas a single band on an SDS-PAGE gel with an apparent molecularmass of 35 kDa, in good agreement with the theoretical massof 35,239 Da. Zymogram analyses of the purified enzymes indicatedthat they are active pectate lyases (Fig. 3). The intensitiesof the clear zones of the respective enzymes were consistentwith the measured specific activities. The K_m values of thepurified A31G, R236F, and A31G R236F proteins with PGA as asubstrate were determined and found not to differ substantiallyfrom each other and from that of the parent enzyme (Table 2).However, the catalytic efficiencies for both the A31G and A31GR236F variants were about twofold greater than that for theparent enzyme. The activity of the most thermostable variant,the R236F variant, was the same as that of the parent. Overall,the specific activities of the purified enzymes are consistentwith their k_cat/K_m values. Similar results were observed whencitrus pectin was used as a substrate (Table 3). Additionally,these variants were found to be 20 to 40% more effective thanthe parent enzyme in releasing pectin from natural hemp fiber(Fig. 4).

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FIG. 3. Purified X. campestris pectate lyase II and its variants on an SDS-PAGE gel (left) and a ruthenium red-stained zymogram gel containing 0.1% PGA (right). Lanes: M, molecular standard; 1, parent enzyme; 2, A31G variant; 3, R236F variant; 4, A31G R236F variant.

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TABLE 2. Kinetic parameters of the parent PL_Xc and variants with PGA as the substrate

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TABLE 3. Kinetic parameters of the variant pectate lyases with citrus pectin as the substrate

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FIG. 4. Amounts of pectin released from natural hemp fiber by PL_Xc and its variants.

CD study.
The structures of the wild-type enzyme and the A31G, R236F,and A31G R236F variant enzymes showed no discernible differencesin their ${alpha}$ -helical structure contents by CD spectroscopy (seeFig. S1 in the supplemental material). However, the T_ms of thefour proteins were established to be 48, 47.5, 54, and 53°C,respectively (Fig. 5). The 5 to 6°C increase in the T_msof the R236F variant and the double variant are consistent withthe increases in the half-lives of the proteins.

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FIG. 5. T_ms of the wild-type and variant enzymes determined by CD. (A) Original CD data; (B) derivative of CD signal.

Structures of wild-type PL_Xc and variant proteins.
To understand the structural basis for the thermal stabilityof the variant proteins, R236F, A31G R236F, and wild-type PL_Xcwere crystallized and their structures solved by molecular replacements.These structures have the same right-handed parallel β-helixarchitecture as that described for Erwinia chrysanthemi (presentlyclassified as Dickeya dadantii) pectate lyase PelC (43) (Fig.6A; see also Fig. S2, the supplementary note, and Table S3 inthe supplemental material). Residue R236 is partially solventexposed and is located in the T1.6 loop (the 6th loop of turnT1), which stacks against the T1.5 loop, containing a helicalstructure previously shown to enhance the catalytic activityof related pectate lyases (10). Its guanidinium group is 10Å, 17 Å, and 22 Å away from the putative activesites R235, R230, and K199, respectively. This may explain thesimilar activities of the wild-type enzyme and the R236 variant.The R236F substitution caused no structural change in the enzyme.Although bulkier, the F236 side chain occupies the same regionof space as the wild-type R236; no neighboring residues areaffected and no major electrostatic interactions interrupted(Fig. 6B). However, the aromatic side chain in this positionallows hydrophobic interactions with several adjacent side chains,particularly M258 from the T1.7 loop, the aliphatic portionsof T210 and N212 from the T1.5 loop, and the neighboring R235.Hydrophobic stacking between the T1.5 and T1.7 loops appearsto confer higher stability on the T1.6 loop. A structure-basedfirst-principle energetic analysis (13) (see Fig. S3 and TableS4 in the supplemental material) predicts a favorable changein folding free energy of –1.7 kcal/mol for the stabilizingR236F mutation, underscoring the gain afforded by hydrophobicdesolvation (–2.8 kcal/mol) as driving the increase instability. This more than offsets the major destabilizing contribution,i.e., the loss of hydrogen bonding energy upon mutation (1.4kcal/mol), between the carbonyl group of D207 and the guanidiniumgroup of R236.

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FIG. 6. Crystal structures of wild-type PL_Xc and its A31G R236F variant. (A) Mapping of the nine mutation sites of wild-type PL_Xc on its crystal structure. The protein is rendered as a yellow ribbon. Side chains at mutation positions are shown as CPK (Corey Pauling Koltun) models and color coded by mutation outcomes: blue, stabilizing; red, activating; green, others. (B and C) Close-up views of structural changes introduced upon mutation at positions 236 and 31, respectively. Superimposed wild-type PL_Xc and its A31G R236F variant are colored green and cyan, respectively. Mutated residues and local structural changes are represented as sticks.

The double mutation A31G R236F also does not introduce majorvariations relative to PL_Xc wild-type and R236F variant structures(Fig. 6B). A noticeable change is a peptide flip at the A31Gmutation site, bringing the backbone carbonyl of A30 closerto the side chain of K151, which undergoes a concerted conformationalchange (Fig. 6C). The A31G mutation resides in the N-terminalregion of the enzyme on the opposite side of the β-helixrelative to the catalytic site. Thus, the twofold increase incatalytic efficiency upon the A31G mutation cannot be accountedfor by the present structural data. Mutations distant from catalyticsites are documented to be as effective as close mutations inimproving enzymatic activity (25), but the underlying factorsare often subtle and elude structural interpretations. Computationalpredictions yielded a marginal destabilizing effect (0.2 kcal/mol)for the A31G mutation, in agreement with the experimental data(Table 1; see also Fig. S3 in the supplemental material).

The remaining single point mutations had either marginal ordestabilizing effects on thermal stability. A retrospectivestructural and computational analysis can rationalize why thesesingle amino acid substitutions did not materialize into improvedthermostability of the target mesophilic enzyme (see the supplementarynote, Fig. S3, and Table S4 in the supplemental material).

Concluding remarks.
This study characterized a new bacterial pectate lyase thatotherwise remains a hypothetical sequence in the vast microbialgenome database. From this basic sequence, more thermostableand active variants were produced that have potential applicationsin the retting of natural fibers. A new approach to protein/enzymethermostabilization was conceived that made selective use ofthose thermophilic sequences that have established T_m valuesto identify invariant residues (besides those of catalytic residuesor binding sites) as possible sites for mutagenesis on a mesophilicsequence. Although based on sequence alignment, this approachdiffers greatly from the "consensus" approach for thermostabilityengineering of proteins whereby a synthetic gene is used toreplace as many as 38 amino acids to arrive at improved thermostability(21). This "consensus" concept for thermostability engineeringof proteins was also based on highly homologous sequences (>80%identity), as opposed to the relatively low degree of sequenceidentity (25% to 36.5%) among the candidate sequences used inthis study. The present strategy also differs from the workof van den Burg et al. (39, 40), where the respective rolesof three amino acid residues, as a result of sequence alignmentof two highly homologous (99% identity) neutral protease sequences,were analyzed by site-directed mutagenesis for their possiblecontributions to thermostability. In the present study, a successrate of at least 11% for identifying "thermosusceptible" residueswas evident. In some directed evolution experiments, for example,a success rate of 0.05 to 0.06% was observed (see Table S2 inthe supplemental material). Our model study is encouraging enoughto be applied to other protein families to provide the firstpossible best candidate for thermostabilization, which may thenrequire the application of additional strategies, includingsaturation mutagenesis (6, 44). Finally, it is acknowledgedthat proteins, even among members of the same families, areindividuals (17). Until universal rules governing protein thermostabilityemerge, proteins need to be studied individually, and new approachesor strategies such as that described in this study can helpin gaining new understanding and facilitate potential industrialapplications.

ACKNOWLEDGMENTS

This research was largely supported by the Climate Change Technologyand Innovation Biotechnology Program of the Canadian BiomassInnovation Network of Natural Resources Canada.

We thank A. Matte for critical reading of the manuscript.

This article is issued as NRCC publication 49095.

FOOTNOTES

* Corresponding author. Mailing address: Biotechnology Research Institute, National Research Council Canada, 6100 Royalmount Avenue, Montreal, Quebec H4P 2R2, Canada. Phone: (514) 496-6325. Fax: (514) 496-6265. E-mail: peter.lau{at}cnrc-nrc.gc.ca

Published ahead of print on 21 December 2007.

Supplemental material for this article may be found at http://aem.asm.org/.

REFERENCES

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