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Molecular and Cellular Biology, January 2006, p. 381-386, Vol. 26, No. 1
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.1.381-386.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Replication past a trans-4-Hydroxynonenal Minor-Groove Adduct by the Sequential Action of Human DNA Polymerases and

William T. Wolfle,¹ Robert E. Johnson,¹ Irina G. Minko,² R. Stephen Lloyd,² Satya Prakash,¹ and Louise Prakash^1*

Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, Texas 77555-1061,¹ Center for Research in Occupational and Environmental Toxicology, Oregon Health and Science University, Portland, Oregon 97239²

Received 8 September 2005/ Returned for modification 7 October 2005/ Accepted 13 October 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
The X-ray crystal structure of human DNA polymerase (Pol) has shown that it differs from all known Pols in its dependence upon Hoogsteen base pairing for synthesizing DNA. Hoogsteen base pairing provides an elegant mechanism for synthesizing DNA opposite minor-groove adducts that present a severe block to synthesis by replicative DNA polymerases. Germane to this problem, a variety of DNA adducts form at the N² minor-groove position of guanine. Previously, we have shown that proficient and error-free replication through the -HOPdG (-hydroxy-1,N²-propano-2'-deoxyguanosine) adduct, which is formed from the reaction of acrolein with the N² of guanine, is mediated by the sequential action of human Pol and Pol, in which Pol incorporates the nucleotide opposite the lesion site and Pol carries out the subsequent extension reaction. To test the general applicability of these observations to other adducts formed at the N² position of guanine, here we examine the proficiency of human Pol and Pol to synthesize past stereoisomers of trans-4-hydroxy-2-nonenal-deoxyguanosine (HNE-dG). Even though HNE- and acrolein-modified dGs share common structural features, due to their increased size and other structural differences, HNE adducts are potentially more blocking for replication than -HOPdG. We show here that the sequential action of Pol and Pol promotes efficient and error-free synthesis through the HNE-dG adducts, in which Pol incorporates the nucleotide opposite the lesion site and Pol performs the extension reaction.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Lipid peroxidation is a chain reaction process that initiates from free radical attack on polyunsaturated fatty acids in membranes and results in the generation of a variety of highly reactive aldehydes: acrolein, crotonaldehyde, malonaldehyde, and trans-4-hydroxy-2-nonenal (HNE) (2, 3, 5, 23). The N² group of guanine in DNA conjugates with these various aldehydes, resulting in adducts that are highly inhibitory to synthesis by replicative DNA polymerases (Pols) (12).

The reaction of acrolein, an ,ß-unsaturated aldehyde, with the N² group of guanine in DNA followed by ring closure at N¹ results in the formation of the cyclic adduct -hydroxy-1,N²-propano-2'-deoxyguanosine (-HOPdG) (Fig. 1). Previously, we have shown that efficient and error-free replication past the -HOPdG adduct can be mediated by the sequential action of human Y family DNA polymerases and , wherein Pol incorporates the nucleotide opposite the lesion site and Pol carries out the subsequent extension step (28).

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FIG. 1. Chemical structures of dG adducts of acrolein and 4-HNE. -HOPdG is formed from the reaction of acrolein with dG. Chemically stable (r) -HOPdG and PdG correspond to the open and closed isomers of -HOPdG, respectively. The chemical structures of two stereoisomers resulting from the reaction of 4-HNE with dG, the 11S HNE-dG, and the 11R HNE-dG are shown.

Since -HOPdG can adopt a ring-closed cyclic form or a ring-open form in DNA, we also examined the ability of human Y family Pol (17) and Pol and Pol (30) to replicate through the structural analogs of -HOPdG which permanently stay in the ring-closed or the ring-open form (Fig. 1). Nuclear magnetic resonance studies of the ring-closed form, 1,N²-propano-2'-deoxyguanosine (PdG), have shown that, in duplex DNA, the adducted base assumes a syn conformation and forms a PdG (syn) · dC⁺ (anti) Hoogsteen base pair with a C (22, 29). The ring-open form of -HOPdG, N²-(3-hydroxypropyl), remains in the anti conformation and retains the ability to form a normal Watson-Crick base pair with the C (4, 13, 14).

Our studies with -HOPdG and its ring-closed and ring-open structural analogs, PdG and reduced -HOPdG [(r) -HOPdG], respectively (Fig. 1), have yielded the following observations and conclusions (28, 30). (i) The ring-open form of -HOPdG is not a block to synthesis by human Pol, Pol, or Pol at either the nucleotide incorporation step or the extension step. Thus, although in the ring-open form, the N²-propyl chain would project into the minor groove, these Y family Pols can replicate through the minor-groove distortions imposed upon DNA by this adduct, presumably because the adducted G can form a normal Watson-Crick base pair with a C (4). (ii) The ring-closed analog of -HOPdG, PdG, is very inhibitory to synthesis by human Pol, Pol, and Pol. Although Pol proficiently incorporates nucleotides opposite the PdG adduct, it lacks the ability to extend from the incorporated nucleotide, whereas Pol and Pol are strongly inhibited at both the nucleotide incorporation and extension steps.

These studies have led us to formulate the following mechanism for the efficient and accurate bypass of -HOPdG by the combined action of Pol and Pol. Since -HOPdG is expected to be in the closed cyclic form when it is the templating residue (15, 21), Pol can incorporate a C or a T opposite this adduct because of its ability to accommodate the closed cyclic form and utilize the Hoogsteen edge of the modified base that would be in a syn conformation (22, 29). Pol proficiently extends from the dCMP residue, but not from the dTMP residue, incorporated by Pol opposite -HOPdG, presumably because the incorporation of dCMP but not of dTMP favors the ring-opening reaction of -HOPdG (4, 16), and the ability of the ring-opened adduct to form a normal Watson-Crick pair with the C (4) enables Pol to extend from such a primer terminus.

Similar to acrolein, HNE (Fig. 1) reacts with N² and N1 of guanine to yield cyclic propano dG adducts. Four stereoisomeric HNE-dG adducts have been detected as endogenous DNA lesions in animal and human tissues (3). Although the structures of the HNE adducts in DNA have not yet been determined, these adducts are hypothesized to resemble -HOPdG in some respects in the structures they adopt in DNA. Thus, in single-stranded DNA and as a templating residue, the HNE-dG adducts are expected to be predominantly in a ring-closed form and to adopt a syn conformation that would place the propano moiety in the major groove. When HNE-d-G pairs with a C, the equilibrium is likely to be shifted towards the ring-open form, with the modified base assuming an anti conformation, projecting the adduct into the minor groove. This model is supported by data of the peptide-trapping experiments (14) that revealed the presence of the ring-open HNE-dGsinoligodeoxynucleotides and suggested higher levels of the ring-open adducts in duplex DNA relative to the single-stranded DNA.

Since HNE-dG adducts are larger and less reactive in peptide-DNA cross-linking reactions (14), we hypothesize that these adducts represent a more significant block to DNA polymerases than -HOPdG. To elaborate on the roles of Y family polymerases in promoting replication through minor-groove DNA lesions, here we have examined the ability of human Pol, Pol, and Pol to replicate through the 6S, 8R, 11S and 6S, 8R, 11R isomers of HNE-dG (Fig. 1), henceforth referred to as 11S and 11R, respectively. We find that, whereas Pol was inhibited at both the nucleotide incorporation and extension steps opposite the HNE-dG adducts, Pol was able to carry out the nucleotide incorporation reaction but did not extend, while Pol was unable to insert the nucleotide opposite the lesion but could perform the extension reaction. These observations support the premise that the sequential action of Pol and Pol can promote proficient and error-free replication through the HNE-dG adducts.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Purification of DNA polymerases. Saccharomyces cerevisiae strain BJ5464 was transformed with plasmid pPOL42, pPOL114, or pBJ765; these plasmids carry the genes encoding human Pol, Pol, and Pol, respectively, fused in frame with glutathione S-transferase (GST) (7-11). Frozen yeast cells were suspended in cell breakage buffer (50 mM Tris-HCl, pH 7.5; 10% sucrose; 300 mM NaCl; 1 mM EDTA; 10 mM ß-mercaptoethanol; 0.5 mM benzamidine; 0.5 mM phenylmethylsulfonyl fluoride; and 5 µg/µl each of aprotinin, chymostatin, pepstatin A, and leupeptin) and lysed by a French press prior to centrifugation (100,000 x g, 60 min). For each enzyme, the respective cell extract was passed over a glutathione-Sepharose 4B column (Amersham Pharmacia) at 4°C and the column subsequently washed with 10 column volumes of cell breakage buffer containing 1 M NaCl. After the column was equilibrated with 3 volumes of elution buffer (100 mM Tris-HCl, pH 8.0, 100 mM NaCl, 10% glycerol, 0.01% Nonidet P-40), the GST tags were cleaved with PreScission protease (Amersham Pharmacia) and the polymerases were eluted. The final enzymes contained a seven-amino-acid N-terminal leader peptide. Protein concentrations were determined by the Bio-Rad protein assay using bovine serum albumin as a standard and by UV absorbance at 280 nM under denaturing conditions (8 M urea) using the molar extinction coefficient calculated from the amino acid composition. The purified polymerases were stored in 5-µl aliquots at –80°C.

Nucleotides and DNA substrates. 5'-Deoxynucleoside triphosphates (dNTPs; 100 mM) were purchased from Roche Diagnostics and stored in aliquots at –20°C. The synthetic oligodeoxynucleotide template and primers were used to prepare the nondamaged DNA substrates. The modified 12-mer DNA oligodeoxynucleotides were synthesized by Carmelo Rizzo and Hao Wang (Department of Chemistry, Vanderbilt University) (25) and assembled into 38-mers as described previously (12). Template sequences were as follows: 5'-GCTAGC GAG TCC GCG CCA AGC TTG GGC TGC AGC AGG TC-3', where the underlined G indicates either an undamaged G, 11S HNE-dG, or 11R HNE-dG residue. For the standing-start DNA polymerase assays, the primer 5'-A GCC CAA GCT TGG CGC GGA CT-3' was annealed to the template, resulting in a partial-duplex DNA substrate with the 3' OH one nucleotide before the lesion. To assay extension from a C · G or T · G primer terminal base pair, the primer 5'-GCC CAA GCT TGG CGC GGA CTC-3' or 5'-GCC CAA GCT TGG CGC GGA CTT-3' was used. ³²P-5'-end-labeled primers (1.0 µM) were annealed to the templates (1.5 µM) in 50 mM Tris-HCl (pH 7.5) and 100 mM NaCl by heating to 95°C for 2 min, followed by slow cooling to room temperature.

DNA polymerase assays. The standard DNA polymerase reaction mixture contained 25 mM Tris-HCl (pH 7.5), 5 mM dithiothreitol, 0.1 mg/ml bovine serum albumin, 5 mM MgCl₂, 10 nM DNA, 1 nM DNA polymerase, and 50 µM of each of four dNTPs (dGTP, dATP, dTTP, and dCTP). Reactions were carried out at 24°C and subsequently quenched with 4 volumes of 95% formamide loading buffer (95% formamide, 0.3% bromophenol blue, 0.3% cyanol FF) and placed on ice. Quenched reaction mixtures were heat denatured at 95°C for 3 min, and product formation was monitored using 12% polyacrylamide gel electrophoresis (8 M urea) followed by PhosphorImager analysis (Molecular Dynamics).

For the steady-state kinetic analyses, the standard DNA polymerase reaction conditions were used with the exception that a single dNTP was used, and concentrations were in a range appropriate for K_m determinations. Product formation was monitored using 12% polyacrylamide-8 M urea gel electrophoresis, and the respective gel band intensities were quantified using the PhosphorImager (Molecular Dynamics). Deoxynucleotide incorporation was measured at multiple time points for each dNTP concentration, and the observed rate of nucleotide incorporation was determined by linear regression. Values of the steady-state parameters k_cat and K_m for nucleotide incorporation were then determined from the best fit to the Michaelis-Menten equation (Sigma Plot 7.0). The reported kinetic parameters for each substrate are the averages of at least three independent experiments.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Nucleotide incorporation opposite the HNE-dG adduct by Pol. The ability of Pol to replicate through the 11S and 11R HNE-dG lesions was examined using a standing-start DNA substrate, and DNA synthesis reactions were carried out in the presence of 10 nM DNA, 1 nM Pol, and 50 µM of each of four dNTPs for 20 min at 24°C. Under these conditions, Pol incorporated a nucleotide opposite both the 11S and 11R HNE-dG lesions, but the subsequent extensions were inhibited (Fig. 2A, lanes 1 to 4).

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FIG. 2. DNA synthesis by Pol and Pol on a template containing either an unmodified G or an 11S or 11R HNE-dG isomer. (A) Nucleotide incorporation opposite the HNE-dG lesion by Pol (lanes 1 to 4) and Pol (lanes 5 to 7). (B) Extension from a C opposite the HNE-dG lesion by Pol (lanes 8 to 10) and Pol (lanes 11 to 13). DNA polymerase (1 nM) was incubated with the primer template DNA (10 nM) and either no nucleotide (lane 1) or 50 µM of each of four dNTPs (lanes 2 to 13) for 20 min at 24°C in a standard reaction buffer. The position of the modified G in the template (shown on top) is indicated by an asterisk. ND, nondamaged DNA.

To determine the identity of the nucleotide(s) inserted by Pol opposite the 11S and the 11R HNE-dG lesions, DNA synthesis assays in the presence of only a single dNTP were performed. Pol primarily incorporated the correct nucleotide, C, opposite both the HNE-dG lesions; a T, however, was also incorporated, but to a lesser extent than a C (data not shown). Next, we determined the efficiency of C and T incorporation by Pol opposite these lesions under steady-state conditions. As shown in Table 1, Pol incorporated a C opposite the 11S and the 11R HNE-dG lesions with an efficiency approximately 25% to 30% of that observed for incorporation opposite the undamaged G. Pol also incorporated a T opposite both the HNE-dG lesions with about 10% of the efficiency observed for the incorporation of a C opposite an undamaged G, comparable to the efficiency for T misincorporation opposite the undamaged G. Thus, Pol can incorporate nucleotides opposite both of the HNE-dG adducts, but the efficiency of C incorporation was somewhat reduced.

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TABLE 1. Steady-state kinetic parameters for nucleotide incorporation opposite the 11S or 11R HNE-dG lesion by Pol

Although, in the presence of all four dNTPs, Pol was unable to continue synthesis beyond the HNE-dG lesions (Fig. 2A, lanes 3 and 4), this observation was confirmed by examining whether Pol could extend a DNA primer that has a C paired with either the 11S or the 11R isomer of the lesion. Under these conditions, no extension by Pol was detected (Fig. 2B, lanes 8 to 10).

Primer extension opposite the HNE-dG adduct by Pol. Synthesis by Pol was severely inhibited by the 11S isomer, whereas some synthesis occurred with the 11R isomer (Fig. 2A, lanes 5 to 7). The ability of Pol to incorporate a C opposite the 11S and 11R isomers was also examined by steady-state kinetic analyses. Compared to the incorporation of a C opposite an undamaged G, C incorporation opposite either lesion was significantly reduced, with the 11S isomer being more inhibitory than the 11R isomer (Table 2).

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TABLE 2. Steady-state kinetic parameters for nucleotide incorporation opposite the 11S or 11R HNE-dG lesion by Pol

Pol extends from a C opposite both the 11S and 11R HNE-dG lesions (Fig. 2B, lanes 11 to 13). As determined from steady-state kinetic analyses, Pol extends from the C opposite the 11S isomer about 25% as efficiently as it extends from the C opposite the undamaged G, whereas the efficiency of extension from a C opposite the 11R isomer was the same as that of extension from opposite the undamaged G (Table 3).

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TABLE 3. Steady-state kinetic parameters for extension from a C or a T opposite the 11S or 11R HNE-dG lesion by Pol

In view of the fact that Pol misincorporates a T opposite both of the HNE-dG stereoisomers, we also examined the ability of Pol to extend from a T placed opposite these lesions. Pol has previously been demonstrated to more proficiently extend mismatched base pairs than either Pol or Pol (24, 26, 27); accordingly, the efficiency of extension by Pol from a T opposite an undamaged G was only 20-fold less than that of extension from a C opposite the undamaged G (Table 3). However, the proficiency of Pol extension from a T opposite the 11S isomer was lowered by a further 80-fold, whereas that of extension from a T opposite the 11R isomer was reduced by 10-fold, compared to the efficiency of extension from a T opposite the undamaged G (Table 3). Thus, Pol extends from a C opposite both the HNE isomers much more efficiently than from a T.

Synthesis by Pol is inhibited at the HNE-dG adduct. Since Pol is capable of replicative bypass of an (r) -HOPdG lesion (17), this polymerase was assayed for its ability to replicate through the 11S and 11R isomers of HNE-dG. The HNE-dG lesions, however, were very inhibitory to synthesis by Pol. Moreover, Pol showed a propensity for nucleotide misincorporation opposite both these lesions. DNA synthesis reactions performed in the presence of only one of the four dNTPs indicated that Pol inserted an A or a G opposite the 11S isomer and that it inserted an A, a G, or a C opposite the 11R isomer, and the level of nucleotide incorporation was much reduced over that seen opposite the undamaged G (data not shown). These qualitative observations were verified by steady-state kinetic analyses. As shown in Table 4, Pol incorporated a G or A with nearly equal efficiencies opposite either the undamaged G or the 11S isomer. Opposite the 11R lesion, however, Pol was able to incorporate a C in addition to an A or a G. The incorporation of an A or a G opposite the 11R isomer occurred almost as infrequently as their misincorporation opposite the undamaged G, whereas the incorporation of a C opposite 11R was reduced by 100-fold compared to the incorporation of a C opposite the undamaged G. Overall, the 11S isomer presents a strong block to nucleotide incorporation by Pol, whereas the 11R isomer is less blocking. We also examined the ability of Pol to extend from a C opposite the two HNE-dG stereoisomers (Table 5). Pol is greatly impaired at extending opposite the 11S isomer, since the efficiency of extension opposite this lesion was reduced by 500-fold compared to extension from the normal G · C primer terminus. Pol, however, could extend from the C opposite the 11R isomer with only an 20-fold reduction in efficiency (Table 5).

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TABLE 4. Steady-state kinetic parameters for nucleotide incorporation opposite the 11S or 11R HNE-dG lesion by Pol

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TABLE 5. Steady-state kinetic parameters for extension from a C opposite from the 11S or 11R HNE-dG lesion by Pol

Top Abstract Introduction Materials and Methods Results Discussion References

 
This investigation has demonstrated that Pol inserts a C or a T opposite the 11S and 11R isomers of HNE-dG; the efficiency of incorporation of a C opposite both the lesions is reduced only by approximately threefold compared to the incorporation of a C opposite the undamaged G, whereas the efficiency of T incorporation opposite the HNE-dG lesions was approximately equal to that measured opposite the undamaged G. Pol was inhibited at the extension step of lesion bypass, and this reaction was performed by Pol. In contrast to Pol, Pol was strongly inhibited at the nucleotide incorporation step opposite the 11S isomer and to a lesser extent opposite the 11R isomer. Pol extended from a C opposite both the HNE-dG isomers, but the efficiency of extension from a C opposite the 11S isomer was reduced approximately fivefold, whereas extension from a C opposite the 11R isomer was as efficient as from a C opposite the undamaged G. Pol was strongly inhibited at both the steps of lesion bypass by the 11S isomer, whereas the 11R isomer was less inhibitory to Pol, particularly, at the extension step. Overall, the 11S isomer was more inhibitory to synthesis by these translesion synthesis polymerases than the 11R isomer. In spite of this, the sequential action of Pol and Pol can promote error-free bypass through the 11S isomer, wherein, following the incorporation of a C by Pol, Pol performs the extension reaction. The combined action of Pol and Pol would also promote synthesis through the 11R isomer.

The structural basis for Pol's incorporation of nucleotides opposite the 11S isomer may reside in its ability to accommodate the template purine in its active site in the syn conformation (18). As a templating residue, the 11S isomer may predominantly exist in the closed cyclic form and, similarly to a PdG adduct, adopt a syn conformation in DNA. It is hypothesized that only Pol could accommodate such a structure, and thus only this polymerase would then be able to incorporate nucleotides opposite this isomer.

The 11R isomer is less inhibitory to nucleotide incorporation by Pol and Pol than the 11S isomer, perhaps because, as a templating residue, it undergoes ring opening more readily. While Pol would be able to incorporate nucleotides opposite both the ring-closed and the ring-open forms of the 11R isomer, presumably Pol and Pol perform this reaction only opposite the ring-open form, and their reduced efficiencies reflect the infrequency of ring opening.

We presume that the incorporation of a C opposite the ring-closed 11S isomer of HNE-dG by Pol triggers the chemical change to a ring-open conformation, as has been deduced from the nuclear magnetic resonance studies of a malondialdehyde 1,N² exocyclic guanine adduct (16). In this conformation the adduct has the potential to form a normal Watson-Crick hydrogen-bonded pair with the C (4, 16), from which Pol could then extend. The poor efficiency of Pol at extending from a T opposite the 11S isomer suggests that this mispair does not efficiently trigger the ring-opening reaction. Pol would then be inhibited from extending from such a ring-closed structure, which presumably stays in the syn conformation. The observation of a more proficient extension by Pol from a C opposite the 11R isomer than from a C opposite the 11S isomer may be because of the greater prevalence of the ring-open configuration in the 11R isomer than the 11S isomer, and that could also account for Pol's ability to extend from a C opposite the 11R isomer. The assumption that ring opening is somewhat more efficient for the 11R isomer is in a good agreement with two observations from other studies: (i) melting experiments showed that a duplex oligodeoxynucleotide containing the 11S HNE-dG is significantly more destabilized than the 11R-containing duplex (25) and (ii) whereas the 11R isomer was essentially nonmutagenic in COS-7 cells, the 11S adduct yielded approximately 4% mutations (6).

Our previous studies with the N² guanine adducts of acrolein (17, 28, 30), taken together with the present studies with the HNE adducts, suggest that Pol, Pol, and Pol can perform a hierarchy of reactions opposite different N² guanine adducts. Thus, the ring-open form of -HOPdG, in spite of the fact that it would project into the minor groove, is not a significant block to synthesis by any of these Y family polymerases. However, when the ring-closed form of -HOPdG is the templating base, it would adopt a syn conformation in DNA, being very inhibitory to nucleotide incorporation by Pol and Pol; only Pol would be able to incorporate a C or a T opposite this lesion. These observations have suggested that, while all three polymerases can accommodate minor-groove perturbations imposed upon DNA by the open form of -HOPdG, only Pol can incorporate nucleotides opposite the closed cyclic form of -HOPdG that adopts a syn conformation in DNA. Since the incorporation of a C by Pol opposite the closed cyclic form of -HOPdG initiates the ring-opening reaction and/or stabilizes the adduct in its ring-open form, Pol is then able to proficiently extend from such a primer terminus.

Here we show that the sequential action of Pol and Pol can promote replication through the HNE-dG adducts, and, overall, the pattern of replication through the HNE adducts by Pol, Pol, and Pol resembles that opposite -HOPdG. Based upon our studies with the -HOPdG and the HNE-dG adducts, we suggest that the sequential action of Pol and Pol would promote replication through the large variety of N² adducts of guanine that form from free radical attack upon polyunsaturated fatty acids and which have been shown to be present at significant levels in the DNAs of human and rodent tissues (1-3, 19, 20).

 
This work was supported by National Institute of Environmental Health Sciences grants ES012411 (L.P., principal investigator [P.I.]) and ES05355 (Michael Stone, P.I., and R.S.L. [P.I. Project 2]).

We acknowledge C. J. Rizzo and H. Wang (Department of Chemistry, Center in Molecular Toxicology, Vanderbilt University, Nashville, TN) for a generous gift of HNE-dG-containing oligodeoxynucleotides.

 
* Corresponding author. Mailing address: Sealy Center for Molecular Science, University of Texas Medical Branch at Galveston, 6.104 Blocker Medical Research Building, 11th and Mechanic Streets, Galveston, TX 77555-1061. Phone: (409) 747-8601. Fax: (409) 747-8608. E-mail: l.prakash{at}utmb.edu.

Top Abstract Introduction Materials and Methods Results Discussion References

 

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Molecular and Cellular Biology, January 2006, p. 381-386, Vol. 26, No. 1
0270-7306/06/$08.00+0     doi:10.1128/MCB.26.1.381-386.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

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