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J Bacteriol. Jul 2009; 191(13): 4451–4457.
Published online May 1, 2009. doi:  10.1128/JB.01582-08
PMCID: PMC2698477

The Iron-Hydrogenase of Thermotoga maritima Utilizes Ferredoxin and NADH Synergistically: a New Perspective on Anaerobic Hydrogen Production[down-pointing small open triangle]


The hyperthermophilic and anaerobic bacterium Thermotoga maritima ferments a wide variety of carbohydrates, producing acetate, CO2, and H2. Glucose is degraded through a classical Embden-Meyerhof pathway, and both NADH and reduced ferredoxin are generated. The oxidation of these electron carriers must be coupled to H2 production, but the mechanism by which this occurs is unknown. The trimeric [FeFe]-type hydrogenase that was previously purified from T. maritima does not use either reduced ferredoxin or NADH as a sole electron donor. This problem has now been resolved by the demonstration that this hydrogenase requires the presence of both electron carriers for catalysis of H2 production. The enzyme oxidizes NADH and ferredoxin simultaneously in an approximately 1:1 ratio and in a synergistic fashion to produce H2. It is proposed that the enzyme represents a new class of bifurcating [FeFe] hydrogenase in which the exergonic oxidation of ferredoxin (midpoint potential, −453 mV) is used to drive the unfavorable oxidation of NADH (E0′ = −320 mV) to produce H2 (E0′ = −420 mV). From genome sequence analysis, it is now clear that there are two major types of [FeFe] hydrogenases: the trimeric bifurcating enzyme and the more well-studied monomeric ferredoxin-dependent [FeFe] hydrogenase. Almost one-third of the known H2-producing anaerobes appear to contain homologs of the trimeric bifurcating enzyme, although many of them also harbor one or more homologs of the simpler ferredoxin-dependent hydrogenase. The discovery of the bifurcating hydrogenase gives a new perspective on our understanding of the bioenergetics and mechanism of H2 production and of anaerobic metabolism in general.

The order Thermotogales is characterized by the ability of its members to utilize a wide variety of carbohydrates (8). All of these organisms ferment sugars predominantly to acetate, CO2, and H2 (23). They thrive mainly at elevated temperatures, although a new subclass of mesophilic “mesotoga” has also been proposed (19). These properties also make the Thermotoga species excellent candidates for biohydrogen production from plant-based biomass. The genome of the type strain, T. maritima, was one of the first to be sequenced, and this revealed a high degree of lateral gene transfer between archaea and bacteria (17, 18). In addition, T. maritima is part of a structural genomics effort, and the structures of over 100 of its proteins have been determined (20, 21). The organism degrades a wide variety of both simple and complex carbohydrates (4, 5), and the glucose that is produced is oxidized by both classical Embden-Meyerhof (85%) and Entner-Douderhoff (15%) pathways (23). The generation of H2 is accomplished by the enzyme hydrogenase. However, little is known about the bioenergetics of the reaction and the pathways of electron flow from carbohydrate oxidation to H2 formation.

Although hydrogenases catalyze the simplest of chemical reactions, the reversible interconversion of protons, electrons, and H2, they are surprisingly complex proteins, some more so than others (33). They can be divided into two major groups, the [NiFe]- and [FeFe]-type hydrogenases, based on the presence of nickel and iron or only iron in their active sites. In general, the physiological roles of the [FeFe] hydrogenases are to evolve H2, while the roles of the [NiFe] enzymes are to oxidize it (33). For example, several Clostridium spp. evolve H2 via a cytoplasmic, monomeric [FeFe] hydrogenase that uses the low-potential redox protein ferredoxin (Fd) (midpoint potential [Em], <−400 mV) as the electron donor (15). In contrast, H2 production using NAD(P)H (E0′ = −320 mV) as the electron donor is thermodynamically unfavorable under physiological conditions because of the more positive redox potential of the pyridine nucleotides (30). Nevertheless, cytoplasmic NAD(P)H-dependent [FeFe] hydrogenases have been reported, although how the endergonic reaction of NAD(P)H-dependent H2 production is accomplished under physiological conditions is not clear (13, 28).

During the oxidation of glucose by T. maritima, both Fd and NAD function as physiological electron acceptors (1, 26, 34). NADH is generated via the glyceraldehyde-3-phosphate dehydrogenase reaction of glycolysis, while the pyruvate that is generated by this pathway is oxidized by pyruvate Fd oxidoreductase (POR) to acetyl coenzyme A (acetyl-CoA), producing reduced Fd. Acetyl-CoA is converted to acetate by phosphotransacetylase and acetate kinase with the concomitant production of ATP. This pathway leads to the production of four moles of H2 per mole of glucose, with reductant provided by two moles of NADH and four moles of reduced Fd, together with two moles of acetate and two moles of CO2 (23). The oxidation of reduced Fd and NADH must be directly or indirectly coupled to the reduction of protons to H2 by hydrogenase, but the trimeric cytoplasmic [FeFe] hydrogenase characterized from T. maritima more than a decade ago does not use either T. maritima Fd or NADH as the sole electron donor (10, 31). Consequently, the mechanism by which the oxidation of Fd and NADH is coupled in vivo to H2 production is not known. In this study, we have resolved this long-standing problem by showing that this cytoplasmic enzyme represents a novel type of hydrogenase that requires both physiological electron carriers to be present for the efficient catalysis of H2 production in which both serve as electron donors.


Growth of the organism and preparation of cell extracts.

T. maritima (DSM 3109) was grown in 500-liter cultures at 80°C with 0.4% (wt/vol) maltose as the carbon source in a medium described previously (31). Cells (25 g total) were lysed anaerobically by osmotic shock in 200 ml 50 mM Tris-HCl, pH 8.0, containing DNase (2.5 mg; Sigma, St. Louis, MO) and sodium dithionite (1 mM). The mixture was sonicated (10 min, power setting 4; Branson Sonifier) under an argon flow. The soluble, cytoplasmic fraction was separated from the insoluble, membrane fraction by centrifugation at 120,000 × g (Optima L-90K; Beckman-Coulter, Ramsey, MN) for 2 h. The membrane fractions were washed twice with 50 mM Tris-HCl, pH 8.0, in order to minimize cytoplasmic contamination.

Purification of hydrogenase, POR, and Fd.

By using published procedures (1, 2, 31), we purified hydrogenase, POR, and Fd from the cytoplasmic fraction derived from 25 g of cells (wet weight) by anaerobic multistep chromatography using an Akta Basic system (GE Healthcare, Piscataway, NY). Unless otherwise stated, 50 mM Tris-HCl, pH 8.0, containing 0.5 mM sodium dithionite was used in all column chromatography steps and all materials were obtained from GE Healthcare. The active hydrogenase fractions were pooled and yielded 18 mg protein with a specific activity of 1.2 U/mg/min. The Fd fractions were pooled and concentrated using ultrafiltration to about 3.5 ml. The 390/280-nm absorbance ratio was 0.8. The active POR fractions were pooled and yielded 65 mg protein with a specific activity of 310 U/mg/min. The Em of T. maritima Fd was previously determined to be −453 mV at 80°C. The standard E0′ values of −420 and −320 mV were used for the H+/H2 and NAD/NADH redox couples, respectively, and these are assumed not to be significantly affected by temperature (27, 29). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of proteins was performed using the Criterion system with 4 to 20% (wt/vol) Tris-HCl gels (Bio-Rad, Hercules, CA). Protein estimations were performed using the Bradford assay (Bio-Rad).

Enzyme assays.

For the H2 production assay, the reaction mixture (500 μl) in 8-ml stoppered serum vials contained 50 mM potassium phosphate, pH 7.5, 100 μM flavin mononucleotide (FMN), 1 mM NADH, 10 mM pyruvate, 100 μM thiamine pyrophosphate, 1 mM coenzyme A, 50 μg T. maritima POR, and ~5.0 μM T. maritima Fd. The mixture was incubated at 80°C, and the amount of H2 produced in the headspace over 6 min was measured using a gas chromatograph (Shimadzu GC-8A). A smaller amount of POR (20 μg) was used for the kinetic analyses of Fd. To determine the source of the reductant for H2 production, known amounts of NADH were added to the standard assay. The amount of H2 produced once the reaction reached completion was measured and was corrected for the amount produced in the absence of added NADH. Hydrogenase activity was also measured using methyl viologen (2 mM) reduced with sodium dithionite (4 mM) as the electron donor by using the same buffer system. One unit of activity is defined as the production of 1 μmol of H2 evolved/min in the indicated assay system. POR activity was measured as described previously (24), and the Fd concentration was determined at 390 nm using a molar absorbance of 17,400 M−1 cm−1 (29). Membrane-bound NADH oxidoreductase was measured using washed membranes in a 2-ml reaction mixture using NADH (1 mM) as the electron donor and benzyl viologen (1 mM; epsilon = 7,400 M−1 cm−1 at 600 nm) as the acceptor.

Genome searches.

Genomes in the NCBI microbial genome database (http://www.ncbi.nlm.nih.gov/genomes/MICROBES/microbial_taxtree.html) were searched for homologs of all three of the subunits of the trimeric [FeFe] hydrogenase of T. maritima and for the catalytic subunit (the TTE0127 protein) of the [NiFe] hydrogenase of Thermoanaerobacter tengcongensis (28). Putative catalytic subunits were also examined for the presence of the conserved residues coordinating the active metal sites of the [FeFe] and [NiFe] enzymes (15, 33).


The electron donors for T. maritima [FeFe] hydrogenase.

Analysis of the genome sequence of T. maritima indicated the presence of two genes that could potentially encode the catalytic subunit of an [FeFe] hydrogenase, TM0201 and TM1426. TM1426 is part of the operon that encodes the heterotrimeric hydrogenase (TM1424-TM1426) that was previously purified (31) while TM0201 does not appear to be part of an operon. Genome-wide transcriptional data indicate that TM0201 is expressed, albeit at a relatively low level, in cells grown on various carbohydrates, although it is not differentially regulated by the growth substrates that were examined (5). It was therefore possible that T. maritima contained two types of [FeFe] hydrogenases. However, fractionation of the cell extract of glucose-grown cells by anion-exchange column chromatography gave rise to only a single peak of hydrogenase activity with the use of dithionite-reduced methyl viologen as the electron donor, and there was no significant hydrogenase activity in the membrane fraction (<1% of the cytoplasmic activity). It is therefore concluded that the in vivo oxidation of the physiological electron carriers, reduced Fd and NADH, must somehow be linked to the production of hydrogen and the multisubunit [FeFe] hydrogenase that was previously characterized (encoded by TM1424-TM1426) must be a key enzyme.

Cell extracts of T. maritima were analyzed for their H2 evolution activity using Fd (purified from T. maritima) that was reduced enzymatically by its physiological partner POR (also purified from T. maritima). However, neither this Fd/POR system nor NAD(P)H (up to 5 mM, with and without flavin adenine dinucleotide or FMN, up to 1 mM) supported sufficient rates of H2 production to allow purification of the activity from an extract. Surprisingly, however, the rate of H2 production by the cell extract increased by more than an order of magnitude (to 0.4 units/mg) when both NADH and the POR/Fd system were present. The activity was located in the cytoplasm (<1% in the membrane fraction) and was stimulated about twofold by the presence of FMN. A standard H2 evolution assay that contained Fd, POR, NADH, and FMN was developed, and with this assay, the enzyme responsible for catalyzing H2 production from a combination of reduced Fd and NADH was purified from the cytoplasmic extract. Sodium dodecyl sulfate-gel electrophoresis showed that this corresponded to the heterotrimeric hydrogenase previously purified from cell extracts by using dithionite-reduced methyl viologen as the electron donor (31). As shown in Fig. Fig.1,1, the hydrogenase displayed similar synergistic uses of Fd and NADH in the purified state and in the cytoplasmic extract. However, in the case of the purified enzyme, the synergistic production of H2 was observed only when FMN was added, indicating that this cofactor is easily lost during purification. This could be replaced by flavin adenine dinucleotide but with a loss of 50% of the activity.

FIG. 1.
Substrate dependence of the bifurcating [FeFe] hydrogenase of T. maritima. The specific activity in the standard H2 production assay containing Fd/POR, NADH, or Fd/POR and NADH was determined using the cytoplasmic extract (dark bars) and the purified ...

Determining the true extent of the synergistic effect of Fd and NADH on T. maritima hydrogenase was complicated by two factors. First, it was previously shown that the enzyme is irreversibly inactivated by exposure to oxygen, with a half-life in air of seconds (10), and must be purified anaerobically with all buffers containing the oxygen scavenger sodium dithionite. However, the synergistic effect of Fd and NADH on the H2 production activity was observed only if the sodium dithionite concentration in the assay mixture was very low (<~20 μM). This was achieved by adding small volumes of concentrated enzyme samples. Presumably, sodium dithionite short-circuits the electron pathways to the catalytic site from the two carriers (see below). Second, another complication is that the H2 evolution-specific activity of the hydrogenase in the Fd/POR/NADH-based assay decreases with increasing amounts of hydrogenase (see Fig. S1 in the supplemental material). The specific activity (~10 units/mg) at extremely low protein concentrations in this assay is about 10% of that measured with dithionite-reduced methyl viologen (~100 units/mg) as the electron carrier. This is consistent with the activities of the Fd-dependent [FeFe] hydrogenases, which are typically an order of magnitude less with Fd than with the artificial carrier (15, 32). The concentration-dependent activity of the T. maritima enzyme may be related to its ability to form higher-order structures (31). Interestingly, a similar effect of enzyme concentration was observed with the butyryl-CoA/Etf complex from Clostridium kluyveri, an enzyme that also simultaneously utilizes Fd and NADH as electron carriers, albeit in a different fashion (11). A constant and relatively high concentration of T. maritima hydrogenase was therefore used in all assays to minimize variations in specific activity (hence, the specific activity values for the purified enzyme are low relative to those measured with the cell extracts).

These modifications to the enzyme assay allowed kinetic parameters to be determined for NADH, Fd, and FMN in the Fd/POR/NADH-based assay. Both NADH and the cofactor FMN gave rise to classical Michaelis-Menten-type kinetics, with apparent Km and Kd (dissociation constant) values of 14 μM (using 100 μM FMN) and 0.9 μM (using 1 mM NADH), respectively (see Fig. S2A and C in the supplemental material). Kinetic analyses conducted with reduced Fd were complicated by the fact that the Fd-reducing enzyme POR was found to directly donate electrons to the hydrogenase, presumably through the Fd-like delta subunit of POR (14). However, no activity was observed when any of the POR substrates or the POR enzyme itself was omitted. Analysis of the cytoplasmic extract by size exclusion chromatography showed that POR and hydrogenase elute independently and do not form a complex in vitro (data not shown), and this is assumed to be the case in vivo. Therefore, in order to estimate the Km for Fd, the concentration of POR was lower than that used in the standard assay. Under these conditions, Michaelis-Menten-type kinetics were observed for Fd, with an apparent Km of 0.25 μM (see Fig. S2B in the supplemental material).

In order to confirm the proposed synergistic effect of NADH and reduced Fd on H2 production, limiting amounts of NADH were added to the standard assay mixture. As shown in Fig. Fig.2,2, in the presence of the POR/Fd generation system, the amount of H2 produced was proportional to the amount of NADH added, even though in the absence of POR/Fd, virtually no H2 is produced (Fig. (Fig.1).1). The results also enabled an estimate of the stoichiometry of the reductant supply to the hydrogenase to be made. From Fig. Fig.2,2, the ratio of H2 produced to NADH added is 1.8 ± 0.2, indicating that T. maritima hydrogenase uses electrons from both Fd and NADH in an approximately 1:1 ratio.

FIG. 2.
Hydrogen production using NADH as the electron donor in the presence of the POR/Fd system. The amounts of NADH added to the standard H2 production assay and the amounts of H2 produced are indicated.

Definition of bifurcating enzymes.

The evidence indicates, therefore, that T. maritima hydrogenase requires both reduced Fd and NADH simultaneously in order to catalyze H2 production efficiently. An enzyme complex that catalyzes a similar pair of linked redox reactions was very recently identified in the ethanol/acetate-fermenting Clostridium kluyveri, in which an exergonic reaction drives an endergonic reaction without the involvement of an ion gradient (7). In this case, the cytoplasmic enzyme complex butyryl-CoA dehydrogenase (or Etf complex) catalyzes the exergonic reduction of crotonyl-CoA (E0′ = −10 mV) to butyryl-CoA with NADH (E0′ = −320 mV) as the electron donor, and this is coupled to the endergonic reduction of Fd (Em = −410 mV) by NADH (7, 11). The reduced Fd that is generated can then couple in an energetically favorable reaction to a “conventional” [FeFe] hydrogenase to produce H2 (E0′ = −420 mV). As a consequence of this pair of linked redox reactions, the oxidation of NADH can be coupled to H2 production, and this is now defined as a bifurcating system (11, 25).

Electron bifurcation is proposed to be the third type of energy conservation, in addition to substrate-level phosphorylation and electron transport phosphorylation (7). This discovery can explain catabolic reactions in several anaerobic bacteria, especially in cases in which butyrate formation is involved (7). We propose that this concept can be extended to include the formation of H2 from NADH and that the heterotrimeric [FeFe] hydrogenase of T. maritima should also be classified as a bifurcating enzyme. It utilizes the exergonic oxidation of Fd (Em = −453 mV) (27, 29) to drive the unfavorable oxidation of NADH (E0′ = −320 mV) to produce H2 (E0′ = −420 mV). The overall hydrogenase reaction can be described as follows: NADH + 2Fdred + 3H+ → 2H2 + NAD+ + 2Fdox (where Fdred indicates reduced Fd and Fdox indicates oxidized Fd).

That T. maritima hydrogenase is part of this novel class of [FeFe] hydrogenases is consistent with the metabolism of this organism. During oxidation of glucose to acetate, T. maritima produces reducing equivalents in the form of NADH and reduced Fd in a 1:1 ratio (Fig. (Fig.3).3). Small amounts of lactate are also produced during glucose fermentation (23), but it is important to note that lactate production does not affect the ratio of NADH to reduced Fd that is available for H2 production. As shown in Fig. Fig.3,3, pyruvate is reduced to lactate by using NADH as the electron donor, and therefore, this pyruvate (that generates lactate) cannot be oxidized to acetate and cannot generate reduced Fd. On the other hand, the NADH/reduced Fd ratio would be affected by biosynthesis and the oxidation state of growth substrates. The appropriate Fd/NADH ratio for the bifurcating [FeFe] hydrogenase and other cell processes is presumably maintained by a membrane-bound, ion-translocating Fd:NADH oxidoreductase of the type identified in some other anaerobes (3, 9, 25). The genome of T. maritima contains a cluster of genes (TM0244-TM0249) that could potentially encode such a membrane complex, and membrane preparations had high NADH oxidoreductase activity by using benzyl viologen as the electron acceptor (~10 U/mg). Further characterization of this enzyme is under way.

FIG. 3.
The role of the bifurcating [FeFe] hydrogenase in the pathway of glucose-to-acetate conversion in T. maritima. Several consecutive enzymatic steps in the pathway are not shown and these are indicated by dotted arrows. GAP, glyceraldehyde-3-phosphate; ...

For unknown reasons, [FeFe] hydrogenases from the archaeal domain have yet to be identified, and so the fermentative archaea must have other solutions to the problem of producing H2 from all of the reducing equivalents generated during carbohydrate fermentation. One example occurs in the hyperthermophile Pyrococcus furiosus. This organism also converts glucose-based substrates to acetate, CO2, and H2 (6), but its glycolytic pathway generates only reduced Fd and not NADH. The expected phosphate- and NAD-dependent glyceraldehyde-3-phosphate dehydrogenase is replaced by a phosphate-independent and Fd-dependent glyceraldehyde-3-phosphate oxidoreductase (16). Although glyceraldehyde-3-phosphate oxidoreductase produces 3-phosphoglycerate such that the ATP-generating phosphoglycerate kinase step is bypassed, a novel ion-gradient-forming, membrane-bound [NiFe] hydrogenase couples Fd oxidation to H2 production, and energy is ultimately conserved in the form of ATP (22). In this case, NAD(P)H for biosynthesis appears to be generated by the recycling of H2 by a cytoplasmic [NiFe] hydrogenase (12).

The potential mechanism of the bifurcating [FeFe] hydrogenase.

The proposed bifurcating [FeFe] hydrogenase of T. maritima therefore uses electrons from reduced Fd and NADH in a 1:1 ratio in such a way that all reductant can be disposed of as H2. The requirement of this hydrogenase for both NADH and reduced Fd as simultaneous electron donors is consistent with the complexity of this trimeric enzyme, which is much greater than that of the typical Fd-dependent, single-subunit [FeFe] hydrogenase found in Clostridium spp. (15, 32). Specifically, as shown in Fig. Fig.4,4, in addition to the subunit (alpha; TM1426 protein) that contains the catalytic H cluster and five Fe-S clusters, the T. maritima enzyme contains two other subunits with redox-active centers, termed beta (TM1424 protein) and gamma (TM1425 protein). These contain four clusters and one cluster of Fe-S, respectively, bringing the total number of Fe-S clusters in the holoenzyme to 10 (Fig. (Fig.4)4) (31). The beta subunit also contains flavin- and NAD-binding sites and is potentially the site for NADH oxidation, with the subsequent shuttling of electrons to the alpha subunit. The gamma subunit potentially coordinates a 2Fe-2S cluster and might be the site where reduced Fd is oxidized (Fig. (Fig.4).4). Considering the fact that the T. maritima enzyme clearly has the capacity to catalyze multiple electron transfer events simultaneously, this intriguing bifurcating mechanism would be rather complex, and determination of the exact mechanism is currently under study.

FIG. 4.
The proposed pathway of electron flow during the oxidation of reduced Fd and NADH by T. maritima hydrogenase. The iron-sulfur cluster composition of each subunit and the FMN- and NAD-binding sites are indicated and are taken from reference 31. Fdred, ...

Diversity of bifurcating [FeFe] hydrogenases.

The sequences of the three subunits of T. maritima hydrogenase were used to search the database of available genome sequences. Of the more than 100 bacterial species that contain a homolog of the catalytic subunit of [FeFe] hydrogenases (32), 30 of them contain gene clusters that encode homologs of all three subunits of the T. maritima enzyme, indicating that these organisms possess a bifurcating hydrogenase of the type shown in Fig. Fig.4.4. Several of them contain additional genes that appear to be part of the same gene cluster (Table (Table1),1), although it is not clear what their relationship is to the putative trimeric hydrogenase. In addition, many of these species contain the simpler single-subunit clostridial type of [FeFe] hydrogenase, which is assumed to be Fd dependent. In fact, some contain four and even five examples of such enzymes (Table (Table1).1). In addition, some species with a bifurcating [FeFe] enzyme also contain as many as three [NiFe] hydrogenases rather than the conventional single-subunit [FeFe] hydrogenase (Table (Table1).1). These organisms clearly have multiple pathways for H2 metabolism, and presumably, which of the alternate pathways of electron transfer that are used depend upon the growth conditions. For example, other than T. maritima, the only organism listed in Table Table11 whose putative bifurcating hydrogenase has been characterized is Thermoanaerobacter tengcongensis. This was purified as a NADH-dependent enzyme, and its presence enables predictions about the hydrogen metabolism of this organism to be made (28). We propose that this bifurcating enzyme oxidizes both reduced Fd and NADH under low partial pressure of H2, while under high partial pressure of H2, NADH is oxidized to produce ethanol and any H2 production is catalyzed by the membrane-bound [NiFe] hydrogenase also present in this organism, using reduced Fd as the electron donor. This is consistent with the downregulation of the [FeFe] hydrogenase but not of the NiFe enzyme under high H2 pressure (28).

Distribution of potential bifurcating [FeFe] hydrogenases in bacteria whose genome sequences are availablea

The discovery of this new type of bifurcating [FeFe] hydrogenase that requires multiple electron carriers dramatically changes our perspective on how anaerobic organisms can produce H2. It provides them with a mechanism to regenerate NAD from NADH by producing H2, even though a conventional reaction of this type is thermodynamically unfavorable. In fact, the general use of a bifurcating mechanism to overcome limiting bioenergetic barriers might be of particular utility to anaerobes that produce a variety of reduced end products, such as H2, ethanol, and butyrate, since it allows them to fine-tune their electron flux based on the environmental pressures. This presumably has distinct evolutionary advantages for all of the organisms listed in Table Table1,1, although elucidating the functions of the multitude of bifurcating and conventional hydrogenases found in these species is a daunting task.

Supplementary Material

[Supplemental material]


This research was funded by a grant from the National Science Foundation (BES-061723).

We thank Frank E. Jenney, Jr., Sung-Jae Yang, and Angeli L. Menon for helpful discussions.


[down-pointing small open triangle]Published ahead of print on 1 May 2009.

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


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