Putative hya and hyb operons of G. sulfurreducens.
The G. sulfurreducens
) was searched for the presence of respiratory uptake hydrogenases with the following sequences as probes: the catalytic subunit of the periplasmic Fe hydrogenase of D. fructosovorans
), an Fe hydrogenase H cluster consensus sequence (pfam02906) (4
), the large and small subunits (hydA
) of the respiratory NiFe hydrogenase of W. succinogenes
), and the large and small subunits (hynA
) of the NiFe hydrogenase of D. fructosovorans
). Two potential operons with significant similarity to proteobacterial respiratory uptake NiFe hydrogenases, hya
, were identified.
The putative hya
operon (Fig. ) consists of four genes, hyaS
, and hyaP
, encoding a large subunit, a small subunit with an N-terminal twin-arginine motif (50
) (SRRDFLK), a hydrophobic type b
cytochrome, and a maturation protease, respectively. Thus, Hya appears to be a member of a family of heterotrimeric membrane-bound uptake hydrogenases with periplasmic active sites (48
) that includes the respiratory hydrogenases of W. succinogenes
), R. eutropha
), and a variety of nitrogen-fixing members of the class Proteobacteria
. The HyaS, HyaL, and HyaB subunits of G. sulfurreducens
Hya are 53 to 57, 63 to 65, and 46 to 51% similar to their respective homologs in R. eutropha
and R. capsulatus
), a nitrogen-fixing member of the alpha subdivision of the class Proteobacteria
. They are slightly less similar, 58.2, 59.8, and 39.3%, to the subunits of HydABC of W. succinogenes
. (Global pairwise alignments were performed with the algorithm of Needleman and Wunsch [36
] and scored with the blosum62 matrix [15
FIG. 1. Structures and mutagenesis of putative hya and hyb operons. (A) Structure of putative hya operon. Twin-arginine motifs are indicated as vertical black bars. (B) Structure of disrupted hya operon and confirmation of Hya-deficient mutant genotype by Southern (more ...)
A search of the partial genome sequence of the closely related species G. metallireducens
(available at www.jgi.doe.gov
) revealed the presence of a homologous hya
operon. The three subunits of G. metallireducens
Hya are 90 to 95% similar to their G. sulfurreducens
counterparts. Because G. metallireducens
does not appear to be capable of growth with hydrogen as an electron donor (27
), this result suggested that the physiological function of Hya might not be related to hydrogen-dependent energy generation.
The subunits and maturation proteases of the three Geobacter
Hya hydrogenases have sizes and features typical of the family of heterotrimeric uptake hydrogenases (48
), with one notable exception. The small subunits of the periplasmically oriented heterotrimeric uptake hydrogenases, as well as those of the periplasmic heterodimeric NiFe hydrogenases, of Desulfovibrio
species contain 10 conserved cysteine residues and 1 conserved histidine residue that serve as ligands for three iron-sulfur clusters (37
). Surprisingly, both of the Geobacteraceae
family HyaS subunits contain an aspartate residue in place of one of the conserved cysteine residues that in other hydrogenases ligates the proximal [4Fe-4S] cluster (CXXD versus CXXC). This cysteine-to-aspartate substitution may have implications for the catalytic activity of Hya. When the analogous cysteine residue of the small subunit of the heterotrimeric respiratory hydrogenase of Azotobacter vinelandii
(HoxGKZ) was mutated to serine, the ability of the hydrogenase to catalyze hydrogen oxidation was nearly eliminated (2% of that of the wild type), whereas its ability to catalyze hydrogen evolution was relatively unaffected (22% of that of the wild type) (32
The second putative G. sulfurreducens
hydrogenase-encoding operon, hyb
, appears to encode a periplasmically oriented membrane-bound NiFe hydrogenase with four subunits: (i) HybS, a small subunit with an N-terminal twin-arginine motif (SRRDFMK); (ii) HybA, a second iron-sulfur cluster-containing subunit with an N-terminal twin-arginine motif (TRRDFLK); (iii) HybB, an integral membrane subunit; and (iv) HybL, a large subunit. Similar gene clusters encoding heterotetrameric NiFe hydrogenases can be found in the genomes of two members of the alpha subdivision of the class Proteobacteria
sp. MC-1 and M. magnetotacticum
, as well as at least three members of the gamma subdivision of the class Proteobacteria
, E. coli
, S. enterica
serovar Typhimurium, and A. pleuropneumoniae
. However, to date, only one heterotetrameric membrane-bound NiFe hydrogenase, Hyd2 of E. coli
, has been genetically and biochemically characterized (3
). E. coli
Hyd2 is a membrane-bound respiratory hydrogenase with a periplasmically oriented active site that is required for the hydrogen-dependent reduction of a variety of electron acceptors and is essential for growth in the presence of hydrogen and fumarate. The four G. sulfurreducens
Hyb subunits are ca. 50 to 70% similar to the subunits of the heterotetrameric hydrogenases encoded in the five gene clusters listed above, with the highest degree of similarity occurring between the various large (70 to 74%) and small (60 to 68%) subunits. Overall, the subunits of G. sulfurreducens
Hyb are slightly more similar to those of their counterparts in the alpha subdivision of the class Proteobacteria
The organization of the 5′ ends of the various gene clusters encoding heterotetrameric hydrogenases is the same; genes encoding the four hydrogenase subunits are followed by a maturation protease gene. In the E. coli hyb
operon, three additional processing genes, hybE
, and hybG
, are present (10
). In contrast, the maturation protease gene, hybP
, of G. sulfurreducens
is followed by a short open reading frame, hybT
, and a putative Rho-independent transcriptional terminator (identified with Transterm software [13
]). On the basis of the presence of a conserved MttA domain (pfam02416 [4
]) at its N terminus, we propose that hybT
may encode a homolog of TatA, a critical component of the Tat secretory pathway (50
). A homologous hyb
operon was not identified in the partial genome sequence of G. metallireducens
Alignment of the various subunits of G. sulfurreducens
Hyb with those of E. coli
Hyd2 revealed that all of the defining features of the subunits of Hyd2 (33
) were present. However, HybB, the integral membrane subunit of G. sulfurreducens
Hyb, contains a hydrophilic 35-amino-acid insertion that is not found in the integral membrane subunit of E. coli
Hyd2 or any of other heterotetrameric hydrogenases examined.
Construction and preliminary analysis of Hya- and Hyb-deficient mutants.
In order to elucidate the roles of Hya and Hyb during hydrogen-dependent growth, Hya and Hyb knockout mutants were constructed via homologous recombination (Fig. ). A Hya-deficient mutant (DLMC1 [ΔhyaSLB::kan]) was constructed by replacing a 3.25-kb stretch of sequence encompassing 90% of hyaS, all of hyaL, and 83% of hyaB with a kanamycin resistance cassette. A Hyb-deficient mutant (DLMC2 [ΔhybL::cam]) was constructed by replacing the N-terminal 77% of the HybL coding sequence with a chloramphenicol resistance cassette. The genotypes of these two mutants were confirmed by Southern blotting (Fig. ) and PCR screening (data not shown).
In-gel hydrogenase assays were performed on membrane fractions prepared from acetate-fumarate-grown wild-type and hydrogenase-deficient cultures (Fig. ). Electrophoresis and solubilization conditions were optimized to maximize the rate of hydrogen-dependent staining. All of the hydrogen-dependent staining in the membrane fraction was found to be dependent on the presence of Hyb (lane 1 versus lane 3). Hyb was found to migrate as three species, a very minor diffuse band with low electrophoretic mobility, and two intense, rapidly migrating species. The relative intensity of these three species was found to be strongly dependent on the solubilization conditions and the presence or absence of specific detergents during electrophoresis (data not shown), suggesting that Hyb may dissociate either in gel or during solubilization. In the case of Hyd2 of E. coli
, multiple species were visualized in nondenaturing gels until it was purified to near homogeneity, when it was found to be a heterodimer of the large and small subunits (3
We have been unable to detect Hya-specific hydrogen uptake activity in either the soluble (data not shown) or the membrane fraction (Fig. ) of acetate-fumarate-grown cells either in gel or in vitro. This may be due either to insufficient expression of Hya during growth on acetate-fumarate medium or because the enzyme is inactive under the conditions tested to date.
Phenotype of the Hya- and Hyb-deficient mutants.
Resting cell suspensions were prepared from acetate-fumarate cultures of wild-type and Hya- and Hyb-deficient G. sulfurreducens
and tested for the ability to reduce three electron acceptors: Fe(III)-NTA, AQDS, and fumarate (Table ). Surprisingly, the rate of fumarate reduction by the wild-type strain, measured as succinate production, was significantly lower than that of either Fe(III)-NTA or AQDS reduction. In addition, in contrast to Fe(III) and AQDS reduction, succinate production was higher in the presence of acetate than in the presence of hydrogen. One factor that may have contributed to the latter finding is that in the presence of acetate, a portion of the succinate (25%) can be produced by oxidation of acetate via the tricarboxylic acid cycle instead of direct reduction of fumarate (14
Reduction of Fe(III)NTA, AQDS, and fumarate by cell suspensions
In the presence of hydrogen, Hya-deficient suspensions reduced the three acceptors at rates comparable to those of the wild type, whereas the Hyb-deficient suspensions could not reduce any of the three acceptors (Table ). Thus, Hyb was found to be essential for hydrogen-dependent reduction of a variety of acceptors by acetate-fumarate-grown cell suspensions.
Longer-term growth studies yielded results that were analogous to those of the cell suspension studies. Both of the hydrogenase-deficient strains could be cultured in medium containing acetate as the electron donor and either Fe(III)-citrate, AQDS, or fumarate as the electron acceptor (data not shown). The wild-type and Hya-deficient strains were capable of sustained hydrogen-dependent growth in medium containing a small amount of acetate as a source of organic carbon and either Fe(III)-citrate, AQDS, or fumarate as the electron acceptor (Fig. and ). When Fe(III)-citrate was provided as the electron acceptor, the final cell yield for the Hya-deficient mutant was ~25% less that than that of the wild type. The Hyb-deficient mutant was incapable of hydrogen-dependent growth in the presence of these three electron acceptors (Fig. and ). In addition, the Hyb-deficient mutant was also found to be unable to grow with hydrogen and poorly crystalline Fe(III) oxide as the electron acceptor (data not shown). To confirm that the phenotype of the Hyb-deficient mutant was due to the absence of the HybL subunit, a hybL expression vector was constructed. Expression of the hybL gene in trans restored the ability of the mutant to grow with hydrogen and AQDS as the electron donor (data not shown). Thus, Hyb was essential for hydrogen-dependent growth in the presence of three electron acceptors.
FIG. 2. Growth of wild-type and hydrogenase-deficient strains with Fe(III)-citrate as the electron acceptor. (A) Hydrogen-dependent growth of the wild-type and Hya-deficient strains. (B) Hydrogen-dependent growth of the wild-type and Hyb-deficient strains. The (more ...)
FIG. 3. Hydrogen-dependent growth of wild-type and hydrogenase-deficient strains in the presence of AQDS and fumarate. Acetate-fumarate-grown cultures were washed in isotonic basal wash medium (22) prior to inoculation to eliminate carryover of acetate and fumarate. (more ...) Insights into the physiological function of Hya and Hyb.
The finding that Hyb was required for hydrogen-dependent reduction of Fe(III), AQDS, and fumarate indicates that Hyb plays a central role in hydrogen-dependent respiration in G. sulfurreducens. Thus, the physiological function of Hyb may be to transfer electrons from hydrogen to the menaquinone pool, where they can be redistributed to a variety of reductases. Examination of the hydrogenase content of the genome of the closely related species G. metallireducens further supports this hypothesis. G. metallireducens is unable to grow with hydrogen as an electron donor, and its genome contains homologs of each of the hydrogenases encoded in the G. sulfurreducens genome except for Hyb. This result confirms the importance of Hyb for growth with hydrogen as the electron donor and suggests that it may be possible to use the presence or absence of a Hyb ortholog as a criterion for predicting whether other members of the family Geobacteraceae have the ability to couple hydrogen oxidation to cell growth.
The genetic studies presented herein indicate that Hya, unlike Hyb, is not essential for coupling hydrogen oxidation to the reduction of Fe(III), AQDS, or fumarate under the conditions tested. These findings are consistent with the presence of a Hya ortholog in the genome of G. metallireducens
, which cannot grow with hydrogen as an electron donor. Elucidation of the physiological role of Hya requires extensive further investigation, including a detailed study of Hya expression, defining growth conditions and media that maximize Hya expression, and creating an in vitro or in vivo assay for Hya activity. Our failure to detect Hya activity or a clear Hya phenotype may be due to a lack of Hya expression or to a low Hya expression level under the growth conditions used in this study. It is also possible that Hya is involved in the reduction of electron acceptors that were not tested in this study. Hyd1 of E. coli
, also a heterotrimeric periplasmically oriented uptake hydrogenase, was found to preferentially reduce high potential electron acceptors such as ferricyanide and oxygen and to be unable to reduce low potential acceptors such as benzyl and methyl viologen (20
In summary, the studies presented herein indicate that Hyb, which is closely related to the Hyd2 respiratory hydrogenase of E. coli, may serve as the principal respiratory hydrogenase of G. sulfurreducens. The physiological role of Hya, in contrast, remains unclear.