Requirements for Heterologous Production of a Complex Metalloenzyme: the Membrane-Bound [NiFe] Hydrogenase

Construction of broad-host-range plasmids with differently regulated MBH operons.

In order to express the MBH of R. eutropha independently of the SH, the MBH operon, comprising 21 genes, was cloned into the broad-host-range vector pEDY309 (21). Bacterial strains and plasmids used for this construction are listed in Table 1. First, the hyp gene region from pCH104 was inserted as an 8.33-kbp EcoRI fragment into a LITMUS 28 derivative in which the SacI site had been eliminated. The resulting plasmid pCH782 was extended by 7.77-kbp BamHI-XbaI fragments of pCH664 and pCH665, harboring the regulatory hydrogenase gene regions of R. eutropha H16 (HoxJ⁻) and its derivative HF433 (HoxJ⁺) (26), respectively; this step yielded plasmids pCH783 and pCH784, respectively. In parallel, a pGE195-derived 8.95-kbp PstI-SacI fragment, containing the 5′ region of the MBH operon, was cloned into LITMUS 28, resulting in pCH781. Finally, the MBH operons on plasmids pCH785 (HoxJ⁻) and pCH786 (HoxJ⁺) were completely reconstituted by combining a 9.00-kbp SpeI-SacI fragment from pCH781 with the appropriately cut fragments of pCH783 and pCH784, respectively. The broad-host-range plasmids pLO6 and pLO8 (Fig. 1) were constructed by ligating the 21.56-kbp SpeI-XbaI-digested fragments of pCH785 and pCH786 into the corresponding restriction sites of vector pEDY309. Plasmids pLO6 and pLO8 differ in a single marker (Fig. 1): pLO8 bears the H₂-responsive operon (HoxJ⁺), whereas plasmid pLO6 carries an H₂-nonresponsive MBH operon due to a point mutation in hoxJ. The latter plasmid confers the H₂-independent regulation of R. eutropha H16 in which a spontaneously occurring alteration in HoxJ had blocked autophosphorylation of the kinase, which in turn prevents phosphorylation of HoxA. HoxA belongs to an unorthodox two-component regulatory system and activates transcription in the nonphosphorylated form (26). Plasmids with differently controlled MBH operons enabled us to study the expression of the MBH under various growth conditions and to explore the H₂-sensing system in a heterologous host.

Functional expression of the MBH in a megaplasmid-free derivative of R. eutropha.

Plasmids pLO6 and pLO8 were transferred via conjugation (44) to the megaplasmid-free derivative R. eutropha HF210 to examine whether they restore MBH activity. Megaplasmid pHG1 encodes the entire information necessary for H₂ oxidation; thus, the pHG1-free recipient is unable to grow on H₂ (22, 43). Both transconjugants recovered lithoautotrophic growth in mineral medium (42) under an atmosphere of hydrogen, carbon dioxide, and oxygen (8:1:1 [vol/vol/vol]). The doubling times (5.0 h) resembled that of the corresponding control strains R. eutropha H16 and HF433 (4.5 h). This was unexpected, since a mutational knockout of the second energy-conserving hydrogenase, SH, generally leads to a significant retardation of growth (19). The enhanced growth rate of the pLO6/pLO8-harboring transconjugants indicated an increased level of MBH compensating the lack of the SH.

When cultivated under heterotrophic hydrogenase-derepressing conditions in mineral salt medium containing a mixture of 0.2% fructose and 0.2% glycerol (FGN) (42), membrane fractions of strain HF210(pLO6) contained about fourfold higher levels of MBH activity than extracts of the megaplasmid-harboring control HF387 (Table 2). Preparation of extracts and MBH assays were done as described before (4). Since hydrogenase gene expression in the H₂-responsive transconjugant HF210(pLO8) and that in its corresponding control HF512 strictly depend on H₂, these cells failed to produce MBH in the absence of hydrogen. Nevertheless, when H₂ was added to FGN medium (atmosphere of 80% H₂-10% CO₂-10% O₂) high levels of MBH activity were obtained for both transconjugants (Table 2). Under the same conditions, the corresponding SH-free controls HF387 and HF512 displayed significantly lower levels of MBH activity, corroborating the observation that the recombinant plasmids confer MBH overproduction.

Immunoblot analysis conducted as previously described (6, 46) confirmed that the addition of H₂ led to an increase of the MBH protein level in soluble and membrane extracts of the transconjugants (Fig. 2a and b). This is in line with the elevated levels of MBH activity measured under these conditions (Table 2). It was obvious, however, that in cells grown in the presence of H₂, substantial amounts of the MBH subunits HoxG and HoxK occurred in the soluble fraction. Moreover, HoxK was observed as a fast-migrating mature form and as a slow-migrating precursor, with sizes of 39.5 and 34.6 kDa, respectively (Fig. 2). This suggests that under MBH-overproducing conditions, the Tat-mediated export of MBH across the membrane (5, 6) is a rate-limiting step, resulting in the arrest of a portion of the dimeric MBH in the cytoplasm.

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FIG. 2.

Identification of the MBH subunits HoxK and HoxG. Membrane (a and c) and soluble (b and d) fractions of R. eutropha HF210 (a and b) and P. stutzeri (c and d) cells harboring pLO6 and pLO8, respectively, were analyzed immunologically. Cells were grown heterotrophically in fructose- or glucose-containing minimal medium, either in the absence (−H₂) or presence (+H₂) of H₂. Twenty micrograms of membrane and 40 μg of soluble proteins, respectively, were applied per lane on 8% (for detection of HoxG) and 10% (for detection of HoxK) sodium dodecyl sulfate-polyacrylamide gel electrophoresis gels.

The fact that recombinant plasmid-harboring derivatives of R. eutropha had extremely high levels of MBH activity when cultivated mixotrophically in the presence of H₂ raised the question of whether this is due to an elevated level of MBH transcription or to processes occurring at the posttranslational level. To monitor MBH gene expression unambiguously, a Φ[hoxK′-lacZ] reporter gene fusion was inserted into the chromosomal norR2A2B2 locus of R. eutropha HF210. norB2 encodes the NO reductase, an enzyme of denitrification, which is dispensable under aerobic conditions (13, 34). For this construction, the Φ[hoxK′-lacZ] cassette was removed from pGE301 as a 3.7-kbp ScaI-SmaI fragment, end polished, and inserted into StuI-digested pCH525. A 6.3-kbp XbaI-Acc65I fragment from the resulting plasmid pCH871 carrying the Δnor(R2A2B2)::Φ[hoxK′-lacZ] cassette was treated with Klenow polymerase and subsequently ligated into PmeI-digested pLO2, yielding the conditionally lethal suicide plasmid pCH872. pCH872 was used for the introduction of the Φ(hoxK′-lacZ) gene fusion into the respective chromosomal locus of strain HF210 by allelic exchange based on the conditionally lethal sacB gene (27). The resulting strain HF631, bearing the H₂-responsive MBH operon on pLO8, showed only traces of β-galactosidase activity when cultivated heterotrophically in the absence of H₂ (Table 2). The addition of H₂ led to a 30-fold increase of β-galactosidase activity. A similar level of hydrogenase gene expression was obtained for the H₂-nonresponsive strain carrying pLO6. However, by far the highest MBH gene expression was obtained with cells of HF631(pLO6) under H₂-free heterotrophic conditions (Table 2). The results indicate that H₂ affects MBH synthesis on two distinct levels. First, H₂ stabilizes the MBH in cells cultivated in the presence of H₂ by a yet unknown mechanism. Secondly, H₂ negatively controls MBH gene transcription in an H₂-nonresponsive strain, presumably due to a catabolite-control-like effect exerted by this powerful energy source.

Active MBH synthesized in Pseudomonas stutzeri.

P. stutzeri was selected as a recipient to explore whether the recombinant plasmids provide sufficient genetic information to produce active MBH in a background natively free of hydrogenase activity. P. stutzeri, a predominantly respiratory bacterium, shares the ability of R. eutropha to grow anaerobically via denitrification (54). On the other hand, P. stutzeri is unable to assimilate carbon dioxide autotrophically, and no evidence for hydrogen-metabolizing activity has ever been reported for this species (31).

Transconjugants of P. stutzeri, harboring pLO6 or pLO8, were cultivated under conditions similar to those used for R. eutropha, except that fructose was replaced by glucose. Membranes of heterotrophically grown cells harboring pLO6 contained about 30% of the R. eutropha H₂-dependent methylene blue-reducing activity (Table 2). Only traces of MBH activity were found in membrane extracts of P. stutzeri (pLO8) cultivated without H₂, indicating that the H₂-sensing apparatus is functional in this transconjugant. The addition of H₂ enhanced the MBH activity in the membranes of both transconjugants to values between 17 and 19 units per mg of protein, which is almost equivalent to the specific activities obtained with R. eutropha (Table 2). Immunological analysis revealed that, although most of the HoxG antigen was localized in the membrane, a significant portion was present in the soluble fraction (Fig. 2c and d). Both fractions contained the mature form of the small subunit HoxK in addition to its nonprocessed precursor. In R. eutropha, significant quantities of the HoxK precursor were observed only in mixotrophically grown pLO6-harboring cells (Fig. 2b). These results indicate a general retardation of MBH translocation in P. stutzeri.

The data clearly demonstrate that (i) the MBH of R. eutropha can be actively synthesized in P. stutzeri, (ii) most of the protein is located in the membrane, (iii) the regulatory system of R. eutropha is instrumental in a heterologous host, and (iv) the level of MBH activity is enhanced by H₂ independently of the transcriptional regulation, as observed for the donor strain. Hence, plasmids pLO6/pLO8 encode all specific functions necessary for the heterologous synthesis of catalytically active membrane-bound [NiFe] hydrogenase in P. stutzeri.

This investigation defines the genetic requirement for the production of active MBH involving structural genes (hoxKGZ) (4), MBH-specific accessory genes (hoxMLOQRTV) (6), seven hyp genes (hypABFCDEX) (11, 14, 51), and regulatory genes (hoxABCJ) (26). The study clearly showed that a truncated version of HypF (HypF1) is sufficient to allow Ni-Fe center biogenesis. HypF, a key enzyme of cyanide ligand synthesis, usually contains three conserved signatures: an acylphosphate phosphatase domain, a cysteine-rich zinc-finger-like region, and an O-carbamoyl transferase motif. All three motifs were shown to be essential for the E. coli HypF protein (32, 33). R. eutropha H16 encodes such a HypF protein (HypF2) in the SH operon (51). The MBH operon, however, codes for a HypF variant that contains only the O-carbamoyl transferase motif. This truncated HypF1 protein is not only fully functional in hydrogenase maturation of R. eutropha HF210 but also in P. stutzeri when provided with the pLO6/pLO8 plasmids. A BLAST search (1) of the National Center for Biotechnology Information database revealed that the monodomain form of HypF is also encoded in the genomes of Burkholderia cepacia, Magnetospirillum magnetotacticum, Ralstonia metallidurans, and Rubrivivax gelatinosus. This points to at least partially different pathways for cyanide ligand synthesis in these organisms.