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Viral protein U (Vpu) is a type-III integral membrane protein encoded by Human Immunodeficiency Virus-1 (HIV- 1). It is expressed in infected host cells and plays several roles in viral progeny escape from infected cells, including down-regulation of CD4 receptors. But key structure/function questions remain regarding the mechanisms by which the Vpu protein contributes to HIV-1 pathogenesis. Here we describe expression of Vpu in bacteria, its purification and characterization. We report the successful expression of PelB-Vpu in Escherichia coli using the leader peptide pectate lyase B (PelB) from Erwinia carotovora. The protein was detergent extractable and could be isolated in a very pure form. We demonstrate that the PelB signal peptide successfully targets Vpu to the cell membranes and inserts it as a type I membrane protein. PelB-Vpu was biophysically characterized by circular dichroism and dynamic light scattering experiments and was shown to be an excellent candidate for elucidating structural models.
Syngas fermentation, the bioconversion of CO, CO[subscript 2], and H[subscript 2] to biofuels and chemicals, has undergone considerable optimization for industrial applications. Even more, full-scale plants for ethanol production from syngas fermentation by pure cultures are being built worldwide. The composition of syngas depends on the feedstock gasified and the gasification conditions. However, it remains unclear how different syngas mixtures affect the metabolism of carboxidotrophs, including the ethanol/acetate ratios. In addition, the potential application of mixed cultures in syngas fermentation and their advantages over pure cultures have not been deeply explored. In this work, the effects of CO[subscript 2] and H[subscript 2] on the CO metabolism by pure and mixed cultures were studied and compared. For this, a CO-enriched mixed culture and two isolated carboxidotrophs were grown with different combinations of syngas components (CO, CO:H[subscript 2], CO:CO[subscript 2], or CO:CO[subscript 2]:H[subscript 2]).
Results
The CO metabolism of the mixed culture was somehow affected by the addition of CO[subscript 2] and/or H[subscript 2], but the pure cultures were more sensitive to changes in gas composition than the mixed culture. CO[subscript 2] inhibited CO oxidation by the Pleomorphomonas-like isolate and decreased the ethanol/acetate ratio by the Acetobacterium-like isolate. H[subscript 2] did not inhibit ethanol or H[subscript 2] production by the Acetobacterium and Pleomorphomonas isolates, respectively, but decreased their CO consumption rates. As part of the mixed culture, these isolates, together with other microorganisms, consumed H[subscript 2] and CO[subscript 2] (along with CO) for all conditions tested and at similar CO consumption rates (2.6 ± 0.6 mmol CO L[superscript −1] day[superscript −1]), while maintaining overall function (acetate production). Providing a continuous supply of CO by membrane diffusion caused the mixed culture to switch from acetate to ethanol production, presumably due to the increased supply of electron donor. In parallel with this change in metabolic function, the structure of the microbial community became dominated by Geosporobacter phylotypes, instead of Acetobacterium and Pleomorphomonas phylotypes.
Conclusions
These results provide evidence for the potential of mixed-culture syngas fermentation, since the CO-enriched mixed culture showed high functional redundancy, was resilient to changes in syngas composition, and was capable of producing acetate or ethanol as main products of CO metabolism.