Expression of Alternative Nitrogenases in Rhodopseudomonas palustris Is Enhanced Using an Optimized Genetic Toolset for Rapid, Markerless Modifications.

The phototrophic bacterium Rhodopseudomonas palustris is emerging as a promising biotechnological chassis organism, due to its resilience to a range of harsh conditions, a wide metabolic repertoire, and the ability to quickly regenerate ATP using light. However, realization of this promise is impeded by a lack of efficient, rapid methods for genetic modification. Here, we present optimized tools for generating chromosomal insertions and deletions employing electroporation as a means of transformation. Generation of markerless strains can be completed in 12 days, approximately half the time for previous conjugation-based methods. This system was used for overexpression of alternative nitrogenase isozymes with the aim of improving biohydrogen productivity. Insertion of the pucBa promoter upstream of vnf and anf nitrogenase operons drove robust overexpression up to 4000-fold higher than wild-type. Transcript quantification was facilitated by an optimized high-quality RNA extraction protocol employing lysis using detergent and heat. Overexpression resulted in increased nitrogenase protein levels, extending to superior hydrogen productivity in bioreactor studies under nongrowing conditions, where promoter-modified strains better utilized the favorable energy state created by reduced competition from cell division. Robust heterologous expression driven by the pucBa promoter is thus attractive for energy-intensive biosyntheses suited to the capabilities of R. palustris. Development of this genetic modification toolset will accelerate the advancement of R. palustris as a biotechnological chassis organism, and insights into the effects of nitrogenase overexpression will guide future efforts in engineering strains for improved hydrogen production.

Immune control of Mycobacterium tuberculosis is dependent on both soluble TNFRp55 and soluble TNFRp75.

Tuberculosis presents a global health challenge, and tumour necrosis factor (TNF) signalling is required for host immunity against Mycobacterium tuberculosis (Mtb). TNF receptor shedding, however, compromises effective immunity by reducing bioactive TNF through the formation of inactive complexes. In this study, we first compared the effect of total soluble TNF receptors using a transgenic p55ΔNS /p75-/- murine strain on host protection during a low-dose aerosol Mtb H37Rv challenge. We report that the presence of membrane-bound TNFRp55 alone in the absence of TNFRp75 results in superior control of a primary Mtb infection where p55ΔNS /p75-/- hyperactive dendritic cells displayed an increased capacity to induce a hyperactive Mtb-specific CD4+ T-cell response. p55ΔNS /p75-/- dendritic cells expressed a higher frequency of MHCII and increased MFIs for both CD86 and MHCII, while CD4+ T cells had higher expression of CD44 and IFN-γ. Next, the relative contributions of soluble TNFRp55 and soluble TNFRp75 to host protection against either primary Mtb infection or during reactivation of latent tuberculosis were delineated by comparing the experimental outcomes of control C57BL/6 mice to transgenic p55ΔNS /p75-/- , p55ΔNS and p75-/- mouse strains. We found that soluble TNFRp55 is redundant for immune regulation during the chronic stages of a primary Mtb infection. However, TNFRp55 together with soluble TNFRp75 has a crucial role in immune regulation of reactivation of latent tuberculosis.

Transparent polyvinyl-alcohol cryogel as immobilisation matrix for continuous biohydrogen production by phototrophic bacteria.

BACKGROUND: Phototrophic purple non-sulfur bacteria (PNSB) have gained attention for their ability to produce a valuable clean energy source in the form biohydrogen via photofermentation of a wide variety of organic wastes. For maturation of these phototrophic bioprocesses towards commercial feasibility, development of suitable immobilisation materials is required to allow continuous production from a stable pool of catalytic biomass in which energy is not diverted towards biomass accumulation, and optimal hydrogen production rates are realised. Here, the application of transparent polyvinyl-alcohol (PVA) cryogel beads to immobilisation of Rhodopseudomonas palustris for long-term hydrogen production is described. PVA cryogel properties are characterised and demonstrated to be well suited to the purpose of continuous photofermentation. Finally, analysis of the long-term biocompatibility of the material is illustrated. RESULTS: The addition of glycerol co-solvent induces favourable light transmission properties in normally opaque PVA cryogels, especially well-suited to the near-infrared light requirements of PNSB. Material characterisation showed high mechanical resilience, low resistance to diffusion of substrates and high biocompatibility of the material and immobilisation process. The glycerol co-solvent in transparent cryogels offered additional benefit by reinforcing physical interactions to the extent that only a single freeze-thaw cycle was required to form durable cryogels, extending utility beyond only phototrophic bioprocesses. In contrast, conventional PVA cryogels require multiple cycles which compromise viability of entrapped organisms. Hydrogen production studies of immobilised Rhodopseudomonas palustris in batch photobioreactors showed higher specific hydrogen production rates which continued longer than planktonic cultures. Continuous cultivation yielded hydrogen production for at least 67 days from immobilised bacteria, demonstrating the suitability of PVA cryogel immobilisation for long-term phototrophic bioprocesses. Imaged organisms immobilised in cryogels showed a monolithic structure to PVA cryogels, and demonstrated a living, stable, photofermentative population after long-term immobilisation. CONCLUSION: Transparent PVA cryogels offer ideal properties as an immobilisation matrix for phototrophic bacteria and present a low-cost photobioreactor technology for the further advancement of biohydrogen from waste as a sustainable energy source, as well as development of alternative photo-bioprocesses exploiting the unique capabilities of purple non-sulfur bacteria.