A thermostable shikimate 5-dehydrogenase from the archaeon Archaeoglobus fulgidus

Shikimate 5-dehydrogenase (SKDH; EC 1.1.1.25) catalyzes the reversible reduction of 3-dehydroshikimate to shikimate and is a key enzyme in the aromatic amino acid biosynthesis pathway. The shikimate 5-dehydrogenase gene, aroE, from Archaeoglobus fulgidus was cloned and overexpressed in Escherichia coli. The recombinant enzyme purified as a homodimer and yielded a maximum specific activity of 732 U/mg at 87 degrees C (with NADP+ as coenzyme). Apparent Km values for shikimate, NADP+, and NAD+ were estimated at 0.17+/-0.03 mM, 0.19+/-0.01 mM, and 11.4+/-0.4 mM, respectively. The half-life of the A. fulgidus SKDH is 2 h at the assay temperature (87 degrees C) and 17 days at 60 degrees C. Addition of 1 M NaCl or KCl stabilized the enzyme's half-life to approximately 70 h at 87 degrees C and approximately 50 days at 60 degrees C. This work presents the first kinetic analysis of an archaeal SKDH.     

Assessing environmental consequences of using co-products in animal feed

Purpose

The livestock sector has a major impact on the environment. This environmental impact may be reduced by feeding agricultural co-products (e.g. beet tails) to livestock, as this transforms inedible products for humans into edible products, e.g. pork or beef. Nevertheless, co-products have different applications such as bioenergy production. Based on a framework we developed, we assessed environmental consequences of using co-products in diets of livestock, including the alternative application of that co-product.

Methods

We performed a consequential life cycle assessment, regarding greenhouse gas emissions (including emissions related to land use change) and land use, for two case studies. Case 1 includes increasing the use of wheat middlings in diets of dairy cattle at the expense of using it in diets of pigs. The decreased use of wheat middlings in diets of pigs was substituted with barley, the marginal product. Case 2 includes increasing the use of beet tails in diets of dairy cattle at the expense of using it to produce bioenergy. During the production of biogas, electricity, heat and digestate (that is used as organic fertilizer) were produced. The decrease of electricity and heat was substituted with fossil fuel, and digestate was substituted with artificial fertilizer.

Results and discussion

Using wheat middlings in diets of dairy cattle instead of using it in diets of pigs resulted in a reduction of 329 kg CO₂ eq per ton wheat middlings and a decrease of 169 m² land. Using beet tails in diets of dairy cattle instead of using it as a substrate for anaerobic digestion resulted in a decrease of 239 kg CO₂ eq per ton beet tails and a decrease of 154 m² land. Emissions regarding land use change contributed significantly in both cases but had a high uncertainty factor, ±170 ton CO₂ ha^?1. Excluding emissions from land use change resulted in a decrease of 9 kg CO₂ eq for case 1 ‘wheat middlings’ and an increase of 50 kg CO₂ eq for case 2 ‘beet tails’.

Conclusions

Assessing the use of co-products in the livestock sector is of importance because shifting its application can reduce the environmental impact of the livestock sector. A correct assessment of the environmental consequences of using co-products in animal feed should also include potential changes in impacts outside the livestock sector, such as the impact in the bioenergy sector.     

Exaggerated root respiration accounts for growth retardation in a starchless mutant of Arabidopsis thaliana

The knock‐out mutation of plastidial phosphoglucomutase (pgm) causes a starchless phenotype in Arabidopsis thaliana, and results in a severe growth reduction of plants cultivated under diurnal conditions. It has been speculated that high soluble sugar levels accumulating during the light phase in leaf mesophyll might cause a reduction of photosynthetic activity or that shortage of reduced carbon during the night is the reason for the slow biomass gain of pgm. Separate simultaneous measurements of leaf net photosynthesis and root respiration demonstrate that photosynthetic activity per unit fresh weight is not reduced in pgm, whereas root respiration is strongly elevated. Comparison with a mutant defective in the dominating vacuolar invertase (AtβFruct4) revealed that high sucrose concentration in the cytosol, but not in the vacuole, of leaf cells is responsible for elevated assimilate transport to the root. Increased sugar supply to the root, as observed in pgm mutants, forces substantial respiratory losses. Because root respiration accounts for 80% of total plant respiration under long‐day conditions, this gives rise to retarded biomass formation. In contrast, reduced vacuolar invertase activity leads to reduced net photosynthesis in the shoot and lowered root respiration, and affords an increased root/shoot ratio. The results demonstrate that roots have very limited capacity for carbon storage but exert rigid control of supply for their maintenance metabolism.     

Systemic and Mucosal Immune Reactivity upon Mycobacterium avium ssp. paratuberculosis Infection in Mice

Mycobacterium avium ssp. paratuberculosis (MAP) is the cause of Johne''s disease, an inflammatory bowel disorder of ruminants. Due to the similar pathology, MAP was also suggested to cause Crohn''s disease (CD). Despite of intensive research, this question is still not settled, possibly due to the lack of versatile mouse models. The aim of this study was to identify basic immunologic mechanisms in response to MAP infection. Immune compromised C57BL/6 Rag2 ^−/− mice were infected with MAP intraperitoneally. Such chronically infected mice were then reconstituted with CD4⁺ and CD8⁺ T cells 28 days after infection. A systemic inflammatory response, detected as enlargement of the spleen and granuloma formation in the liver, was observed in mice infected and reconstituted with CD4⁺ T cells. Whereby inflammation in infected and CD4⁺CD45RB^hi T cell reconstituted animals was always higher than in the other groups. Reconstitution of infected animals with CD8⁺ T cells did not result in any inflammatory signs. Interestingly, various markers of inflammation were strongly up-regulated in the colon of infected mice reconstituted with CD4⁺CD45RB^lo/int T cells. We propose, the usual non-colitogenic CD4⁺CD45RB^lo/int T cells were converted into inflammatory T cells by the interaction with MAP. However, the power of such cells might be not sufficient for a fully established inflammatory response in the colon. Nevertheless, our model system appears to mirror aspects of an inflammatory bowel disease (IBD) like CD and Johne''s diseases. Thus, it will provide an experimental platform on which further knowledge on IBD and the involvement of MAP in the induction of CD could be acquired.     

Specific Interaction of the Unmodified Bacteriocin Lactococcin 972 with the Cell Wall Precursor Lipid II

Lactococcin 972 (Lcn972) is a nonlantibiotic bacteriocin that inhibits septum biosynthesis in Lactococcus lactis rather than forming pores in the cytoplasmic membrane. In this study, a deeper analysis of the molecular basis of the mode of action of Lcn972 was performed. Of several lipid cell wall precursors, only lipid II antagonized Lcn972 inhibitory activity in vivo. Likewise, Lcn972 only coprecipitated with lipid II micelles. This bacteriocin inhibited the in vitro polymerization of lipid II by the recombinant S. aureus PBP2 and the addition to lipid II of the first glycine catalyzed by FemX. These experiments demonstrate that Lcn972 specifically interacts with lipid II, the substrate of both enzymes. In the presence of Lcn972, nisin pore formation was partially hindered in whole cells. However, binding of Lcn972 to lipid II could not compete with nisin in lipid II-doped 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) liposomes, possibly indicating a distinct binding site. The existence of a putative cotarget for Lcn972 activity is discussed in the context of its narrow inhibitory spectrum and the localized action at the division septum. To our knowledge, this is the first unmodified bacteriocin that binds to the cell wall precursor lipid II.     

The membraneous nitrite reductase involved in the electron transport of Wolinella succinogenes

Wolinella succinogenes grown with nitrate as terminal electron acceptor contains two nitrite reductases as measured with the donor viologen radical, one in the cytoplasm and the other integrated in the cytoplasmic membrane. The fumarate-grown bacteria contain only the membraneous species.The isolated membraneous enzyme consists of a single polypeptide chain (M _r 63,000) carrying 4 hemeC groups and probably an iron-sulphur cluster as prosthetic groups. The enzyme amounts to about 1% of the total membrane protein.The isolated enzyme catalyses the reduction of nitrite to ammonium without accumulation of significant amounts of intermediates or alternative products. The Michaelis constant for nitrite was 0.1 mM and the turnover number of the hemeC 1.5 · 10⁵ electrons per min at 37°C.The viologen-reactive site of the enzyme in the membrane is oriented towards the cytoplasm. When the isolated enzyme is incorporated into liposomes, the viologen-as well as the nitrite-reactive site is exposed to thooutside.The cytoplasmic membrane contains a second hemeC protein (M _r 22,000) which may represent a cytochrome c.Abbreviations NQNO 2-(n-nonyl)-4-hydroxyquinoline-N-oxide - MES 2-(N-morpholino)ethanesulfonate - MOPS 3-(N-morpholino)propanesulfonate - HEPES N-2-Hydroxyethylpiperazine-N-2-ethanesulfonate - TES N-tris(hydroxymethyl)methyl-2-aminoethanesulfonate - MK menaquinone     

Defining a land boundary for sustainable livestock consumption

The need for more sustainable production and consumption of animal source food (ASF) is central to the achievement of the sustainable development goals: within this context, wise use of land is a core challenge and concern. A key question in feeding the future world is: how much ASF should we eat? We demonstrate that livestock raised under the circular economy concept could provide a significant, nonnegligible part (9–23 g/per capita) of our daily protein needs (~50–60 g/per capita). This livestock then would not consume human‐edible biomass, such as grains, but mainly convert leftovers from arable land and grass resources into valuable food, implying that production of livestock feed is largely decoupled from arable land. The availability of these biomass streams for livestock then determines the boundaries for livestock production and consumption. Under this concept, the competition for land for feed or food would be minimized and compared to no ASF, including some ASF in the human diet could free up about one quarter of global arable land. Our results also demonstrate that restricted growth in consumption of ASF in Africa and Asia would be feasible under these boundary conditions, while reductions in the rest of the world would be necessary to meet land use sustainability criteria. Managing this expansion and contraction of future consumption of ASF is essential for achieving sustainable nutrition security.     

Quantifying the climatic niche of symbiont partners in a lichen symbiosis indicates mutualist‐mediated niche expansions

The large distributional areas and ecological niches of many lichenized fungi may in part be due to the plasticity in interactions between the fungus (mycobiont) and its algal or cyanobacterial partners (photobionts). On the one hand, broad‐scale phylogenetic analyses show that partner compatibility in lichens is rather constrained and shaped by reciprocal selection pressures and codiversification independent of ecological drivers. On the other hand, sub‐species‐level associations among lichen symbionts appear to be environmentally structured rather than phylogenetically constrained. In particular, switching between photobiont ecotypes with distinct environmental preferences has been hypothesized as an adaptive strategy for lichen‐forming fungi to broaden their ecological niche. The extent and direction of photobiont‐mediated range expansions in lichens, however, have not been examined comprehensively at a broad geographic scale. Here we investigate the population genetic structure of Lasallia pustulata symbionts at sub‐species‐level resolution across the mycobiont's Europe‐wide range, using fungal MCM7 and algal ITS rDNA sequence markers. We show that variance in occurrence probabilities in the geographic distribution of genetic diversity in mycobiont‐photobiont interactions is closely related to changes in climatic niches. Quantification of niche extent and overlap based on species distribution modeling and construction of Hutchinsonian climatic hypervolumes revealed that combinations of fungal–algal interactions change at the sub‐species level along latitudinal temperature gradients and in Mediterranean climate zones. Our study provides evidence for symbiont‐mediated niche expansion in lichens. We discuss our results in the light of symbiont polymorphism and partner switching as potential mechanisms of environmental adaptation and niche evolution in mutualisms.     

Assessing broad life cycle impacts of daily onboard decision-making,annual strategic planning,and fisheries management in a northeast Atlantic trawl fishery

Purpose

Capture fisheries are the only industrial-scale harvesting of a wild resource for food. Temporal variability in environmental performance of fisheries has only recently begun to be explored, but only between years, not within a year. Our aim was to better understand the causes of temporal variability within and between years and to identify improvement options through management at a company level and in fisheries management.

Methods

We analyzed the variability in broad environmental impacts of a demersal freeze trawler targeting cod, haddock, saithe, and shrimp, mainly in the Norwegian Sea and in the Barents Sea. The analysis was based on daily data for fishing activities between 2011 and 2014 and the functional unit was a kilo of landing from one fishing trip. We used biological indicators in a novel hierarchic approach, depending on data availability, to quantify biotic impacts. Landings were categorized as target (having defined target reference points) or bycatch species (classified as threatened or as data-limited). Indicators for target and bycatch impacts were quantified for each fishing trip, as was the seafloor area swept.

Results and discussion

No significant difference in fuel use was found between years, but variability was considerable within a year, i.e., between fishing trips. Trips targeting shrimp were more fuel intensive than those targeting fish, due to a lower catch rate. Steaming to and from port was less important for fuel efficiency than steaming between fishing locations. A tradeoff was identified between biotic and abiotic impacts. Landings classified as main target species generally followed the maximum sustainable yield (MSY) framework, and proportions of threatened species were low, while proportions of data-limited bycatch were larger. This improved considerably when reference points were defined for saithe in 2014.

Conclusions

The variability between fishing trips shows that there is room for improvement through management. Fuel use per landing was strongly influenced by target species, fishing pattern, and fisheries management. Increased awareness about the importance of onboard decision-making can lead to improved performance. This approach could serve to document performance over time helping fishing companies to better understand the effect of their daily and more long-term decision-making on the environmental performance of their products.

Recommendations

Fishing companies should document their resource use and production on a detailed level. Fuel use should be monitored as part of the management system. Managing authorities should ensure that sufficient data is available to evaluate the sustainability of exploitation levels of all harvested species.