Biochemical and Structural Insights into the Preference of Nairoviral DeISGylases for Interferon-Stimulated Gene Product 15 Originating from Certain Species.

UNLABELLED: The regulation of the interferon type I (IFN-I) response has been shown to rely on posttranslational modification by ubiquitin (Ub) and Ub-like interferon-stimulated gene product 15 (ISG15) to stabilize, or activate, a variety of IFN-I signaling and downstream effector proteins. Unlike Ub, which is almost perfectly conserved among eukaryotes, ISG15 is highly divergent, even among mammals. Since zoonotic viruses rely on viral proteins to recognize, or cleave, ISG15 conjugates in order to evade, or suppress, innate immunity, the impact of ISG15 biodiversity on deISGylating proteases of the ovarian tumor family (vOTU) from nairoviruses was evaluated. The enzymatic activities of vOTUs originating from the Crimean-Congo hemorrhagic fever virus, Erve virus, and Nairobi sheep disease virus were tested against ISG15s from humans, mice, shrews, sheep, bats, and camels, which are mammalian species known to be infected by nairoviruses. This along with investigation of binding by isothermal titration calorimetry illustrated significant differences in the abilities of nairovirus deISGylases to accommodate certain species of ISG15. To investigate the molecular underpinnings of species preferences of these vOTUs, a structure was determined to 2.5 Å for a complex of Erve virus vOTU protease and a mouse ISG15 domain. This structure revealed the molecular basis of Erve virus vOTU's preference for ISG15 over Ub and the first structural insight into a nonhuman ISG15. This structure also revealed key interactions, or lack thereof, surrounding three amino acids that may drive a viral deISgylase to prefer an ISG15 from one species over that of another. IMPORTANCE: Viral ovarian tumor domain proteases (vOTUs) are one of the two principal classes of viral proteases observed to reverse posttranslational modification of host proteins by ubiquitin and interferon-stimulated gene product 15 (ISG15), subsequently facilitating downregulation of IFN-I signaling pathways. Unlike the case with ubiquitin, the amino acid sequences of ISG15s from various species are notably divergent. We illustrate that vOTUs have clear preferences for ISG15s from certain species. In addition, these observations are related to the molecular insights acquired via the first X-ray structure of the vOTU from the Erve nairovirus in complex with the first structurally resolved nonhuman ISG15. This information implicates certain amino acids that drive the preference of vOTUs for ISG15s from certain species.

Enhancement and regulation of extracellular protein production by Bacillus brevis 47 through manipulation of cell culture conditions.

Bacillus brevis 47 was cultivated in 2 liter fermentors in semidefined media containing polypeptone with or without glucose or fructose. Neither sugar was essential for growth or extracellular (S-layer) protein production, and 2.5 to 3.0 g/L protein was accumulated in the medium. When present, glucose was used very slowly, however, fructose was used much more quickly. Dramatic changes in metabolic indicators (dissolved oxygen and pH) were seen when fructose became depleted, and protease was produced, decreasing the amount of protein ultimatelv accumulated in the medium. Using the change in dissolved oxygen as a marker for the time of addition, polypeptone, fructose, or both were used to stimulate protein production. With the addition of polypeptone, on stimulation was achieved, but protease production was suppressed. Addition of fructose did result in a small stimulation of protein production (to 5 g/L) if added once. Further additions resulted in more growth, but no increase in protein production. Various combinations of polypeptone and fructose were also used, with the most effective combination (fructose added early, fructose and polypeptone added later) resulting in an accumulation of 15 g/L protein in the medium. This is comparable to that seen when B. brevis 47 is grown in a complex glucose medium and stimulated with polypeptone addition at 21 hours. These results are discussed with respect to the structure and function of S-layer proteins, as well as the use of this organism for the production of heterologous proteins.

Induction of T4 DNA ligase in a recombinant strain of Escherichia coli.

The kinetics of cell growth and foreign protein production, as well as factors affecting protein stability, were studied and optimized in batch and fed-batch fermentations of a recombinant strain of Escherichia coli. The pL promoter from bacteriophage lambda under the control of a temperature-sensitive cl represser, with the entire construct integrated into the E. coli chromosome through the use of a defective bacteriophage lambda lysogen, was used to direct the synthesis of T4 DNA ligase. The biphasic fermentations consisted of a primary growth phase at 30 degrees C followed by an induction phase which was initiated by shifting the temperature to 42 degrees C. In the fed-batch fermentations, additional nutrients were added at the time of initiating induction. Maintenance of sufficiently high concentrations of the organic substrates (glucose and casamino acids) during the induction phase was required for continued cell growth at 42 degrees C. Such growth was essential for T4 DNA ligase formation and in vivo stability. Hence, fed-batch fermentations produced the highest yield of the foreign protein Commensurate with providing lower total amounts of substrates. In such cases, high cell densities (6 g dry wt/L) with substantial intracellular levels of T4 DNA ligase (4.6% total cellular protein, or 2.7% of the dry biomass) were achieved.

Susceptibility of Saccharomyces spp. and Schwanniomyces spp. to the aminoglycoside antibiotic G418.

Industrially useful polyploid yeasts such as the brewing yeasts do not possess any auxotrophic genetic markers and hence are not easily amenable to plasmid-mediated DNA transformations. In an attempt to obtain genetic markers, a number of useful Saccharomyces sp. strains and some amylolytic Schwanniomyces sp. strains were tested for their susceptibility to the antibiotic Geneticin G418 , a 2-deoxystreptamine reported to be active against bacteria, yeasts, and plant and animal cells. All of the Saccharomyces sp. strains, including the brewing strains, were found to be susceptible to G418 in the concentration range of 150 to 500 micrograms/ml. Of the three Schwanniomyces species investigated, only Schwanniomyces castellii (strain 1402) was found to be resistant to G418 at concentrations up to 1 mg/ml. Resistance was exhibited both in liquid media and on glycerol-peptone-yeast extract agar plates. This finding is interesting in view of the possibility of using this strain as a DNA donor for transformations aimed at introducing the amylolytic capability into brewing yeasts.