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Clostridium acetobutylicum was metabolically engineered to produce a biofuel consisting of an isopropanol/butanol/ethanol mixture. For this purpose, different synthetic isopropanol operons were constructed and introduced on plasmids in a butyrate minus mutant strain (C acetobutylicum ATCC 824 Delta cac15 Delta upp Delta buk). The best strain expressing the isopropanol operon from the thl promoter was selected from batch experiments at pH 5. By further optimizing the pH of the culture, a biofuel mixture with almost no by-products was produced at a titer, a yield and productivity never reached before, opening the opportunities to develop an industrial process for alternative biofuels with Clostridia! species. Furthermore, by performing in vivo and in vitro flux analysis of the synthetic isopropanol pathway, this flux was identified to be limited by the [acetate](int) and the high Km of CoA-transferase for acetate. Decreasing the Km of this enzyme using a protein engineering approach would be a good target for improving isopropanol production and avoiding acetate accumulation in the culture medium. (C) 2013 Elsevier Inc. All rights reserved.

Streptococcus uberis is a highly prevalent causative agent of bovine mastitis, which leads to large economic losses in the dairy industry. The aim of this study was to examine the host response during acute inflammation after experimental challenge with capsulated Strep. uberis. Gene expression in response to Strep. uberis was compared between infected and control quarters in 3 animals. All quarters (n=16) were sampled at 16 different locations. Microarray data showed that 239 genes were differentially expressed between infected and control quarters. No differences in gene expression were observed between the different locations. Microarray data were confirmed for several genes using quantitative PCR analysis. Genes differentially expressed due to early Strep. uberis mastitis represented several stages of the process of infection: (1) pathogen recognition; (2) chemoattraction of neutrophils; (3) tissue repair mechanisms; and (4) bactericidal activity. Three different pathogen recognition genes were induced: ficolins, lipopolysaccharide binding protein, and toll-like receptor 2. Calgranulins were found to be the most strongly upregulated genes during early inflammation. By histology and immunohistochemistry, we demonstrated that changes in gene expression in response to Strep. uberis were induced both in infiltrating somatic milk cells and in mammary epithelial cells, demonstrating that the latter cell type plays a role in milk production as well as immune responsiveness. Given the rapid development of inflammation or mastitis after infection, early diagnosis of (Strep. uberis) mastitis is required for prevention of disease and spread of the pathogen. Insight into host responses could help to design immunomodulatory therapies to dampen inflammation after (early) diagnosis of Strep. uberis mastitis. Future research should focus on development of these early diagnostics and immunomodulatory components for mastitis treatment.

Although increasing concentrations of atmospheric CO2 are predicted to have substantial impacts on plant growth and functioning of ecosystems, there is insufficient understanding of the responses of belowground processes to such increases. We investigated the effects of different dark septate root endophytic (DSE) fungi on growth and nutrient acquisition by Pinus sylvestris seedlings under conditions of N limitation and at ambient and elevated CO2 (350 or 700 μ1 CO2 l−1). Each seedling was inoculated with one of the following species: Phialocephala fortinii (two strains), Cadophora finlandica, Chloridium paucisporum, Scytalidium vaccinii, Meliniomyces variabilis and M. vraolstadiae. The trial lasted 125 days. During the final 27 days, the seedlings were labeled with 14CO2 and 15NH4+. We measured extraradical hyphal length, internal colonization, plant biomass, 14C allocation, and plant N and 15N content. Under elevated CO2, the biomass of seedlings inoculated with DSE fungi was on average 17% higher than in control seedlings. Simultaneously, below-ground respiration doubled or trebled, and as a consequence carbon use efficiency by the DSE fungi significantly decreased. Shoot N concentration decreased on average by 57% under elevated CO2 and was lowest in seedlings inoculated with S. vaccinii. Carbon gain by the seedlings despite reduced shoot N concentration indicates that DSE fungi increase plant nutrient use efficiency and are therefore more beneficial to the plant under elevated CO2.