Pseudomonas aeruginosa was studied for its effects on T-cell-mediated responses in mice, as exemplified by anti- Listeria immunity and delayed-type hypersensitivity to sheep erythrocytes. Immunity to Listeria monocytogenes was measured by quantitation of bacteria in spleens and mortality; delayed hypersensitivity to sheep erythrocytes was tested by the footpad reaction. Three different preparations of P. aeruginosa were used: the supernatant of a heat-killed culture, living bacteria, and heat-killed organisms. Similar results were obtained with the three preparations. Administration of P. aeruginosa 24 h before Listeria infection reduced the resistance to the secondary challenge, as measured by increased bacterial multiplication in the spleen and rate of mortality. Cell transfer experiments showed that pretreatment of normal recipient mice with P. aeruginosa prevented them from being adoptively immunized against a Listeria challenge infection with spleen cells from immune donors. They also showed that treatment of donors with P. aeruginosa before immunization affected the capacity of their spleen cells to protect normal recipients against Listeria. Furthermore, spleen and peritoneal exudate cells obtained from mice given P. aeruginosa were capable of preventing immunization of normal recipients against Listeria. Similar results were obtained when the delayed hypersensitivity response to sheep erythrocytes was studied. The suppressive activity of P. aeruginosa -treated spleen cells was lost by removing adherent cells. Conversely, the adherent, heat-killed, anti-immunoglobulin-treated spleen cells exerted a suppressor effect. It thus appears that P. aeruginosa injection changes macrophage and T-lymphocyte activities and results in the development of adherent, macrophage-like suppressor cells in the spleen and peritoneal cavity.

Recently, several approaches have been published in order to develop a functional biosynthesis route for the non-natural compound 1,4-butanediol (BDO) in E. coli using glucose as a sole carbon source or starting from xylose. Among these studies, there was reported as high as 18 g/L product concentration achieved by industrial strains, however BDO production varies greatly in case of the reviewed studies. Our motivation was to build a simple heterologous pathway for this compound in E. coli and to design an appropriate cellular chassis based on a systemic biology approach, using constraint-based flux balance analysis and bi-level optimization for gene knock-out prediction. Thus, the present study reports, at the "proof-of concept" level, our findings related to model-driven development of a metabolically engineered E. coli strain lacking key genes for ethanol, lactate and formate production (ΔpflB, ΔldhA and ΔadhE), with a three-step biosynthetic pathway. We found this strain to produce a limited quantity of 1,4-BDO (.89 mg/L BDO under microaerobic conditions and .82 mg/L under anaerobic conditions). Using glycerol as carbon source, an approach, which to our knowledge has not been tackled before, our results suggest that further metabolic optimization is needed (gene-introductions or knock-outs, promoter fine-tuning) to address the redox potential imbalance problem and to achieve development of an industrially sustainable strain. Our experimental data on culture conditions, growth dynamics and fermentation parameters can consist a base for ongoing research on gene expression profiles and genetic stability of such metabolically engineered E. coli strains.

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Bio-based, environmentally benign production of commodity chemicals such as 1,4-butanediol (BDO) from renewable feedstocks is highly challenging due to the lack of natural synthesis pathways. Herein, we present a systematic model-driven evaluation of the production potential for Escherichia coli to produce BDO from renewable carbohydrates (glucose, glycerol). Computational analysis was carried out in order to decipher the metabolic characteristics under various genetic and environmental conditions. Optimal strain designs were achieved using only two (adhE2- alcohol dehydrogenase and cat/sucCD- 4-hydroxybutyrate-CoA transferase/4-hydroxybutyryl-CoA ligase) heterologous reactions; highest yields were attained for: glucose ~0.37 g g-1 (3 knockouts, anaerobically) and glycerol ~0.43 g g-1 (4 knockouts, microaerobically). The maximum achievable production yield was over 95% of the theoretical maximum potential for glucose and over 75% for glycerol. In regards to the genome-scale metabolic model predictions, a metabolically engineered E. coli was created to analyze the new biosynthetic pathway stability and functionality. Considering the preliminary outcomes the strain and pathway is stable under fermentative conditions and a limited quantity of BDO ~1 mg L-1 was obtained, therefore long-term adaptive evolution is mandatory. This study outlines a strain design and analysis pipeline -systems biology-based approach- for non-native compounds production strains.

ABSTRACT. New proteins as molecular targets in development of therapies are discovered every day. However, study of their interactions with other proteins or binding partners in complex cellular environments has its limits. Therefore, high-yield production of these proteins in heterologous systems is a valid necessity, while obtaining these proteins linked to suitable fluorescent markers represents a step ahead in protein-protein interaction studies and cellular or subcellular localization.In this study, we present production of human SMAC/Diablo recombinant protein with EGFP as a fusion partner. High-yield expression of the fusion protein was carried out in E. coli Rosetta™(DE3)pLysS strain, and an acceptable purity of the protein was obtained after affinity chromatography purification and gel filtration. The obtained protein can be further used in protein-protein interaction studies, whereas our method represents a cost-effective and efficient production method for EGFP-fused proteins, applicable for a number of therapeutically important polypeptides.