Investigation of global regulators influencing styrene metabolism and bioplastic synthesis in Pseudomonas putida CA-3

The genetics and biochemistry involved in the biodegradation of styrene and the production of polyhydroxyalkanoates in Pseudomonas putida CA-3 have been well characterised to date. Knowledge of the role played by global regulators in controlling these pathways currently represents a critical knowledge gap in this area. Here we report on our efforts to identify such regulators using mini-Tn5 transposon mutagenesis of the P. putida CA-3 genome. The library generated was subjected to phenotypic screening to identify mutants exhibiting a reduced sensitivity to the effects of carbon catabolite repression of aromatic pathway activity. Our efforts identified a clpX disrupted mutant which exhibited wild-type levels of growth on styrene but significantly reduced growth on phenylacetic acid. RT-PCR analysis of key PACoA catabolon genes necessary for phenylacetic acid metabolism, and SDS-PAGE protein profile analyses suggest that no direct alteration of PACoA pathway transcriptional or translational activity was involved. The influence of global regulators affecting the accumulation of PHAs in P. putida CA-3 was also studied. Phenotypic screening of the mini-Tn5 library revealed a gacS sensor kinase gene disruption resulting in the loss of PHA accumulation capacity in P. putida CA-3. Subsequent SDS-PAGE protein analyses of the wild type and gacS mutant strains identified post-transcriptional control of phaC1 synthase as a key point of control of PHA synthesis in P. putida CA-3. Disruption of the gacS gene in another PHA accumulating organism, P. putida S12, also demonstrated a reduction of PHA accumulation capacity. PHA accumulation was observed to be disrupted in the CA-3 gacS mutant under phosphorus limited growth conditions. Over-expression studies in both wild type CA-3 and gacS mutant demonstrated that rsmY over-expression in gacS disrupted P. putida CA-3 is insufficient to restore PHA accumulation in the cell however in wild type cells, over-expression of rsmY results in an altered PHA monomer compositions.

Chitosan gel film bandages: correlating structure, composition, and antimicrobial properties

Chitosan gel films were successfully obtained by evaporation cast from chitosan solutions in aqueous acidic solutions of organic acids (lactic and acetic acid) as gel film bandages, with a range of additives that directly influence film morphology and porosity. We show that the structure and composition of a wide range of 128 thin gel films, is correlated to the antimicrobial properties, their biocompatibility and resistance to biodegradation. Infrared spectroscopy and solid-state 13C nuclear magnetic resonance spectroscopy was used to correlate film molecular structure and composition to good antimicrobial properties against 10 of the most prevalent Gram positive and Gram negative bacteria. Chitosan gel films reduce the number of colonies after 24 h of incubation by factors of ∼105–107 CFU/mL, compared with controls. For each of these films, the structure and preparation condition has a direct relationship to antimicrobial activity and effectiveness. These gel film bandages also show excellent stability against biodegradation with lysozyme under physiological conditions (5% weight loss over a period of 1 month, 2% in the first week), allowing use during the entire healing process. These chitosan thin films and subsequent derivatives hold potential as low-cost, dissolvable bandages, or second skin, with antimicrobial properties that prohibit the most relevant intrahospital bacteria that infest burn injuries.

Evaluation of a gelatin modified poly(ɛ-caprolactone) film as a scaffold for lung disease

Lung transplantation is a necessary step for the patients with the end-stage of chronic obstructive pulmonary disease. The use of artificial lungs is a promising alternative to natural lung transplantation which is complicated and is restricted by low organ donations. For successful lung engineering, it is important to choose the correct combination of specific biological cells and a synthetic carrier polymer. The focus of this study was to investigate the interactions of human lung epithelial cell line NCl-H292 that is involved in lung tissue development with the biodegradable poly(ϵ-caprolactone) before and after its chemical modification to evaluate potential for use in artificial lung formation. Also, the effect of polymer chemical modification on its mechanical and surface properties has been investigated. The poly(ϵ-caprolactone) surface was modified using aminolysis followed by immobilization of gelatine. The unmodified and modified polymer surfaces were characterized for roughness, tensile strength, and NCl-H292 metabolic cell activity. The results showed for the first time the possibility for NCI-H292 cells to adhere on this polymeric material. The Resazurin assay showed that the metabolic activity at 24 hours post seeding of 80% in the presence of the unmodified and greater than 100% in the presence of the modified polymer was observed. The roughness of the poly(ϵ-caprolactone) increased from 4 nm to 26 nm and the film strength increased from 0.01 kN to 0.045 kN when the material was chemically modified. The results obtained to date show potential for using modified poly(ϵ-caprolactone) as a scaffold for lung tissue engineering.