Comparative studies to assess bacterial communities on the clover phylloplane using MLST, DGGE and T-RFLP

Denaturing gradient gel electrophoresis (DGGE) and terminal restriction fragment length polymorphism (T-RFLP) were used to characterise the changes that occurred in Bacillus cereus group strains present in the phylloplane of clover Trifolium hybridum over 4 months. These strains had previously been analysed by multiple locus sequence typing (MLST). DGGE displayed many equally intense bands which indicated many equally abundant ribotypes. The bacterial community composition was variable and the leaves sampled as little as a week apart were found to have some temporal variability, indicating that diverse phylloplane bacterial communities follow sequential patterns from time to time. The B. cereus group community clearly clustered into early, mid and late branches, possibly due to multiple successional sequences occurring during growing seasons. The functionally and phylogenetically diverse microbial communities appeared to exhibit predictable successional patterns over shorter time scales. DGGE analysis with the molecular marker rpoB gave better resolution than 16S rRNA amplicons. There were no strong similarities between the dendrograms produced by DGGE, MLST and T-RFLP and the clustering produced by the automated T-RFLP method was variable even between the three restriction enzymes used. The DGGE–MLST method emerged as a superior method to T-RFLP–MLST for rapid typing of bacterial communities.     

Optimization of DNA delivery by three classes of hybrid nanoparticle/DNA complexes

Plasmid DNA encoding a luciferase reporter gene was complexed with each of six different hybrid nanoparticles (NPs) synthesized from mixtures of poly (D, L-lactide-co-glycolide acid) (PLGA 50:50) and the cationic lipids DOTAP (1, 2-Dioleoyl-3-Trimethyammonium-Propane) or DC-Chol {3β-[N-(N', N'-Dimethylaminoethane)-carbamyl] Cholesterol}. Particles were 100-400 nm in diameter and the resulting complexes had DNA adsorbed on the surface (out), encapsulated (in), or DNA adsorbed and encapsulated (both). A luciferase reporter assay was used to quantify DNA expression in 293 cells for the uptake of six different NP/DNA complexes. Optimal DNA delivery occurred for 10⁵ cells over a range of 500 ng - 10 μg of NPs containing 20-30 μg DNA per 1 mg of NPs. Uptake of DNA from NP/DNA complexes was found to be 500-600 times as efficient as unbound DNA. Regression analysis was performed and lines were drawn for DNA uptake over a four week interval. NP/DNA complexes with adsorbed NPs (out) showed a large initial uptake followed by a steep slope of DNA decline and large angle of declination; lines from uptake of adsorbed and encapsulated NPs (both) also exhibited a large initial uptake but was followed by a gradual slope of DNA decline and small angle of declination, indicating longer times of luciferase expression in 293 cells. NPs with encapsulated DNA only (in), gave an intermediate activity. The latter two effects were best seen with DOTAP-NPs while the former was best seen with DC-Chol-NPs. These results provide optimal conditions for using different hybrid NP/DNA complexes in vitro and in the future, will be tested in vivo.