Poly-γ-Glutamic Acid: Biodegradable Polymer for Potential Protection of Beneficial Viruses.

Poly-γ-glutamic acid (γ-PGA) is a naturally occurring polymer, which due to its biodegradable, non-toxic and non-immunogenic properties has been used successfully in the food, medical and wastewater industries. A major hurdle in bacteriophage application is the inability of phage to persist for extended periods in the environment due to their susceptibility to environmental factors such as temperature, sunlight, desiccation and irradiation. Thus, the aim of this study was to protect useful phage from the harmful effect of these environmental factors using the γ-PGA biodegradable polymer. In addition, the association between γ-PGA and phage was investigated. Formulated phage (with 1% γ-PGA) and non-formulated phage were exposed to 50 °C. A clear difference was noticed as viability of non-formulated phage was reduced to 21% at log10 1.3 PFU/mL, while phage formulated with γ-PGA was 84% at log10 5.2 PFU/mL after 24 h of exposure. In addition, formulated phage remained viable at log10 2.5 PFU/mL even after 24 h of exposure at pH 3 solution. In contrast, non-formulated phages were totally inactivated after the same time of exposure. In addition, non-formulated phages when exposed to UV irradiation died within 10 min. In contrast also phages formulated with 1% γ-PGA had a viability of log10 4.1 PFU/mL at the same exposure time. Microscopy showed a clear interaction between γ-PGA and phages. In conclusion, the results suggest that γ-PGA has an unique protective effect on phage particles.

Cross-species chimeras reveal BamA POTRA and ß-barrel domains must be fine-tuned for efficient OMP insertion.

BAM is a conserved molecular machine, the central component of which is BamA. Orthologues of BamA are found in all Gram-negative bacteria, chloroplasts and mitochondria where it is required for the folding and insertion of ß-barrel containing integral outer membrane proteins (OMPs) into the outer membrane. BamA binds unfolded ß-barrel precursors via the five polypeptide transport-associated (POTRA) domains at its N-terminus. The C-terminus of BamA folds into a ß-barrel domain, which tethers BamA to the outer membrane and is involved in OMP insertion. BamA orthologues are found in all Gram-negative bacteria and appear to function in a species-specific manner. Here we investigate the nature of this species-specificity by examining whether chimeric Escherichia coli BamA fusion proteins, carrying either the ß-barrel or POTRA domains from various BamA orthologues, can functionally replace E. coli BamA. We demonstrate that the ß-barrel domains of many BamA orthologues are functionally interchangeable. We show that defects in the orthologous POTRA domains can be rescued by compensatory mutations within the ß-barrel. These data reveal that the POTRA and barrel domains must be precisely aligned to ensure efficient OMP insertion.

Mutational and topological analysis of the Escherichia coli BamA protein.

The multi-protein ß-barrel assembly machine (BAM) of Escherichia coli is responsible for the folding and insertion of ß-barrel containing integral outer membrane proteins (OMPs) into the bacterial outer membrane. An essential component of this complex is the BamA protein, which binds unfolded ß-barrel precursors via the five polypeptide transport-associated (POTRA) domains in its N-terminus. The C-terminus of BamA contains a ß-barrel domain, which tethers BamA to the outer membrane and is also thought to be involved in OMP insertion. Here we mutagenize BamA using linker scanning mutagenesis and demonstrate that all five POTRA domains are essential for BamA protein function in our experimental system. Furthermore, we generate a homology based model of the BamA ß-barrel and test our model using insertion mutagenesis, deletion analysis and immunofluorescence to identify ß-strands, periplasmic turns and extracellular loops. We show that the surface-exposed loops of the BamA ß-barrel are essential.