Characterization of antibiotic resistomes by reprogrammed bacteriophage-enabled functional metagenomics in clinical strains.

Functional metagenomics is a powerful experimental tool to identify antibiotic resistance genes (ARGs) in the environment, but the range of suitable host bacterial species is limited. This limitation affects both the scope of the identified ARGs and the interpretation of their clinical relevance. Here we present a functional metagenomics pipeline called Reprogrammed Bacteriophage Particle Assisted Multi-species Functional Metagenomics (DEEPMINE). This approach combines and improves the use of T7 bacteriophage with exchanged tail fibres and targeted mutagenesis to expand phage host-specificity and efficiency for functional metagenomics. These modified phage particles were used to introduce large metagenomic plasmid libraries into clinically relevant bacterial pathogens. By screening for ARGs in soil and gut microbiomes and clinical genomes against 13 antibiotics, we demonstrate that this approach substantially expands the list of identified ARGs. Many ARGs have species-specific effects on resistance; they provide a high level of resistance in one bacterial species but yield very limited resistance in a related species. Finally, we identified mobile ARGs against antibiotics that are currently under clinical development or have recently been approved. Overall, DEEPMINE expands the functional metagenomics toolbox for studying microbial communities.

Enhancing the Translational Capacity of E. coli by Resolving the Codon Bias.

Escherichia coli is a well-established and popular host for heterologous expression of proteins. The preference in the choice of synonymous codons (codon bias), however, might differ for the host and the original source of the recombinant protein, constituting a potential bottleneck in production. Codon choice affects the efficiency of translation by a complex and poorly understood mechanism. The availability of certain tRNA species is one of the factors that may curtail the capacity of translation. Here we provide a tRNA-overexpressing strategy that allows the resolution of the codon bias, and boosts the translational capacity of the popular host BL21(DE3) when rare codons are encountered. In the BL21(DE3)-derived strain, called SixPack, copies of the genes corresponding to the six least abundant tRNA species have been assembled in a synthetic fragment and inserted into a rRNA operon. This arrangement, while not interfering with the growth properties of the new strain, allows dynamic control of the transcription of the extra tRNA genes, providing significantly elevated levels of the rare tRNAs in the exponential growth phase. Results from expression assays of a panel of recombinant proteins of diverse origin and codon composition showed that the performance of SixPack surpassed that of the parental BL21(DE3) or a related strain equipped with a rare tRNA-expressing plasmid.