Borges, Adair L. | Search NASA

DOE OSTI2022

Experimental validation that human microbiome phages use alternative genetic coding

Previous bioinformatic analyses of metagenomic data have indicated that bacteriophages can use genetic codes different from those of their host bacteria. In particular, reassignment of stop codon TAG to glutamine (a variation known as ‘genetic code 15’) has been predicted. Here, we use LC-MS/MS-based metaproteomics of human fecal samples to provide experimental evidence of the use of genetic code 15 in two crAss-like phages. Furthermore, the proteomic data from several phage structural proteins supports the reassignment of the TAG stop codon to glutamine late in the phage infection cycle. Thus, our work experimentally validates the expression of genetic code 15 in human microbiome phages.

59 BASIC BIOLOGICAL SCIENCES↗

DOE OSTI2021

Species- and site-specific genome editing in complex bacterial communities

Knowledge of microbial gene functions comes from manipulating the DNA of individual strains in isolation from their natural communities. While this approach to microbial genetics has been foundational, its requirement for culturable microorganisms has left the majority of microbes and their interactions genetically unexplored. Here, we describe a generalizable strategy for editing the genomes of specific organisms within microbial communities. We identified genetically tractable bacteria within a community using Environmental Transformation Sequencing (ET-Seq), an approach in which non-targeted transposon integrations are mapped and quantified following community delivery. We next developed and used DNA-editing All-in-one RNA-guided CRISPR-Cas Transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-Seq, enabling organism- and locus-specific genetic manipulation within the community context. To illustrate the utility of our approach, we selectively edited closely related strains, measured gene fitness, and enriched targeted members within soil and infant gut microbiota. These results establish a new paradigm for targeted community editing relevant to research and applications on medical, agricultural, and industrial microbiomes.

59 BASIC BIOLOGICAL SCIENCES↗