The genomic revolution of the last twenty years has launched the biosciences into a new frontier. For scientists working on many organisms, the availability of a genome sequence has dramatically accelerated research. The effects have been profound: Genetic engineering is now standard laboratory practice; evolutionary biologists can trace entire genomes; and genetic risk factors have been defined for many human conditions. However, our rapidly accelerating ability to collect and assemble DNA sequence information has greatly outpaced our ability to assign biological meaning to it. This imbalance creates a need for high-throughput methods aimed at developing leads to gene function: phenomic technologies. Phenomic technologies seek to associate DNA sequences with phenotypes in a high-throughput manner to gain a functional understanding of genetic code.

Bacteria are ideal candidates for phenomic analyses because of their amenability to genetic manipulation and ease at which they can be grown and analyzed under varying parameters in-vitro. In addition, even the best-studied prokaryotes like E. coli lack functional annotation for a large fraction of their genes. Therefore, bacteria present excellent experimental systems to establish the power of phenomic approaches to generate testable hypotheses of gene function en masse.

Using E. coli as proof-of-principle, we show that combining large-scale phenomics with quantitative fitness measurements provides a high quality dataset, rich in discovery. The identification of >10,000 growth phenotypes allowed us to study gene essentiality, discover leads for gene function and drug action, and understand higher-order organization of the bacterial chromosome. We highlight insights concerning a gene involved in multiple antibiotic resistance and provide information about synergy of a broadly used combinatory antibiotic therapy, trimethoprim and sulfonamides. This dataset, publicly available at http://ecoliwiki.net/tools/chemgen/, is a valuable resource for both the microbiological and bioinformatic communities as it provides high confidence associations between hundreds of annotated and uncharacterized genes as well as inferences about the mode-of-action of several poorly understood drugs. This approach can be used for all culturable microbes with available ordered mutant libraries.

For many decades, it was thought that the phenomenon of cellular compartmentalization was exclusive to eukaryotes. Advances in cell imaging techniques have revealed that the presence of subcellular compartments is widespread throughout prokaryotes and that there exists a great diversity of organelles in bacteria and archaea. Recently, a new class of prokaryotic organelle has been discovered – the protein-bounded prokaryotic nanocompartments, also called encapsulins. Encapsulins are among the simplest of the prokaryotic organelles, often comprised of a two gene system; a shell-encoding gene and a cargo-encoding gene. Despite their simplicity, little is known about the biochemistry and physiological function of the encapsulins.

Here we investigate the structure and function of the encapsulin nanocompartments from various prokaryotes. First, we demonstrate that the existence of prokaryotic nanocompartments extends beyond the sequence homologs of the previously characterized encapsulins and that there exist additional, evolutionarily distinct families of encapsulins with unique cargo systems. We have characterized one of these new encapsulin families, which we name Family 2A, from the cyanobacteria Synechococcus elongatus PCC 7942. This encapsulin system hosts a cysteine desulfurase cargo enzyme that is directed to the interior of the nanocompartment via an N-terminal targeting sequence. We have determined the structure of the Family 2A encapsulin using cryo- electron microscopy to 2.2 Å resolution. This structure has yielded insights into the cargo- binding site within the organelle and the potential size and charge selectivity of the compartment. Additionally, we have characterized the cysteine desulfurase activity of the Family 2A encapsulin and show an increase in cargo enzyme activity upon encapsulation.

Finally, we examine the biochemistry and physiological role of a previously discovered encapsulin from Mycobacterium tuberculosis (Mtb). We demonstrate that the Mtb encapsulin is stable and active under the harsh conditions of the phagolysosome – the ecological niche we implicate the Mtb encapsulin is localized. Furthermore, we show Mtb encapsulin plays a functional role in Mtb’s resistance to oxidative stress.

Taken together, our work provides critical insight as to how the structure of the prokaryotic nanocompartments informs their function in vivo and show that this emerging class of protein-bounded organelle represents a new paradigm within the field of prokaryotic cell biology.