Optimized APEX2 peroxidase-mediated proximity labeling in fast- and slow-growing mycobacteria.

Proximity labeling is a technology for tagging proteins and other biomolecules in living cells. These methods use enzymes that generate reactive species whose properties afford high spatial resolution for the localization of proteins to subcellular compartments and the identification of endogenous interaction partners. Here we present the adaptation of the engineered peroxidase APEX2 to proximity labeling in mycobacteria, including the human pathogen Mycobacterium tuberculosis. APEX2 is uniquely suited for general use in bacteria because unlike other proximity labeling enzymes, it does not depend on metabolites like ATP that are found in the cytoplasm, but are absent from the bacterial periplasm. Importantly, we found that in slow-growing mycobacteria like M. tuberculosis, codon usage optimization is required for APEX2 export into the periplasm via fusion to an N-terminal secretion signal. APEX2 expressed from codon-optimized genes affords robust, compartment-specific protein labeling in the cytoplasm and the periplasm of both fast- and slow-growing species. Here we detail these updated constructs and provide an optimized protocol for APEX2-mediated protein labeling in mycobacteria. We expect this approach to be broadly useful for determining the localization of specific proteins, cataloging subcellular proteomes, and identifying interaction partners of 'bait' proteins expressed as fusions to APEX2.

Genomics analysis of hexanoic acid exposure in Drosophila species.

Drosophila sechellia is a dietary specialist endemic to the Seychelles islands that has evolved to consume the fruit of Morinda citrifolia. When ripe, the fruit of M. citrifolia contains octanoic acid and hexanoic acid, two medium-chain fatty acid volatiles that deter and are toxic to generalist insects. Drosophila sechellia has evolved resistance to these volatiles allowing it to feed almost exclusively on this host plant. The genetic basis of octanoic acid resistance has been the focus of multiple recent studies, but the mechanisms that govern hexanoic acid resistance in D. sechellia remain unknown. To understand how D. sechellia has evolved to specialize on M. citrifolia fruit and avoid the toxic effects of hexanoic acid, we exposed adult D. sechellia, D. melanogaster and D. simulans to hexanoic acid and performed RNA sequencing comparing their transcriptional responses to identify D. sechellia specific responses. Our analysis identified many more genes responding transcriptionally to hexanoic acid in the susceptible generalist species than in the specialist D. sechellia. Interrogation of the sets of differentially expressed genes showed that generalists regulated the expression of many genes involved in metabolism and detoxification whereas the specialist primarily downregulated genes involved in the innate immunity. Using these data, we have identified interesting candidate genes that may be critically important in aspects of adaptation to their food source that contains high concentrations of HA. Understanding how gene expression evolves during dietary specialization is crucial for our understanding of how ecological communities are built and how evolution shapes trophic interactions.