# Cappable-Seq reveals the transcriptional landscape of stress responses in the bacterial endosymbiont Wolbachia

## TL;DR

This study uses Cappable-Seq to explore how Wolbachia bacteria respond to stress, revealing detailed insights into their transcriptional activity.

## Contribution

The study provides the first detailed analysis of Wolbachia's transcriptional start sites under stress using Cappable-Seq.

## Key findings

- Cappable-Seq revealed differential regulation of transcriptional start sites in Wolbachia under stress conditions.
- Doxycycline had the greatest impact on gene expression from primary transcriptional start sites.
- The study resolved the organization of the cifA/cifB operon linked to cytoplasmic incompatibility.

## Abstract

Bacterial endosymbionts are highly prevalent among invertebrate animals, in which they can confer fitness benefits such as pathogen defence and/or act as reproductive manipulators, inducing phenotypes including cytoplasmic incompatibility (CI). For the alpha-proteobacterium Wolbachia, its wide distribution among macroparasites and disease-transmitting arthropods coupled with mutualistic roles, reduction of vector competence and CI has found recent applications in the control of several vector-borne tropical diseases. However, in common with other bacterial endosymbionts, which often lose regulatory elements during genomic erosion, the degree to which Wolbachia can respond to environmental or pharmacological stressors is poorly understood. Here, we apply Cappable-Seq methodology to achieve unprecedented depth and resolution of transcriptional start sites (TSS) in two Wolbachia strains (wMelPop-CLA and wAlbB) that have been used to transinfect mosquitoes for arbovirus control. We exposed Wolbachia in mosquito cell lines to temperature stress (both strains) or antibiotics (wAlbB only) and observed that all classes of TSS (including antisense) exhibited differential regulation, some of which were associated with mobile elements and may control ncRNA expression. Of the three antibiotics used as pharmacological stressors (doxycycline, rifampicin and moxifloxacin), doxycycline had the greatest impact on differential expression from primary TSS. Cappable-Seq also resolved the organization of the bicistronic cifA/cifB operon that is responsible for inducing CI in Wolbachia hosts. The use of Cappable-Seq in this study enabled the resolution of the primary transcriptome of an obligate intracellular bacterium in unparalleled detail. Moreover, this methodology shows great promise for revealing regulation of symbiont functions in whole invertebrates.